Preface

Despite significant advances in our understanding of the pathological and biochemical changes that are associated with the motor neuron disease Amyotrophic Lateral Sclerosis (ALS), no cure currently exists. Although available treatments can temporarily slow disease progression, they are unable to prevent neuronal death. The groundbreaking discoveries in 2006 that implicated toxicity of the RNA/DNA-binding proteins TDP-43 and FUS in ALS triggered a flurry of research activities towards understanding the neurobiology and pathology of these proteins. Resultantly, the involvement of more than a dozen additional factors (e.g., C9ORF72, profilin, senataxin, etc.) was subsequently reported. Still, how these factors trigger neuronal dysfunction remains unclear, which has been a roadblock in the development of more effective therapeutics. The relative contribution of 'gain-of-toxicity' or 'loss-of-function' of ALS-linked factors is a key question. While initial research focused on RNA processing defects, recent studies demonstrated a widespread imbalance in genome damage versus repair rates, thus opening avenues for potential DNA repair-based therapeutics. This book debates recent advances in our understanding of the ALS group of diseases and outlines future directions for research activities towards finding a cure for these debilitating brain diseases. The diverse but complementary chapters highlight the need for an overarching approach to unravel the fundamental mechanisms of disease initiation and progression that will ultimately allow scientists and clinicians to design effective ways to develop improved treatment protocols for ALS patients.

The book contains eight chapters that cover both basic research/methodologies and therapeutic advances in the area of ALS, with a particular focus on emerging science and concepts. Both common and rare subsets of ALS are covered, including sporadic and inherited disease. Chapters address various topics, such as ALS-associated etiological factors TDP-43, FUS, and senataxin alongside emerging research on genome repair defects, mitochondrial dysfunction, exosomes, non-coding RNA/ biomarker discovery, R-loop as well as the recent advances in ALS therapy and utilization of axonal transport for drug delivery. These chapters are contributed by esteemed scientists from the United States, Italy, Sweden, Belgium and Costa Rica.

The editor would like to express his sincere gratitude to the chapter authors for their invaluable contributions.

**II**

**Section 3**

Novel Therapeutics and Challenges **113**

**Chapter 7 115**

**Chapter 8 135**

RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis *by Orietta Pansarasa, Stella Gagliardi, Daisy Sproviero and Cristina Cereda*

Targeting Axonal Transport: A New Therapeutic Avenue for ALS

*by Wenting Guo, Laura Fumagalli and Ludo Van Den Bosch*

**Dr. Muralidhar L. Hegde, PhD, FABAP**

Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist Research Institute, Affiliate of Weill Medical College, New York, Houston, Texas, USA

**1**

Section 1

New Paradigm of Genome

Instability and DNA

Repair Defects in ALS

### Section 1

New Paradigm of Genome Instability and DNA Repair Defects in ALS

**3**

**Chapter 1**

**Abstract**

The Role of TDP-43 in

*Joy Mitra and Muralidhar L. Hegde*

development of neuroprotective therapies.

**1. TDP-43 pathology: a predominant player in ALS**

Genome Repair and beyond in

Amyotrophic Lateral Sclerosis

The pathology of the RNA-/DNA-binding protein TDP-43, first implicated a decade ago in the motor neuron disease amyotrophic lateral sclerosis (ALS), has been subsequently linked to a wide spectrum of neurodegenerative diseases, including frontotemporal dementia (FTD), Alzheimer's disease (AD), and related dementia-associated disorders. ALS, also known as Lou Gehrig's disease, is a progressive, degenerative motor neuron disorder, characterized by a diverse etiopathology. TDP-43 pathology, mediated by a combination of several mutations in the *TARDBP* gene and stress factors, has been linked to more than 97% of ALS patients. We recently identified, for the first time, the critical involvement of TDP-43 in neuronal genome maintenance and the repair of DNA double-strand breaks (DSBs). Our studies showed that TDP-43 regulates the DNA break-sealing activities of the XRCC4-DNA Ligase 4 (LIG4) complex in DSB repair, suggesting that loss of genomic integrity in TDP-43-associated neurodegeneration may be amenable to a DNA repair-based intervention. In this chapter, we discuss the broader aspects of TDP-43 toxicityinduced pathomechanisms, including the emerging role of TDP-43 in neuronal DSB repair and its synergistic genotoxic effects with other neurodegeneration-associated etiologies that contribute significantly to neuronal dysfunction. We also discuss potential future perspectives and underscore how unraveling the molecular basis and implications of TDP-43-induced genome instability in ALS could guide the

**Keywords:** TDP-43, DNA damage, DNA double-strand breaks, nonhomologous end joining, XRCC4-DNA ligase 4, ALS, stress granule, Rab11, neurodegeneration

Transactive response DNA-binding protein 43 (TDP-43) is a versatile 43 kDa DNA-/RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. TDP-43's cytosolic aggregation and inclusion body formation are the key pathologic hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [1–3]. In these progressive motor neuron diseases, TDP-43 pathology manifests as the tau- and synuclein- negative and ubiquitinpositive inclusions in the anterior horn, spinal cord, neocortex, and hippocampus regions of the brain. In the last decade, tremendous scientific investigations have been carried out to understand the etiopathologies of TDP-43 toxicity in ALS and FTD. These studies demonstrated that TDP-43 pathology-linked ALS is one of the

#### **Chapter 1**

## The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis

*Joy Mitra and Muralidhar L. Hegde*

#### **Abstract**

The pathology of the RNA-/DNA-binding protein TDP-43, first implicated a decade ago in the motor neuron disease amyotrophic lateral sclerosis (ALS), has been subsequently linked to a wide spectrum of neurodegenerative diseases, including frontotemporal dementia (FTD), Alzheimer's disease (AD), and related dementia-associated disorders. ALS, also known as Lou Gehrig's disease, is a progressive, degenerative motor neuron disorder, characterized by a diverse etiopathology. TDP-43 pathology, mediated by a combination of several mutations in the *TARDBP* gene and stress factors, has been linked to more than 97% of ALS patients. We recently identified, for the first time, the critical involvement of TDP-43 in neuronal genome maintenance and the repair of DNA double-strand breaks (DSBs). Our studies showed that TDP-43 regulates the DNA break-sealing activities of the XRCC4-DNA Ligase 4 (LIG4) complex in DSB repair, suggesting that loss of genomic integrity in TDP-43-associated neurodegeneration may be amenable to a DNA repair-based intervention. In this chapter, we discuss the broader aspects of TDP-43 toxicityinduced pathomechanisms, including the emerging role of TDP-43 in neuronal DSB repair and its synergistic genotoxic effects with other neurodegeneration-associated etiologies that contribute significantly to neuronal dysfunction. We also discuss potential future perspectives and underscore how unraveling the molecular basis and implications of TDP-43-induced genome instability in ALS could guide the development of neuroprotective therapies.

**Keywords:** TDP-43, DNA damage, DNA double-strand breaks, nonhomologous end joining, XRCC4-DNA ligase 4, ALS, stress granule, Rab11, neurodegeneration

#### **1. TDP-43 pathology: a predominant player in ALS**

Transactive response DNA-binding protein 43 (TDP-43) is a versatile 43 kDa DNA-/RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. TDP-43's cytosolic aggregation and inclusion body formation are the key pathologic hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [1–3]. In these progressive motor neuron diseases, TDP-43 pathology manifests as the tau- and synuclein- negative and ubiquitinpositive inclusions in the anterior horn, spinal cord, neocortex, and hippocampus regions of the brain. In the last decade, tremendous scientific investigations have been carried out to understand the etiopathologies of TDP-43 toxicity in ALS and FTD. These studies demonstrated that TDP-43 pathology-linked ALS is one of the most complex neurological diseases due to TDP-43 pathomechanisms overlapping with other ALS-causing genes, such as C9orf72 [4–6], valosin-containing protein (VCP) [7–9], fused in sarcoma (FUS) [10], optineurin (OPTN) [11], ubiquilin 2 (UBQLN2) [12, 13], and ataxin 2 (ATXN2) [14, 15]. Strikingly, TDP-43 pathology has been detected in more than 97% of sporadic ALS cases (>90%), making it a predominant player in most ALS patients.

#### **1.1 Familial and sporadic ALS**

Due to highly overlapping symptoms, determining the sporadic or familial nature of the ALS disease at its time of onset is difficult based on clinical features alone. Familial ALS (FALS) is predicted based on genetic screening for *TARDBP* mutation(s) in the patient and matching the same mutation in the family member(s) with a positive clinical/medical history of neurodegeneration and dementia in the juvenile or early stage of life. Race, age, and gender are risk factors for progression of FALS. FALS incidences vary significantly from 5 to 10% among all ALS cases. The disease is more common in Caucasian men and women than African, Asian, and Hispanic populations [16]. Notably, clusters of ALS incidences have been associated with socio-economic conditions along with race. According to Centers for Disease Control and Prevention, ALS prevalence is highest in the Midwest and Northeast of US main land (5.7 and 5.5 per 100,000 population, respectively) [17]. In Europe, the prevalence of FALS was lower in the southern part [18]. Studies show that men are at slightly higher risk than women, with an average age of onset ~60 years. Patients usually survive for 1.5–4 years after diagnosis, depending on the disease aggressiveness [19].

FALS associated pathology affects anterior horn motor neurons in the cervical and lumbosacral regions in the spinal cord, frontal cortex, and cranial nerve motor neurons in the pons and medulla segments of brainstem. Both sporadic ALS (SALS) and FALS pathologies are associated with upper and/or lower motor neurons of the spinal cord. Upper motor neuron diseases accompany degeneration of lateral corticospinal tracts leading to hyperreflexia/spasticity and cardiac arrest. Conversely, lower motor neuron death leads to muscle atrophy and denervation.

FALS cases fall into to three categories, namely, autosomal dominant, autosomal recessive, and X-linked dominant. Autosomal dominance is the most common inherited pattern among the FALS cases, where a mutation in one copy of the ALS-linked gene is sufficient to develop ALS conditions. These patients usually have a strong positive family history and their children bear 50% risk of developing ALS. However, autosomal dominance can also happen in apparently sporadic ALS (because the genetic inheritance is unknown or poor medical history) with de novo dominant mutation(s). In such cases, patients' siblings may have very low risk but children may have up to 50% risk of developing ALS. To date, more than 50 mutations in *TARDBP* gene have been identified in ALS etiopathologies [20]. In addition, a study on a German cohort of non-SOD1 FALS patients revealed mutations in *TARDBP*, suggesting *TARDBP* gene mutation screening should be crucial among non-SOD1 FALS patients [21]. The known *TARDBP* mutations in FALS are G290A, G298S, A315T, M337V, N345K, A382T, and I383V; some of the common mutations identified in SALS are D169G, G287S, G294A, Q331K, G348C, R361S, and N390S/D (**Figure 1**) [21–27]. Most of these *TARDBP* mutations are located within exon 6, as commonly found in Caucasian and European ALS patients; however, a large cohort of Han Chinese population with non-SOD1 SALS did not show any such mutations in exon 6 [28].

SALS has been reported in an increasing number among people exposed to lead or other heavy metals [29–31], smoking [32, 33], and pesticides [34]. Increased exposure to organochlorine pesticides, biphenyls, and brominated flame retardants

**5**

populations [49].

**Figure 1.**

**and gain of toxicity**

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis*

used in household furniture is associated with higher risk for ALS [35–37]. Interestingly, while smoking has been shown to predispose individuals to ALS risk, alcohol consumption did not show any effect on ALS development [38]. This suggests that diet and exposure to various environmental toxins could increase the ALS susceptibility in the vulnerable population. Most notably, US veterans from the Gulf War [39, 40] and those with head injury [41, 42] are also at a greater risk for developing SALS. By targeted sequencing of exon 6 of *TARDBP* from brain samples of US veterans with SALS, we observed several missense mutations (both reported and novel) and the association of TDP-43 Q331K mutation with DNA DSB repair impairment [22]. Another well-known incidence of sporadic ALS is the Guamanian ALS, which falls on the Parkinsonism-dementia complex spectrum (ALS/PDC). This particular type of ALS in people living in Guam and Rota islands is caused by the consumption of cyanobacteria-infested cycad fruits containing the neurotoxin β-methylamino-L-alanine (BMAA) [43, 44]. BMAA exerts neurotoxicity by incorporating into amino acid sequences and inducing tau pathology, oxidative stress, glutathione depletion, and protein aggregation [45–48]. Genome-wide analyses for ALS-/PDC-associated chromosomal loci revealed three disease-specific loci—two regions on chromosome 12 and the MAPT region on chromosome 17—in these

*ALS-related mutations are indicated in red, and sporadic ALS-linked mutations are in blue color.*

*TDP-43 protein's structural organization. Schematic representation of TDP-43 protein structure shows N-terminal domain of TDP-43 consists of highly ordered nucleic acid binding domains, namely, RNA recognition motifs (RRM) 1 and 2. RRM1 domain follows a bifurcated nuclear localization signal sequence (82–98aa)—NLS1 and NLS2. RRM1 domain spans from 104 to 176aa. RRM2 domain ranges from 192aa to 262aa including bifurcated nuclear export signal sequence (239–250aa)—NES1 and NES2. Later part in the C-terminal domain is mostly disordered and contains majority of the pathogenic mutational hotspots. Familial* 

**1.2 Unique feature of TDP-43 pathology: dual role of loss of function** 

Given that TDP-43 contains a prion-like domain, its cellular content and distribution are two contributing factors for preventing the protein aggregation leading to the onset of disease. These two conditions are maintained by TDP-43's autoregulatory feedback loop mechanism. TDP-43 is mainly a nuclear protein, but it also shuttles to the cytosol, mitochondria, and other cellular organelles in response to various stimuli. In ALS/FTD pathology, TDP-43 mislocalization, due to pathogenic mutations and/or protein aggregation, is known as the primary culprit for the onset of neurodegeneration. Studies suggest that initiation of caspase-3/7 activation by TDP-43 toxicity facilitates the disease progression by cleaving mislocalized TDP-43 into 35 and 25 kDa fragments. These highly aggregation-prone fragments promote inclusion body formations with the hyperphosphorylated and polyubiquitinated C-terminal domain of TDP-43 [50, 51]. In addition to the C-terminal fragment, full-length and homo-dimerized TDP-43 also has been identified in the spinal cord

*DOI: http://dx.doi.org/10.5772/intechopen.92696*

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.92696*

#### **Figure 1.**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

predominant player in most ALS patients.

depending on the disease aggressiveness [19].

**1.1 Familial and sporadic ALS**

most complex neurological diseases due to TDP-43 pathomechanisms overlapping with other ALS-causing genes, such as C9orf72 [4–6], valosin-containing protein (VCP) [7–9], fused in sarcoma (FUS) [10], optineurin (OPTN) [11], ubiquilin 2 (UBQLN2) [12, 13], and ataxin 2 (ATXN2) [14, 15]. Strikingly, TDP-43 pathology has been detected in more than 97% of sporadic ALS cases (>90%), making it a

Due to highly overlapping symptoms, determining the sporadic or familial nature of the ALS disease at its time of onset is difficult based on clinical features alone. Familial ALS (FALS) is predicted based on genetic screening for *TARDBP* mutation(s) in the patient and matching the same mutation in the family member(s) with a positive clinical/medical history of neurodegeneration and dementia in the juvenile or early stage of life. Race, age, and gender are risk factors for progression of FALS. FALS incidences vary significantly from 5 to 10% among all ALS cases. The disease is more common in Caucasian men and women than African, Asian, and Hispanic populations [16]. Notably, clusters of ALS incidences have been associated with socio-economic conditions along with race. According to Centers for Disease Control and Prevention, ALS prevalence is highest in the Midwest and Northeast of US main land (5.7 and 5.5 per 100,000 population, respectively) [17]. In Europe, the prevalence of FALS was lower in the southern part [18]. Studies show that men are at slightly higher risk than women, with an average age of onset ~60 years. Patients usually survive for 1.5–4 years after diagnosis,

FALS associated pathology affects anterior horn motor neurons in the cervical and lumbosacral regions in the spinal cord, frontal cortex, and cranial nerve motor neurons in the pons and medulla segments of brainstem. Both sporadic ALS (SALS) and FALS pathologies are associated with upper and/or lower motor neurons of the spinal cord. Upper motor neuron diseases accompany degeneration of lateral corticospinal tracts leading to hyperreflexia/spasticity and cardiac arrest. Conversely,

FALS cases fall into to three categories, namely, autosomal dominant, autosomal recessive, and X-linked dominant. Autosomal dominance is the most common inherited pattern among the FALS cases, where a mutation in one copy of the ALS-linked gene is sufficient to develop ALS conditions. These patients usually have a strong positive family history and their children bear 50% risk of developing ALS. However, autosomal dominance can also happen in apparently sporadic ALS (because the genetic inheritance is unknown or poor medical history) with de novo dominant mutation(s). In such cases, patients' siblings may have very low risk but children may have up to 50% risk of developing ALS. To date, more than 50 mutations in *TARDBP* gene have been identified in ALS etiopathologies [20]. In addition, a study on a German cohort of non-SOD1 FALS patients revealed mutations in *TARDBP*, suggesting *TARDBP* gene mutation screening should be crucial among non-SOD1 FALS patients [21]. The known *TARDBP* mutations in FALS are G290A, G298S, A315T, M337V, N345K, A382T, and I383V; some of the common mutations identified in SALS are D169G, G287S, G294A, Q331K, G348C, R361S, and N390S/D (**Figure 1**) [21–27]. Most of these *TARDBP* mutations are located within exon 6, as commonly found in Caucasian and European ALS patients; however, a large cohort of Han Chinese population with non-SOD1 SALS did not show any such mutations in exon 6 [28]. SALS has been reported in an increasing number among people exposed to lead or other heavy metals [29–31], smoking [32, 33], and pesticides [34]. Increased exposure to organochlorine pesticides, biphenyls, and brominated flame retardants

lower motor neuron death leads to muscle atrophy and denervation.

**4**

*TDP-43 protein's structural organization. Schematic representation of TDP-43 protein structure shows N-terminal domain of TDP-43 consists of highly ordered nucleic acid binding domains, namely, RNA recognition motifs (RRM) 1 and 2. RRM1 domain follows a bifurcated nuclear localization signal sequence (82–98aa)—NLS1 and NLS2. RRM1 domain spans from 104 to 176aa. RRM2 domain ranges from 192aa to 262aa including bifurcated nuclear export signal sequence (239–250aa)—NES1 and NES2. Later part in the C-terminal domain is mostly disordered and contains majority of the pathogenic mutational hotspots. Familial ALS-related mutations are indicated in red, and sporadic ALS-linked mutations are in blue color.*

used in household furniture is associated with higher risk for ALS [35–37]. Interestingly, while smoking has been shown to predispose individuals to ALS risk, alcohol consumption did not show any effect on ALS development [38]. This suggests that diet and exposure to various environmental toxins could increase the ALS susceptibility in the vulnerable population. Most notably, US veterans from the Gulf War [39, 40] and those with head injury [41, 42] are also at a greater risk for developing SALS. By targeted sequencing of exon 6 of *TARDBP* from brain samples of US veterans with SALS, we observed several missense mutations (both reported and novel) and the association of TDP-43 Q331K mutation with DNA DSB repair impairment [22]. Another well-known incidence of sporadic ALS is the Guamanian ALS, which falls on the Parkinsonism-dementia complex spectrum (ALS/PDC). This particular type of ALS in people living in Guam and Rota islands is caused by the consumption of cyanobacteria-infested cycad fruits containing the neurotoxin β-methylamino-L-alanine (BMAA) [43, 44]. BMAA exerts neurotoxicity by incorporating into amino acid sequences and inducing tau pathology, oxidative stress, glutathione depletion, and protein aggregation [45–48]. Genome-wide analyses for ALS-/PDC-associated chromosomal loci revealed three disease-specific loci—two regions on chromosome 12 and the MAPT region on chromosome 17—in these populations [49].

#### **1.2 Unique feature of TDP-43 pathology: dual role of loss of function and gain of toxicity**

Given that TDP-43 contains a prion-like domain, its cellular content and distribution are two contributing factors for preventing the protein aggregation leading to the onset of disease. These two conditions are maintained by TDP-43's autoregulatory feedback loop mechanism. TDP-43 is mainly a nuclear protein, but it also shuttles to the cytosol, mitochondria, and other cellular organelles in response to various stimuli. In ALS/FTD pathology, TDP-43 mislocalization, due to pathogenic mutations and/or protein aggregation, is known as the primary culprit for the onset of neurodegeneration. Studies suggest that initiation of caspase-3/7 activation by TDP-43 toxicity facilitates the disease progression by cleaving mislocalized TDP-43 into 35 and 25 kDa fragments. These highly aggregation-prone fragments promote inclusion body formations with the hyperphosphorylated and polyubiquitinated C-terminal domain of TDP-43 [50, 51]. In addition to the C-terminal fragment, full-length and homo-dimerized TDP-43 also has been identified in the spinal cord

and brain of ALS patients [52, 53]. Furthermore, recent findings have highlighted that this protein aggregation enhances the aggregation propensities of other prion-like domain containing proteins. The interaction of these prion-like proteins increasing the disease complexity severalfold higher than that of single-protein toxicities [54]. TDP-43's cellular shuttling is regulated by the bifurcated nuclear localization signal (NLS) and nuclear export signal (NES) sequences (**Figure 1**), and loss of either of these signal sequences could be deleterious for cell survival. In vitro studies expressing NLS-deleted TDP-43 found accelerated cytoplasmic aggregate formation along with aberrant RNA processing defects [55, 56]. This phenomenon was further supported by in vivo studies with a transgenic mouse model and transcriptomic analysis in ALS/FTD human brain samples, which revealed significantly altered mRNA splicing of histones and aberrant chromatin remodeling following cytosolic accumulation of toxic TDP-43 [57]. Almost all of the pathogenic sporadic and familial mutations in TDP-43 induce its nuclear clearance and cytosolic sequestration to varying extents. However, in vitro studies with the FALS-linked, NLS-specific A90V mutation in TDP-43 found disease-like detergent insoluble cytosolic aggregates, confirming the crucial role of NLS/NES sequences in TDP-43 homeostasis, and further suggesting cytosolic aggregation of TDP-43 is a determining factor in motor neuron death [58, 59].

Persistent neuroinflammation is considered one of the leading causes for motor neuron death in ALS/FTD and also affects patients' therapeutic response. Innate immune responses, including microglial hyperactivation and astrogliosis, were consistently documented in human postmortem ALS human brain and spinal cord, as well as brains from ALS-model mice [60–63]. TDP-43 plays a vital role in regulation of neuroinflammation by inhibiting NF-κB activation, possibly through competitive binding to importin α3 [64]. TDP-43 depletion or nuclear loss induces extracellular secretion of TNF-α and enhanced nuclear localization of NF-κB p65 in glia and neurons [65–67]. Furthermore, inflammatory responses increase nuclear loss of TDP-43 and progression of ALS/FTD pathology.

#### **2. Broad functions of TDP-43 in central nervous system (CNS)**

#### **2.1 RNA transactions**

Extensive research in the last decade on TDP-43 toxicity in ALS has established that TDP-43 has multiple functions, including autoregulation of its levels through a negative feedback loop and controlling a diverse set of RNA-associated mechanisms, including pre-mRNA processing and splicing, micro RNA biogenesis, and RNA transport, transcription, and translation. Importantly, TDP-43 also regulates protein levels of critical RNA-binding proteins involved in RNA splicing, including SRSF1, PTB, and hnRNP L [68, 69]. TDP-43's autoregulation is solely controlled by the TDP-43-binding region (TDPBR) domain, which is comprised of several polyadenylation (pA) sites. The silent intron-7 linked to the TDPBR regulates the alternative splicing of pA sites based on the TDP-43 cellular concentration. In the steady state, pA1 allows cytoplasmic shuttling of TDP-43 mRNA for the appropriate amount of protein production. When overexpressed, TDP-43 binds threefold more to TDPBR and increases polymerase II binding at this sequence, which then processes intron-7 to remove the pA1 signal sequence and enforces the use of pA2 and pA4 sites [70]. The use of pA2/A4 sites makes the TDP-43 mRNA partially retained in the nucleus and leads to reduced production of TDP-43 protein in the cell [71]. Moreover, a recent study on TDP-43 autoregulation using the *Drosophila* model has identified human homolog transcription elongation regulator 1 (TCGER1) of

**7**

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis*

*Drosophila* protein GC42724 as having a role in TDP-43 mRNA alternative splicing and nucleo-cytoplasmic shuttling. TCGER1 regulates TDP-43 production through

TDP-43 contains two RNA recognition motifs (RRMs): RRM1 and RRM2 (**Figure 1**). Each RRM contains two highly conserved RNA recognition segments: octameric ribonucleoprotein 1 and hexameric ribonucleoprotein 2 [73]. TDP-43 RRMs bind a minimum of six UG/TG repeats, and its binding affinity increases with increasing numbers of repeats [74]. However, the binding preference is quite different between two motifs; unlike RRM1, RRM2 prefers a short stretch of UG

Amino acid sequence analysis reveals a high degree of sequence similarities in the N-terminal domains of TDP-43 among human, mouse, rat, *Drosophila melanogaster*, and *Caenorhabditis elegans*, suggesting that TDP-43's function is crucial in these organisms. The C-terminal region of TDP-43 is predominantly disordered in its native structure, comprising a prion-like domain and several glycine-asparagine rich patches that contribute to the exon-skipping activity of TDP-43 (**Figure 1**). In vivo studies have revealed that TDP-43 regulates its splicing mechanism via its C-terminal glycine-rich domains [76]. A recent study of TDP-43 knockdown in a *Drosophila* model expressing chimeric repressor proteins demonstrated that TDP-43 in motor neurons regulates RNA splicing fidelity through splicing repression [77]. These findings are in agreement with previous studies reporting TDP-43's interaction with more than 6000 mRNAs in the mouse brain and TDP-43 depletioninduced altered splicing of ~900 mRNAs causing neuronal vulnerability (**Figure 2**) [78]. Furthermore, a recent study with ALS-TDP-43 M337V mutant knock-in mouse model has revealed mRNA splicing deregulation as the major pathological hallmark of mutant TDP-43, even in the absence of any noticeable motor deficits [79]. The mammalian TDP-43 primary transcript produces 11 alternatively spliced variants of mRNA, further supporting the functional complexity of TDP-43 [80]. As part of the hnRNP family, TDP-43 has a similar domain organization to hnRNP A1 and A2/B1 [81]. TDP-43's C-terminal domain interacts with a number of hnRNP family proteins, particularly hnRNP A2/B1 and A1, to form the hnRNP rich complex that is crucial for splicing inhibition [82]. HnRNP A1, a 34 kDa protein, is abundant in motor neurons and has been implicated in the pathomechanism of spino-muscular atrophy [83]. Mislocalized TDP-43 modulates the inclusion of exon 7B in the alternatively spliced hnRNP A1 transcript, leading to the production of an isoform with an extended prion-like domain [84–86]. Because TDP-43 and hnRNP A1 interact directly, increasing the aggregation propensity of hnRNP A1 could

strongly influence TDP-43's aggregation in a synergistic pattern [87].

In addition to pre-mRNA splicing regulation, the N-terminal domain (NTD) of TDP-43 plays an essential role in protein stabilization and prevention of pathogenic cytoplasmic aggregation. In vitro studies have shown that N-terminal Leu71 and Val72 in the β7 strand region at the interface are crucial for its homodimerization into dimers and/or tetramers in a concentration-dependent manner [88]. Furthermore, single amino acid substitutions at V31R and T32R disrupt TDP-43's splicing activity and induce aggregation. The L28A mutation strongly destabilizes the TDP-43 NTD and promotes its mislocalization, and the L27A mutation increases its monomeric forms [89]. Recent findings by Chen et al. show that the K181E mutation near the TDP-43 RRM1 domain disrupts TDP-43's ability to process mRNA, induces mutant TDP-43 hyperphosphorylation, enhances detergent-resistant aggregation propensity by several fold, and leads to more wild-type TDP-43 in the nuclear and cytoplasmic inclusion bodies [90]. TDP-43 oligomerization has been reported to be the first toxic intermediate in TDP-43 proteinopathies [91]. An RNA-mediated intervention strategy showed inhibition of TDP-43 misfolding in

*DOI: http://dx.doi.org/10.5772/intechopen.92696*

repeats over long repeats ((UG)3 > (UG)6) [75].

its TDPBR region [72].

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.92696*

*Drosophila* protein GC42724 as having a role in TDP-43 mRNA alternative splicing and nucleo-cytoplasmic shuttling. TCGER1 regulates TDP-43 production through its TDPBR region [72].

TDP-43 contains two RNA recognition motifs (RRMs): RRM1 and RRM2 (**Figure 1**). Each RRM contains two highly conserved RNA recognition segments: octameric ribonucleoprotein 1 and hexameric ribonucleoprotein 2 [73]. TDP-43 RRMs bind a minimum of six UG/TG repeats, and its binding affinity increases with increasing numbers of repeats [74]. However, the binding preference is quite different between two motifs; unlike RRM1, RRM2 prefers a short stretch of UG repeats over long repeats ((UG)3 > (UG)6) [75].

Amino acid sequence analysis reveals a high degree of sequence similarities in the N-terminal domains of TDP-43 among human, mouse, rat, *Drosophila melanogaster*, and *Caenorhabditis elegans*, suggesting that TDP-43's function is crucial in these organisms. The C-terminal region of TDP-43 is predominantly disordered in its native structure, comprising a prion-like domain and several glycine-asparagine rich patches that contribute to the exon-skipping activity of TDP-43 (**Figure 1**). In vivo studies have revealed that TDP-43 regulates its splicing mechanism via its C-terminal glycine-rich domains [76]. A recent study of TDP-43 knockdown in a *Drosophila* model expressing chimeric repressor proteins demonstrated that TDP-43 in motor neurons regulates RNA splicing fidelity through splicing repression [77]. These findings are in agreement with previous studies reporting TDP-43's interaction with more than 6000 mRNAs in the mouse brain and TDP-43 depletioninduced altered splicing of ~900 mRNAs causing neuronal vulnerability (**Figure 2**) [78]. Furthermore, a recent study with ALS-TDP-43 M337V mutant knock-in mouse model has revealed mRNA splicing deregulation as the major pathological hallmark of mutant TDP-43, even in the absence of any noticeable motor deficits [79]. The mammalian TDP-43 primary transcript produces 11 alternatively spliced variants of mRNA, further supporting the functional complexity of TDP-43 [80]. As part of the hnRNP family, TDP-43 has a similar domain organization to hnRNP A1 and A2/B1 [81]. TDP-43's C-terminal domain interacts with a number of hnRNP family proteins, particularly hnRNP A2/B1 and A1, to form the hnRNP rich complex that is crucial for splicing inhibition [82]. HnRNP A1, a 34 kDa protein, is abundant in motor neurons and has been implicated in the pathomechanism of spino-muscular atrophy [83]. Mislocalized TDP-43 modulates the inclusion of exon 7B in the alternatively spliced hnRNP A1 transcript, leading to the production of an isoform with an extended prion-like domain [84–86]. Because TDP-43 and hnRNP A1 interact directly, increasing the aggregation propensity of hnRNP A1 could strongly influence TDP-43's aggregation in a synergistic pattern [87].

In addition to pre-mRNA splicing regulation, the N-terminal domain (NTD) of TDP-43 plays an essential role in protein stabilization and prevention of pathogenic cytoplasmic aggregation. In vitro studies have shown that N-terminal Leu71 and Val72 in the β7 strand region at the interface are crucial for its homodimerization into dimers and/or tetramers in a concentration-dependent manner [88]. Furthermore, single amino acid substitutions at V31R and T32R disrupt TDP-43's splicing activity and induce aggregation. The L28A mutation strongly destabilizes the TDP-43 NTD and promotes its mislocalization, and the L27A mutation increases its monomeric forms [89]. Recent findings by Chen et al. show that the K181E mutation near the TDP-43 RRM1 domain disrupts TDP-43's ability to process mRNA, induces mutant TDP-43 hyperphosphorylation, enhances detergent-resistant aggregation propensity by several fold, and leads to more wild-type TDP-43 in the nuclear and cytoplasmic inclusion bodies [90]. TDP-43 oligomerization has been reported to be the first toxic intermediate in TDP-43 proteinopathies [91]. An RNA-mediated intervention strategy showed inhibition of TDP-43 misfolding in

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

determining factor in motor neuron death [58, 59].

loss of TDP-43 and progression of ALS/FTD pathology.

**2.1 RNA transactions**

**2. Broad functions of TDP-43 in central nervous system (CNS)**

and brain of ALS patients [52, 53]. Furthermore, recent findings have highlighted that this protein aggregation enhances the aggregation propensities of other prion-like domain containing proteins. The interaction of these prion-like proteins increasing the disease complexity severalfold higher than that of single-protein toxicities [54]. TDP-43's cellular shuttling is regulated by the bifurcated nuclear localization signal (NLS) and nuclear export signal (NES) sequences (**Figure 1**), and loss of either of these signal sequences could be deleterious for cell survival. In vitro studies expressing NLS-deleted TDP-43 found accelerated cytoplasmic aggregate formation along with aberrant RNA processing defects [55, 56]. This phenomenon was further supported by in vivo studies with a transgenic mouse model and transcriptomic analysis in ALS/FTD human brain samples, which revealed significantly altered mRNA splicing of histones and aberrant chromatin remodeling following cytosolic accumulation of toxic TDP-43 [57]. Almost all of the pathogenic sporadic and familial mutations in TDP-43 induce its nuclear clearance and cytosolic sequestration to varying extents. However, in vitro studies with the FALS-linked, NLS-specific A90V mutation in TDP-43 found disease-like detergent insoluble cytosolic aggregates, confirming the crucial role of NLS/NES sequences in TDP-43 homeostasis, and further suggesting cytosolic aggregation of TDP-43 is a

Persistent neuroinflammation is considered one of the leading causes for motor neuron death in ALS/FTD and also affects patients' therapeutic response. Innate immune responses, including microglial hyperactivation and astrogliosis, were consistently documented in human postmortem ALS human brain and spinal cord, as well as brains from ALS-model mice [60–63]. TDP-43 plays a vital role in regulation of neuroinflammation by inhibiting NF-κB activation, possibly through competitive binding to importin α3 [64]. TDP-43 depletion or nuclear loss induces extracellular secretion of TNF-α and enhanced nuclear localization of NF-κB p65 in glia and neurons [65–67]. Furthermore, inflammatory responses increase nuclear

Extensive research in the last decade on TDP-43 toxicity in ALS has established that TDP-43 has multiple functions, including autoregulation of its levels through a negative feedback loop and controlling a diverse set of RNA-associated mechanisms, including pre-mRNA processing and splicing, micro RNA biogenesis, and RNA transport, transcription, and translation. Importantly, TDP-43 also regulates protein levels of critical RNA-binding proteins involved in RNA splicing, including SRSF1, PTB, and hnRNP L [68, 69]. TDP-43's autoregulation is solely controlled by the TDP-43-binding region (TDPBR) domain, which is comprised of several polyadenylation (pA) sites. The silent intron-7 linked to the TDPBR regulates the alternative splicing of pA sites based on the TDP-43 cellular concentration. In the steady state, pA1 allows cytoplasmic shuttling of TDP-43 mRNA for the appropriate amount of protein production. When overexpressed, TDP-43 binds threefold more to TDPBR and increases polymerase II binding at this sequence, which then processes intron-7 to remove the pA1 signal sequence and enforces the use of pA2 and pA4 sites [70]. The use of pA2/A4 sites makes the TDP-43 mRNA partially retained in the nucleus and leads to reduced production of TDP-43 protein in the cell [71]. Moreover, a recent study on TDP-43 autoregulation using the *Drosophila* model has identified human homolog transcription elongation regulator 1 (TCGER1) of

**6**

#### **Figure 2.**

*TDP-43 proteinopathy causes RNA processing defects. TDP-43 being a major functional subunit of spliceosomal complex regulates the RNA maturation processes of hundreds of target genes. Many of which are directly associated with amyotrophic lateral sclerosis (ALS) and FTD. TDP-43, through its RRM domains, binds mainly to intronic and 3*ʹ *UTR-located UG-repeat sequences of pre-mRNA to modulate their splicing events. In healthy condition, TDP-43 strongly promotes normal splicing events and inhibits disease-causing aberrant alternative splicing. However, ALS/FTD-TDP-43 pathology induces aberrant splicing several folds higher than normal splicing leading to mRNA dysregulation-associated proteinopathies in motor neurons and glia. Moreover, exposure to various environmental toxins like heavy metals (Lead, iron, zinc), pesticides (organochloride compounds), and food toxins (BMAA) could cause pathogenic alterations in TDP-43 leading to its mislocalization and dysregulation of its splicing activity as a result causing sporadic ALS.*

wild-type and pathogenic ALS-TDP-43 mutants, further suggesting a critical role for RRM domains in TDP-43 pathology.

MicroRNAs (miRNAs) are the master regulators of a number of vital cellular mechanisms and diseases, including neurodegeneration and genomic instability. TDP-43 regulates a subset of miRNAs by direct interaction and modulates their biogenesis through Drosha and Dicer complexes [92]. In the brain, neurexin-1 (NRXN1) controls vesicular trafficking between synaptic junctions. TDP-43 has been shown to bind mir-NID1 to suppress NRXN1 expression and thereby inhibit neuronal development and functionality [93]. Given that long noncoding RNA (lncRNA) confers a higher degree of gene expression regulation, TDP-43's increased interaction with two crucial lncRNAs, MALAT1 and NEAT1, possibly modulates the RNA metabolism dysregulation of ALS- and FTD-associated TDP-43 pathology [94, 95]. Furthermore, lncRNA gadd7, which is involved in cell cycle regulation and DDR signaling, orchestrates TDP-43's interaction with CDK6 mRNA that leads to its controlled decay [96].

#### **2.2 Stress granules**

Stress granule (SG) assembly is a dynamic, reversible process that promotes cell survival when stress factors are present. Membraneless SG organelles vary in their morphology and building composition in a cell type-specific manner [97, 98]. As long as the assembly/disassembly ratio is maintained in healthy cells, SGs act as the emergency store house for certain classes of RNA and protein molecules to protect

**9**

**Figure 3.**

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis*

them from the various stress stimuli (**Figure 3**). However, altered SG assembly processes are associated with a number of human diseases, including neurodegenerative disorders and dementia [99]. Recent discoveries indicate SGs are critical players in modulating signal circuits determining cell death versus survival in response to

Emerging evidence suggests that TDP-43, but not FUS, regulates SG dynamics and secondary polymerization of TIA-1, which is essential for SG assembly (**Figure 3**) [100]. TIA-1 is an RNA-binding protein and classical SG marker and has more recently been considered as a candidate gene for ALS and ALS/FTD due to its rare heterozygous mutations (P362L and E384K) in the conserved amino acid residues within its low complexity domain [101]. TDP-43 increases its association with TIA-1 in a time-dependent manner in response to the osmotic and oxidative stressor, Sorbitol, in primary glial cells or other stressor-induced SGs in primary cortical neurons and astrocytes [102]. Studies have also shown aging as a crucial modulator of SG dynamics in neurodegenerative conditions. Notably, wild-type and ALS-linked pathogenic TDP-43 mutants show distinct patterns of stress formation rates and morphology. Mutant TDP-43 exhibits faster stress granule incorporation along with rapid increases in granule-size, while wild-type TDP-43 forms increasing numbers of granules with consistent size over time [99]. Apart from the TIA-1 aggregation regulation, TDP-43 also controls the mRNA level of G3BP1, an essential SG initiation factor. The loss of functional TDP-43 or pathogenic TDP-43-mediated G3BP1 mRNA depletion perturbs the interaction between SG components and processing bodies, leading to impaired storage of polyadenylated mRNAs [103]. However, different TDP-43 ALS-mutant variants exhibit differential regulation mechanisms. For instance, TDP-43 D169G does not affect SG formation mechanism, but R361S and A382T variants show loss-of-function phenotypes with

*TDP-43 is a critical component of stress granule. Stress granule formation, in normal condition, is a very important cellular defense mechanism to overcome stress responses. Stress granule assembly and disassembly is a reversible process in healthy cells. It's a membraneless structure consisting of stress granule marker proteins TIA-1, G3BP-1, and TDP-43 as the major structural component. Given that ALS-/FTD-associated TDP-43 pathology induces aberrant cytosolic sequestration of toxic TDP-43, it impairs the reversible nature of stress* 

*granule formation mechanism, thereby inducing the seeding mechanism for protein aggregation.*

*DOI: http://dx.doi.org/10.5772/intechopen.92696*

stress exposure.

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.92696*

them from the various stress stimuli (**Figure 3**). However, altered SG assembly processes are associated with a number of human diseases, including neurodegenerative disorders and dementia [99]. Recent discoveries indicate SGs are critical players in modulating signal circuits determining cell death versus survival in response to stress exposure.

Emerging evidence suggests that TDP-43, but not FUS, regulates SG dynamics and secondary polymerization of TIA-1, which is essential for SG assembly (**Figure 3**) [100]. TIA-1 is an RNA-binding protein and classical SG marker and has more recently been considered as a candidate gene for ALS and ALS/FTD due to its rare heterozygous mutations (P362L and E384K) in the conserved amino acid residues within its low complexity domain [101]. TDP-43 increases its association with TIA-1 in a time-dependent manner in response to the osmotic and oxidative stressor, Sorbitol, in primary glial cells or other stressor-induced SGs in primary cortical neurons and astrocytes [102]. Studies have also shown aging as a crucial modulator of SG dynamics in neurodegenerative conditions. Notably, wild-type and ALS-linked pathogenic TDP-43 mutants show distinct patterns of stress formation rates and morphology. Mutant TDP-43 exhibits faster stress granule incorporation along with rapid increases in granule-size, while wild-type TDP-43 forms increasing numbers of granules with consistent size over time [99]. Apart from the TIA-1 aggregation regulation, TDP-43 also controls the mRNA level of G3BP1, an essential SG initiation factor. The loss of functional TDP-43 or pathogenic TDP-43-mediated G3BP1 mRNA depletion perturbs the interaction between SG components and processing bodies, leading to impaired storage of polyadenylated mRNAs [103]. However, different TDP-43 ALS-mutant variants exhibit differential regulation mechanisms. For instance, TDP-43 D169G does not affect SG formation mechanism, but R361S and A382T variants show loss-of-function phenotypes with

#### **Figure 3.**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

wild-type and pathogenic ALS-TDP-43 mutants, further suggesting a critical role

*to its mislocalization and dysregulation of its splicing activity as a result causing sporadic ALS.*

*TDP-43 proteinopathy causes RNA processing defects. TDP-43 being a major functional subunit of spliceosomal complex regulates the RNA maturation processes of hundreds of target genes. Many of which are directly associated with amyotrophic lateral sclerosis (ALS) and FTD. TDP-43, through its RRM domains, binds mainly to intronic and 3*ʹ *UTR-located UG-repeat sequences of pre-mRNA to modulate their splicing events. In healthy condition, TDP-43 strongly promotes normal splicing events and inhibits disease-causing aberrant alternative splicing. However, ALS/FTD-TDP-43 pathology induces aberrant splicing several folds higher than normal splicing leading to mRNA dysregulation-associated proteinopathies in motor neurons and glia. Moreover, exposure to various environmental toxins like heavy metals (Lead, iron, zinc), pesticides (organochloride compounds), and food toxins (BMAA) could cause pathogenic alterations in TDP-43 leading* 

MicroRNAs (miRNAs) are the master regulators of a number of vital cellular mechanisms and diseases, including neurodegeneration and genomic instability. TDP-43 regulates a subset of miRNAs by direct interaction and modulates their biogenesis through Drosha and Dicer complexes [92]. In the brain, neurexin-1 (NRXN1) controls vesicular trafficking between synaptic junctions. TDP-43 has been shown to bind mir-NID1 to suppress NRXN1 expression and thereby inhibit neuronal development and functionality [93]. Given that long noncoding RNA (lncRNA) confers a higher degree of gene expression regulation, TDP-43's increased interaction with two crucial lncRNAs, MALAT1 and NEAT1, possibly modulates the RNA metabolism dysregulation of ALS- and FTD-associated TDP-43 pathology [94, 95]. Furthermore, lncRNA gadd7, which is involved in cell cycle regulation and DDR signaling, orchestrates TDP-43's interaction with CDK6 mRNA that leads to its

Stress granule (SG) assembly is a dynamic, reversible process that promotes cell survival when stress factors are present. Membraneless SG organelles vary in their morphology and building composition in a cell type-specific manner [97, 98]. As long as the assembly/disassembly ratio is maintained in healthy cells, SGs act as the emergency store house for certain classes of RNA and protein molecules to protect

for RRM domains in TDP-43 pathology.

controlled decay [96].

**2.2 Stress granules**

**Figure 2.**

**8**

*TDP-43 is a critical component of stress granule. Stress granule formation, in normal condition, is a very important cellular defense mechanism to overcome stress responses. Stress granule assembly and disassembly is a reversible process in healthy cells. It's a membraneless structure consisting of stress granule marker proteins TIA-1, G3BP-1, and TDP-43 as the major structural component. Given that ALS-/FTD-associated TDP-43 pathology induces aberrant cytosolic sequestration of toxic TDP-43, it impairs the reversible nature of stress granule formation mechanism, thereby inducing the seeding mechanism for protein aggregation.*

respect to SG dynamics [104, 105]. Interestingly, chronic TDP-43 liquid-liquid phase separation (LLPS) has been observed in neuronal cells and ALS-iPSc-derived motor neurons, even with only a transient stress induction from sonicated amyloid fibrils or arsenite. Early stage LLPS TDP-43 droplets are formed in the nucleus independent of conventional SG mechanisms. Cytosolic TDP-43 droplets formed by transient stress induction gradually incorporate importin-α and Nup62, leading to the mislocalization of nuclear pore complex proteins Nup107, Ran, and RanGap1. This situation inhibits nucleo-cytoplasmic shuttling and clearance of toxic TDP-43 and results in neuronal cell death [106]. Hans et al. have shown that TDP-43 hyperubiquitylation-mediated insolubility could happen by multiple distinct mechanisms independent of its translocation to cytosolic SGs [107]. This study shows neither endoplasmic reticulum kinase inhibitors nor translation blockers could prevent TDP-43 ubiquitylation. Moreover, the sorbitol-induced stress response involves impaired TDP-43 splicing activity, whereas sodium arsenite-induced SG formation occurs through oxidative stress, which can be quenched by the treatment with antioxidant like N-acetylcysteine. High-content screening of inhibitors for blocking pathogenic TDP-43 accumulation in SGs from ALS patient-derived iPSc-motor neurons has identified planar aromatic moieties with DNA intercalation properties as the potent small molecule therapy for ALS/FTD-TDP-43 pathology [108]. In this context, it is important to mention that TDP-43 aggregates/inclusions do not completely overlap with TDP-43 associated SGs, rather a subpopulation of those TDP-43 aggregates enter into SGs, and ALS-pathology-linked TDP-43 inclusion bodies are devoid of SGs. Notably, SGs indirectly exhibit positive feedback regulation of TDP-43 aggregation by disrupting HDAC6-mediated pathogenic TDP-43 clearance from ALS neurons and thereby accelerating TDP-43's cytosolic aggregation [109].

#### **2.3 Novel roles of GTPases: Rab11 and RGNEF in neurodegeneration**

A common feature in a number of neurodegenerative diseases, including ALS, Alzheimer's, and Parkinson's disease, is the impaired clearance and recycling of damaged and aggregated proteins from cells. Moreover, perturbation of physiological vesicle trafficking systems affects several vital mechanisms in the cells, such as efficient nutrient absorption, failure of cell-to-cell communication, the immune response, and loss of synaptic transmission [110, 111]. Rab-GTPases, first discovered in brain tissue, belong to the major subset of the Ras superfamily [112]. In neurons, Rab-GTPases primarily orchestrate vesicle sorting and trafficking between target membranes through their interactions with effector proteins (coat proteins, kinesins, and dyneins), in addition to tethering and SNARE complexes [113, 114]. Among the Rab-GTPases, Rab11 plays critical roles in trafficking, sorting, and recycling endosomal vesicles around the perinuclear region. Rab11 is transported to the cellular periphery via recycling vesicles traveling along the microtubules and directly regulates vesicular exocytosis at the plasma membrane (**Figure 4**) [115, 116]. Furthermore, Rab11 has been identified as the master regulator for the transport of neurotrophin receptors and β-integrin via axonal junctions in dorsal root ganglion neurons and is critical for their maturation, functionality, and survival [117]. Rab11 participates in different cell survival pathways in neurodegenerative diseases in response prion/prion-like protein toxicity. In the ALS-TDP-43 *Drosophila* model, TDP-43 toxicity reduces levels of the synaptic growth promoting bone morphogenetic protein (BMP) at the neuromuscular junction and increases BMP receptors in recycling endosomes and at the neuromuscular junction. This pathogenic condition leads to larval crawling defects in ALS fruit flies, which are rescued by the overexpression of Rab11 [118]. Schwenk et al. has shown that TDP-43 regulates the number of Rab11-positive recycling endosomes in dendrites (**Figure 4**) [119]. These recycling

**11**

**Figure 4.**

*apoptosis.*

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis*

defects in turn affect transferrin recycling in neurons and its decrease in cerebrospinal fluid of patients. In *Drosophila*, a Rab11 mutation induces JNK and MAPK/ERK signaling activation and apoptosis, resulting in defective eye and wing phenotypes [120, 121]. TDP-43 can be phosphorylated at threonine 153 and tyrosine 155 by MEK kinase, a central player in MAPK/ERK signaling pathway, in response to heat shock [122]. However, this unique phospho-TDP-43 variant is not involved in aggregate formation but is instead recruited to nucleoli for processing nucleolar-associated RNA. Further supporting the *Drosophila* model results, we have observed that sporadic ALS-TDP-43 mutations induce the loss of Rab11 in patients' brain and spinal cord compared to age-matched control samples [123]. In contrast to MAPK/ERK and Akt signaling pathways operating in parallel to activate mTORC signaling [124], we have shown that Akt and ERK signaling pathways work in a competitive manner to determine the cell fate. Moreover, in Parkinson's disease, Rab11 co-localizes with toxic α-synuclein inclusion bodies in dopaminergic neurons, and overexpression of Rab11 prevents the affected neurons from degeneration and rescues behavioral deficits [125]. In Huntington's disease, Rab11 overexpression restores synaptic dysfunction and prevents glutamate-induced cell death in neurons [126, 127]. Rab11 has also been found to co-localize with Rab7 and C9orf72 in postmortem ALS brain samples and primary cortical neurons exhibiting C9orf72-induced disrupted vesicular trafficking system in ALS [128]. These findings suggest a broader impact of TDP-43 mutationinduced Rab11 dysfunction on cellular function and survival signaling cascades.

*TDP-43 modulates synaptic vesicle trafficking. Synaptic vesicle trafficking is a crucial mechanism for transporting macromolecules and neurotransmitters across the neuronal axon junctions. Defect in this mechanism may lead to accumulation of toxic waste products in the cells and activation of neurodegenerative conditions. Ras-GTPases like Rab11 plays a critical role in endosomal vesicle trafficking, sorting, and recycling. A study with sporadic ALS patients' brain and spinal cord samples show loss of Rab11 could be closely linked to TDP-43 proteinopathies in ALS and FTD. Loss of Rab11 affects the cargo load capacity and the size of the vesicles as well. This condition in turn activates JNK and MAPK/ERK signaling cascades leading to neuronal* 

Apart from the Rab-GTPases of Ras superfamily, small RhoA GTPases also regulate broad spectrum of cellular mechanisms including cell-to-cell communication, migration, and proliferation. Given that the mode of GTPase functionality relies on the rate of GTP to GDP turn over and vice versa, dysregulation of guanine nucleotide exchange factor (GNEF) that is responsible for such turnover has been linked to a number of diseases including neurodegeneration. Among this class of proteins with enzymatic activity, Rho GNEF (RGNEF/p190) is directly associated with neurodegenerative disease like ALS, where it not only regulates the function of GTPases

*DOI: http://dx.doi.org/10.5772/intechopen.92696*

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.92696*

#### **Figure 4.**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

respect to SG dynamics [104, 105]. Interestingly, chronic TDP-43 liquid-liquid phase separation (LLPS) has been observed in neuronal cells and ALS-iPSc-derived motor neurons, even with only a transient stress induction from sonicated amyloid fibrils or arsenite. Early stage LLPS TDP-43 droplets are formed in the nucleus independent of conventional SG mechanisms. Cytosolic TDP-43 droplets formed by transient stress induction gradually incorporate importin-α and Nup62, leading to the mislocalization of nuclear pore complex proteins Nup107, Ran, and RanGap1. This situation inhibits nucleo-cytoplasmic shuttling and clearance of toxic TDP-43 and results in neuronal cell death [106]. Hans et al. have shown that TDP-43 hyperubiquitylation-mediated insolubility could happen by multiple distinct mechanisms independent of its translocation to cytosolic SGs [107]. This study shows neither endoplasmic reticulum kinase inhibitors nor translation blockers could prevent TDP-43 ubiquitylation. Moreover, the sorbitol-induced stress response involves impaired TDP-43 splicing activity, whereas sodium arsenite-induced SG formation occurs through oxidative stress, which can be quenched by the treatment with antioxidant like N-acetylcysteine. High-content screening of inhibitors for blocking pathogenic TDP-43 accumulation in SGs from ALS patient-derived iPSc-motor neurons has identified planar aromatic moieties with DNA intercalation properties as the potent small molecule therapy for ALS/FTD-TDP-43 pathology [108]. In this context, it is important to mention that TDP-43 aggregates/inclusions do not completely overlap with TDP-43 associated SGs, rather a subpopulation of those TDP-43 aggregates enter into SGs, and ALS-pathology-linked TDP-43 inclusion bodies are devoid of SGs. Notably, SGs indirectly exhibit positive feedback regulation of TDP-43 aggregation by disrupting HDAC6-mediated pathogenic TDP-43 clearance from ALS neurons and thereby accelerating TDP-43's cytosolic aggregation [109].

**2.3 Novel roles of GTPases: Rab11 and RGNEF in neurodegeneration**

A common feature in a number of neurodegenerative diseases, including ALS, Alzheimer's, and Parkinson's disease, is the impaired clearance and recycling of damaged and aggregated proteins from cells. Moreover, perturbation of physiological vesicle trafficking systems affects several vital mechanisms in the cells, such as efficient nutrient absorption, failure of cell-to-cell communication, the immune response, and loss of synaptic transmission [110, 111]. Rab-GTPases, first discovered in brain tissue, belong to the major subset of the Ras superfamily [112]. In neurons, Rab-GTPases primarily orchestrate vesicle sorting and trafficking between target membranes through their interactions with effector proteins (coat proteins, kinesins, and dyneins), in addition to tethering and SNARE complexes [113, 114]. Among the Rab-GTPases, Rab11 plays critical roles in trafficking, sorting, and recycling endosomal vesicles around the perinuclear region. Rab11 is transported to the cellular periphery via recycling vesicles traveling along the microtubules and directly regulates vesicular exocytosis at the plasma membrane (**Figure 4**) [115, 116]. Furthermore, Rab11 has been identified as the master regulator for the transport of neurotrophin receptors and β-integrin via axonal junctions in dorsal root ganglion neurons and is critical for their maturation, functionality, and survival [117]. Rab11 participates in different cell survival pathways in neurodegenerative diseases in response prion/prion-like protein toxicity. In the ALS-TDP-43 *Drosophila* model, TDP-43 toxicity reduces levels of the synaptic growth promoting bone morphogenetic protein (BMP) at the neuromuscular junction and increases BMP receptors in recycling endosomes and at the neuromuscular junction. This pathogenic condition leads to larval crawling defects in ALS fruit flies, which are rescued by the overexpression of Rab11 [118]. Schwenk et al. has shown that TDP-43 regulates the number of Rab11-positive recycling endosomes in dendrites (**Figure 4**) [119]. These recycling

**10**

*TDP-43 modulates synaptic vesicle trafficking. Synaptic vesicle trafficking is a crucial mechanism for transporting macromolecules and neurotransmitters across the neuronal axon junctions. Defect in this mechanism may lead to accumulation of toxic waste products in the cells and activation of neurodegenerative conditions. Ras-GTPases like Rab11 plays a critical role in endosomal vesicle trafficking, sorting, and recycling. A study with sporadic ALS patients' brain and spinal cord samples show loss of Rab11 could be closely linked to TDP-43 proteinopathies in ALS and FTD. Loss of Rab11 affects the cargo load capacity and the size of the vesicles as well. This condition in turn activates JNK and MAPK/ERK signaling cascades leading to neuronal apoptosis.*

defects in turn affect transferrin recycling in neurons and its decrease in cerebrospinal fluid of patients. In *Drosophila*, a Rab11 mutation induces JNK and MAPK/ERK signaling activation and apoptosis, resulting in defective eye and wing phenotypes [120, 121]. TDP-43 can be phosphorylated at threonine 153 and tyrosine 155 by MEK kinase, a central player in MAPK/ERK signaling pathway, in response to heat shock [122]. However, this unique phospho-TDP-43 variant is not involved in aggregate formation but is instead recruited to nucleoli for processing nucleolar-associated RNA. Further supporting the *Drosophila* model results, we have observed that sporadic ALS-TDP-43 mutations induce the loss of Rab11 in patients' brain and spinal cord compared to age-matched control samples [123]. In contrast to MAPK/ERK and Akt signaling pathways operating in parallel to activate mTORC signaling [124], we have shown that Akt and ERK signaling pathways work in a competitive manner to determine the cell fate. Moreover, in Parkinson's disease, Rab11 co-localizes with toxic α-synuclein inclusion bodies in dopaminergic neurons, and overexpression of Rab11 prevents the affected neurons from degeneration and rescues behavioral deficits [125]. In Huntington's disease, Rab11 overexpression restores synaptic dysfunction and prevents glutamate-induced cell death in neurons [126, 127]. Rab11 has also been found to co-localize with Rab7 and C9orf72 in postmortem ALS brain samples and primary cortical neurons exhibiting C9orf72-induced disrupted vesicular trafficking system in ALS [128]. These findings suggest a broader impact of TDP-43 mutationinduced Rab11 dysfunction on cellular function and survival signaling cascades.

Apart from the Rab-GTPases of Ras superfamily, small RhoA GTPases also regulate broad spectrum of cellular mechanisms including cell-to-cell communication, migration, and proliferation. Given that the mode of GTPase functionality relies on the rate of GTP to GDP turn over and vice versa, dysregulation of guanine nucleotide exchange factor (GNEF) that is responsible for such turnover has been linked to a number of diseases including neurodegeneration. Among this class of proteins with enzymatic activity, Rho GNEF (RGNEF/p190) is directly associated with neurodegenerative disease like ALS, where it not only regulates the function of GTPases for stress survival [129] but also acts as the RNA-binding protein to modulate the stability of the crucial low molecular weight cytoskeleton protein neurofilament's mRNA by binding to its 3′ untranslated region [130]. Emerging studies on GNEF's involvement in ALS has revealed that RGNEF co-exists with TDP-43 and FUS in the neuronal cytoplasmic inclusion bodies in spinal motor neurons indicating a cross talk of protein aggregation and dysregulated cell signaling pathways in the ALS pathogenesis [131]. Micronuclei structures, containing small DNA fragments with clustered DSBs and surrounded by one lipid layer, are a strong hallmark of cellular metabolic stress, exposure to genotoxic agents, and genomic instability leading to apoptosis [132–134]. Notably, micronuclei have been observed in the ALS patient brain and spinal cord tissue samples. Studies have shown that TDP-43 interacts with RGNEF through its leucine-rich domain and forms co-aggregated structures [135]. Taken together, these findings further support the fact that genomic instability is one of the major outcomes in ALS-TDP-43 pathology leading to neuronal death.

#### **3. DNA binding and role in DNA transactions**

TDP-43 was first identified and characterized as the transcriptional regulator of HIV-1 gene expression by binding to long terminal repeat TAR DNA motifs [136]. In contrast, a separate study found that when exposed to HIV-1 infection, HIV-1 viral production could occur in T cells and macrophages, even in the absence of TDP-43 protein [137]. As an RNA-/DNA-binding protein, TDP-43 also interacts with specific TG and non-TG repeat containing DNA sequences through its RRM domains, with each domain having specific interaction affinities and DNA conformation requirements [138]. TDP-43 has been found to regulate the cyclin-dependent kinase 6 (CDK6) transcript and protein levels through its binding to the highly conserved long-stretch of GT repeats in CDK6 gene sequence. This binding leads to CDK6 upregulation and thereby increases phosphorylation of retinoblastoma proteins pRb and pRb2/p130 [139]. TDP-43 also acts as the transcriptional repressor and/or insulation regulator for the spatiotemporal regulation of the ACRV1 (SP-10) gene [140, 141]. TDP-43 binds to two GTGTGT motifs in the promoter core region through its N-terminal RRM1 domain during spermatogenesis. In vivo studies reveal that, unlike the wild-type variant, mutations in the GTGTGT motifs in the −186/+28 promoter region leads to premature reporter gene expression in the meiotic spermatocytes [142]. Previously, TDP-43 has been shown to bind both double-stranded DNA as well as single-stranded DNA, with a higher affinity toward single-stranded DNA through binding its RRM1 domain [143, 144]. Qin et al. reported that TDP-43's N-terminal domain is indispensable for its physiological and proteinopathy functions and showed that the N-terminus maintains a highly ordered structure that equilibrates the C-terminal disordered structure by acquiring a novel ubiquitin-like fold that directly binds single-strand DNA [145]. More recently, we also documented in vitro studies on TDP-43's affinity for binding free double-stranded DNA ends, instead of binding a partially or completely blocked terminus [146].

#### **4. TDP-43 in DNA damage response (DDR) and repair**

Induction of DNA damage and dysregulated damage response are critical factors for neuronal death in ALS and other neurodegenerative diseases [147, 148]. Studies also suggest endogenous DNA breaks, including DSBs, are routinely generated and repaired in healthy neurons and are essential for the regulation of neuronal gene

**13**

**Figure 5.**

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis*

markers (γH2AX and TUNEL) than their age-matched controls [146].

*TDP-43's scaffolding role in NHEJ repair mechanism of nuclear genome. In response to DNA damage induction, TDP-43 gets recruited at the DSB sites and participates in relaying DDR signaling through interaction with p-ATM (Serine1981), p-histone H2AX (Serine139), p-53BP1 (Serine1778), and Ku70/80 heterodimer. More importantly, TDP-43 regulates the most rate-limiting step of the DNA DSB repair pathway—DNA DSB end ligation, through its interaction with DNA ligation complex XRCC4-ligase 4 at the break sites. However, pathogenic mutations in TDP-43 related to ALS and FTD cause enhanced cytosolic mislocalization of TDP-43, thereby inhibiting the DNA damage-induced translocation of XRCC4-ligase 4 complex from cytosol to nucleus. Either of TDP-43 loss-of-function or ligation complex translocation inhibition causes DNA DSB repair impairment leading to nuclear genome instability in ALS/FTD motor neurons.*

expression [149, 150]. Besides the transcriptional regulation by RRM domains of TDP-43, no other DNA interactions have been reported for TDP-43. Based on an interactome study targeting TDP-43 in human cells, Freibaum et al. identified the DNA repair protein, Ku70, as one of the interacting partners of TDP-43 [151]. Furthermore, we found that TDP-43's interaction with Ku70 was modulated by DNA damage induction in neuronal cells. Notably, TDP-43 showed pathway-specific roles in DNA DSB repair via direct interaction with the classical nonhomologous end joining (NHEJ) factors XRCC4 and DNA Ligase 4 (LIG4) complex, but not with singlestrand break repair factors XRCC1 and DNA Ligase 3 (LIG3) complex. Further experimental studies revealed TDP-43's interaction with a number of NHEJ proteins, including DNA-PKcs, 53BP1, polymerase lambda, XRCC4-like factor (XLF), and the DNA damage response (DDR) factors phosphorylated ATM and histone H2AX (γH2AX). Interestingly, TDP-43 depletion in neurons elicited hyperactivation of DDR signaling without affecting the recruitment of activated DDR proteins to genome damage sites and inhibited the docking, but not assembly, of DNA ligation complex factors (XRCC4, LIG4, and XLF) at the break sites. This suggests that TDP-43 has a crucial role in maintenance of genomic integrity by efficiently completing the rate-limiting DSB sealing step (**Figure 5**). Consistent with these results, neuronal cells with TDP-43 downregulation exhibited delayed DSB repair kinetics and a higher population of apoptotic cells due to the persistent accumulation of unrepaired DSBs than controls. In a correlative study of postmortem human sporadic ALS brain and cervical spinal cord samples, we determined that samples with extensive TDP-43 aggregation and/or fragmentation had greater staining for DSB

*DOI: http://dx.doi.org/10.5772/intechopen.92696*

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.92696*

expression [149, 150]. Besides the transcriptional regulation by RRM domains of TDP-43, no other DNA interactions have been reported for TDP-43. Based on an interactome study targeting TDP-43 in human cells, Freibaum et al. identified the DNA repair protein, Ku70, as one of the interacting partners of TDP-43 [151]. Furthermore, we found that TDP-43's interaction with Ku70 was modulated by DNA damage induction in neuronal cells. Notably, TDP-43 showed pathway-specific roles in DNA DSB repair via direct interaction with the classical nonhomologous end joining (NHEJ) factors XRCC4 and DNA Ligase 4 (LIG4) complex, but not with singlestrand break repair factors XRCC1 and DNA Ligase 3 (LIG3) complex. Further experimental studies revealed TDP-43's interaction with a number of NHEJ proteins, including DNA-PKcs, 53BP1, polymerase lambda, XRCC4-like factor (XLF), and the DNA damage response (DDR) factors phosphorylated ATM and histone H2AX (γH2AX). Interestingly, TDP-43 depletion in neurons elicited hyperactivation of DDR signaling without affecting the recruitment of activated DDR proteins to genome damage sites and inhibited the docking, but not assembly, of DNA ligation complex factors (XRCC4, LIG4, and XLF) at the break sites. This suggests that TDP-43 has a crucial role in maintenance of genomic integrity by efficiently completing the rate-limiting DSB sealing step (**Figure 5**). Consistent with these results, neuronal cells with TDP-43 downregulation exhibited delayed DSB repair kinetics and a higher population of apoptotic cells due to the persistent accumulation of unrepaired DSBs than controls. In a correlative study of postmortem human sporadic ALS brain and cervical spinal cord samples, we determined that samples with extensive TDP-43 aggregation and/or fragmentation had greater staining for DSB markers (γH2AX and TUNEL) than their age-matched controls [146].

#### **Figure 5.**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

**3. DNA binding and role in DNA transactions**

**4. TDP-43 in DNA damage response (DDR) and repair**

Induction of DNA damage and dysregulated damage response are critical factors for neuronal death in ALS and other neurodegenerative diseases [147, 148]. Studies also suggest endogenous DNA breaks, including DSBs, are routinely generated and repaired in healthy neurons and are essential for the regulation of neuronal gene

for stress survival [129] but also acts as the RNA-binding protein to modulate the stability of the crucial low molecular weight cytoskeleton protein neurofilament's mRNA by binding to its 3′ untranslated region [130]. Emerging studies on GNEF's involvement in ALS has revealed that RGNEF co-exists with TDP-43 and FUS in the neuronal cytoplasmic inclusion bodies in spinal motor neurons indicating a cross talk of protein aggregation and dysregulated cell signaling pathways in the ALS pathogenesis [131]. Micronuclei structures, containing small DNA fragments with clustered DSBs and surrounded by one lipid layer, are a strong hallmark of cellular metabolic stress, exposure to genotoxic agents, and genomic instability leading to apoptosis [132–134]. Notably, micronuclei have been observed in the ALS patient brain and spinal cord tissue samples. Studies have shown that TDP-43 interacts with RGNEF through its leucine-rich domain and forms co-aggregated structures [135]. Taken together, these findings further support the fact that genomic instability is one of the major outcomes in ALS-TDP-43 pathology leading to neuronal death.

TDP-43 was first identified and characterized as the transcriptional regulator of HIV-1 gene expression by binding to long terminal repeat TAR DNA motifs [136]. In contrast, a separate study found that when exposed to HIV-1 infection, HIV-1 viral production could occur in T cells and macrophages, even in the absence of TDP-43 protein [137]. As an RNA-/DNA-binding protein, TDP-43 also interacts with specific TG and non-TG repeat containing DNA sequences through its RRM domains, with each domain having specific interaction affinities and DNA conformation requirements [138]. TDP-43 has been found to regulate the cyclin-dependent kinase 6 (CDK6) transcript and protein levels through its binding to the highly conserved long-stretch of GT repeats in CDK6 gene sequence. This binding leads to CDK6 upregulation and thereby increases phosphorylation of retinoblastoma proteins pRb and pRb2/p130 [139]. TDP-43 also acts as the transcriptional repressor and/or insulation regulator for the spatiotemporal regulation of the ACRV1 (SP-10) gene [140, 141]. TDP-43 binds to two GTGTGT motifs in the promoter core region through its N-terminal RRM1 domain during spermatogenesis. In vivo studies reveal that, unlike the wild-type variant, mutations in the GTGTGT motifs in the −186/+28 promoter region leads to premature reporter gene expression in the meiotic spermatocytes [142]. Previously, TDP-43 has been shown to bind both double-stranded DNA as well as single-stranded DNA, with a higher affinity toward single-stranded DNA through binding its RRM1 domain [143, 144]. Qin et al. reported that TDP-43's N-terminal domain is indispensable for its physiological and proteinopathy functions and showed that the N-terminus maintains a highly ordered structure that equilibrates the C-terminal disordered structure by acquiring a novel ubiquitin-like fold that directly binds single-strand DNA [145]. More recently, we also documented in vitro studies on TDP-43's affinity for binding free double-stranded DNA ends, instead of binding a partially or completely blocked terminus [146].

**12**

*TDP-43's scaffolding role in NHEJ repair mechanism of nuclear genome. In response to DNA damage induction, TDP-43 gets recruited at the DSB sites and participates in relaying DDR signaling through interaction with p-ATM (Serine1981), p-histone H2AX (Serine139), p-53BP1 (Serine1778), and Ku70/80 heterodimer. More importantly, TDP-43 regulates the most rate-limiting step of the DNA DSB repair pathway—DNA DSB end ligation, through its interaction with DNA ligation complex XRCC4-ligase 4 at the break sites. However, pathogenic mutations in TDP-43 related to ALS and FTD cause enhanced cytosolic mislocalization of TDP-43, thereby inhibiting the DNA damage-induced translocation of XRCC4-ligase 4 complex from cytosol to nucleus. Either of TDP-43 loss-of-function or ligation complex translocation inhibition causes DNA DSB repair impairment leading to nuclear genome instability in ALS/FTD motor neurons.*

#### **4.1 Mutant TDP-43 and defective DSB repair in neurons**

Efficient sealing of DNA DSBs, which are the most lethal form of DNA damage, is critical for maintaining genome integrity and fidelity. In the majority of the SALS cases, the total amount of TDP-43 does not change significantly, but a significant proportion of the protein mislocalizes to the cytosol and causes ALS/FTD pathophysiology similar to disease-linked mutant TDP-43. We recently linked the TDP-43 Q331K mutation with genome instability and DSB repair defects. In studies with conditionally expressed TDP-43 Q331K mutant, cultured neurons exhibited a strong nuclear clearance phenotype and a higher amount of DNA damage at basal level without the effect of any external DNA damaging agents than control cells. Further investigations showed that TDP-43 Q331K trapped XRCC4 and LIG4 in the cytosol and inhibited their translocation to the nucleus in response to DNA damage induction. These findings suggest that TDP-43 is not only involved in the recruitment of the DNA ligation complex at genomic break sites but also regulates the damage-dependent nuclear translocation of DNA repair proteins (**Figure 5**). These impaired TDP-43 functions in the disease condition leading to genomic instability and neurodegeneration [22].

#### **5. A double whammy of DNA damage induction and defects in their repair in TDP-43-ALS**

We and others have observed that the most salient hallmarks of ALS/FTD-TDP-43 pathology, the protein aggregation and inclusion body formation in the cytosol, increase oxidative stress in affected neurons [22, 48, 152, 153]. 4-hydroxynonenal (HNE) increases as an oxidative stress indicator in sporadic ALS patients' brain, spinal cord, and serum. HNE induces oxidative damage to a broad range of cysteine-containing proteins, including TDP-43, through cysteine oxidation. Oxidized TDP-43 mislocalizes from the nucleus to form insoluble cytosolic aggregates in ALS [154]. Such nuclear clearance and cytosolic increase may cause a gain-of-toxicity, leading to aberrant mRNA processing, which in turn exerts a direct DNA destabilization through impaired DSB repair and an indirect repair inhibition by negatively regulating mRNA splicing of DNA repair proteins. Furthermore, the unrepaired DNA DSBs leads to persistent DDR signaling activation through an ATM-mediated signaling cascade that promotes neuroinflammation. This inflammatory response may induce oxidative genome damage and enhanced TDP-43's nuclear clearance, exacerbating the ALS/FTD conditions in a feed forward pattern. Moreover, dysregulation of metal homeostasis has been observed in ALS patients' brain/spinal cord tissues. In the presence of cellular oxidative stress, zinc induces TDP-43's mislocalization and cytoplasmic inclusions [155, 156]. Given that zinc is a crucial metal co-activator for several transcription factors, trapping of zinc in TDP-43 aggregates could globally affect gene activation patterns, including those for DNA repair and response-associated factors, leading to genomic instability.

#### **6. Conclusions: TDP-43-ALS, a case for DNA repair-targeted therapy?**

Emerging studies from our laboratory and other groups have shown that in addition to its RNA processing and miRNA biogenesis functions, TDP-43 acts as a key component of the NHEJ pathway for DSB repair in neurons, and its pathological clearance from the nucleus leads to the DSB repair defects seen in ALS and other TDP-43-linked neurodegenerative diseases. These newly discovered paradigms link

**15**

**Author details**

**Acknowledgements**

\* and Muralidhar L. Hegde1,2\*

ameliorating the genome instability of ALS-TDP-43.

2 Weill Medical College of Cornell University, New York, USA

\*Address all correspondence to: jmitra@houstonmethodist.org

Research Institute, Houston, Texas, USA

and mlhegde@houstonmethodist.org

provided the original work is properly cited.

1 Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Research in Hegde laboratory is supported by grants from the National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Aging (NIA) of the National Institute of Health (NIH) under the award numbers R01NS088645, RF1NS112719, and R03AG064266 and the Houston Methodist Research Institute funds. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. The authors thank members of the Hegde Laboratory—Haibo Wang, Manohar Kodavati,

Pavana Hegde, Velmarini Vasquez, and Erika Guerrero for assistance.

Joy Mitra1

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis*

TDP-43 pathology to impaired DNA repair and suggest potential avenues for DNA repair-targeted therapies for TDP-43-ALS and related motor neuron diseases. Future studies should focus on a comprehensive delineation of the molecular mechanisms involved in order to develop efficiently targeted interventions. In addition, the implications of these defects in neuronal and glial functions in the CNS of TDP-43-ALS patients and the role of TDP-43 in maintaining genome integrity in non-neuronal brain cells, including glia and astrocytes, are important lines of investigation. By comparing the DNA repair role of TDP-43 in post-mitotic vs. cycling cells, we could learn important mechanistic insights on the selective vulnerability of neurons in ALS. Recently, an increasing number of studies have demonstrated the role of RNA/ DNA-binding proteins in DNA repair. We previously documented the involvement of hnRNP-U, a member of the hnRNP family, in oxidative damage repair in the human genome and its role as a molecular switch between DSB repair and oxidative damage repair when these complex damages occur in a clustered fashion [157, 158]. We also discovered that another RNA-/DNA-binding protein, fused in sarcoma/translocated in liposarcoma (FUS/TLS), linked to the ALS-FUS subtype, participates in break sealing during DNA SSB repair [10]. Because both TDP-43 and FUS influence the final DNA break/sealing step of repair, further investigations in DNA repair mechanisms are critical to developing clinically effective strategies for

*DOI: http://dx.doi.org/10.5772/intechopen.92696*

#### *The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.92696*

TDP-43 pathology to impaired DNA repair and suggest potential avenues for DNA repair-targeted therapies for TDP-43-ALS and related motor neuron diseases. Future studies should focus on a comprehensive delineation of the molecular mechanisms involved in order to develop efficiently targeted interventions. In addition, the implications of these defects in neuronal and glial functions in the CNS of TDP-43-ALS patients and the role of TDP-43 in maintaining genome integrity in non-neuronal brain cells, including glia and astrocytes, are important lines of investigation. By comparing the DNA repair role of TDP-43 in post-mitotic vs. cycling cells, we could learn important mechanistic insights on the selective vulnerability of neurons in ALS.

Recently, an increasing number of studies have demonstrated the role of RNA/ DNA-binding proteins in DNA repair. We previously documented the involvement of hnRNP-U, a member of the hnRNP family, in oxidative damage repair in the human genome and its role as a molecular switch between DSB repair and oxidative damage repair when these complex damages occur in a clustered fashion [157, 158]. We also discovered that another RNA-/DNA-binding protein, fused in sarcoma/translocated in liposarcoma (FUS/TLS), linked to the ALS-FUS subtype, participates in break sealing during DNA SSB repair [10]. Because both TDP-43 and FUS influence the final DNA break/sealing step of repair, further investigations in DNA repair mechanisms are critical to developing clinically effective strategies for ameliorating the genome instability of ALS-TDP-43.

#### **Acknowledgements**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

Efficient sealing of DNA DSBs, which are the most lethal form of DNA damage, is critical for maintaining genome integrity and fidelity. In the majority of the SALS cases, the total amount of TDP-43 does not change significantly, but a significant proportion of the protein mislocalizes to the cytosol and causes ALS/FTD pathophysiology similar to disease-linked mutant TDP-43. We recently linked the TDP-43 Q331K mutation with genome instability and DSB repair defects. In studies with conditionally expressed TDP-43 Q331K mutant, cultured neurons exhibited a strong nuclear clearance phenotype and a higher amount of DNA damage at basal level without the effect of any external DNA damaging agents than control cells. Further investigations showed that TDP-43 Q331K trapped XRCC4 and LIG4 in the cytosol and inhibited their translocation to the nucleus in response to DNA damage induction. These findings suggest that TDP-43 is not only involved in the recruitment of the DNA ligation complex at genomic break sites but also regulates the damage-dependent nuclear translocation of DNA repair proteins (**Figure 5**). These impaired TDP-43 functions in the disease condition leading to genomic instability

**5. A double whammy of DNA damage induction and defects in their** 

We and others have observed that the most salient hallmarks of ALS/FTD-TDP-43 pathology, the protein aggregation and inclusion body formation in the cytosol, increase oxidative stress in affected neurons [22, 48, 152, 153].

4-hydroxynonenal (HNE) increases as an oxidative stress indicator in sporadic ALS patients' brain, spinal cord, and serum. HNE induces oxidative damage to a broad range of cysteine-containing proteins, including TDP-43, through cysteine oxidation. Oxidized TDP-43 mislocalizes from the nucleus to form insoluble cytosolic aggregates in ALS [154]. Such nuclear clearance and cytosolic increase may cause a gain-of-toxicity, leading to aberrant mRNA processing, which in turn exerts a direct DNA destabilization through impaired DSB repair and an indirect repair inhibition by negatively regulating mRNA splicing of DNA repair proteins. Furthermore, the unrepaired DNA DSBs leads to persistent DDR signaling activation through an ATM-mediated signaling cascade that promotes neuroinflammation. This inflammatory response may induce oxidative genome damage and enhanced TDP-43's nuclear clearance, exacerbating the ALS/FTD conditions in a feed forward pattern. Moreover, dysregulation of metal homeostasis has been observed in ALS patients' brain/spinal cord tissues. In the presence of cellular oxidative stress, zinc induces TDP-43's mislocalization and cytoplasmic inclusions [155, 156]. Given that zinc is a crucial metal co-activator for several transcription factors, trapping of zinc in TDP-43 aggregates could globally affect gene activation patterns, including those for DNA repair and response-associated factors, leading to genomic instability.

**6. Conclusions: TDP-43-ALS, a case for DNA repair-targeted therapy?**

Emerging studies from our laboratory and other groups have shown that in addition to its RNA processing and miRNA biogenesis functions, TDP-43 acts as a key component of the NHEJ pathway for DSB repair in neurons, and its pathological clearance from the nucleus leads to the DSB repair defects seen in ALS and other TDP-43-linked neurodegenerative diseases. These newly discovered paradigms link

**4.1 Mutant TDP-43 and defective DSB repair in neurons**

and neurodegeneration [22].

**repair in TDP-43-ALS**

**14**

Research in Hegde laboratory is supported by grants from the National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Aging (NIA) of the National Institute of Health (NIH) under the award numbers R01NS088645, RF1NS112719, and R03AG064266 and the Houston Methodist Research Institute funds. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies. The authors thank members of the Hegde Laboratory—Haibo Wang, Manohar Kodavati, Pavana Hegde, Velmarini Vasquez, and Erika Guerrero for assistance.

#### **Author details**

Joy Mitra1 \* and Muralidhar L. Hegde1,2\*

1 Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist Research Institute, Houston, Texas, USA

2 Weill Medical College of Cornell University, New York, USA

\*Address all correspondence to: jmitra@houstonmethodist.org and mlhegde@houstonmethodist.org

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[24] Gitcho MA et al. TDP-43 A315T mutation in familial motor neuron disease. Annals of Neurology. 2008;**63**(4):535-538

[25] Sreedharan J et al. TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science. 2008;**319**(5870):1668-1672

[26] Van Deerlin VM et al. TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: A genetic and histopathological analysis. Lancet Neurology. 2008;**7**(5):409-416

[27] Kabashi E et al. TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nature Genetics. 2008;**40**(5):572-574

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[29] Fang F et al. Association between blood lead and the risk of amyotrophic lateral sclerosis. American Journal of Epidemiology. 2010;**171**(10):1126-1133

[30] Campbell AM, Williams ER, Barltrop D. Motor neurone disease and exposure to lead. Journal of Neurology, Neurosurgery, and Psychiatry. 1970;**33**(6):877-885

[31] Gresham LS et al. Amyotrophic lateral sclerosis and occupational heavy metal exposure: A case-control study. Neuroepidemiology. 1986;**5**(1):29-38

[32] Wang H et al. Smoking and risk of amyotrophic lateral sclerosis: A pooled analysis of 5 prospective cohorts. Archives of Neurology. 2011;**68**(2):207-213

[33] Gallo V et al. Smoking and risk for amyotrophic lateral sclerosis: Analysis of the EPIC cohort. Annals of Neurology. 2009;**65**(4):378-385

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**16**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

toxicity and turnover. Molecular and Cellular Biology. 2020:**40**(4):e00256-19

[9] Neumann M et al. TDP-43 in the ubiquitin pathology of frontotemporal dementia with VCP gene mutations.

[10] Wang H et al. Mutant FUS causes DNA ligation defects to inhibit oxidative damage repair in amyotrophic lateral sclerosis. Nature Communications.

[11] Hortobagyi T et al. Optineurin inclusions occur in a minority of TDP-43 positive ALS and FTLD-TDP cases and are rarely observed in other neurodegenerative disorders. Acta Neuropathologica. 2011;**121**(4):519-527

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sclerosis: A resolution in sight? Brain. 2019;**142**(5):1176-1194

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L-alanine (BMAA) and beta-Noxalylamino-L-alanine (BOAA) on cultured cortical neurons. Brain Research. 1989;**497**(1):64-71

Neurology. 2014;**261**:1-9

2016;**145-146**:78-97

2009;**18**(19):3725-3738

[47] Yin HZ et al. Intrathecal infusion of BMAA induces selective motor neuron damage and astrogliosis in the ventral horn of the spinal cord. Experimental

[48] Guerrero EN et al. TDP-43/FUS in motor neuron disease: Complexity and challenges. Progress in Neurobiology.

[49] Sieh W et al. Identification of novel susceptibility loci for Guam neurodegenerative disease: Challenges of genome scans in genetic isolates. Human Molecular Genetics.

[50] Li Q et al. The cleavage pattern of TDP-43 determines its rate of clearance and cytotoxicity. Nature Communications. 2015;**6**:6183

[51] Asakawa K, Handa H, Kawakami K. Optogenetic modulation of TDP-43 oligomerization accelerates ALS-related

[52] Zhang YJ et al. Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proceedings of the National Academy of Sciences of the United States of America.

[53] Shiina Y et al. TDP-43 dimerizes in human cells in culture. Cellular and Molecular Neurobiology.

[54] Hergesheimer RC et al. The debated toxic role of aggregated TDP-43 in amyotrophic lateral

pathologies in the spinal motor neurons. Nature Communications.

2020;**11**(1):1004

2009;**106**(18):7607-7612

2010;**30**(4):641-652

[36] Malek AM et al. Exposure to hazardous air pollutants and the risk of amyotrophic lateral sclerosis. Environmental Pollution.

[37] Yu Y et al. Environmental risk factors and amyotrophic lateral sclerosis (ALS): A case-control study of ALS in Michigan. PLoS One. 2014;**9**(6):e101186

[38] de Jong SW et al. Smoking, alcohol consumption, and the risk of amyotrophic lateral sclerosis: A population-based study.

American Journal of Epidemiology.

[39] Haley RW. Excess incidence of ALS in young gulf war veterans. Neurology.

[40] Weisskopf MG et al. Prospective study of military service and mortality from ALS. Neurology. 2005;**64**(1):32-37

[41] Chen H et al. Head injury and amyotrophic lateral sclerosis. American Journal of Epidemiology.

[42] Schmidt S et al. Association of ALS with head injury, cigarette smoking and APOE genotypes. Journal of the Neurological Sciences.

[43] Cox PA, Sacks OW. Cycad neurotoxins, consumption of flying foxes, and ALS-PDC disease in Guam. Neurology. 2002;**58**(6):956-959

of Neurology. 1980;**8**(6):612-619

[45] Arif M et al. Tau pathology involves protein phosphatase 2A in parkinsonism-dementia of Guam. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**(3):1144-1149

[44] Garruto RM, Gajdusek C, Chen KM. Amyotrophic lateral sclerosis among Chamorro migrants from Guam. Annals

2012;**176**(3):233-239

2003;**61**(6):750-756

2007;**166**(7):810-816

2010;**291**(1-2):22-29

2015;**197**:181-186

**18**

[55] Winton MJ et al. Disturbance of nuclear and cytoplasmic TAR DNA-binding protein (TDP-43) induces disease-like redistribution, sequestration, and aggregate formation. The Journal of Biological Chemistry. 2008;**283**(19):13302-13309

[56] Shodai A et al. Conserved acidic amino acid residues in a second RNA recognition motif regulate assembly and function of TDP-43. PLoS One. 2012;**7**(12):e52776

[57] Amlie-Wolf A et al. Transcriptomic changes due to cytoplasmic TDP-43 expression reveal Dysregulation of histone transcripts and nuclear chromatin. PLoS One. 2015;**10**(10):e0141836

[58] Winton MJ et al. A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro. FEBS Letters. 2008;**582**(15):2252-2256

[59] Barmada SJ et al. Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis. The Journal of Neuroscience. 2010;**30**(2):639-649

[60] Jara JH et al. Evidence for an early innate immune response in the motor cortex of ALS. Journal of Neuroinflammation. 2017;**14**(1):129

[61] Jara JH et al. MCP1-CCR2 and neuroinflammation in the ALS motor cortex with TDP-43 pathology. Journal of Neuroinflammation. 2019;**16**(1):196

[62] Beers DR et al. Neuroinflammation modulates distinct regional and temporal clinical responses in ALS mice. Brain, Behavior, and Immunity. 2011;**25**(5):1025-1035

[63] Puentes F et al. Non-neuronal cells in ALS: Role of glial, immune cells and

blood-CNS barriers. Brain Pathology. 2016;**26**(2):248-257

[64] Zhu J, Cynader MS, Jia W. TDP-43 inhibits NF-kappaB activity by blocking p65 nuclear translocation. PLoS One. 2015;**10**(11):e0142296

[65] Frakes AE et al. Microglia induce motor neuron death via the classical NF-kappaB pathway in amyotrophic lateral sclerosis. Neuron. 2014;**81**(5):1009-1023

[66] Correia AS et al. Inflammation induces TDP-43 Mislocalization and aggregation. PLoS One. 2015;**10**(10):e0140248

[67] Swarup V et al. Deregulation of TDP-43 in amyotrophic lateral sclerosis triggers nuclear factor kappaBmediated pathogenic pathways. The Journal of Experimental Medicine. 2011;**208**(12):2429-2447

[68] Buratti E, Baralle FE. Multiple roles of TDP-43 in gene expression, splicing regulation, and human disease. Frontiers in Bioscience. 2008;**13**:867-878

[69] Buratti E, Baralle FE. TDP-43: Gumming up neurons through proteinprotein and protein-RNA interactions. Trends in Biochemical Sciences. 2012;**37**(6):237-247

[70] Avendano-Vazquez SE et al. Autoregulation of TDP-43 mRNA levels involves interplay between transcription, splicing, and alternative polyA site selection. Genes & Development. 2012;**26**(15):1679-1684

[71] Bembich S et al. Predominance of spliceosomal complex formation over polyadenylation site selection in TDP-43 autoregulation. Nucleic Acids Research. 2014;**42**(5):3362-3371

[72] Pons M et al. Identification of TCERG1 as a new genetic modulator of TDP-43 production in drosophila. Acta Neuropathologica Communications. 2018;**6**(1):138

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[83] Kashima T, Manley JL. A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy. Nature Genetics. 2003;**34**(4):460-463

[84] Buvoli M et al. Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein. The EMBO Journal. 1990;**9**(4):1229-1235

[85] Bekenstein U, Soreq H. Heterogeneous nuclear ribonucleoprotein A1 in health and neurodegenerative disease: From structural insights to posttranscriptional regulatory roles. Molecular and Cellular Neurosciences. 2013;**56**:436-446

[86] Deshaies JE et al. TDP-43 regulates the alternative splicing of hnRNP A1 to yield an aggregation-prone variant in amyotrophic lateral sclerosis. Brain. 2018;**141**(5):1320-1333

[87] Honda H et al. Loss of hnRNPA1 in ALS spinal cord motor neurons with TDP-43-positive inclusions. Neuropathology. 2015;**35**(1):37-43

[88] Jiang LL et al. The N-terminal dimerization is required for TDP-43 splicing activity. Scientific Reports. 2017;**7**(1):6196

[89] Mompean M et al. Point mutations in the N-terminal domain of transactive response DNA-binding protein 43 kDa (TDP-43) compromise its stability,

**21**

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis*

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2006;**26**(15):5744-5758

2011;**31**(5):1098-1108

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[102] Khalfallah Y et al. TDP-43 regulation of stress granule dynamics

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relevant cell types. Scientific Reports.

[103] Aulas A et al. G3BP1 promotes stress-induced RNA granule

interactions to preserve polyadenylated mRNA. The Journal of Cell Biology.

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[106] Gasset-Rosa F et al. Cytoplasmic TDP-43 De-mixing independent of stress granules drives inhibition of nuclear import, loss of nuclear

2017;**95**(4):808-816.e9

2018;**8**(1):7551

2015;**209**(1):73-84

2011;**20**(7):1400-1410

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dimerization, and functions. The Journal of Biological Chemistry. 2017;**292**(28):11992-12006

[90] Chen HJ et al. RRM adjacent TARDBP mutations disrupt RNA binding and enhance TDP-43 proteinopathy. Brain. 2019;**142**(12):3753-3770

[91] French RL et al. Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation. The Journal of Biological Chemistry.

[92] Kawahara Y, Mieda-Sato A. TDP-43 promotes microRNA biogenesis as a component of the Drosha and dicer complexes. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(9):3347-3352

Transcriptome-wide analysis of TDP-43 binding small RNAs identifies miR-NID1 (miR-8485), a novel miRNA that represses NRXN1 expression. Genomics.

[94] Tollervey JR et al. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nature Neuroscience. 2011;**14**(4):452-458

[95] Beltran M, Garcia de Herreros A. Antisense non-coding RNAs and regulation of gene transcription. Transcription. 2016;**7**(2):39-43

[96] Liu X et al. Long non-coding RNA gadd7 interacts with TDP-43 and regulates Cdk6 mRNA decay. The EMBO Journal. 2012;**31**(23):4415-4427

[97] Anderson P, Kedersha N. Stress granules: The Tao of RNA triage. Trends in Biochemical Sciences.

[98] Guil S, Long JC, Caceres JF. hnRNP A1 relocalization to the stress granules

2008;**33**(3):141-150

2019;**294**(17):6696-6709

[93] Fan Z, Chen X, Chen R.

2014;**103**(1):76-82

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.92696*

dimerization, and functions. The Journal of Biological Chemistry. 2017;**292**(28):11992-12006

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

mRNA. Annual Review of Biochemistry.

1993;**62**:289-321

[82] Buratti E et al. TDP-43 binds heterogeneous nuclear ribonucleoprotein a/B through its C-terminal tail: An important region for the inhibition of cystic fibrosis transmembrane conductance

regulator exon 9 splicing. The Journal of Biological Chemistry. 2005;**280**(45):37572-37584

[84] Buvoli M et al. Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein. The EMBO Journal.

1990;**9**(4):1229-1235

2013;**56**:436-446

2018;**141**(5):1320-1333

2017;**7**(1):6196

[85] Bekenstein U, Soreq H. Heterogeneous nuclear

ribonucleoprotein A1 in health and neurodegenerative disease: From structural insights to posttranscriptional regulatory roles. Molecular and Cellular Neurosciences.

[86] Deshaies JE et al. TDP-43 regulates the alternative splicing of hnRNP A1 to yield an aggregation-prone variant in amyotrophic lateral sclerosis. Brain.

[87] Honda H et al. Loss of hnRNPA1 in

ALS spinal cord motor neurons with TDP-43-positive inclusions. Neuropathology. 2015;**35**(1):37-43

[88] Jiang LL et al. The N-terminal dimerization is required for TDP-43 splicing activity. Scientific Reports.

[89] Mompean M et al. Point mutations in the N-terminal domain of transactive response DNA-binding protein 43 kDa (TDP-43) compromise its stability,

[83] Kashima T, Manley JL. A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy. Nature Genetics. 2003;**34**(4):460-463

Neuropathologica Communications.

Allain FH. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. The FEBS Journal.

[73] Maris C, Dominguez C,

2005;**272**(9):2118-2131

[74] Buratti E, Baralle FE. Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9. The Journal of Biological Chemistry.

2001;**276**(39):36337-36343

[75] Kuo PH et al. Structural insights into TDP-43 in nucleic-acid binding and domain interactions. Nucleic Acids

[76] Ayala YM et al. Human, drosophila, and C.elegans TDP43: Nucleic acid binding properties and splicing regulatory function. Journal of

Molecular Biology. 2005;**348**(3):575-588

[77] Donde A et al. Splicing repression is a major function of TDP-43 in motor neurons. Acta Neuropathologica.

[78] Polymenidou M et al. Long premRNA depletion and RNA missplicing contribute to neuronal vulnerability

from loss of TDP-43. Nature Neuroscience. 2011;**14**(4):459-468

[79] Watanabe S et al. ALS-linked TDP-43(M337V) knock-in mice exhibit splicing deregulation without neurodegeneration. Molecular Brain.

[80] Wang HY et al. Structural diversity and functional implications of the eukaryotic TDP gene family. Genomics.

2019;**138**(5):813-826

2020;**13**(1):8

2004;**83**(1):130-139

[81] Dreyfuss G et al. hnRNP proteins and the biogenesis of

Research. 2009;**37**(6):1799-1808

2018;**6**(1):138

**20**

[90] Chen HJ et al. RRM adjacent TARDBP mutations disrupt RNA binding and enhance TDP-43 proteinopathy. Brain. 2019;**142**(12):3753-3770

[91] French RL et al. Detection of TAR DNA-binding protein 43 (TDP-43) oligomers as initial intermediate species during aggregate formation. The Journal of Biological Chemistry. 2019;**294**(17):6696-6709

[92] Kawahara Y, Mieda-Sato A. TDP-43 promotes microRNA biogenesis as a component of the Drosha and dicer complexes. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(9):3347-3352

[93] Fan Z, Chen X, Chen R. Transcriptome-wide analysis of TDP-43 binding small RNAs identifies miR-NID1 (miR-8485), a novel miRNA that represses NRXN1 expression. Genomics. 2014;**103**(1):76-82

[94] Tollervey JR et al. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Nature Neuroscience. 2011;**14**(4):452-458

[95] Beltran M, Garcia de Herreros A. Antisense non-coding RNAs and regulation of gene transcription. Transcription. 2016;**7**(2):39-43

[96] Liu X et al. Long non-coding RNA gadd7 interacts with TDP-43 and regulates Cdk6 mRNA decay. The EMBO Journal. 2012;**31**(23):4415-4427

[97] Anderson P, Kedersha N. Stress granules: The Tao of RNA triage. Trends in Biochemical Sciences. 2008;**33**(3):141-150

[98] Guil S, Long JC, Caceres JF. hnRNP A1 relocalization to the stress granules

reflects a role in the stress response. Molecular and Cellular Biology. 2006;**26**(15):5744-5758

[99] Dewey CM et al. TDP-43 is directed to stress granules by sorbitol, a novel physiological osmotic and oxidative stressor. Molecular and Cellular Biology. 2011;**31**(5):1098-1108

[100] Aulas A, Stabile S, Vande Velde C. Endogenous TDP-43, but not FUS, contributes to stress granule assembly via G3BP. Molecular Neurodegeneration. 2012;**7**:54

[101] Mackenzie IR et al. TIA1 mutations in amyotrophic lateral sclerosis and Frontotemporal dementia promote phase separation and Alter stress granule dynamics. Neuron. 2017;**95**(4):808-816.e9

[102] Khalfallah Y et al. TDP-43 regulation of stress granule dynamics in neurodegenerative diseaserelevant cell types. Scientific Reports. 2018;**8**(1):7551

[103] Aulas A et al. G3BP1 promotes stress-induced RNA granule interactions to preserve polyadenylated mRNA. The Journal of Cell Biology. 2015;**209**(1):73-84

[104] McDonald KK et al. TAR DNA-binding protein 43 (TDP-43) regulates stress granule dynamics via differential regulation of G3BP and TIA-1. Human Molecular Genetics. 2011;**20**(7):1400-1410

[105] Orru S et al. Reduced stress granule formation and cell death in fibroblasts with the A382T mutation of TARDBP gene: Evidence for loss of TDP-43 nuclear function. Human Molecular Genetics. 2016;**25**(20):4473-4483

[106] Gasset-Rosa F et al. Cytoplasmic TDP-43 De-mixing independent of stress granules drives inhibition of nuclear import, loss of nuclear

TDP-43, and cell death. Neuron. 2019;**102**(2):339-357.e7

[107] Hans F, Glasebach H, Kahle PJ. Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules. The Journal of Biological Chemistry. Jan 17 2020;**295**(3):673-689

[108] Fang MY et al. Small-molecule modulation of TDP-43 recruitment to stress granules prevents persistent TDP-43 accumulation in ALS/FTD. Neuron. 2019;**103**(5):802-819.e11

[109] Chen Y, Cohen TJ. Aggregation of the nucleic acid-binding protein TDP-43 occurs via distinct routes that are coordinated with stress granule formation. The Journal of Biological Chemistry. 2019;**294**(10):3696-3706

[110] Vagnozzi AN, Pratico D. Endosomal sorting and trafficking, the retromer complex and neurodegeneration. Molecular Psychiatry. 2019;**24**(6):857-868

[111] Bouchet J et al. Rab11-FIP3 regulation of Lck Endosomal traffic controls TCR signal transduction. Journal of Immunology. 2017;**198**(7):2967-2978

[112] Touchot N, Chardin P, Tavitian A. Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: Molecular cloning of YPT-related cDNAs from a rat brain library. Proceedings of the National Academy of Sciences of the United States of America. 1987;**84**(23):8210-8214

[113] Cai H, Reinisch K, Ferro-Novick S. Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. Developmental Cell. 2007;**12**(5):671-682

[114] Grosshans BL, Ortiz D, Novick P. Rabs and their effectors: Achieving specificity in membrane traffic. Proceedings of the National Academy of Sciences of the United States of America. 2006;**103**(32):11821-11827

[115] Takahashi S et al. Rab11 regulates exocytosis of recycling vesicles at the plasma membrane. Journal of Cell Science. 2012;**125**(Pt 17):4049-4057

[116] Goldenring JR. Recycling endosomes. Current Opinion in Cell Biology. 2015;**35**:117-122

[117] Bucci C, Alifano P, Cogli L. The role of Rab proteins in neuronal cells and in the trafficking of neurotrophin receptors. Membranes. 2014;**4**(4):642-677

[118] Deshpande M et al. Role of BMP receptor traffic in synaptic growth defects in an ALS model. Molecular Biology of the Cell. 2016;**27**(19):2898-2910

[119] Schwenk BM et al. TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons. The EMBO Journal. 2016;**35**(21):2350-2370

[120] Tiwari AK, Roy JK. Mutation in Rab11 results in abnormal organization of ommatidial cells and activation of JNK signaling in the drosophila eye. European Journal of Cell Biology. 2009;**88**(8):445-460

[121] Bhuin T, Roy JK. Rab11 regulates JNK and Raf/MAPK-ERK signalling pathways during drosophila wing development. Cell Biology International. 2010;**34**(11):1113-1118

[122] Li W et al. Heat shock-induced phosphorylation of TAR DNA-binding protein 43 (TDP-43) by MAPK/ERK kinase regulates TDP-43 function.

**23**

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis*

inclusions in amyotrophic lateral sclerosis. Neurobiology of Aging.

[131] Keller BA et al. Co-aggregation of RNA binding proteins in ALS spinal motor neurons: Evidence of a common pathogenic mechanism. Acta Neuropathologica. 2012;**124**(5):733-747

[132] Utani K et al. Emergence of

[133] Kiraly G et al. Micronucleus formation during chromatin condensation and under

apoptotic conditions. Apoptosis.

[134] Xu B et al. Replication stress induces micronuclei comprising of aggregated DNA double-strand breaks.

[135] Droppelmann CA et al. TDP-43 aggregation inside micronuclei reveals a potential mechanism for protein inclusion formation in ALS. Scientific

PLoS One. 2011;**6**(4):e18618

Reports. 2019;**9**(1):19928

2014;**9**(8):e105478

[136] Ou SH et al. Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs. Journal of Virology. 1995;**69**(6):3584-3596

[137] Nehls J et al. HIV-1 replication in human immune cells is independent of TAR DNA binding protein 43 (TDP-43) expression. PLoS One.

[138] Furukawa Y et al. A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43.

[139] Ayala YM, Misteli T, Baralle FE. TDP-43 regulates retinoblastoma protein phosphorylation through the repression of cyclin-dependent

Scientific Reports. 2016;**6**:20576

One. 2010;**5**(4):e10089

2017;**22**(2):207-219

micronuclei and their effects on the fate of cells under replication stress. PLoS

2013;**34**(1):248-262

*DOI: http://dx.doi.org/10.5772/intechopen.92696*

[123] Mitra J, Hegde PM, Hegde ML. Loss of endosomal recycling factor RAB11 coupled with complex regulation of MAPK/ERK/AKT signaling in postmortem spinal cord specimens of sporadic amyotrophic lateral sclerosis patients. Molecular Brain. 2019;**12**(1):55

The Journal of Biological Chemistry.

2017;**292**(12):5089-5100

[124] Winter JN, Jefferson LS, Kimball SR. ERK and Akt signaling pathways function through parallel mechanisms to promote mTORC1 signaling. American Journal of Physiology. Cell Physiology. 2011;**300**(5):C1172-C1180

[125] Breda C et al. Rab11 modulates alpha-synuclein-mediated defects in synaptic transmission and behaviour.

[126] Steinert JR et al. Rab11 rescues synaptic dysfunction and behavioural deficits in a drosophila model of

Genetics. 2012;**21**(13):2912-2922

Disease. 2009;**36**(2):374-383

[128] Farg MA et al. C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Human Molecular Genetics. 2017;**26**(20):4093-4094

[129] Cheung K et al. Rho guanine nucleotide exchange factor (RGNEF) is a prosurvival factor under stress conditions. Molecular and Cellular Neurosciences. 2017;**82**:88-95

[130] Droppelmann CA et al. Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic

[127] Li X et al. Disruption of Rab11 activity in a knock-in mouse model of Huntington's disease. Neurobiology of

Huntington's disease. Human Molecular

Human Molecular Genetics. 2015;**24**(4):1077-1091

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.92696*

The Journal of Biological Chemistry. 2017;**292**(12):5089-5100

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

[114] Grosshans BL, Ortiz D, Novick P. Rabs and their effectors: Achieving specificity in membrane

traffic. Proceedings of the National Academy of Sciences of the United States of America. 2006;**103**(32):11821-11827

[115] Takahashi S et al. Rab11 regulates exocytosis of recycling vesicles at the plasma membrane. Journal of Cell Science. 2012;**125**(Pt 17):4049-4057

[116] Goldenring JR. Recycling endosomes. Current Opinion in Cell

[117] Bucci C, Alifano P, Cogli L. The role of Rab proteins in neuronal cells and in the trafficking of

neurotrophin receptors. Membranes.

[118] Deshpande M et al. Role of BMP receptor traffic in synaptic growth defects in an ALS model. Molecular Biology of the Cell. 2016;**27**(19):2898-2910

[119] Schwenk BM et al. TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons. The EMBO Journal.

[120] Tiwari AK, Roy JK. Mutation in Rab11 results in abnormal organization of ommatidial cells and activation of JNK signaling in the drosophila eye. European Journal of Cell Biology.

[121] Bhuin T, Roy JK. Rab11 regulates JNK and Raf/MAPK-ERK signalling

[122] Li W et al. Heat shock-induced phosphorylation of TAR DNA-binding protein 43 (TDP-43) by MAPK/ERK kinase regulates TDP-43 function.

pathways during drosophila wing development. Cell Biology International. 2010;**34**(11):1113-1118

2016;**35**(21):2350-2370

2009;**88**(8):445-460

Biology. 2015;**35**:117-122

2014;**4**(4):642-677

TDP-43, and cell death. Neuron.

into stress granules. The Journal of Biological Chemistry. Jan 17

[108] Fang MY et al. Small-molecule modulation of TDP-43 recruitment to stress granules prevents persistent TDP-43 accumulation in ALS/FTD. Neuron.

[109] Chen Y, Cohen TJ. Aggregation of the nucleic acid-binding protein TDP-43 occurs via distinct routes that are coordinated with stress granule formation. The Journal of Biological Chemistry. 2019;**294**(10):3696-3706

[110] Vagnozzi AN, Pratico D. Endosomal sorting and trafficking,

[111] Bouchet J et al. Rab11-FIP3 regulation of Lck Endosomal traffic controls TCR signal

transduction. Journal of Immunology.

[112] Touchot N, Chardin P, Tavitian A. Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: Molecular cloning of YPT-related cDNAs from a rat brain library. Proceedings of the National Academy of Sciences of the United States of America.

the retromer complex and neurodegeneration. Molecular Psychiatry. 2019;**24**(6):857-868

2017;**198**(7):2967-2978

1987;**84**(23):8210-8214

2007;**12**(5):671-682

[113] Cai H, Reinisch K, Ferro-Novick S. Coats, tethers, Rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle. Developmental Cell.

[107] Hans F, Glasebach H, Kahle PJ. Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation

2019;**102**(2):339-357.e7

2020;**295**(3):673-689

2019;**103**(5):802-819.e11

**22**

[123] Mitra J, Hegde PM, Hegde ML. Loss of endosomal recycling factor RAB11 coupled with complex regulation of MAPK/ERK/AKT signaling in postmortem spinal cord specimens of sporadic amyotrophic lateral sclerosis patients. Molecular Brain. 2019;**12**(1):55

[124] Winter JN, Jefferson LS, Kimball SR. ERK and Akt signaling pathways function through parallel mechanisms to promote mTORC1 signaling. American Journal of Physiology. Cell Physiology. 2011;**300**(5):C1172-C1180

[125] Breda C et al. Rab11 modulates alpha-synuclein-mediated defects in synaptic transmission and behaviour. Human Molecular Genetics. 2015;**24**(4):1077-1091

[126] Steinert JR et al. Rab11 rescues synaptic dysfunction and behavioural deficits in a drosophila model of Huntington's disease. Human Molecular Genetics. 2012;**21**(13):2912-2922

[127] Li X et al. Disruption of Rab11 activity in a knock-in mouse model of Huntington's disease. Neurobiology of Disease. 2009;**36**(2):374-383

[128] Farg MA et al. C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Human Molecular Genetics. 2017;**26**(20):4093-4094

[129] Cheung K et al. Rho guanine nucleotide exchange factor (RGNEF) is a prosurvival factor under stress conditions. Molecular and Cellular Neurosciences. 2017;**82**:88-95

[130] Droppelmann CA et al. Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic

inclusions in amyotrophic lateral sclerosis. Neurobiology of Aging. 2013;**34**(1):248-262

[131] Keller BA et al. Co-aggregation of RNA binding proteins in ALS spinal motor neurons: Evidence of a common pathogenic mechanism. Acta Neuropathologica. 2012;**124**(5):733-747

[132] Utani K et al. Emergence of micronuclei and their effects on the fate of cells under replication stress. PLoS One. 2010;**5**(4):e10089

[133] Kiraly G et al. Micronucleus formation during chromatin condensation and under apoptotic conditions. Apoptosis. 2017;**22**(2):207-219

[134] Xu B et al. Replication stress induces micronuclei comprising of aggregated DNA double-strand breaks. PLoS One. 2011;**6**(4):e18618

[135] Droppelmann CA et al. TDP-43 aggregation inside micronuclei reveals a potential mechanism for protein inclusion formation in ALS. Scientific Reports. 2019;**9**(1):19928

[136] Ou SH et al. Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs. Journal of Virology. 1995;**69**(6):3584-3596

[137] Nehls J et al. HIV-1 replication in human immune cells is independent of TAR DNA binding protein 43 (TDP-43) expression. PLoS One. 2014;**9**(8):e105478

[138] Furukawa Y et al. A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43. Scientific Reports. 2016;**6**:20576

[139] Ayala YM, Misteli T, Baralle FE. TDP-43 regulates retinoblastoma protein phosphorylation through the repression of cyclin-dependent

kinase 6 expression. Proceedings of the National Academy of Sciences of the United States of America. 2008;**105**(10):3785-3789

[140] Lalmansingh AS, Urekar CJ, Reddi PP. TDP-43 is a transcriptional repressor: The testis-specific mouse acrv1 gene is a TDP-43 target in vivo. The Journal of Biological Chemistry. 2011;**286**(13):10970-10982

[141] Abhyankar MM, Urekar C, Reddi PP. A novel CpG-free vertebrate insulator silences the testis-specific SP-10 gene in somatic tissues: Role for TDP-43 in insulator function. The Journal of Biological Chemistry. 2007;**282**(50):36143-36154

[142] Acharya KK et al. Cisrequirement for the maintenance of round spermatid-specific transcription. Developmental Biology. 2006;**295**(2):781-790

[143] Kitamura A et al. Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy. Biochemistry and Biophysics Reports. 2018;**14**:58-63

[144] Kuo PH et al. The crystal structure of TDP-43 RRM1-DNA complex reveals the specific recognition for UG- and TG-rich nucleic acids. Nucleic Acids Research. 2014;**42**(7):4712-4722

[145] Qin H et al. TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**(52):18619-18624

[146] Mitra J et al. Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects. Proceedings of the National Academy of Sciences of the United States of America. 2019;**116**(10):4696-4705

[147] Vasquez V et al. Chromatin-bound oxidized alpha-Synuclein causes Strand breaks in neuronal genomes in in vitro models of Parkinson's disease. Journal of Alzheimer's Disease. 2017;**60**(s1):S133-s150

[148] Hou Y et al. NAD(+) supplementation normalizes key Alzheimer's features and DNA damage responses in a new AD mouse model with introduced DNA repair deficiency. Proceedings of the National Academy of Sciences of the United States of America. 2018;**115**(8):E1876-e1885

[149] Madabhushi R et al. Activityinduced DNA breaks govern the expression of neuronal early-response genes. Cell. 2015;**161**(7):1592-1605

[150] Su Y, Ming GL, Song H. DNA damage and repair regulate neuronal gene expression. Cell Research. 2015;**25**(9):993-994

[151] Freibaum BD et al. Global analysis of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery. Journal of Proteome Research. 2010;**9**(2):1104-1120

[152] Nagase M et al. Increased oxidative stress in patients with amyotrophic lateral sclerosis and the effect of edaravone administration. Redox Report. 2016;**21**(3):104-112

[153] Niedzielska E et al. Oxidative stress in neurodegenerative diseases. Molecular Neurobiology. 2016;**53**(6):4094-4125

[154] Kabuta C et al. 4-Hydroxynonenal induces persistent insolubilization of TDP-43 and alters its intracellular localization. Biochemical and Biophysical Research Communications. 2015;**463**(1-2):82-87

[155] Zinszner H, Albalat R, Ron D. A novel effector domain from the

**25**

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis*

*DOI: http://dx.doi.org/10.5772/intechopen.92696*

RNA-binding protein TLS or EWS is required for oncogenic transformation by CHOP. Genes & Development.

1994;**8**(21):2513-2526

2010;**48**(9):1152-1161

[156] Caragounis A et al. Zinc induces depletion and aggregation of endogenous TDP-43. Free Radical Biology & Medicine.

[157] Hegde ML et al. Scaffold attachment factor a (SAF-A) and Ku temporally regulate repair of radiationinduced clustered genome lesions. Oncotarget. 2016;**7**(34):54430-54444

nuclear ribonucleoprotein U (hnRNP-U) via direct interaction. The Journal of Biological Chemistry.

2012;**287**(41):34202-34211

[158] Hegde ML et al. Enhancement of NEIL1 protein-initiated oxidized DNA base excision repair by heterogeneous

*The Role of TDP-43 in Genome Repair and beyond in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.92696*

RNA-binding protein TLS or EWS is required for oncogenic transformation by CHOP. Genes & Development. 1994;**8**(21):2513-2526

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

[147] Vasquez V et al. Chromatin-bound oxidized alpha-Synuclein causes Strand breaks in neuronal genomes in in vitro models of Parkinson's disease. Journal of Alzheimer's Disease.

2017;**60**(s1):S133-s150

[148] Hou Y et al. NAD(+) supplementation normalizes key Alzheimer's features and DNA damage responses in a new AD mouse model with introduced DNA repair deficiency. Proceedings of the National Academy of Sciences of the United States of America. 2018;**115**(8):E1876-e1885

[149] Madabhushi R et al. Activityinduced DNA breaks govern the expression of neuronal early-response genes. Cell. 2015;**161**(7):1592-1605

[150] Su Y, Ming GL, Song H. DNA damage and repair regulate neuronal gene expression. Cell Research.

[151] Freibaum BD et al. Global analysis

[152] Nagase M et al. Increased oxidative stress in patients with amyotrophic lateral sclerosis and the effect of edaravone administration. Redox Report. 2016;**21**(3):104-112

[153] Niedzielska E et al. Oxidative stress in neurodegenerative diseases. Molecular Neurobiology.

[154] Kabuta C et al. 4-Hydroxynonenal induces persistent insolubilization of TDP-43 and alters its intracellular localization. Biochemical and

Biophysical Research Communications.

[155] Zinszner H, Albalat R, Ron D. A novel effector domain from the

of TDP-43 interacting proteins reveals strong association with RNA splicing and translation machinery. Journal of Proteome Research.

2015;**25**(9):993-994

2010;**9**(2):1104-1120

2016;**53**(6):4094-4125

2015;**463**(1-2):82-87

kinase 6 expression. Proceedings of the National Academy of Sciences of the United States of America.

[140] Lalmansingh AS, Urekar CJ, Reddi PP. TDP-43 is a transcriptional repressor: The testis-specific mouse acrv1 gene is a TDP-43 target in vivo. The Journal of Biological Chemistry.

2008;**105**(10):3785-3789

2011;**286**(13):10970-10982

2007;**282**(50):36143-36154

[142] Acharya KK et al. Cisrequirement for the maintenance of round spermatid-specific

2006;**295**(2):781-790

transcription. Developmental Biology.

[143] Kitamura A et al. Analysis of the substrate recognition state of TDP-43 to single-stranded DNA using fluorescence correlation spectroscopy. Biochemistry and Biophysics Reports. 2018;**14**:58-63

[144] Kuo PH et al. The crystal structure of TDP-43 RRM1-DNA complex reveals the specific recognition for UG- and TG-rich nucleic acids. Nucleic Acids Research. 2014;**42**(7):4712-4722

[145] Qin H et al. TDP-43 N terminus encodes a novel ubiquitin-like fold and its unfolded form in equilibrium that can be shifted by binding to ssDNA. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**(52):18619-18624

[146] Mitra J et al. Motor neuron disease-associated loss of nuclear TDP-43 is linked to DNA double-strand break repair defects. Proceedings of the National Academy of Sciences of the United States of America.

2019;**116**(10):4696-4705

[141] Abhyankar MM, Urekar C, Reddi PP. A novel CpG-free vertebrate insulator silences the testis-specific SP-10 gene in somatic tissues: Role for TDP-43 in insulator function. The Journal of Biological Chemistry.

**24**

[156] Caragounis A et al. Zinc induces depletion and aggregation of endogenous TDP-43. Free Radical Biology & Medicine. 2010;**48**(9):1152-1161

[157] Hegde ML et al. Scaffold attachment factor a (SAF-A) and Ku temporally regulate repair of radiationinduced clustered genome lesions. Oncotarget. 2016;**7**(34):54430-54444

[158] Hegde ML et al. Enhancement of NEIL1 protein-initiated oxidized DNA base excision repair by heterogeneous nuclear ribonucleoprotein U (hnRNP-U) via direct interaction. The Journal of Biological Chemistry. 2012;**287**(41):34202-34211

**27**

**Chapter 2**

Target

**Abstract**

Molecular Basis of DNA Repair

Defects in FUS-Associated ALS:

Implications of a New Paradigm

and Its Potential as Therapeutic

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disorder, characterized by a diverse etiopathology. While ALS is predominantly sporadic, mutations in one or more of a dozen risk factors have been linked to approximately 10% of familial ALS patients. The multifunctional RNA/DNA-binding protein fused in sarcoma (FUS) is one such protein whose autosomal dominant missense mutations were identified in a subset of familial and sporadic ALS patients. Initial studies linked FUS with both RNA-related and genome maintenance functions, yet the mechanisms and potential implications to neurodegeneration were not completely understood. We recently identified a novel function of FUS in repairing single-strand break (SSB) in the genome. FUS directly interacts and recruits XRCC1/DNA Ligase IIIα (LigIII) to DNA oxidative damage sites in a PARP1 activitydependent manner, which facilitates optimal oxidative genome damage repair. Besides, FUS regulates DNA strand break sealing by enhancing ligation activity of LigIII. The mutation of FUS induces accumulation of oxidative DNA damage as well as DNA repair deficiency in ALS patients. The novel findings provide insights into a previously undescribed mechanism of DNA repair defect in FUS-associated neurodegeneration, and raise the pentientials of developing neuroprotective therapies by

**Keywords:** FUS, neurodegeneration, DNA repair, oxidative genome damage,

**1. Fused in sarcoma (FUS): pathophysiology and links to familial** 

The FUS/TLS (Fused in sarcoma Protein/Translocated in liposarcoma) gene encodes a 526-amino acid protein that belongs to the TET (TAF15, EWS, and TLS) family, which is implicated in multiple aspects of RNA metabolism. The FUS gene was initially identified as an oncogene in multiple cancers. FUS fuses with the

*Haibo Wang and Muralidhar L. Hegde*

targeting DNA break ligating defects.

**1.1 FUS and its physiological functions**

XRCC1-DNA ligase III

**and sporadic ALS**

### **Chapter 2**

## Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New Paradigm and Its Potential as Therapeutic Target

*Haibo Wang and Muralidhar L. Hegde*

### **Abstract**

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disorder, characterized by a diverse etiopathology. While ALS is predominantly sporadic, mutations in one or more of a dozen risk factors have been linked to approximately 10% of familial ALS patients. The multifunctional RNA/DNA-binding protein fused in sarcoma (FUS) is one such protein whose autosomal dominant missense mutations were identified in a subset of familial and sporadic ALS patients. Initial studies linked FUS with both RNA-related and genome maintenance functions, yet the mechanisms and potential implications to neurodegeneration were not completely understood. We recently identified a novel function of FUS in repairing single-strand break (SSB) in the genome. FUS directly interacts and recruits XRCC1/DNA Ligase IIIα (LigIII) to DNA oxidative damage sites in a PARP1 activitydependent manner, which facilitates optimal oxidative genome damage repair. Besides, FUS regulates DNA strand break sealing by enhancing ligation activity of LigIII. The mutation of FUS induces accumulation of oxidative DNA damage as well as DNA repair deficiency in ALS patients. The novel findings provide insights into a previously undescribed mechanism of DNA repair defect in FUS-associated neurodegeneration, and raise the pentientials of developing neuroprotective therapies by targeting DNA break ligating defects.

**Keywords:** FUS, neurodegeneration, DNA repair, oxidative genome damage, XRCC1-DNA ligase III

#### **1. Fused in sarcoma (FUS): pathophysiology and links to familial and sporadic ALS**

#### **1.1 FUS and its physiological functions**

The FUS/TLS (Fused in sarcoma Protein/Translocated in liposarcoma) gene encodes a 526-amino acid protein that belongs to the TET (TAF15, EWS, and TLS) family, which is implicated in multiple aspects of RNA metabolism. The FUS gene was initially identified as an oncogene in multiple cancers. FUS fuses with the

transcription repressor C/EBP homologous protein 10 (CHOP) in myxoid liposarcomas. It was identified for its role as an activator of ETS-related gene (ERG) in acute myeloid leukemia [1, 2] and in Ewing's sarcoma tumors [3]. Subsequently, mutations in FUS were linked to motor neuron diseases, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) [4, 5].

FUS protein is ubiquitously expressed in both the nucleus and cytoplasm of many cell types; however, it is predominantly found in the nucleus of glial cells and neurons in the central nervous system. A small fraction of FUS shuttles between the nucleus and cytoplasm under various stimuli, for example, sodium arseniteinduced oxidative stress [6, 7]. A previous study revealed that FUS binds RNA *in vivo* to engage in nucleo-cytoplasmic shuttling [8]. Another study showed that FUS is localized to dendritic spines as an RNA-protein complex and associates with mRNA encoding an actin-stabilizer protein, which indicates a regulatory role of FUS in actin/cytoskeleton processes in dendrites [9]. Interestingly, in response to stress-induced by sorbitol, FUS redistributes to the cytoplasm and localizes to cytoplasmic stress granules [10], a complex comprised of mRNA, ribosomes, RNA-binding proteins, transcription factors and nucleases that form in response to induced stress, such as oxidative stress and heat shock to maintain the stability of selected mRNAs and protein activities [11, 12]. The redistribution of FUS on stress granules is regulated by posttranslational modification, methylation, and highly related with cell survival from the stress [10]. FUS protein contains an SYGQ-rich region at its N terminal, followed by a RGG box (RGG1), an RRM motif, a second RGG box (RGG2), a zinc finger (ZnF) motif, and a third RGG box (RGG3). The C-terminus contains a nonclassical nuclear localization signal (NLS) with conserved proline and tyrosine residues (PY-NLS). The 13 terminal amino acids (514–526) containing the NLS sequence were shown to be necessary, but not sufficient, for nuclear import of FUS [13]. FUS can bind with RNA and both singlestranded (ss) as well as double-stranded (ds) DNA [14]. FUS is associated with multiple roles in RNA processing, including splicing, transcription, and transport. Studies have highlighted that FUS has a transcriptional regulatory role in global or specialized components of transcriptional machinery [15]. A Clip-seq based study revealed that FUS regulates alternative splicing of pre-mRNAs and processing of long-intron containing transcripts, and the RNAs targeted by FUS are associated with neurogenesis and gene expression regulation; interestingly, some of FUS' mRNA targets are involved in DNA damage response and repair pathways [16].

#### **1.2 FUS is mutated in both familial and sporadic ALS**

The prevalence of ALS has been observed to be non-uniform geographically but ranges between 0.6 and 3.8 per 100,000 population worldwide. The rate of ALS incidence appears to be rising according to the recent epidemiological studies, despite the geographical differences [17]. Notably, the incidence and prevalence of ALS are greater in men than in women [18]. It has been reported that the male: female ratio is between 1 and 3 and changes in an age-dependent manner [19]. In approximately 90% of ALS cases the cause is unknown, and, as such, they are considered sporadic (SALS); while, around 10% of ALS patients have a clear family history (FALS). In 2009, two independent studies identified that FUS R521 is mutated in FALS cases and that mutation is characterized pathologically with enhanced cytoplasmic inclusions of FUS and motor neuron degeneration [4, 5]. FUS mutations have also been linked to cases of SALS [20–22]. The FUS gene is composed of 15 exons and most mutations are clustered in exon15. Exon15 encodes the NLS domain, implying a possible involvement of its nuclear import defects. These mutations/truncations include 495X, R521C/G/H/L/S, R522G, P525L, and so

**29**

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New…*

on. R521 is the most commonly mutated site in ALS, whereas P525 mutations are highly related with early onset and severe progress of ALS [23–25]. Mutations also occur in domains other than NLS. For example, G187S within Gly-rich domain, G399V in RGG domain, and P431L in ZnF domain [26], which is believed to induce functional defects of FUS. Autosomal dominant mutations in the gene encoding the FUS protein have been detected in approximately 5% of FALS patients and a small

It is important to note that many ALS patients (36–51%) also exhibit cognitive impairment, with about 20% developing FTD, also called frontotemporal lobar degeneration (FTLD) [27], a disorder characterized by cognitive, behavioral, and linguistic dysfunction. The reverse is also seen, wherein patients with FTD can develop ALS [28]. FTD accounts for 10–15% of dementias, making it the second most common type of dementia for people under the age of 65, after Alzheimer's disease. The overlap between ALS and FTD indicates a likely common molecular basis between FUS and cognitive deficits. After the discovery of FUS mutation in ALS, a novel subtype of FTD with FUS pathology was identified, although no FUS mutation was seen [29]. In 2010, Oriane Broustal et al. identified three exonic FUS variants, c. 1562G > A (p.Arg521His), c. 1566G > A (p.Arg522Arg), and c.188A > G (pAsn63Ser) from 317 patients including 144 patients with familial FTD and 173 patients with FTD-ALS. Interestingly, the three variants were found

*DOI: http://dx.doi.org/10.5772/intechopen.92637*

only in patients with both FTD and ALS [27].

ALS-linked FUS mutations [37, 38].

**2. Multifaceted role of FUS in RNA and DNA transactions**

The molecular mechanisms of FUS-ALS are complex and ambiguous, typically described by both loss of function and gain of toxicity hypotheses. Although controversial, loss of function is not entirely supported by animal models: FUS knockout mice and zebrafish do not develop ALS-like phenotypes [30–32]. As mentioned in the previous section, FUS plays multiple roles in RNA metabolism. In fact, thousands of RNA targets have been identified that bind to FUS in various cell lines and brain tissue from both patients and animal models [33]. The dysregulation and disturbance of RNA processing are considered to be one mechanism that leads to neurodegeneration. Depletion of FUS in mouse nervous system has been shown to alter the levels of splicing of over 950 mRNAs [34]; FUS knockout in neuroblastoma cells disturbs the splicing of more than 400 introns [35]. Expression of FUS P525L mutant was shown to inhibit splicing of minor introns by trapping U11 and U12 small nuclear RNAs (snRNAs) in these aggregates [35], and expression of R521G and R522G mutations influence RNA transcription and splicing but in a different way [36]. Besides, mRNA transport and stabilization are also affected by

The majority of the mutations occur in the NLS region of FUS, which induces its cytoplasmic accumulation. The widespread FUS mislocalization has been considered a hallmark of ALS [39]. Mutant, but not the wild-type FUS was shown to be assembled into stress granules in cytoplasm in response to oxidative stress or heat shock [7, 40], which potentially contributes to neurotoxicity by impairing mRNA translation [41, 42]. In fact, mutations in RNA-binding proteins (RBPs) are highly related to ALS. The interaction between mistranslocated FUS and other RBPs was recently investigated. These studies show that the cytoplasmic mislocalization caused by FUS P525L mutation impairs its interaction with other ALS-associated

subset (~1%) of SALS cases.

**1.3 FUS pathology in FTD**

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New… DOI: http://dx.doi.org/10.5772/intechopen.92637*

on. R521 is the most commonly mutated site in ALS, whereas P525 mutations are highly related with early onset and severe progress of ALS [23–25]. Mutations also occur in domains other than NLS. For example, G187S within Gly-rich domain, G399V in RGG domain, and P431L in ZnF domain [26], which is believed to induce functional defects of FUS. Autosomal dominant mutations in the gene encoding the FUS protein have been detected in approximately 5% of FALS patients and a small subset (~1%) of SALS cases.

#### **1.3 FUS pathology in FTD**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

sis (ALS), and frontotemporal dementia (FTD) [4, 5].

**1.2 FUS is mutated in both familial and sporadic ALS**

The prevalence of ALS has been observed to be non-uniform geographically but ranges between 0.6 and 3.8 per 100,000 population worldwide. The rate of ALS incidence appears to be rising according to the recent epidemiological studies, despite the geographical differences [17]. Notably, the incidence and prevalence of ALS are greater in men than in women [18]. It has been reported that the male: female ratio is between 1 and 3 and changes in an age-dependent manner [19]. In approximately 90% of ALS cases the cause is unknown, and, as such, they are considered sporadic (SALS); while, around 10% of ALS patients have a clear family history (FALS). In 2009, two independent studies identified that FUS R521 is mutated in FALS cases and that mutation is characterized pathologically with enhanced cytoplasmic inclusions of FUS and motor neuron degeneration [4, 5]. FUS mutations have also been linked to cases of SALS [20–22]. The FUS gene is composed of 15 exons and most mutations are clustered in exon15. Exon15 encodes the NLS domain, implying a possible involvement of its nuclear import defects. These mutations/truncations include 495X, R521C/G/H/L/S, R522G, P525L, and so

transcription repressor C/EBP homologous protein 10 (CHOP) in myxoid liposarcomas. It was identified for its role as an activator of ETS-related gene (ERG) in acute myeloid leukemia [1, 2] and in Ewing's sarcoma tumors [3]. Subsequently, mutations in FUS were linked to motor neuron diseases, amyotrophic lateral sclero-

FUS protein is ubiquitously expressed in both the nucleus and cytoplasm of many cell types; however, it is predominantly found in the nucleus of glial cells and neurons in the central nervous system. A small fraction of FUS shuttles between the nucleus and cytoplasm under various stimuli, for example, sodium arseniteinduced oxidative stress [6, 7]. A previous study revealed that FUS binds RNA *in vivo* to engage in nucleo-cytoplasmic shuttling [8]. Another study showed that FUS is localized to dendritic spines as an RNA-protein complex and associates with mRNA encoding an actin-stabilizer protein, which indicates a regulatory role of FUS in actin/cytoskeleton processes in dendrites [9]. Interestingly, in response to stress-induced by sorbitol, FUS redistributes to the cytoplasm and localizes to cytoplasmic stress granules [10], a complex comprised of mRNA, ribosomes, RNA-binding proteins, transcription factors and nucleases that form in response to induced stress, such as oxidative stress and heat shock to maintain the stability of selected mRNAs and protein activities [11, 12]. The redistribution of FUS on stress granules is regulated by posttranslational modification, methylation, and highly related with cell survival from the stress [10]. FUS protein contains an SYGQ-rich region at its N terminal, followed by a RGG box (RGG1), an RRM motif, a second RGG box (RGG2), a zinc finger (ZnF) motif, and a third RGG box (RGG3). The C-terminus contains a nonclassical nuclear localization signal (NLS) with conserved proline and tyrosine residues (PY-NLS). The 13 terminal amino acids (514–526) containing the NLS sequence were shown to be necessary, but not sufficient, for nuclear import of FUS [13]. FUS can bind with RNA and both singlestranded (ss) as well as double-stranded (ds) DNA [14]. FUS is associated with multiple roles in RNA processing, including splicing, transcription, and transport. Studies have highlighted that FUS has a transcriptional regulatory role in global or specialized components of transcriptional machinery [15]. A Clip-seq based study revealed that FUS regulates alternative splicing of pre-mRNAs and processing of long-intron containing transcripts, and the RNAs targeted by FUS are associated with neurogenesis and gene expression regulation; interestingly, some of FUS' mRNA targets are involved in DNA damage response and repair pathways [16].

**28**

It is important to note that many ALS patients (36–51%) also exhibit cognitive impairment, with about 20% developing FTD, also called frontotemporal lobar degeneration (FTLD) [27], a disorder characterized by cognitive, behavioral, and linguistic dysfunction. The reverse is also seen, wherein patients with FTD can develop ALS [28]. FTD accounts for 10–15% of dementias, making it the second most common type of dementia for people under the age of 65, after Alzheimer's disease. The overlap between ALS and FTD indicates a likely common molecular basis between FUS and cognitive deficits. After the discovery of FUS mutation in ALS, a novel subtype of FTD with FUS pathology was identified, although no FUS mutation was seen [29]. In 2010, Oriane Broustal et al. identified three exonic FUS variants, c. 1562G > A (p.Arg521His), c. 1566G > A (p.Arg522Arg), and c.188A > G (pAsn63Ser) from 317 patients including 144 patients with familial FTD and 173 patients with FTD-ALS. Interestingly, the three variants were found only in patients with both FTD and ALS [27].

#### **2. Multifaceted role of FUS in RNA and DNA transactions**

The molecular mechanisms of FUS-ALS are complex and ambiguous, typically described by both loss of function and gain of toxicity hypotheses. Although controversial, loss of function is not entirely supported by animal models: FUS knockout mice and zebrafish do not develop ALS-like phenotypes [30–32]. As mentioned in the previous section, FUS plays multiple roles in RNA metabolism. In fact, thousands of RNA targets have been identified that bind to FUS in various cell lines and brain tissue from both patients and animal models [33]. The dysregulation and disturbance of RNA processing are considered to be one mechanism that leads to neurodegeneration. Depletion of FUS in mouse nervous system has been shown to alter the levels of splicing of over 950 mRNAs [34]; FUS knockout in neuroblastoma cells disturbs the splicing of more than 400 introns [35]. Expression of FUS P525L mutant was shown to inhibit splicing of minor introns by trapping U11 and U12 small nuclear RNAs (snRNAs) in these aggregates [35], and expression of R521G and R522G mutations influence RNA transcription and splicing but in a different way [36]. Besides, mRNA transport and stabilization are also affected by ALS-linked FUS mutations [37, 38].

The majority of the mutations occur in the NLS region of FUS, which induces its cytoplasmic accumulation. The widespread FUS mislocalization has been considered a hallmark of ALS [39]. Mutant, but not the wild-type FUS was shown to be assembled into stress granules in cytoplasm in response to oxidative stress or heat shock [7, 40], which potentially contributes to neurotoxicity by impairing mRNA translation [41, 42]. In fact, mutations in RNA-binding proteins (RBPs) are highly related to ALS. The interaction between mistranslocated FUS and other RBPs was recently investigated. These studies show that the cytoplasmic mislocalization caused by FUS P525L mutation impairs its interaction with other ALS-associated

RBPs including shnRNPA1, hnRNPA2B1, EWSR1, and TAF15, which facilitates the nucleation of toxic cytoplasmic FUS aggregates. In addition, high cytoplasmic FUS levels exhibit defects in protein degradation and reduced protein levels of RBPs, shedding lights on the FUS-ALS pathology linked to the homeostasis of multiple ALS-associated RBPs [43].

#### **2.1 FUS toxicity and impaired DNA damage response (DDR) signaling**

In addition to RNA dysregulation, there has been a lot of focus on the genome instability caused by FUS mutation in ALS patients since FUS was first linked to DNA damage repair by multiple studies. In human cells, one major source of genome damage is the reactive oxygen species (ROS) accumulation-induced oxidative stress. ROS that defined as a group of reactive molecules derived from oxygen including but not limited to free radicals (superoxide, O2 <sup>−</sup>), hydroxyl radical (·OH), or non-radicals (hydrogen peroxide) can be balanced by various antioxidant systems, while the imbalance between ROS production and antioxidant defenses causes oxidative stress, leading to oxidation of lipid, protein, and DNA in cells [44]. Accumulation of oxidative DNA damge has been linked to multiple neurodegenerative diseases like Parkinson's disease (PD) and Huntington's disease (HD) [45, 46]. In addition to DNA damage, mutation in the genes of specific DNA repair pathways that lead to DNA damage repair (DDR) is another challenge for the central nervous system [47]. For example, nucleotide excision repair (NER) is defective in xeroderma pigmentosum (XP) and Cockayne syndrome (CS), base excision/single-strand break repair (BER/SSBR) defects in ataxia with oculomotor apraxia type 1 (AOA1) [48], spinocerebellar ataxia with axonal neuropathy (SCAN1) [49] and ALS [50], and defective DDR signaling and DNA double-strand break repair (DSBR) in ataxia telangiectasia (A-T) and Nijmegen break-age syndrome. FUS is found to be phosphorylated by DDR proteins ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) in response to DSB-inducing agents. FUS was identified to interact with histone deacetylase 1 (HDAC1) in primary mouse cortical neurons, and the interaction is believed to modulate the homologous recombination (HR) and non-homologous end joining (NHEJ), two major pathways for DSB repair. Besides, FUS was shown to be recruited at DNA damage tracks induced by microirradiation (MIR) at wavelengths of 405 or 351 nm, in a PARP1-depedent manner and accompanied with PARylation by PARP1 [51–53]. Notably, MIR causes clusters of different types of DNA damage including oxidized base lesions, single-strand breaks (SSBs), and DSBs. It is generally believed that UVA (wavelength between 320 and 400 nm) predominantly induces SSBs via elevated ROS, while UVA may also induce secondary DSBs due to clustered SSBs [54]. Thus, recruitment of FUS at MIR with wavelength of 351 nm suggests its potential role in the repair of SSBs.

#### **2.2 FUS and repair of oxidative genome damage: mechanistic insights**

A comprehensive investigation by our group described the mechanistic role of FUS in BER/SSBR, a major pathway to repair oxidative DNA damage. Our study utilized multiple *in vitro* and *in vivo* model systems, including, CRISPR/Cas9 mediated FUS knockout (KO) human embryonic kidney (HEK)293 cells, FALS patient-derived induced pluripotent stem cells (iPSCs) with FUS mutations R521H and P525L, motor neurons induced from these iPSC lines, and ALS patients spinal cord tissues with FUS pathology. We discovered that the DNA integrity is substantially affected in the spinal cord tissues and the motor neurons derived from ALS patients. Both downregulation and pathological mutation of FUS were associated

**31**

**Figure 1.**

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New…*

with DNA SSB accumulation and SSBR defects. Finally, we identified that FUS directly interacts with PARP1, XRCC1, and LigIII in response to oxidative stress and FUS facilitates the recruitment of XRCC1/LigIII to DNA damage sites in a PARP1 activity-dependent manner. FUS enhances the ligation activity of LigIII, which is critical for an optimal SSBR in neurons (**Figure 1**). The SSBR deficiency due to ALS-linked FUS mutations can be rescued by the correction of those mutations via Crisper/Cas9 technology [50]. Furthermore, FUS regulates PARP1's PARylation activity in motor neurons and thus could affect neuronal energy metabolism by

**2.3 FUS-PARP-1 interactions: a new paradigm with diverse implications**

During DDR, PARP1 plays an important role in regulating DNA damage repair. In response to DNA damage, PARP1 is self-activated by its auto-PARylation and transfers ADP-ribose to create long and branched poly(ADP-ribose) on target DNA repair proteins to facilitate their recruitment; for example, PARP1 recruits XRCC1 by its PARylation. A study showed that PARP1 likely plays a major role for the poly(ADP-ribose) synthesis induced by alkylating agents, since the amount of poly(ADP-ribose) can be reduced for around 10 folds (from 60 pmol per mg of DNA to 5.85 pmol per mg of DNA) in PARP1 KO cells in the presence of MNNG [55], which activates DNA mismatch repair. Consistantly, PARP1 knockout mice are

*Model of FUS involving DNA damage response in the nucleus. In response to DNA SSBs, FUS is PARylated and recruited to DNA damage sites by PARP1 and the recruitment facilitates the loading of XRCC1 and nuclear LigIII (nLigIII) complex, where FUS enhances the ligation activity of LigIII for an optimal SSB ligating. While in response to DSB, FUS is associated with HDAC1, which is required for an efficient NHEJ and HR-mediated DSB repair. FUS is also phosphorylated by ATM and DNA-PK, although the underlying mechanism is not known. Due to its role in the functional activation of LigII, which is involved in MMEJ-mediated DSB repair,* 

*it is likely that FUS also affects MMEJ, which needs to be investigated.*

*DOI: http://dx.doi.org/10.5772/intechopen.92637*

/NADH levels.

uncoupling NAD<sup>+</sup>

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New… DOI: http://dx.doi.org/10.5772/intechopen.92637*

with DNA SSB accumulation and SSBR defects. Finally, we identified that FUS directly interacts with PARP1, XRCC1, and LigIII in response to oxidative stress and FUS facilitates the recruitment of XRCC1/LigIII to DNA damage sites in a PARP1 activity-dependent manner. FUS enhances the ligation activity of LigIII, which is critical for an optimal SSBR in neurons (**Figure 1**). The SSBR deficiency due to ALS-linked FUS mutations can be rescued by the correction of those mutations via Crisper/Cas9 technology [50]. Furthermore, FUS regulates PARP1's PARylation activity in motor neurons and thus could affect neuronal energy metabolism by uncoupling NAD<sup>+</sup> /NADH levels.

#### **2.3 FUS-PARP-1 interactions: a new paradigm with diverse implications**

During DDR, PARP1 plays an important role in regulating DNA damage repair. In response to DNA damage, PARP1 is self-activated by its auto-PARylation and transfers ADP-ribose to create long and branched poly(ADP-ribose) on target DNA repair proteins to facilitate their recruitment; for example, PARP1 recruits XRCC1 by its PARylation. A study showed that PARP1 likely plays a major role for the poly(ADP-ribose) synthesis induced by alkylating agents, since the amount of poly(ADP-ribose) can be reduced for around 10 folds (from 60 pmol per mg of DNA to 5.85 pmol per mg of DNA) in PARP1 KO cells in the presence of MNNG [55], which activates DNA mismatch repair. Consistantly, PARP1 knockout mice are

#### **Figure 1.**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

ALS-associated RBPs [43].

RBPs including shnRNPA1, hnRNPA2B1, EWSR1, and TAF15, which facilitates the nucleation of toxic cytoplasmic FUS aggregates. In addition, high cytoplasmic FUS levels exhibit defects in protein degradation and reduced protein levels of RBPs, shedding lights on the FUS-ALS pathology linked to the homeostasis of multiple

In addition to RNA dysregulation, there has been a lot of focus on the genome instability caused by FUS mutation in ALS patients since FUS was first linked to DNA damage repair by multiple studies. In human cells, one major source of genome damage is the reactive oxygen species (ROS) accumulation-induced oxidative stress. ROS that defined as a group of reactive molecules derived from

radical (·OH), or non-radicals (hydrogen peroxide) can be balanced by various antioxidant systems, while the imbalance between ROS production and antioxidant defenses causes oxidative stress, leading to oxidation of lipid, protein, and DNA in cells [44]. Accumulation of oxidative DNA damge has been linked to multiple neurodegenerative diseases like Parkinson's disease (PD) and Huntington's disease (HD) [45, 46]. In addition to DNA damage, mutation in the genes of specific DNA repair pathways that lead to DNA damage repair (DDR) is another challenge for the central nervous system [47]. For example, nucleotide excision repair (NER) is defective in xeroderma pigmentosum (XP) and Cockayne syndrome (CS), base excision/single-strand break repair (BER/SSBR) defects in ataxia with oculomotor apraxia type 1 (AOA1) [48], spinocerebellar ataxia with axonal neuropathy (SCAN1) [49] and ALS [50], and defective DDR signaling and DNA double-strand break repair (DSBR) in ataxia telangiectasia (A-T) and Nijmegen break-age syndrome. FUS is found to be phosphorylated by DDR proteins ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) in response to DSB-inducing agents. FUS was identified to interact with histone deacetylase 1 (HDAC1) in primary mouse cortical neurons, and the interaction is believed to modulate the homologous recombination (HR) and non-homologous end joining (NHEJ), two major pathways for DSB repair. Besides, FUS was shown to be recruited at DNA damage tracks induced by microirradiation (MIR) at wavelengths of 405 or 351 nm, in a PARP1-depedent manner and accompanied with PARylation by PARP1 [51–53]. Notably, MIR causes clusters of different types of DNA damage including oxidized base lesions, single-strand breaks (SSBs), and DSBs. It is generally believed that UVA (wavelength between 320 and 400 nm) predominantly induces SSBs via elevated ROS, while UVA may also induce secondary DSBs due to clustered SSBs [54]. Thus, recruitment of FUS at MIR with wavelength of 351 nm

<sup>−</sup>), hydroxyl

**2.1 FUS toxicity and impaired DNA damage response (DDR) signaling**

oxygen including but not limited to free radicals (superoxide, O2

suggests its potential role in the repair of SSBs.

**2.2 FUS and repair of oxidative genome damage: mechanistic insights**

A comprehensive investigation by our group described the mechanistic role of FUS in BER/SSBR, a major pathway to repair oxidative DNA damage. Our study utilized multiple *in vitro* and *in vivo* model systems, including, CRISPR/Cas9 mediated FUS knockout (KO) human embryonic kidney (HEK)293 cells, FALS patient-derived induced pluripotent stem cells (iPSCs) with FUS mutations R521H and P525L, motor neurons induced from these iPSC lines, and ALS patients spinal cord tissues with FUS pathology. We discovered that the DNA integrity is substantially affected in the spinal cord tissues and the motor neurons derived from ALS patients. Both downregulation and pathological mutation of FUS were associated

**30**

*Model of FUS involving DNA damage response in the nucleus. In response to DNA SSBs, FUS is PARylated and recruited to DNA damage sites by PARP1 and the recruitment facilitates the loading of XRCC1 and nuclear LigIII (nLigIII) complex, where FUS enhances the ligation activity of LigIII for an optimal SSB ligating. While in response to DSB, FUS is associated with HDAC1, which is required for an efficient NHEJ and HR-mediated DSB repair. FUS is also phosphorylated by ATM and DNA-PK, although the underlying mechanism is not known. Due to its role in the functional activation of LigII, which is involved in MMEJ-mediated DSB repair, it is likely that FUS also affects MMEJ, which needs to be investigated.*

highly susceptible to DNA damaging agents and are accumulated with DNA strand breaks accompanied with genomic instability in them [56, 57].

In response to DNA damage, FUS is recruited to DNA damage site in a PARP1 activity-dependent manner [51, 52]. FUS directly binds to PAR synthesized by activated PARP1 leading to the formation of damaged DNA-rich compartments contributed by its N-terminal low-complexity domain (LCD) and C-terminal RGG domains, and the compartments are dissociated by the hydrolysis of PAR by PARG [58], which indicates that PAR polymers play an essential role for the recruitment, likely through enhancing the FUS droplet formation [59]. While the activation of PARP1 can induce shuttling of FUS from nucleus to cytoplasm, which in turn enhances FUS aggregate formation and neurodegeneration [58, 60]. Furthermore, like FUS, the other two members in TET family, TAF15 and EWSR1 are also recruited to MIR-induced DNA damage tracks depending on PARP1 activity [53], while the molecular mechanism of the cross-talk between TET family proteins and PARP-1 has not been investigated.

#### **3. Indirect role of FUS in DDR: FUS-directed RNA transactions in DNA repair**

Although, recent reports from our group and others demonstrate a complex, but the direct role of FUS in maintaining genome integrity, whether FUS RNAbinding activity plays a role in regulating the expression of DDR factors has not been investigated. FUS regulates several key steps of RNA metabolism that impairs various biological processes. CLIP-seq has been used to identify RNAs that FUS targeted in multiple studies [16, 34, 61–65], which reveals an indirect role of FUS involving in DDR by regulating RNA transcription. One report shows that FUS regulates alternative splicing of pre-mRNAs and processing of long-intron containing transcripts in HeLa cells, and FUS binds RNA encoding proteins important for DNA damage response and repair pathways. By comparing with other CLIP-based assays, a map of FUS RNA targets to DNA DSBR by NHEJ and HR is generated in the report and in which, a number of key DDR factors such as ATM, 53BP1, MRN11, NBS1 are included [16]. We recently performed RT<sup>2</sup> PCR arrays for DNA repair and DDR signaling pathways in CRISPR/cas9 FUS knockout (KO) cells, patient-derived FUS-mutant cells, as well as FUS-ALS patient spinal cord autopsy tissue, which revealed significant downregulation of DDR factors BRCA1, MSH complex, RAD23B, and DNA ligase 4. Notably, BRCA1 depletion has been linked to neuronal DNA DSB accumulation and cognitive defects in Alzheimer's disease. The ubiquitin receptor RAD23 functions both in nucleotide excision repair and the proteasomal protein clearance pathway and is thus linked to amyloid load in neurodegeneration. This study provides evidence of FUS pathology-mediated perturbation in the expression of DNA repair and DDR signaling factors and thus highlights the intricate connections between FUS, genome instability, and neurodegeneration.

#### **4. Conclusions and perspectives**

As early as 1982, the defective DNA repair and its possible role in ALS pathology was first proposed by Bradley et al. [66], where they hypothesize that abnormal DNA in ALS may arise from a deficiency of an isozyme of a DNA repair factor. Subsequently, growing evidence suggests that defective DNA repair is a common

**33**

activity defects caused by FUS mutation.

Addressing these critical follow-up questions is an unmet need in the FUS-ALS field, which will help to develop a mechanism-based DNA-repair-targeted therapy.

With recent emerging studies, the stage is set for such a paradigm shift.

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New…*

feature of not only ALS but also several other neurodegenerative diseases, underscoring the needs of studying the implications of unrepaired DNA damage in neurons affected by neurodegeneration [67], which may lead to novel therapeutic

Our recent studies made a critical breakthrough in this direction by first time shedding lights on the molecular insights into the involvement of ALS and FTD-associated FUS and other RNA/DNA-binding proteins in specific DNA repair failure mechanisms, such as DNA ligation deficiency. While this study suggests a potential for DNA ligase complementation strategy, several key questions should be addressed to develop DNA repair-based interventions for ALS. These are: (1) The role of FUS in mitochondrial genome stability maintaining. The linkage between FUS and mitochondrial integrity has been established by a number of studies. Deng et al. demonstrated interactions between FUS and two mitochondrial proteins, mitochondrial chaperonin HSP60 and ATP synthase beta subunit ATP5B, in different studies. HSP60 mediates the translocation of FUS into mitochondria, and downregulating of HSP60 rescues mitochondrial defects and neurodegenerative phenotypes in FUS transgenic flies. While interaction between FUS and ATP5B indicates a involvement of FUS in the dysregulation of mitochondrial ATP synthesis: expression of wild-type or FUS P525L mutant disrupts the formation of the mitochondrial ATP synthase supercomplexes and suppresses the activity of ATP synthase, resulting in mitochondrial cristae loss followed by mitochondrial fragmentation [68, 69]. Nakaya and Maragkakis et al. found that expression of human FUS R495X in mouse embryonic stem cell-differentiated neurons disturbs the translation efficiency of mitochondria-associated genes and results in significant reduction of mitochondrial size [70]. Although ALS-FUS has been linked with the dysfunction of mitochondria, the role of FUS in mitochondrial genome integrity has not been explored. (2) The role of FUS in microhomology-mediated end joining (MMEJ) repair. We have established a relationship between FUS and XRCC1/LigIII, which is required for an optimal SSBR. While LigIII, together with XRCC1 and PARP1, also participates in the MMEJ-mediated DSB repair pathway, the role of which in primary neurons is unknown (**Figure 1**). We hypothesized that the MMEJ contributes DSBR in motor neurons and the loss of FUS may affect MMEJ and lead to genomic instability, which we are currently pursuing. (3) The role of FUS in maintaining genome integrity in astrocytes. Recent studies have shown that expression of ALS-linked mutant FUS and other ALS causative factors in astrocytes induces motor neuron death [71–73]. It will, therefore, be critical to investigate FUS toxicity-induced ligation activity defects in astrocytes and the collateral influence on motor neurons. (4) Whether DNA ligase I (LigI) rescues FUS mutant-mediated LigIII defects. Mammalian cells express three DNA ligases including ligase IV (LigIV), LigIII, and LigI. LigIV specifically participates in NHEJ-mediated DSB repair; LigIII has nuclear and mitochondrial isoforms. Both the nuclear and mitochondrial isoforms have ~98% similarity and function in BER/single-strand break repair (SSBR); LigI has been shown to functionally overlap with LigIII and is believed a back-up of LigIII, however, the level of LigI expression in non-cycling, postmitotic cells like neurons is negligible due to lack of replication-associated repair. Very likely, the induction of LigI into motor neurons with FUS pathology can rescue the LigIII ligation

*DOI: http://dx.doi.org/10.5772/intechopen.92637*

strategies.

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New… DOI: http://dx.doi.org/10.5772/intechopen.92637*

feature of not only ALS but also several other neurodegenerative diseases, underscoring the needs of studying the implications of unrepaired DNA damage in neurons affected by neurodegeneration [67], which may lead to novel therapeutic strategies.

Our recent studies made a critical breakthrough in this direction by first time shedding lights on the molecular insights into the involvement of ALS and FTD-associated FUS and other RNA/DNA-binding proteins in specific DNA repair failure mechanisms, such as DNA ligation deficiency. While this study suggests a potential for DNA ligase complementation strategy, several key questions should be addressed to develop DNA repair-based interventions for ALS. These are: (1) The role of FUS in mitochondrial genome stability maintaining. The linkage between FUS and mitochondrial integrity has been established by a number of studies. Deng et al. demonstrated interactions between FUS and two mitochondrial proteins, mitochondrial chaperonin HSP60 and ATP synthase beta subunit ATP5B, in different studies. HSP60 mediates the translocation of FUS into mitochondria, and downregulating of HSP60 rescues mitochondrial defects and neurodegenerative phenotypes in FUS transgenic flies. While interaction between FUS and ATP5B indicates a involvement of FUS in the dysregulation of mitochondrial ATP synthesis: expression of wild-type or FUS P525L mutant disrupts the formation of the mitochondrial ATP synthase supercomplexes and suppresses the activity of ATP synthase, resulting in mitochondrial cristae loss followed by mitochondrial fragmentation [68, 69]. Nakaya and Maragkakis et al. found that expression of human FUS R495X in mouse embryonic stem cell-differentiated neurons disturbs the translation efficiency of mitochondria-associated genes and results in significant reduction of mitochondrial size [70]. Although ALS-FUS has been linked with the dysfunction of mitochondria, the role of FUS in mitochondrial genome integrity has not been explored. (2) The role of FUS in microhomology-mediated end joining (MMEJ) repair. We have established a relationship between FUS and XRCC1/LigIII, which is required for an optimal SSBR. While LigIII, together with XRCC1 and PARP1, also participates in the MMEJ-mediated DSB repair pathway, the role of which in primary neurons is unknown (**Figure 1**). We hypothesized that the MMEJ contributes DSBR in motor neurons and the loss of FUS may affect MMEJ and lead to genomic instability, which we are currently pursuing. (3) The role of FUS in maintaining genome integrity in astrocytes. Recent studies have shown that expression of ALS-linked mutant FUS and other ALS causative factors in astrocytes induces motor neuron death [71–73]. It will, therefore, be critical to investigate FUS toxicity-induced ligation activity defects in astrocytes and the collateral influence on motor neurons. (4) Whether DNA ligase I (LigI) rescues FUS mutant-mediated LigIII defects. Mammalian cells express three DNA ligases including ligase IV (LigIV), LigIII, and LigI. LigIV specifically participates in NHEJ-mediated DSB repair; LigIII has nuclear and mitochondrial isoforms. Both the nuclear and mitochondrial isoforms have ~98% similarity and function in BER/single-strand break repair (SSBR); LigI has been shown to functionally overlap with LigIII and is believed a back-up of LigIII, however, the level of LigI expression in non-cycling, postmitotic cells like neurons is negligible due to lack of replication-associated repair. Very likely, the induction of LigI into motor neurons with FUS pathology can rescue the LigIII ligation activity defects caused by FUS mutation.

Addressing these critical follow-up questions is an unmet need in the FUS-ALS field, which will help to develop a mechanism-based DNA-repair-targeted therapy. With recent emerging studies, the stage is set for such a paradigm shift.

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

breaks accompanied with genomic instability in them [56, 57].

PARP-1 has not been investigated.

genome instability, and neurodegeneration.

**4. Conclusions and perspectives**

**repair**

RT<sup>2</sup>

highly susceptible to DNA damaging agents and are accumulated with DNA strand

In response to DNA damage, FUS is recruited to DNA damage site in a PARP1 activity-dependent manner [51, 52]. FUS directly binds to PAR synthesized by activated PARP1 leading to the formation of damaged DNA-rich compartments contributed by its N-terminal low-complexity domain (LCD) and C-terminal RGG domains, and the compartments are dissociated by the hydrolysis of PAR by PARG [58], which indicates that PAR polymers play an essential role for the recruitment, likely through enhancing the FUS droplet formation [59]. While the activation of PARP1 can induce shuttling of FUS from nucleus to cytoplasm, which in turn enhances FUS aggregate formation and neurodegeneration [58, 60]. Furthermore, like FUS, the other two members in TET family, TAF15 and EWSR1 are also recruited to MIR-induced DNA damage tracks depending on PARP1 activity [53], while the molecular mechanism of the cross-talk between TET family proteins and

**3. Indirect role of FUS in DDR: FUS-directed RNA transactions in DNA** 

Although, recent reports from our group and others demonstrate a complex, but the direct role of FUS in maintaining genome integrity, whether FUS RNAbinding activity plays a role in regulating the expression of DDR factors has not been investigated. FUS regulates several key steps of RNA metabolism that impairs various biological processes. CLIP-seq has been used to identify RNAs that FUS targeted in multiple studies [16, 34, 61–65], which reveals an indirect role of FUS involving in DDR by regulating RNA transcription. One report shows that FUS regulates alternative splicing of pre-mRNAs and processing of long-intron containing transcripts in HeLa cells, and FUS binds RNA encoding proteins important for DNA damage response and repair pathways. By comparing with other CLIP-based assays, a map of FUS RNA targets to DNA DSBR by NHEJ and HR is generated in the report and in which, a number of key DDR factors such as ATM, 53BP1, MRN11, NBS1 are included [16]. We recently performed

 PCR arrays for DNA repair and DDR signaling pathways in CRISPR/cas9 FUS knockout (KO) cells, patient-derived FUS-mutant cells, as well as FUS-ALS patient spinal cord autopsy tissue, which revealed significant downregulation of DDR factors BRCA1, MSH complex, RAD23B, and DNA ligase 4. Notably, BRCA1 depletion has been linked to neuronal DNA DSB accumulation and cognitive defects in Alzheimer's disease. The ubiquitin receptor RAD23 functions both in nucleotide excision repair and the proteasomal protein clearance pathway and is thus linked to amyloid load in neurodegeneration. This study provides evidence of FUS pathology-mediated perturbation in the expression of DNA repair and DDR signaling factors and thus highlights the intricate connections between FUS,

As early as 1982, the defective DNA repair and its possible role in ALS pathology was first proposed by Bradley et al. [66], where they hypothesize that abnormal DNA in ALS may arise from a deficiency of an isozyme of a DNA repair factor. Subsequently, growing evidence suggests that defective DNA repair is a common

**32**

### **Acknowledgements**

The research was supported by grants from the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institute of Health (NIH) under the award numbers R01NS088645, RF1NS112719 and the Houston Methodist Research Institute funds. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

#### **Author details**

Haibo Wang1,2\* and Muralidhar L. Hegde1,2,3\*

1 Department of Radiation Oncology, Houston Methodist Research Institute, Houston, TX, USA

2 Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist Neurological Institute, Institute of Academic Medicine, Houston Methodist Hospital, Houston, TX, USA

3 Weill Medical College, New York, NY, USA

\*Address all correspondence to: hwang@houstonmethodist.org and mlhegde@houstonmethodist.org

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**35**

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New…*

nucleo-cytoplasmic shuttling. Journal of Cell Science. 1997;**110**(Pt 15):1741-1750

[10] Sama RR, Ward CL, Kaushansky LJ, Lemay N, Ishigaki S, Urano F, et al. FUS/TLS assembles into stress granules and is a prosurvival factor during hyperosmolar stress. Journal of Cellular Physiology. 2013;**228**(11):2222-2231

[11] Anderson P, Kedersha N. Stress granules. Current Biology.

[12] Kedersha N, Anderson P. Stress granules: Sites of mRNA triage that regulate mRNA stability and translatability. Biochemical Society Transactions. 2002;**30**(Pt 6):963-969

[13] Dormann D, Rodde R, Edbauer D, Bentmann E, Fischer I, Hruscha A, et al. ALS-associated fused in sarcoma (FUS) mutations disrupt transportin-mediated nuclear import. The EMBO Journal.

2009;**19**(10):R397-R398

2010;**29**(16):2841-2857

Biology. 2009;**1**(2):82-92

2016;**7**(3):330-340

Rare Disease. 2014;**2**:e29515

[14] Tan AY, Manley JL. The TET family of proteins: Functions and roles in disease. Journal of Molecular Cell

[15] Masuda A, Takeda J, Ohno K. FUS-mediated regulation of alternative RNA processing in neurons: Insights from global transcriptome analysis. Wiley Interdisciplinary Reviews: RNA.

[16] Zhou Y, Liu S, Ozturk A, Hicks GG. FUS-regulated RNA metabolism and DNA damage repair: Implications for amyotrophic lateral sclerosis and frontotemporal dementia pathogenesis.

[9] Fujii R, Takumi T. TLS facilitates transport of mRNA encoding an actinstabilizing protein to dendritic spines. Journal of Cell Science. 2005;**118**(Pt 24):

5755-5765

*DOI: http://dx.doi.org/10.5772/intechopen.92637*

[1] Ichikawa H, Shimizu K, Hayashi Y, Ohki M. An RNA-binding protein gene, TLS/FUS, is fused to ERG in human myeloid leukemia with t(16,21) chromosomal translocation. Cancer Research. 1994;**54**(11):2865-2868

[2] Panagopoulos I, Aman P, Fioretos T, Hoglund M, Johansson B, Mandahl N, et al. Fusion of the FUS gene with ERG in acute myeloid leukemia with t(16,21) (p11;q22). Genes, Chromosomes &

[3] Shing DC, McMullan DJ, Roberts P,

Cancer. 1994;**11**(4):256-262

2003;**63**(15):4568-4576

2009;**323**(5918):1205-1208

2013;**22**(13):2676-2688

[5] Vance C, Rogelj B, Hortobagyi T, De Vos KJ, Nishimura AL, Sreedharan J, et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009;**323**(5918):1208-1211

[6] Vance C, Scotter EL, Nishimura AL, Troakes C, Mitchell JC, Kathe C, et al. ALS mutant FUS disrupts nuclear localization and sequesters wildtype FUS within cytoplasmic stress granules. Human Molecular Genetics.

[7] Gal J, Zhang J, Kwinter DM, Zhai J, Jia H, Jia J, et al. Nuclear localization sequence of FUS and induction of stress granules by ALS mutants. Neurobiology of Aging. 2011;**32**(12):e2327-e2340

[8] Zinszner H, Sok J, Immanuel D, Yin Y, Ron D. TLS (FUS) binds RNA in vivo and engages in

Smith K, Chin SF, Nicholson J, et al. FUS/ERG gene fusions in Ewing's tumors. Cancer Research.

[4] Kwiatkowski TJ Jr, Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science.

**References**

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New… DOI: http://dx.doi.org/10.5772/intechopen.92637*

#### **References**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

The research was supported by grants from the National Institute of Neurological Disorders and Stroke (NINDS) of the National Institute of Health (NIH) under the award numbers R01NS088645, RF1NS112719 and the Houston Methodist Research Institute funds. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding

**34**

**Author details**

**Acknowledgements**

agencies.

Houston, TX, USA

Hospital, Houston, TX, USA

mlhegde@houstonmethodist.org

Haibo Wang1,2\* and Muralidhar L. Hegde1,2,3\*

3 Weill Medical College, New York, NY, USA

provided the original work is properly cited.

1 Department of Radiation Oncology, Houston Methodist Research Institute,

\*Address all correspondence to: hwang@houstonmethodist.org and

2 Department of Neurosurgery, Center for Neuroregeneration, Houston Methodist Neurological Institute, Institute of Academic Medicine, Houston Methodist

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

[1] Ichikawa H, Shimizu K, Hayashi Y, Ohki M. An RNA-binding protein gene, TLS/FUS, is fused to ERG in human myeloid leukemia with t(16,21) chromosomal translocation. Cancer Research. 1994;**54**(11):2865-2868

[2] Panagopoulos I, Aman P, Fioretos T, Hoglund M, Johansson B, Mandahl N, et al. Fusion of the FUS gene with ERG in acute myeloid leukemia with t(16,21) (p11;q22). Genes, Chromosomes & Cancer. 1994;**11**(4):256-262

[3] Shing DC, McMullan DJ, Roberts P, Smith K, Chin SF, Nicholson J, et al. FUS/ERG gene fusions in Ewing's tumors. Cancer Research. 2003;**63**(15):4568-4576

[4] Kwiatkowski TJ Jr, Bosco DA, Leclerc AL, Tamrazian E, Vanderburg CR, Russ C, et al. Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science. 2009;**323**(5918):1205-1208

[5] Vance C, Rogelj B, Hortobagyi T, De Vos KJ, Nishimura AL, Sreedharan J, et al. Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009;**323**(5918):1208-1211

[6] Vance C, Scotter EL, Nishimura AL, Troakes C, Mitchell JC, Kathe C, et al. ALS mutant FUS disrupts nuclear localization and sequesters wildtype FUS within cytoplasmic stress granules. Human Molecular Genetics. 2013;**22**(13):2676-2688

[7] Gal J, Zhang J, Kwinter DM, Zhai J, Jia H, Jia J, et al. Nuclear localization sequence of FUS and induction of stress granules by ALS mutants. Neurobiology of Aging. 2011;**32**(12):e2327-e2340

[8] Zinszner H, Sok J, Immanuel D, Yin Y, Ron D. TLS (FUS) binds RNA in vivo and engages in

nucleo-cytoplasmic shuttling. Journal of Cell Science. 1997;**110**(Pt 15):1741-1750

[9] Fujii R, Takumi T. TLS facilitates transport of mRNA encoding an actinstabilizing protein to dendritic spines. Journal of Cell Science. 2005;**118**(Pt 24): 5755-5765

[10] Sama RR, Ward CL, Kaushansky LJ, Lemay N, Ishigaki S, Urano F, et al. FUS/TLS assembles into stress granules and is a prosurvival factor during hyperosmolar stress. Journal of Cellular Physiology. 2013;**228**(11):2222-2231

[11] Anderson P, Kedersha N. Stress granules. Current Biology. 2009;**19**(10):R397-R398

[12] Kedersha N, Anderson P. Stress granules: Sites of mRNA triage that regulate mRNA stability and translatability. Biochemical Society Transactions. 2002;**30**(Pt 6):963-969

[13] Dormann D, Rodde R, Edbauer D, Bentmann E, Fischer I, Hruscha A, et al. ALS-associated fused in sarcoma (FUS) mutations disrupt transportin-mediated nuclear import. The EMBO Journal. 2010;**29**(16):2841-2857

[14] Tan AY, Manley JL. The TET family of proteins: Functions and roles in disease. Journal of Molecular Cell Biology. 2009;**1**(2):82-92

[15] Masuda A, Takeda J, Ohno K. FUS-mediated regulation of alternative RNA processing in neurons: Insights from global transcriptome analysis. Wiley Interdisciplinary Reviews: RNA. 2016;**7**(3):330-340

[16] Zhou Y, Liu S, Ozturk A, Hicks GG. FUS-regulated RNA metabolism and DNA damage repair: Implications for amyotrophic lateral sclerosis and frontotemporal dementia pathogenesis. Rare Disease. 2014;**2**:e29515

[17] Longinetti E, Fang F. Epidemiology of amyotrophic lateral sclerosis: An update of recent literature. Current Opinion in Neurology. 2019;**32**(5):771-776

[18] McCombe PA, Henderson RD. Effects of gender in amyotrophic lateral sclerosis. Gender Medicine. 2010;**7**(6):557-570

[19] Manjaly ZR, Scott KM, Abhinav K, Wijesekera L, Ganesalingam J, Goldstein LH, et al. The sex ratio in amyotrophic lateral sclerosis: A population based study. Amyotrophic Lateral Sclerosis. 2010;**11**(5):439-442

[20] Lai SL, Abramzon Y, Schymick JC, Stephan DA, Dunckley T, Dillman A, et al. FUS mutations in sporadic amyotrophic lateral sclerosis. Neurobiology of Aging. 2011;**32**(3):e551-e554

[21] Rademakers R, Stewart H, Dejesus-Hernandez M, Krieger C, Graff-Radford N, Fabros M, et al. Fus gene mutations in familial and sporadic amyotrophic lateral sclerosis. Muscle & Nerve. 2010;**42**(2):170-176

[22] Zou ZY, Peng Y, Feng XH, Wang XN, Sun Q, Liu MS, et al. Screening of the FUS gene in familial and sporadic amyotrophic lateral sclerosis patients of Chinese origin. European Journal of Neurology. 2012;**19**(7):977-983

[23] Conte A, Lattante S, Zollino M, Marangi G, Luigetti M, Del Grande A, et al. P525L FUS mutation is consistently associated with a severe form of juvenile amyotrophic lateral sclerosis. Neuromuscular Disorders. 2012;**22**(1):73-75

[24] Eura N, Sugie K, Suzuki N, Kiriyama T, Izumi T, Shimakura N, et al. A juvenile sporadic amyotrophic lateral sclerosis case with P525L mutation in

the FUS gene: A rare co-occurrence of autism spectrum disorder and tremor. Journal of the Neurological Sciences. 2019;**398**:67-68

[25] Baumer D, Hilton D, Paine SM, Turner MR, Lowe J, Talbot K, et al. Juvenile ALS with basophilic inclusions is a FUS proteinopathy with FUS mutations. Neurology. 2010;**75**(7):611-618

[26] Harrison AF, Shorter J. RNA-binding proteins with prion-like domains in health and disease. The Biochemical Journal. 2017;**474**(8):1417-1438

[27] Broustal O, Camuzat A, Guillot-Noel L, Guy N, Millecamps S, Deffond D, et al. FUS mutations in frontotemporal lobar degeneration with amyotrophic lateral sclerosis. Journal of Alzheimer's Disease. 2010;**22**(3):765-769

[28] Lipton AM, White CL 3rd, Bigio EH. Frontotemporal lobar degeneration with motor neuron disease-type inclusions predominates in 76 cases of frontotemporal degeneration. Acta Neuropathologica. 2004;**108**(5):379-385

[29] Neumann M, Rademakers R, Roeber S, Baker M, Kretzschmar HA, Mackenzie IR. A new subtype of frontotemporal lobar degeneration with FUS pathology. Brain. 2009;**132** (Pt 11):2922-2931

[30] Kino Y, Washizu C, Kurosawa M, Yamada M, Miyazaki H, Akagi T, et al. FUS/TLS deficiency causes behavioral and pathological abnormalities distinct from amyotrophic lateral sclerosis. Acta Neuropathologica Communications. 2015;**3**:24

[31] Lebedeva S, de Jesus Domingues AM, Butter F, Ketting RF. Characterization of genetic loss-offunction of FUS in zebrafish. RNA Biology. 2017;**14**(1):29-35

**37**

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New…*

amyotrophic lateral sclerosis. Brain.

[40] Bosco DA, Lemay N, Ko HK, Zhou H, Burke C, Kwiatkowski TJ Jr, et al. Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules. Human Molecular Genetics. 2010;**19**(21):4160-4175

[41] Kamelgarn M, Chen J, Kuang L, Jin H, Kasarskis EJ, Zhu H. ALS mutations of FUS suppress protein translation and disrupt the regulation of nonsense-mediated decay. Proceedings of the National Academy of Sciences of the United States of America. 2018;**115**(51):E11904-E11913

[42] Yasuda K, Zhang H, Loiselle D, Haystead T, Macara IG, Mili S. The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules. The Journal of Cell

[43] Marrone L, Drexler HCA, Wang J, Tripathi P, Distler T, Heisterkamp P, et al. FUS pathology in ALS is linked to alterations in multiple ALSassociated proteins and rescued by drugs stimulating autophagy. Acta Neuropathologica. 2019;**138**(1):67-84

Biology. 2013;**203**(5):737-746

[44] Kim GH, Kim JE, Rhie SJ, Yoon S. The role of oxidative stress in neurodegenerative diseases. Experimental Neurobiology.

[45] De Luca G, Russo MT, Degan P, Tiveron C, Zijno A, Meccia E, et al. A role for oxidized DNA precursors in Huntington's disease-like striatal neurodegeneration. PLoS Genetics.

[46] Zhang J, Perry G, Smith MA, Robertson D, Olson SJ, Graham DG, et al. Parkinson's disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra

2015;**24**(4):325-340

2008;**4**(11):e1000266

2019;**142**(9):2572-2580

*DOI: http://dx.doi.org/10.5772/intechopen.92637*

[32] Sharma A, Lyashchenko AK, Lu L, Nasrabady SE, Elmaleh M, Mendelsohn M, et al. ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function. Nature Communications.

[33] Butti Z, Patten SA. RNA

[34] Lagier-Tourenne C,

2012;**15**(11):1488-1497

2016;**35**(14):1504-1521

2013;**8**(4):e60788

2004;**43**(4):513-525

dysregulation in amyotrophic lateral sclerosis. Frontiers in Genetics.

Polymenidou M, Hutt KR, Vu AQ, Baughn M, Huelga SC, et al. Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs. Nature Neuroscience.

[35] Reber S, Stettler J, Filosa G,

[36] van Blitterswijk M, Wang ET, Friedman BA, Keagle PJ, Lowe P, Leclerc AL, et al. Characterization of FUS mutations in amyotrophic lateral sclerosis using RNA-Seq. PLoS One.

[37] Kanai Y, Dohmae N, Hirokawa N. Kinesin transports RNA: Isolation and characterization of an RNAtransporting granule. Neuron.

[38] Udagawa T, Fujioka Y, Tanaka M, Honda D, Yokoi S, Riku Y, et al. FUS regulates AMPA receptor function and FTLD/ALS-associated behaviour via GluA1 mRNA stabilization. Nature Communications. 2015;**6**:7098

[39] Tyzack GE, Luisier R, Taha DM, Neeves J, Modic M, Mitchell JS, et al. Widespread FUS mislocalization is a molecular hallmark of

Colombo M, Jutzi D, Lenzken SC, et al. Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants. The EMBO Journal.

2016;**7**:10465

2018;**9**:712

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New… DOI: http://dx.doi.org/10.5772/intechopen.92637*

[32] Sharma A, Lyashchenko AK, Lu L, Nasrabady SE, Elmaleh M, Mendelsohn M, et al. ALS-associated mutant FUS induces selective motor neuron degeneration through toxic gain of function. Nature Communications. 2016;**7**:10465

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

the FUS gene: A rare co-occurrence of autism spectrum disorder and tremor. Journal of the Neurological Sciences.

[25] Baumer D, Hilton D, Paine SM, Turner MR, Lowe J, Talbot K, et al. Juvenile ALS with basophilic inclusions is a FUS proteinopathy with FUS mutations. Neurology.

[27] Broustal O, Camuzat A, Guillot-Noel L, Guy N, Millecamps S, Deffond D, et al. FUS mutations in frontotemporal lobar degeneration with amyotrophic lateral sclerosis. Journal of Alzheimer's Disease. 2010;**22**(3):765-769

[28] Lipton AM, White CL 3rd, Bigio EH. Frontotemporal lobar degeneration with motor neuron disease-type inclusions predominates in 76 cases of frontotemporal degeneration. Acta Neuropathologica. 2004;**108**(5):379-385

[29] Neumann M, Rademakers R, Roeber S, Baker M, Kretzschmar HA, Mackenzie IR. A new subtype of frontotemporal lobar degeneration with

FUS pathology. Brain. 2009;**132**

[30] Kino Y, Washizu C, Kurosawa M, Yamada M, Miyazaki H, Akagi T, et al. FUS/TLS deficiency causes behavioral and pathological abnormalities distinct from amyotrophic lateral sclerosis. Acta Neuropathologica Communications.

Domingues AM, Butter F, Ketting RF. Characterization of genetic loss-offunction of FUS in zebrafish. RNA

(Pt 11):2922-2931

2015;**3**:24

[31] Lebedeva S, de Jesus

Biology. 2017;**14**(1):29-35

2019;**398**:67-68

2010;**75**(7):611-618

[26] Harrison AF, Shorter J. RNA-binding proteins with prion-like domains in health and disease. The Biochemical Journal.

2017;**474**(8):1417-1438

[17] Longinetti E, Fang F. Epidemiology

of amyotrophic lateral sclerosis: An update of recent literature. Current Opinion in Neurology.

[18] McCombe PA, Henderson RD. Effects of gender in amyotrophic lateral sclerosis. Gender Medicine.

[19] Manjaly ZR, Scott KM, Abhinav K,

[20] Lai SL, Abramzon Y, Schymick JC,

Wijesekera L, Ganesalingam J, Goldstein LH, et al. The sex ratio in amyotrophic lateral sclerosis: A population based study. Amyotrophic Lateral Sclerosis. 2010;**11**(5):439-442

Stephan DA, Dunckley T, Dillman A, et al. FUS mutations in sporadic amyotrophic lateral sclerosis. Neurobiology of Aging.

[21] Rademakers R, Stewart H, Dejesus-Hernandez M, Krieger C, Graff-Radford N, Fabros M, et al. Fus gene mutations in familial and sporadic amyotrophic lateral sclerosis. Muscle &

Nerve. 2010;**42**(2):170-176

2012;**19**(7):977-983

2012;**22**(1):73-75

[22] Zou ZY, Peng Y, Feng XH, Wang XN, Sun Q, Liu MS, et al. Screening of the FUS gene in familial and sporadic amyotrophic lateral sclerosis patients of Chinese origin. European Journal of Neurology.

[23] Conte A, Lattante S, Zollino M, Marangi G, Luigetti M, Del Grande A,

consistently associated with a severe form of juvenile amyotrophic lateral sclerosis. Neuromuscular Disorders.

et al. P525L FUS mutation is

[24] Eura N, Sugie K, Suzuki N,

Kiriyama T, Izumi T, Shimakura N, et al. A juvenile sporadic amyotrophic lateral sclerosis case with P525L mutation in

2011;**32**(3):e551-e554

2019;**32**(5):771-776

2010;**7**(6):557-570

**36**

[33] Butti Z, Patten SA. RNA dysregulation in amyotrophic lateral sclerosis. Frontiers in Genetics. 2018;**9**:712

[34] Lagier-Tourenne C, Polymenidou M, Hutt KR, Vu AQ, Baughn M, Huelga SC, et al. Divergent roles of ALS-linked proteins FUS/TLS and TDP-43 intersect in processing long pre-mRNAs. Nature Neuroscience. 2012;**15**(11):1488-1497

[35] Reber S, Stettler J, Filosa G, Colombo M, Jutzi D, Lenzken SC, et al. Minor intron splicing is regulated by FUS and affected by ALS-associated FUS mutants. The EMBO Journal. 2016;**35**(14):1504-1521

[36] van Blitterswijk M, Wang ET, Friedman BA, Keagle PJ, Lowe P, Leclerc AL, et al. Characterization of FUS mutations in amyotrophic lateral sclerosis using RNA-Seq. PLoS One. 2013;**8**(4):e60788

[37] Kanai Y, Dohmae N, Hirokawa N. Kinesin transports RNA: Isolation and characterization of an RNAtransporting granule. Neuron. 2004;**43**(4):513-525

[38] Udagawa T, Fujioka Y, Tanaka M, Honda D, Yokoi S, Riku Y, et al. FUS regulates AMPA receptor function and FTLD/ALS-associated behaviour via GluA1 mRNA stabilization. Nature Communications. 2015;**6**:7098

[39] Tyzack GE, Luisier R, Taha DM, Neeves J, Modic M, Mitchell JS, et al. Widespread FUS mislocalization is a molecular hallmark of

amyotrophic lateral sclerosis. Brain. 2019;**142**(9):2572-2580

[40] Bosco DA, Lemay N, Ko HK, Zhou H, Burke C, Kwiatkowski TJ Jr, et al. Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules. Human Molecular Genetics. 2010;**19**(21):4160-4175

[41] Kamelgarn M, Chen J, Kuang L, Jin H, Kasarskis EJ, Zhu H. ALS mutations of FUS suppress protein translation and disrupt the regulation of nonsense-mediated decay. Proceedings of the National Academy of Sciences of the United States of America. 2018;**115**(51):E11904-E11913

[42] Yasuda K, Zhang H, Loiselle D, Haystead T, Macara IG, Mili S. The RNA-binding protein Fus directs translation of localized mRNAs in APC-RNP granules. The Journal of Cell Biology. 2013;**203**(5):737-746

[43] Marrone L, Drexler HCA, Wang J, Tripathi P, Distler T, Heisterkamp P, et al. FUS pathology in ALS is linked to alterations in multiple ALSassociated proteins and rescued by drugs stimulating autophagy. Acta Neuropathologica. 2019;**138**(1):67-84

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[57] Shibata A, Kamada N, Masumura K, Nohmi T, Kobayashi S, Teraoka H, et al. Parp-1 deficiency causes an increase of deletion mutations and insertions/ rearrangements in vivo after treatment with an alkylating agent. Oncogene. 2005;**24**(8):1328-1337

[58] Singatulina AS, Hamon L, Sukhanova MV, Desforges B, Joshi V, Bouhss A, et al. PARP-1 activation directs FUS to DNA damage sites to form PARG-reversible compartments enriched in damaged DNA. Cell Reports. 2019;**27**(6):1809-1821

[59] Patel A, Lee HO, Jawerth L, Maharana S, Jahnel M, Hein MY, et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell. 2015;**162**(5):1066-1077

[60] Naumann M, Pal A, Goswami A, Lojewski X, Japtok J, Vehlow A, et al. Impaired DNA damage response signaling by FUS-NLS mutations

**39**

*Molecular Basis of DNA Repair Defects in FUS-Associated ALS: Implications of a New…*

interacts with HSP60 to promote mitochondrial damage. PLoS Genetics.

[70] Nakaya T, Maragkakis M.

Reports. 2018;**8**(1):15575

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Amyotrophic lateral sclerosis associated FUS mutation shortens mitochondria and induces neurotoxicity. Scientific

Krishnamurthy K, Trotti D, Pasinelli P. Astrocytes expressing ALS-linked mutant FUS induce motor neuron death through release of tumor necrosis factor-alpha. Glia. 2018;**66**(5):1016-1033

Chalazonitis A, Jessell TM, Wichterle H, et al. Astrocytes expressing ALS-linked mutated SOD1 release factors selectively

[73] Ragagnin AMG, Shadfar S, Vidal M, Jamali MS, Atkin JD. Motor neuron susceptibility in ALS/FTD. Frontiers in

[72] Nagai M, Re DB, Nagata T,

toxic to motor neurons. Nature Neuroscience. 2007;**10**(5):615-622

Neuroscience. 2019;**13**:532

[69] Deng J, Wang P, Chen X, Cheng H, Liu J, Fushimi K, et al. FUS interacts with ATP synthase beta subunit and induces mitochondrial unfolded protein response in cellular and animal models. Proceedings of the National Academy of Sciences of the United States of America. 2018;**115**(41):E9678-E9686

2015;**11**(9):e1005357

*DOI: http://dx.doi.org/10.5772/intechopen.92637*

leads to neurodegeneration and FUS aggregate formation. Nature Communications. 2018;**9**(1):335

[61] Hoell JI, Larsson E, Runge S, Nusbaum JD, Duggimpudi S, Farazi TA, et al. RNA targets of wild-type and mutant FET family proteins. Nature Structural & Molecular Biology.

[62] Zhou Y, Liu S, Liu G, Ozturk A, Hicks GG. ALS-associated FUS mutations result in compromised FUS alternative splicing and autoregulation. PLoS Genetics. 2013;**9**(10):e1003895

[63] Ishigaki S, Masuda A, Fujioka Y, Iguchi Y, Katsuno M, Shibata A, et al. Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions. Scientific

[64] Nakaya T, Alexiou P, Maragkakis M, Chang A, Mourelatos Z. FUS regulates

genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns. RNA.

[65] Rogelj B, Easton LE, Bogu GK, Stanton LW, Rot G, Curk T, et al. Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain. Scientific Reports.

[66] Bradley WG, Krasin F. A new hypothesis of the etiology of amyotrophic lateral sclerosis. The DNA hypothesis. Archives of Neurology.

1982;**39**(11):677-680

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[68] Deng J, Yang M, Chen Y, Chen X, Liu J, Sun S, et al. FUS

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Pfenning AR, Pan L, Yamakawa S, Seo J, et al. Activity-induced DNA breaks govern the expression of neuronal early-response genes. Cell.

2011;**18**(12):1428-1431

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leads to neurodegeneration and FUS aggregate formation. Nature Communications. 2018;**9**(1):335

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

Grofte M, et al. Liquid demixing of intrinsically disordered proteins is seeded by poly(ADP-ribose). Nature Communications. 2015;**6**:8088

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Cardoso MC, et al. UVA-induced DNA double-strand breaks result from the repair of clustered oxidative DNA damages. Nucleic Acids Research.

[55] Sallmann FR, Vodenicharov MD, Wang ZQ, Poirier GG. Characterization of sPARP-1. An alternative product of PARP-1 gene with poly(ADPribose) polymerase activity

independent of DNA strand breaks. The Journal of Biological Chemistry.

Breitbart EW, Greulich KO,

2012;**40**(20):10263-10273

2000;**275**(20):15504-15511

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2005;**24**(8):1328-1337

[58] Singatulina AS, Hamon L, Sukhanova MV, Desforges B, Joshi V, Bouhss A, et al. PARP-1 activation directs FUS to DNA damage sites to form PARG-reversible compartments enriched in damaged DNA. Cell Reports. 2019;**27**(6):1809-1821

[59] Patel A, Lee HO, Jawerth L, Maharana S, Jahnel M, Hein MY, et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell. 2015;**162**(5):1066-1077

[60] Naumann M, Pal A, Goswami A, Lojewski X, Japtok J, Vehlow A, et al. Impaired DNA damage response signaling by FUS-NLS mutations

[56] Ko HL, Ren EC. Functional aspects of PARP1 in DNA repair and transcription. Biomolecules.

[57] Shibata A, Kamada N, Masumura K, Nohmi T, Kobayashi S, Teraoka H, et al. Parp-1 deficiency causes an increase of deletion mutations and insertions/ rearrangements in vivo after treatment with an alkylating agent. Oncogene.

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[47] Hegde ML, Hegde PM, Rao KS, Mitra S. Oxidative genome damage and its repair in neurodegenerative diseases: Function of transition metals as a double-edged sword. Journal of Alzheimer's Disease. 2011;**24**(Suppl 2):

[48] Castellotti B, Mariotti C, Rimoldi M, Fancellu R, Plumari M, Caimi S, et al. Ataxia with oculomotor apraxia type1 (AOA1): Novel and recurrent aprataxin mutations, coenzyme Q10 analyses, and clinical findings in Italian patients. Neurogenetics. 2011;**12**(3):193-201

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Hegde PM, Vandoorne T, Eckelmann BJ, et al. Mutant FUS causes DNA ligation defects to inhibit oxidative damage repair in amyotrophic lateral sclerosis. Nature Communications. 2018;**9**(1):3683

[51] Mastrocola AS, Kim SH, Trinh AT, Rodenkirch LA, Tibbetts RS. The RNA-binding protein fused in

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[52] Rulten SL, Rotheray A, Green RL, Grundy GJ, Moore DA, Gomez-Herreros F, et al. PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage. Nucleic Acids Research. 2014;**42**(1):307-314

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Teloni F, Pozdnyakova I, Pellegrino S,

183-198

**38**

[61] Hoell JI, Larsson E, Runge S, Nusbaum JD, Duggimpudi S, Farazi TA, et al. RNA targets of wild-type and mutant FET family proteins. Nature Structural & Molecular Biology. 2011;**18**(12):1428-1431

[62] Zhou Y, Liu S, Liu G, Ozturk A, Hicks GG. ALS-associated FUS mutations result in compromised FUS alternative splicing and autoregulation. PLoS Genetics. 2013;**9**(10):e1003895

[63] Ishigaki S, Masuda A, Fujioka Y, Iguchi Y, Katsuno M, Shibata A, et al. Position-dependent FUS-RNA interactions regulate alternative splicing events and transcriptions. Scientific Reports. 2012;**2**:529

[64] Nakaya T, Alexiou P, Maragkakis M, Chang A, Mourelatos Z. FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns. RNA. 2013;**19**(4):498-509

[65] Rogelj B, Easton LE, Bogu GK, Stanton LW, Rot G, Curk T, et al. Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain. Scientific Reports. 2012;**2**:603

[66] Bradley WG, Krasin F. A new hypothesis of the etiology of amyotrophic lateral sclerosis. The DNA hypothesis. Archives of Neurology. 1982;**39**(11):677-680

[67] Madabhushi R, Gao F, Pfenning AR, Pan L, Yamakawa S, Seo J, et al. Activity-induced DNA breaks govern the expression of neuronal early-response genes. Cell. 2015;**161**(7):1592-1605

[68] Deng J, Yang M, Chen Y, Chen X, Liu J, Sun S, et al. FUS interacts with HSP60 to promote mitochondrial damage. PLoS Genetics. 2015;**11**(9):e1005357

[69] Deng J, Wang P, Chen X, Cheng H, Liu J, Fushimi K, et al. FUS interacts with ATP synthase beta subunit and induces mitochondrial unfolded protein response in cellular and animal models. Proceedings of the National Academy of Sciences of the United States of America. 2018;**115**(41):E9678-E9686

[70] Nakaya T, Maragkakis M. Amyotrophic lateral sclerosis associated FUS mutation shortens mitochondria and induces neurotoxicity. Scientific Reports. 2018;**8**(1):15575

[71] Kia A, McAvoy K, Krishnamurthy K, Trotti D, Pasinelli P. Astrocytes expressing ALS-linked mutant FUS induce motor neuron death through release of tumor necrosis factor-alpha. Glia. 2018;**66**(5):1016-1033

[72] Nagai M, Re DB, Nagata T, Chalazonitis A, Jessell TM, Wichterle H, et al. Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nature Neuroscience. 2007;**10**(5):615-622

[73] Ragagnin AMG, Shadfar S, Vidal M, Jamali MS, Atkin JD. Motor neuron susceptibility in ALS/FTD. Frontiers in Neuroscience. 2019;**13**:532

**41**

**Chapter 3**

**Abstract**

**1. Introduction**

Senataxin: A Putative RNA: DNA

Emerging Mechanisms of Genome

Amyotrophic lateral sclerosis type 4 (ALS4) is a rare, autosomal dominant childhood- or adolescent-onset motor neuron disease caused by genetic defects in senataxin (SETX), a putative RNA–DNA helicase. Studies on the yeast SETX ortholog Sen1 revealed its role in small RNA termination pathways. It has been postulated that ALS4-associated neuronal pathologies could stem from defects in RNA metabolism and altered gene expression. Importantly, SETX prevents the accumulation of R-loops, which are potentially pathogenic RNA–DNA hybrids that stem from perturbations in transcription. SETX also interacts with the tumor suppressor BRCA1 that helps promote DNA double-strand break repair by homologous recombination. As such, SETX could contribute toward the removal of harmful R-loops and DSBs in postmitotic neurons. This chapter will visit the plausible mechanistic role of SETX in R-loop removal and DNA break repair that could prevent the activation of

apoptotic cell death in neurons and pathological manifestation of ALS4.

break, homologous recombination, nonhomologous DNA end joining

**Keywords:** spinal muscular atrophy, SETX, transcription, R-loop, DNA double-strand

Amyotrophic lateral sclerosis (ALS) type 4 is a rare form of distal spinal muscular atrophy (SMA) with onset at age 25 years or younger. The disease first manifests itself with weakness of ankles and wrists and gradually paralyzes the limbs due to severe muscle wasting. However, unlike classical ALS, the respiratory and bulbar muscles, sensory abilities, and cognitive functions are largely preserved in ALS4 patients. Hence, ALS4 patients, while having to endure severe disabilities, could expect an otherwise normal life expectancy with proper medical attention.

In 1998, the joint effort of Phillip Chance and David Cornblath described the Mattingly disease, a hereditary peripheral neuropathy as ALS type 4 (ALS4) [1]. The Mattingly disease was first seen among the descendants of a seventeenthcentury English colonist, Thomas Mattingly from Maryland. With collaborations of the Mattingly clan, the work of Chance and Cornblath led to the identification of the disease gene locus located at chromosome 9q34 [1]. The causative gene was

Helicase Mutated in ALS4—

Stability in Motor Neurons

*Arijit Dutta, Robert Hromas and Patrick Sung*

#### **Chapter 3**

## Senataxin: A Putative RNA: DNA Helicase Mutated in ALS4— Emerging Mechanisms of Genome Stability in Motor Neurons

*Arijit Dutta, Robert Hromas and Patrick Sung*

### **Abstract**

Amyotrophic lateral sclerosis type 4 (ALS4) is a rare, autosomal dominant childhood- or adolescent-onset motor neuron disease caused by genetic defects in senataxin (SETX), a putative RNA–DNA helicase. Studies on the yeast SETX ortholog Sen1 revealed its role in small RNA termination pathways. It has been postulated that ALS4-associated neuronal pathologies could stem from defects in RNA metabolism and altered gene expression. Importantly, SETX prevents the accumulation of R-loops, which are potentially pathogenic RNA–DNA hybrids that stem from perturbations in transcription. SETX also interacts with the tumor suppressor BRCA1 that helps promote DNA double-strand break repair by homologous recombination. As such, SETX could contribute toward the removal of harmful R-loops and DSBs in postmitotic neurons. This chapter will visit the plausible mechanistic role of SETX in R-loop removal and DNA break repair that could prevent the activation of apoptotic cell death in neurons and pathological manifestation of ALS4.

**Keywords:** spinal muscular atrophy, SETX, transcription, R-loop, DNA double-strand break, homologous recombination, nonhomologous DNA end joining

#### **1. Introduction**

Amyotrophic lateral sclerosis (ALS) type 4 is a rare form of distal spinal muscular atrophy (SMA) with onset at age 25 years or younger. The disease first manifests itself with weakness of ankles and wrists and gradually paralyzes the limbs due to severe muscle wasting. However, unlike classical ALS, the respiratory and bulbar muscles, sensory abilities, and cognitive functions are largely preserved in ALS4 patients. Hence, ALS4 patients, while having to endure severe disabilities, could expect an otherwise normal life expectancy with proper medical attention.

In 1998, the joint effort of Phillip Chance and David Cornblath described the Mattingly disease, a hereditary peripheral neuropathy as ALS type 4 (ALS4) [1]. The Mattingly disease was first seen among the descendants of a seventeenthcentury English colonist, Thomas Mattingly from Maryland. With collaborations of the Mattingly clan, the work of Chance and Cornblath led to the identification of the disease gene locus located at chromosome 9q34 [1]. The causative gene was identified to be *Senataxin* (SETX), which is a large protein with features that typify RNA–DNA helicases [2]. Three distinct mutations in SETX were found in pedigree analysis of ALS4 patients. Some later studies also reported sporadic mutations in SETX [3, 4], which are summarized in **Table 1**. It should be noted that the other three forms of juvenile ALS (JALS) stem from mutations in different genes, namely, *ALS2* (ALS2), *SPG11* (ALS5), and *SIGMAR1* (ALS16).

Pathological studies of ALS4 have been hampered because of the rarity of the disease, with only about a dozen of diagnosed families around the world. Chen et al. [2] detected degeneration of anterior horn cells in spinal cords and corticospinal tracts in postmortem tissues from two aged individuals of pedigree K7000. Specifically, even though sensory abilities were not significantly affected in these individuals, a significant loss of dorsal root ganglia and posterior columns was detected, along with marked axonal degeneration of motor and sensory roots and peripheral nerves.

In another study, cytosolic mislocalization of the transactive response DNAbinding protein (TDP-43) was observed in spinal cord motor neurons in postmortem tissues from all the ALS4 patients examined [8]. TDP-43 is an RNA metabolism factor and is a well-documented biomarker that forms toxic protein aggregates in multiple neurodegenerative diseases including ALS [9, 10]. Recapitulation of TDP-43 histopathology in motor neurons of mice carrying ALS4 mutations led the authors to imply that dysfunction of SETX converges on TDP-43 pathology causing an ALStype motor neurodegeneration [8], although the mechanism was not identified.

SETX has also been found mutated in another neurodegenerative disorder termed ataxia with oculomotor apraxia type 2 (AOA2) [11]. However, in this case, disease is caused by missense mutations leading to premature termination of the SETX mRNA transcript. AOA2 patients suffer from progressive cerebellar ataxia with peripheral neuropathy, cerebellar atrophy, and occasional oculomotor apraxia. However, unlike in ALS4, the motor neuron functions are largely preserved in AOA2 patients [12]. It has been suggested that distinct pathologies of AOA2 and ALS4 stem from unique alterations in expression of genes regulated via SETX in neuronal cells [13].

In spite of the seminal discovery of SETX mutations as being the root cause of ALS4, the etiopathogenesis of this disease remains largely unknown. In this


**43**

*Senataxin: A Putative RNA: DNA Helicase Mutated in ALS4—Emerging Mechanisms…*

chapter we will consider the properties of SETX and its role in the maintenance of genomic stability that are likely germane for the health of motor neurons and ALS4

**2. Senataxin at the crossroads of RNA metabolism and genomic stability**

Studies on SETX predate its ALS4 association. SETX is the likely ortholog of a budding yeast protein, splicing endonuclease 1 (Sen1), so named because of its suspected role in the endonucleolytic processing of tRNA during its splicing and maturation [14]. However, because Sen1 lacks endonuclease activity, it likely functions as a non-catalytic effecter of the nucleolytic entity within the splicing machinery [15]. Sen1 possesses sequence motifs characteristic of superfamily 1 (SF1B) nucleic acid helicases [16]. Consistent with this, Sen1 possesses a helicase activity capable of unwinding RNA-DNA hybrids [17–19]. Like other SF1B helicases, Sen1

SETX is of low abundance (<500 molecules/cell) predominantly a nuclear protein with some studies reporting its presence in the nucleolus [21, 22]. SETX interacts with RNA polymerase II (pol II) and helps ensure correct termination of transcription and, as such, is important for the processing of noncoding RNAs (ncRNAs) and mRNAs [23, 24]. Importantly, recent studies suggest a role of SETX in maintaining genomic stability across highly transcribed genomic regions via resolution of RNA–DNA hybrids called R-loops, which arise as a consequence of RNA pol II stalling or perturbations of a transcription-coupled process such as mRNA splicing [25]. Moreover, SETX could also clear RNA–DNA hybrids at genomic breaks and promote DNA repair via homologous recombination (HR) [26]. We will explore the various functions of SETX/Sen1 and possible mechanisms

SETX is a large protein of 2677 amino acid residues (303 kDa) and, like yeast Sen1, harbors SF1B-type helicase motifs (**Figure 1A**). It should be noted that even though Sen1 is known to unwind RNA-DNA hybrids [14, 15], such an activity has not yet been demonstrated for SETX. However, ALS4-associated missense mutations (K2029Q, R2136H, and I2547T) are all located in the putative C-terminal helicase domain of SETX (**Figure 1A**). Both SETX and Sen1 have an N-terminal domain that undergoes SUMO modification (**Figure 1A**) and that mediates protein–protein interactions with factors that function in RNA metabolism [27]. SETX likely forms a homodimer via the N-terminal domain*,* but the hereditary ALS4 mutations do not

*In silico* analysis suggests that there is a large IDR in SETX that spans more than 1000 amino acid residues, a structural feature that is absent in the yeast ortholog Sen1. This putative IDR could confer to SETX the ability to interact with different protein partners, to bind nucleic acids [29]. IDRs in nucleic acid-binding proteins are often subject to post-translational modifications and could undergo phase separation, a molecular phenomenon of rearrangement of molecules in a homogenous solution into distinctly concentrated regions of space called condensates [30–33]. However, unrestrained phase separation causes protein aggregation that is observed with FUS [34] and TDP-43 [35], two extensively studied factors associated with

translocates on nucleic acid strands in the 5′ → 3′ direction [20].

by which SETX mutations give rise to ALS4.

appear to affect protein dimerization [28].

*2.1.1 SETX has a large intrinsically disordered region (IDR)*

**2.1 Biochemical and structural features of SETX**

*DOI: http://dx.doi.org/10.5772/intechopen.90215*

pathology.

#### **Table 1.**

*ALS4-associated mutations in SETX.*

chapter we will consider the properties of SETX and its role in the maintenance of genomic stability that are likely germane for the health of motor neurons and ALS4 pathology.

### **2. Senataxin at the crossroads of RNA metabolism and genomic stability**

Studies on SETX predate its ALS4 association. SETX is the likely ortholog of a budding yeast protein, splicing endonuclease 1 (Sen1), so named because of its suspected role in the endonucleolytic processing of tRNA during its splicing and maturation [14]. However, because Sen1 lacks endonuclease activity, it likely functions as a non-catalytic effecter of the nucleolytic entity within the splicing machinery [15]. Sen1 possesses sequence motifs characteristic of superfamily 1 (SF1B) nucleic acid helicases [16]. Consistent with this, Sen1 possesses a helicase activity capable of unwinding RNA-DNA hybrids [17–19]. Like other SF1B helicases, Sen1 translocates on nucleic acid strands in the 5′ → 3′ direction [20].

SETX is of low abundance (<500 molecules/cell) predominantly a nuclear protein with some studies reporting its presence in the nucleolus [21, 22]. SETX interacts with RNA polymerase II (pol II) and helps ensure correct termination of transcription and, as such, is important for the processing of noncoding RNAs (ncRNAs) and mRNAs [23, 24]. Importantly, recent studies suggest a role of SETX in maintaining genomic stability across highly transcribed genomic regions via resolution of RNA–DNA hybrids called R-loops, which arise as a consequence of RNA pol II stalling or perturbations of a transcription-coupled process such as mRNA splicing [25]. Moreover, SETX could also clear RNA–DNA hybrids at genomic breaks and promote DNA repair via homologous recombination (HR) [26]. We will explore the various functions of SETX/Sen1 and possible mechanisms by which SETX mutations give rise to ALS4.

#### **2.1 Biochemical and structural features of SETX**

SETX is a large protein of 2677 amino acid residues (303 kDa) and, like yeast Sen1, harbors SF1B-type helicase motifs (**Figure 1A**). It should be noted that even though Sen1 is known to unwind RNA-DNA hybrids [14, 15], such an activity has not yet been demonstrated for SETX. However, ALS4-associated missense mutations (K2029Q, R2136H, and I2547T) are all located in the putative C-terminal helicase domain of SETX (**Figure 1A**). Both SETX and Sen1 have an N-terminal domain that undergoes SUMO modification (**Figure 1A**) and that mediates protein–protein interactions with factors that function in RNA metabolism [27]. SETX likely forms a homodimer via the N-terminal domain*,* but the hereditary ALS4 mutations do not appear to affect protein dimerization [28].

#### *2.1.1 SETX has a large intrinsically disordered region (IDR)*

*In silico* analysis suggests that there is a large IDR in SETX that spans more than 1000 amino acid residues, a structural feature that is absent in the yeast ortholog Sen1. This putative IDR could confer to SETX the ability to interact with different protein partners, to bind nucleic acids [29]. IDRs in nucleic acid-binding proteins are often subject to post-translational modifications and could undergo phase separation, a molecular phenomenon of rearrangement of molecules in a homogenous solution into distinctly concentrated regions of space called condensates [30–33]. However, unrestrained phase separation causes protein aggregation that is observed with FUS [34] and TDP-43 [35], two extensively studied factors associated with

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

*ALS2* (ALS2), *SPG11* (ALS5), and *SIGMAR1* (ALS16).

peripheral nerves.

**Mutation Amino acid** 

**substitution**

c.4660T → G C1554G Negative United

c.6085C → G K2029Q Negative United

c.7640T → C I2547T Negative United

c.1166T → C L389S Positive

identified to be *Senataxin* (SETX), which is a large protein with features that typify RNA–DNA helicases [2]. Three distinct mutations in SETX were found in pedigree analysis of ALS4 patients. Some later studies also reported sporadic mutations in SETX [3, 4], which are summarized in **Table 1**. It should be noted that the other three forms of juvenile ALS (JALS) stem from mutations in different genes, namely,

Pathological studies of ALS4 have been hampered because of the rarity of the disease, with only about a dozen of diagnosed families around the world. Chen et al. [2] detected degeneration of anterior horn cells in spinal cords and corticospinal tracts in postmortem tissues from two aged individuals of pedigree K7000. Specifically, even though sensory abilities were not significantly affected in these individuals, a significant loss of dorsal root ganglia and posterior columns was detected, along with marked axonal degeneration of motor and sensory roots and

In another study, cytosolic mislocalization of the transactive response DNAbinding protein (TDP-43) was observed in spinal cord motor neurons in postmortem tissues from all the ALS4 patients examined [8]. TDP-43 is an RNA metabolism factor and is a well-documented biomarker that forms toxic protein aggregates in multiple neurodegenerative diseases including ALS [9, 10]. Recapitulation of TDP-43 histopathology in motor neurons of mice carrying ALS4 mutations led the authors to imply that dysfunction of SETX converges on TDP-43 pathology causing an ALStype motor neurodegeneration [8], although the mechanism was not identified.

SETX has also been found mutated in another neurodegenerative disorder termed ataxia with oculomotor apraxia type 2 (AOA2) [11]. However, in this case, disease is caused by missense mutations leading to premature termination of the SETX mRNA transcript. AOA2 patients suffer from progressive cerebellar ataxia with peripheral neuropathy, cerebellar atrophy, and occasional oculomotor apraxia. However, unlike in ALS4, the motor neuron functions are largely preserved in AOA2 patients [12]. It has been suggested that distinct pathologies of AOA2 and ALS4 stem from unique alterations in expression of genes regulated via SETX in neuronal cells [13].

In spite of the seminal discovery of SETX mutations as being the root cause of ALS4, the etiopathogenesis of this disease remains largely unknown. In this

> **Family history**

c.8C → T T3I Positive Austria Chen et al. [2]

Positive

c.6407G → A R2136H Positive Belgium Chen et al. 2004 [2] c.6406C → T R2136C Negative Japan Saiga T et al., 2012 [4]

c.2672T → T V891A Positive Germany Rudnik-Schoneborn et al. [7]

**Origin References**

Rabin et al. [5] Chen et al. [2] Avemaria et al. [6]

Hirano et al. [3]

Hirano et al. [3]

Hirano et al. [3]

United States Italy

States

States

States

**42**

**Table 1.**

*ALS4-associated mutations in SETX.*

#### **Figure 1.**

*(A) Schematic diagram of SETX, indicating N-terminal domain (green), central domain (red), and C-terminal helicase domain (blue); conserved motifs are highlighted: Motif I interacts with Mg2+ and NTP, conserved G maintains a flexible loop, motif 1a binds with substrate nucleic acid and transduces energy from the ATP-binding site to the DNA-binding site, motif II binds and hydrolyses ATP, motif III couples ATP hydrolysis with helicase activity, motif V binds substrate nucleic acid, and motif VI couples ATP hydrolysis with helicase activity; ALS4 mutation residues (red) are T3I, L389S, V891A, C1554G, K2029Q, R1236H/C, and I2647T; predicted sumoylation residues (blue) are K78, K863, and K1051 [119]; and cysteine residues predicted by CYSPRED (reliability*≥*8) to form disulfide bonds (indicated by stars) are C4, C5, C7, C145, C555, C637, C688, C997, C1080, C1123C,1153, C1262, C1277, C1398, C1442, C1509, C1672, C1719, and C2622. (B) Prediction of natural disordered region of SETX (upper panel), and Sen1 (lower panel), with the in silico metapredictor PONDR-VL3 [120]. N-terminal domain (green), central disordered region (red), C-terminal helicase domain (blue).*

**45**

**Figure 2.**

*binding proteins (green), adapted from [40].*

*Senataxin: A Putative RNA: DNA Helicase Mutated in ALS4—Emerging Mechanisms…*

ALS, which are known to form stress granules in diseased neurons. It could be that SETX, through a phase separation mechanism, forms macromolecular complexes with factors associated with transcription and DNA damage repair. Pathological mutations in SETX could then lead to protein aggregation and loss of protein func-

Multiple neurodegenerative diseases including ALS have been classified among protein misfolding disorders, with disruption of protein disulfide isomerases (PDIs) causing aggregation of superoxide dismutase (SOD1) and TDP-43 in ALS neurons [36]. PDIs are a family of proteins that catalyze formation of disulfide bonds and proper folding of proteins, particularly especially those that harbor an IDR [37]. SETX has 31 cysteine residues in its IDR*,* and in silico analysis of this region using two independent neural network based predictors, CYSPRED [38] and DIpro [39], revealed that at least 14 cysteine residues in the SETX IDR could engage in disulfide bonding (**Figure 1A**), which is expected to be catalyzed by a PDI. This notion is supported by a proteomic analysis where PDIA6 was detected as a component of the SETX-interactome (**Figure 2**), [40]. We also note that amino acid residue C1554, expected to engage in disulfide linkage with C1509 (**Figure 1A**), is mutated in a sporadic case of ALS4 [3]. Testing of SETX regulation via redox homeostasis [41, 42]

The role of SETX in regulation of coding and noncoding transcripts is highly

conserved. RNA-seq analysis showed that SETX mutations that cause either ALS4 or AOA2 induce unique changes in gene expression patterns [13]. Studies in yeast have shown that Sen1 interacts directly with RNA pol II and is an integral component of the transcription termination machinery consisting of two other

*SETX interactome: DNA repair factors (red), sumoylation and ubiquitination-associated factors (purple), RNA exosome factors (blue), RNAP II and transcription-associated factors (orange), splicing factor and RNA-*

*DOI: http://dx.doi.org/10.5772/intechopen.90215*

merits the effort of ALS researchers.

*2.2.1 Role in transcription termination*

**2.2 Involvement of SETX in RNA metabolism**

tion in ALS4. This premise awaits experimental testing.

*2.1.2 SETX structure could be regulated via disulfide bonding*

*Senataxin: A Putative RNA: DNA Helicase Mutated in ALS4—Emerging Mechanisms… DOI: http://dx.doi.org/10.5772/intechopen.90215*

ALS, which are known to form stress granules in diseased neurons. It could be that SETX, through a phase separation mechanism, forms macromolecular complexes with factors associated with transcription and DNA damage repair. Pathological mutations in SETX could then lead to protein aggregation and loss of protein function in ALS4. This premise awaits experimental testing.

#### *2.1.2 SETX structure could be regulated via disulfide bonding*

Multiple neurodegenerative diseases including ALS have been classified among protein misfolding disorders, with disruption of protein disulfide isomerases (PDIs) causing aggregation of superoxide dismutase (SOD1) and TDP-43 in ALS neurons [36]. PDIs are a family of proteins that catalyze formation of disulfide bonds and proper folding of proteins, particularly especially those that harbor an IDR [37]. SETX has 31 cysteine residues in its IDR*,* and in silico analysis of this region using two independent neural network based predictors, CYSPRED [38] and DIpro [39], revealed that at least 14 cysteine residues in the SETX IDR could engage in disulfide bonding (**Figure 1A**), which is expected to be catalyzed by a PDI. This notion is supported by a proteomic analysis where PDIA6 was detected as a component of the SETX-interactome (**Figure 2**), [40]. We also note that amino acid residue C1554, expected to engage in disulfide linkage with C1509 (**Figure 1A**), is mutated in a sporadic case of ALS4 [3]. Testing of SETX regulation via redox homeostasis [41, 42] merits the effort of ALS researchers.

#### **2.2 Involvement of SETX in RNA metabolism**

#### *2.2.1 Role in transcription termination*

The role of SETX in regulation of coding and noncoding transcripts is highly conserved. RNA-seq analysis showed that SETX mutations that cause either ALS4 or AOA2 induce unique changes in gene expression patterns [13]. Studies in yeast have shown that Sen1 interacts directly with RNA pol II and is an integral component of the transcription termination machinery consisting of two other

#### **Figure 2.**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

*(A) Schematic diagram of SETX, indicating N-terminal domain (green), central domain (red), and C-terminal helicase domain (blue); conserved motifs are highlighted: Motif I interacts with Mg2+ and NTP, conserved G maintains a flexible loop, motif 1a binds with substrate nucleic acid and transduces energy from the ATP-binding site to the DNA-binding site, motif II binds and hydrolyses ATP, motif III couples ATP hydrolysis with helicase activity, motif V binds substrate nucleic acid, and motif VI couples ATP hydrolysis with helicase activity; ALS4 mutation residues (red) are T3I, L389S, V891A, C1554G, K2029Q, R1236H/C, and I2647T; predicted sumoylation residues (blue) are K78, K863, and K1051 [119]; and cysteine residues predicted by CYSPRED (reliability*≥*8) to form disulfide bonds (indicated by stars) are C4, C5, C7, C145, C555, C637, C688, C997, C1080, C1123C,1153, C1262, C1277, C1398, C1442, C1509, C1672, C1719, and C2622. (B) Prediction of natural disordered region of SETX (upper panel), and Sen1 (lower panel), with the in silico metapredictor PONDR-VL3 [120]. N-terminal domain (green), central disordered region (red), C-terminal* 

**44**

*helicase domain (blue).*

**Figure 1.**

*SETX interactome: DNA repair factors (red), sumoylation and ubiquitination-associated factors (purple), RNA exosome factors (blue), RNAP II and transcription-associated factors (orange), splicing factor and RNAbinding proteins (green), adapted from [40].*

factors (Nrd1 and Nab3) that regulate the generation of small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) [43–45]. During aberrant RNA pol II pausing, the Nrd1-Nab3-Sen1 (NNS) complex is recruited via direct interaction of Sen1 and Nrd1 with the C-terminal domain (CTD) of RNA pol II [46, 47]. Moreover, Pcf11, a component of the cleavage and polyadenylation complex (CPAC), facilitates RNA pol II CTD Ser2 phosphorylation and handoff of Sen1 from the NNS complex to RNA pol II [48]. NNS complex also captures polyadenylated RNAs and channels them to the RNA exosome complex for degradation, which we will discuss further in Section 2.2.2 [49]. Sen1 is also necessary for recruitment of Rat1/Xrn2, a 5′ → 3′ exoribonuclease at G-rich RNA pol II pause sites for degradation of the nascent transcripts and to prevent the accumulation of pathogenic R-loops [18, 50, 51]. Human lymphoblastoid and fibroblast cells with loss of both SETX or XRN2 result in increased R-loops and DNA doublestrand breaks (DSBs) at transcriptional pause sites and hypersensitivity of cells to replications of stress and DNA damage induced by ionizing radiation, ultraviolet light, and oxidative stress [52, 53], which will be discussed further in Section 2.3.3*.* Thus, defects in pathways of RNA metabolism can lead to the induction of DNA damage.

#### *2.2.2 Role in RNA surveillance machinery*

SETX interacts with the RNA exosome, a highly conserved multiprotein ribonuclease complex that processes or degrades a diverse spectrum of RNAs in cells [54]. The exosome removes improperly processed coding and noncoding RNAs (ncRNAs) in the nucleus and regulates mRNA turnover in the cytoplasm. Other critical functions of the exosome include generation of mature ribosomal RNAs, processing of ncRNAs into snRNAs and snoRNAs, and turnover of tRNAs. The human RNA exosome is a ten-subunit complex with a central six-subunit core that constitutes a channel (EXOSC4–9), a three-subunit cap (EXOSC1–3) and a ribonuclease (EXOSC11) subunit located at the bottom of the channel. The nuclear form of the exosome also harbors a riboexonuclease subunit, EXOSC10 [54, 55]. The current model posits that a RNA strand enters the exosome through the cap and is threaded through the channel to be fed to the ribonuclease module for nucleolytic processing [55].

Importantly, SETX interacts with EXOSC9, and complex formation requires SUMOylation of the N-terminal domain of SETX [27]. It has been inferred that SETX-exosome interaction reflects a vital mechanistic axis for resolving co-transcriptional R-loops and preventing genomic instability at heavily transcribed regions in neurons. It might also be surmised that the RNA exosome is recruited via SETX at R-loops to help resolve these pathogenic structures via degradation of the RNA moiety.

Interestingly, the RNA exosome has also been linked to spinal SMA-type neuropathies. Familial missense mutations in EXOSC3 [56] and EXOSC8 [57] are linked to an infantile neuronal disorder, pontocerebellar hypoplasia type 1 (PCH1), that is marked by cerebellar atrophy and progressive microcephaly along with developmental defects and degeneration of spinal motor neurons. Hereditary mutations in EXOSC10 also cause similar neurological defects [58]. Taken together, the available evidence points to a critical role of the exosome in the avoidance of motor neuropathies. Given the interaction noted for SETX and exosome, it might be contemplated that an R-loop removal defect in spinal motor neuronal precursor and differentiated cells could represent the underlying basis for PCH1.

**47**

*Senataxin: A Putative RNA: DNA Helicase Mutated in ALS4—Emerging Mechanisms…*

**2.3 SETX: a guardian of genomic stability across highly transcribed genomic** 

Nucleotide excision repair (NER) is a conserved DNA repair pathway that removes bulky DNA lesions such as those induced by ultraviolet light. When the RNA polymerase II ensemble is obstructed by such a bulky lesion, Cockayne syndrome B (CSB) protein, which interacts with RNA pol II, mediates the recruitment of NER factors such as the Cockayne syndrome A (CSA)-E3-ubiquitin ligase complex [59] and the endonucleases ERCC1-XPF and XPG to mediate the removal of the DNA lesion. In yeast cells, Sen1 has been shown to play a direct role in TC-NER via interaction with Rad2, the yeast XPG ortholog [47]. Whether SETX also functions in TC-NER in humans remains an open question. However, in the absence of SETX, the TC-NER endonucleases XPF and XPG generate DSBs at R-loops, leading to the activation of DNA damage response pathways and repair via HR or the alternate DSB repair pathway of nonhomologous DNA end joining (NHEJ) [60].

R-loops are RNA–DNA hybrid structures with a displaced single DNA strand and are generated upon reannealing of a nascent transcript with the sense strand [61]. R-loops are transiently formed in many regions of the genome, including those transcribed by RNA pol I, II, and III [62]. R-loops are abundant at promoters of RNA pol II-transcribed genes [63–65], at sequences that are prone to forming G-quadruplex or hairpin structures in the non-template DNA strand [66]. Perturbations of transcription-coupled processes, such as mRNA splicing, also result in R-loop formation [61, 67]. R-loops are detected in the genome via DNA– RNA immunoprecipitation (DRIP) [68] and immunofluorescence assays with the monoclonal antibody S9.6, which has high affinity for RNA–DNA hybrids [69]. Typically, to ensure that the signal detected is specific for RNA–DNA hybrids, one would include the expression of RNase H in cells (to digest the hybrids) or pretreat

Depending upon their location and size, R-loops could impart beneficial or harmful effects. R-loops could extend from a few hundred base pairs to kilo base pairs in size. In immunoglobulin (Ig) class switch regions, R-loops serve an important role in Ig class switch recombination by promoting the induction of DNA breaks via the action of activation-induced cytidine deaminase (AID) and base excision repair factors [70]. R-loops also prime DNA replication in the mitochondrial genome [71]. In human fibroblast cells, R-loops can influence the expression of over 1200 genes by facilitating transcription via suppression of DNA methylation [72] and recruitment or eviction of chromatin remodeling complexes [73]. On the contrary, R-loops could interfere with transcription at certain genomic loci like rDNA [62, 74]. The major threat from unscheduled R-loops is the generation of lethal DSBs

because of head-on collisions with the DNA replication machinery [75, 76]*.*

Because of the potential harm that R-loops could cause, their levels are tightly regulated via a variety of mechanisms. In this regard, RNAseH1/2 provides a major means for R-loop clearance via ribonucleolytic cleavage of RNA strand [77, 78]. While topoisomerase I prevents R-loop formation by reducing negative supercoiling behind the elongating RNA pol II, TRanscription EXport (TREX) complex factors (THOC1–7, UAP56) and serine−/arginine-rich splicing factor 1 (SRSF1) suppress R-loops by removing the nascent mRNA [79]. RNA biogenesis factors like Trf4/Air2/Mtr4p polyadenylation

*DOI: http://dx.doi.org/10.5772/intechopen.90215*

*2.3.1 Transcription-coupled (TC) repair*

**landscapes**

*2.3.2 Resolution of R-loops*

samples for sequencing with this enzyme.

#### **2.3 SETX: a guardian of genomic stability across highly transcribed genomic landscapes**

#### *2.3.1 Transcription-coupled (TC) repair*

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

factors (Nrd1 and Nab3) that regulate the generation of small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs) [43–45]. During aberrant RNA pol II pausing, the Nrd1-Nab3-Sen1 (NNS) complex is recruited via direct interaction of Sen1 and Nrd1 with the C-terminal domain (CTD) of RNA pol II [46, 47]. Moreover, Pcf11, a component of the cleavage and polyadenylation complex (CPAC), facilitates RNA pol II CTD Ser2 phosphorylation and handoff of Sen1 from the NNS complex to RNA pol II [48]. NNS complex also captures polyadenylated RNAs and channels them to the RNA exosome complex for degradation, which we will discuss further in Section 2.2.2 [49]. Sen1 is also necessary for recruitment of Rat1/Xrn2, a 5′ → 3′ exoribonuclease at G-rich RNA pol II pause sites for degradation of the nascent transcripts and to prevent the accumulation of pathogenic R-loops [18, 50, 51]. Human lymphoblastoid and fibroblast cells with loss of both SETX or XRN2 result in increased R-loops and DNA doublestrand breaks (DSBs) at transcriptional pause sites and hypersensitivity of cells to replications of stress and DNA damage induced by ionizing radiation, ultraviolet light, and oxidative stress [52, 53], which will be discussed further in Section 2.3.3*.* Thus, defects in pathways of RNA metabolism can lead to the induction of

SETX interacts with the RNA exosome, a highly conserved multiprotein ribonuclease complex that processes or degrades a diverse spectrum of RNAs in cells [54]. The exosome removes improperly processed coding and noncoding RNAs (ncRNAs) in the nucleus and regulates mRNA turnover in the cytoplasm. Other critical functions of the exosome include generation of mature ribosomal RNAs, processing of ncRNAs into snRNAs and snoRNAs, and turnover of tRNAs. The human RNA exosome is a ten-subunit complex with a central six-subunit core that constitutes a channel (EXOSC4–9), a three-subunit cap (EXOSC1–3) and a ribonuclease (EXOSC11) subunit located at the bottom of the channel. The nuclear form of the exosome also harbors a riboexonuclease subunit, EXOSC10 [54, 55]. The current model posits that a RNA strand enters the exosome through the cap and is threaded through the channel to be fed to the ribonuclease module for nucleolytic

Importantly, SETX interacts with EXOSC9, and complex formation requires SUMOylation of the N-terminal domain of SETX [27]. It has been inferred that SETX-exosome interaction reflects a vital mechanistic axis for resolving co-transcriptional R-loops and preventing genomic instability at heavily transcribed regions in neurons. It might also be surmised that the RNA exosome is recruited via SETX at R-loops to help resolve these pathogenic structures via degradation of the RNA

Interestingly, the RNA exosome has also been linked to spinal SMA-type neuropathies. Familial missense mutations in EXOSC3 [56] and EXOSC8 [57] are linked to an infantile neuronal disorder, pontocerebellar hypoplasia type 1 (PCH1), that is marked by cerebellar atrophy and progressive microcephaly along with developmental defects and degeneration of spinal motor neurons. Hereditary mutations in EXOSC10 also cause similar neurological defects [58]. Taken together, the available evidence points to a critical role of the exosome in the avoidance of motor neuropathies. Given the interaction noted for SETX and exosome, it might be contemplated that an R-loop removal defect in spinal motor neuronal precursor and differentiated cells could represent the underlying basis

**46**

for PCH1.

DNA damage.

processing [55].

moiety.

*2.2.2 Role in RNA surveillance machinery*

Nucleotide excision repair (NER) is a conserved DNA repair pathway that removes bulky DNA lesions such as those induced by ultraviolet light. When the RNA polymerase II ensemble is obstructed by such a bulky lesion, Cockayne syndrome B (CSB) protein, which interacts with RNA pol II, mediates the recruitment of NER factors such as the Cockayne syndrome A (CSA)-E3-ubiquitin ligase complex [59] and the endonucleases ERCC1-XPF and XPG to mediate the removal of the DNA lesion. In yeast cells, Sen1 has been shown to play a direct role in TC-NER via interaction with Rad2, the yeast XPG ortholog [47]. Whether SETX also functions in TC-NER in humans remains an open question. However, in the absence of SETX, the TC-NER endonucleases XPF and XPG generate DSBs at R-loops, leading to the activation of DNA damage response pathways and repair via HR or the alternate DSB repair pathway of nonhomologous DNA end joining (NHEJ) [60].

#### *2.3.2 Resolution of R-loops*

R-loops are RNA–DNA hybrid structures with a displaced single DNA strand and are generated upon reannealing of a nascent transcript with the sense strand [61]. R-loops are transiently formed in many regions of the genome, including those transcribed by RNA pol I, II, and III [62]. R-loops are abundant at promoters of RNA pol II-transcribed genes [63–65], at sequences that are prone to forming G-quadruplex or hairpin structures in the non-template DNA strand [66]. Perturbations of transcription-coupled processes, such as mRNA splicing, also result in R-loop formation [61, 67]. R-loops are detected in the genome via DNA– RNA immunoprecipitation (DRIP) [68] and immunofluorescence assays with the monoclonal antibody S9.6, which has high affinity for RNA–DNA hybrids [69]. Typically, to ensure that the signal detected is specific for RNA–DNA hybrids, one would include the expression of RNase H in cells (to digest the hybrids) or pretreat samples for sequencing with this enzyme.

Depending upon their location and size, R-loops could impart beneficial or harmful effects. R-loops could extend from a few hundred base pairs to kilo base pairs in size. In immunoglobulin (Ig) class switch regions, R-loops serve an important role in Ig class switch recombination by promoting the induction of DNA breaks via the action of activation-induced cytidine deaminase (AID) and base excision repair factors [70]. R-loops also prime DNA replication in the mitochondrial genome [71]. In human fibroblast cells, R-loops can influence the expression of over 1200 genes by facilitating transcription via suppression of DNA methylation [72] and recruitment or eviction of chromatin remodeling complexes [73]. On the contrary, R-loops could interfere with transcription at certain genomic loci like rDNA [62, 74]. The major threat from unscheduled R-loops is the generation of lethal DSBs because of head-on collisions with the DNA replication machinery [75, 76]*.*

Because of the potential harm that R-loops could cause, their levels are tightly regulated via a variety of mechanisms. In this regard, RNAseH1/2 provides a major means for R-loop clearance via ribonucleolytic cleavage of RNA strand [77, 78]. While topoisomerase I prevents R-loop formation by reducing negative supercoiling behind the elongating RNA pol II, TRanscription EXport (TREX) complex factors (THOC1–7, UAP56) and serine−/arginine-rich splicing factor 1 (SRSF1) suppress R-loops by removing the nascent mRNA [79]. RNA biogenesis factors like Trf4/Air2/Mtr4p polyadenylation

(TRAMP) complex and, as discussed earlier, the RNA exosome complex also participate in the regulation of R-loop formation or removal [80]. Moreover, R-loops can be dissociated via the nucleic acid unwinding activity of Sen1/SETX [23, 50, 81] and other helicase proteins such as Pif1 [82], DEAH box protein 9 (DHX9) [83], and Fanconi Anemia Complementation factor M (FANCM) [84]. It seems reasonable to postulate that each of the above-named factors operates at specific genomic milieu.

Studies so forth have suggested that SETX is involved in resolving R-loops at paused transcription sites [50] via forming a physiological complex with the tumor suppressor protein BRCA1 to prevent R-loop-associated DNA damage [25]. This warrants further investigation on SETX-BRCA1 axis to reveal the molecular mechanisms of R-loop resolution.

#### *2.3.3 DNA double-strand break repair and replication fork stability*

Occurrence of DSBs and their repair in terminally differentiated neurons were reported in the early 1970s [85, 86]. Recent studies have provided evidence that ALS is associated with a defect in DSB repair [87–89]. DSBs are generated in neurons via endogenous oxidative stress [90, 91] or a topoisomerase IIβ-dependent mechanism that is essential for expression of early genes regulating vital neuronal functions [92]. A recent study showed that neuronal cells from SMA express only low levels of SETX and DNA-PKcs, a highly conserved NHEJ factor. As a result, SMA neurons display higher levels of R-loops that culminate DSB formation, and cellular toxicity [93]. Importantly, these phenotypic manifestations can be corrected by the overexpression of SETX. These observations aptly underscore the genome protective role of SETX in neurons.

Importantly, SETX colocalizes at DSBs with various factors that function in the DNA damage response and repair factors, including γH2AX, 53BP1, and BRCA1, and it forms a co-immunoprecipitable complex with DNA-PKcs, MRE11, RAD50 [24, 25, 53, 94]. AOA2-pateint derived lymphoblastoid cell lines lacking SETX are sensitive toward topoisomerase I inhibitor, camptothecin, DNA crosslinking agent mitomycin C and hydrogen peroxide [53], again indicative of a role of SETX in the DNA damage response. Moreover, *Setx−/−* mice displays a defect in germ cell maturation due to defective meiotic recombination with unrepaired DSBs [81]. Interestingly, recent findings revealed that nascent transcripts or small ncRNAs could accumulate at DSBs via diverse mechanisms, and affect DSB repair via distinct pathways of HR or NHEJ [95–103]. This was further evidenced by the studies demonstrating that both excess removal and impaired clearance of RNA–DNA hybrids result in defective DSB repair [100], suggesting the role of RNA in DSB repair via processes that are yet to be characterized. In this context, SETX has been implicated in enhancing HR-mediated DSB repair that is catalyzed by the recombinase RAD51, via resolving RNA–DNA hybrids at DSBs [26]. Further biochemical investigations are required to dissect the mechanistic underpinnings of this process.

It should be noted that cycling cells face an incessant threat of genomic instability via replication-transcription collisions, wherein the replication and transcription machineries could engage in head-on collisions [104]. This could directly lead to formation of DSBs [76]. Importantly, SETX associates with DNA replication forks and promotes their progression across RNA pol II transcribed regions, a function that appears to be independent of its transcription termination role [105].

#### *2.3.4 Telomere maintenance*

Telomeres, which comprise repetitive DNA sequences, cap the ends of each chromosome, and their attrition leads to cellular senescence. Telomerase reverse

**49**

*Senataxin: A Putative RNA: DNA Helicase Mutated in ALS4—Emerging Mechanisms…*

transcriptase (TERT), the catalytic subunit of telomerase that maintains the normal length of telomeres, is present at a low level in most differentiated cells including neurons [106]. TERT levels appear to be significantly lower in the spinal cord tissues of ALS patients than healthy individuals [107]. Ex vivo studies have suggested that while telomere damage induces neuronal cell death [108], the activation of telomerase can enhance neuronal cell viability [109, 110]. Interestingly, a novel compound that enhances telomerase activity in neurons also appears to ameliorate the symp-

Importantly, SETX is present at telomeres, and AOA2 lymphocytes and lymphoblasts showed reduced telomere length along with higher sensitivity toward oxidative stress and DNA-damaging agents [111]. Another study has implicated SETX in the maintenance of telomeres in *Myotis* bats [112]. These observations should constitute the basis for investigating the mechanistic role of SETX in telomere

Defects in RNA metabolism factors have been associated with multiple motor neuron diseases including ALS and SMA [113, 114]. Despite having distinctive pathological manifestations and clinical onsets, such motor neuropathies could stem from related underlying mechanisms pertaining to RNA homeostasis. In this chapter, we reviewed how defective RNA metabolic pathways could ensue genomic instability, an emerging mechanism in neurodegenerative diseases. Cotranscriptional R-loops are crucial for regulating gene expression in both dividing and postmitotic neurons; however, when not timely resolved, it will lead to genomic instability via generation of DNA strand breaks. While replication-transcription conflicts are the major source of R-loop-induced DSBs in dividing cells that need repair via HR or NHEJ, how R-loops trigger DSBs in postmitotic neurons remains to be investigated. Moreover, small RNAs at DSBs could impede repair and must be cleared nucleolytically or via unwinding of RNA–DNA hybrids. Genetic and cell-based studies of human SETX, together with biochemical characterization of its yeast ortholog Sen1, have suggested that SETX protects genomic stability via resolution of R-loops, assisting replication fork progression across transcribing genomic regions and promoting HR at DSBs. In the light of recent findings on implication of genomic instability in neurodegenerative diseases, as reviewed in [115–118], in-depth studies are required to precisely delineate role of SETX in ALS4. Thus, it is apposite to test if ALS4 gain-of-function mutations affect SETX activities

Treatment of ALS4 or other JALS is currently limited to physical and occupational therapies to promote mobility and independence. While an FDA-approved glutamate inhibitor drug, riluzole, that slows down symptoms and prolongs survival is used clinically to treat ALS, there are currently no specific treatment for juvenile ALS diseases. Further studies are obligatory to recognize SETX as a thera-

Dr. Dutta has been supported by a Brown-Coxe postdoctoral fellowship from Yale University School of Medicine and a Jeane B. Kempner postdoctoral fellowship from the University of Texas Medical Branch, Galveston. The laboratories of Robert Hromas and Patrick Sung have been funded by research grants from the US

*DOI: http://dx.doi.org/10.5772/intechopen.90215*

maintenance in motor neurons and other cell types.

toms of ALS [109].

**3. Conclusion**

pertaining to DSB repair.

**Acknowledgements**

peutic target for treatment of ALS4.

*Senataxin: A Putative RNA: DNA Helicase Mutated in ALS4—Emerging Mechanisms… DOI: http://dx.doi.org/10.5772/intechopen.90215*

transcriptase (TERT), the catalytic subunit of telomerase that maintains the normal length of telomeres, is present at a low level in most differentiated cells including neurons [106]. TERT levels appear to be significantly lower in the spinal cord tissues of ALS patients than healthy individuals [107]. Ex vivo studies have suggested that while telomere damage induces neuronal cell death [108], the activation of telomerase can enhance neuronal cell viability [109, 110]. Interestingly, a novel compound that enhances telomerase activity in neurons also appears to ameliorate the symptoms of ALS [109].

Importantly, SETX is present at telomeres, and AOA2 lymphocytes and lymphoblasts showed reduced telomere length along with higher sensitivity toward oxidative stress and DNA-damaging agents [111]. Another study has implicated SETX in the maintenance of telomeres in *Myotis* bats [112]. These observations should constitute the basis for investigating the mechanistic role of SETX in telomere maintenance in motor neurons and other cell types.

#### **3. Conclusion**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

the above-named factors operates at specific genomic milieu.

*2.3.3 DNA double-strand break repair and replication fork stability*

nisms of R-loop resolution.

of SETX in neurons.

(TRAMP) complex and, as discussed earlier, the RNA exosome complex also participate in the regulation of R-loop formation or removal [80]. Moreover, R-loops can be dissociated via the nucleic acid unwinding activity of Sen1/SETX [23, 50, 81] and other helicase proteins such as Pif1 [82], DEAH box protein 9 (DHX9) [83], and Fanconi Anemia Complementation factor M (FANCM) [84]. It seems reasonable to postulate that each of

Studies so forth have suggested that SETX is involved in resolving R-loops at paused transcription sites [50] via forming a physiological complex with the tumor suppressor protein BRCA1 to prevent R-loop-associated DNA damage [25]. This warrants further investigation on SETX-BRCA1 axis to reveal the molecular mecha-

Occurrence of DSBs and their repair in terminally differentiated neurons were reported in the early 1970s [85, 86]. Recent studies have provided evidence that ALS is associated with a defect in DSB repair [87–89]. DSBs are generated in neurons via endogenous oxidative stress [90, 91] or a topoisomerase IIβ-dependent mechanism that is essential for expression of early genes regulating vital neuronal functions [92]. A recent study showed that neuronal cells from SMA express only low levels of SETX and DNA-PKcs, a highly conserved NHEJ factor. As a result, SMA neurons display higher levels of R-loops that culminate DSB formation, and cellular toxicity [93]. Importantly, these phenotypic manifestations can be corrected by the overexpression of SETX. These observations aptly underscore the genome protective role

Importantly, SETX colocalizes at DSBs with various factors that function in the DNA damage response and repair factors, including γH2AX, 53BP1, and BRCA1, and it forms a co-immunoprecipitable complex with DNA-PKcs, MRE11, RAD50 [24, 25, 53, 94]. AOA2-pateint derived lymphoblastoid cell lines lacking SETX are sensitive toward topoisomerase I inhibitor, camptothecin, DNA crosslinking agent mitomycin C and hydrogen peroxide [53], again indicative of a role of SETX in the DNA damage response. Moreover, *Setx−/−* mice displays a defect in germ cell maturation due to defective meiotic recombination with unrepaired DSBs [81]. Interestingly, recent findings revealed that nascent transcripts or small ncRNAs could accumulate at DSBs via diverse mechanisms, and affect DSB repair via distinct pathways of HR or NHEJ [95–103]. This was further evidenced by the studies demonstrating that both excess removal and impaired clearance of RNA–DNA hybrids result in defective DSB repair [100], suggesting the role of RNA in DSB repair via processes that are yet to be characterized. In this context, SETX has been implicated in enhancing HR-mediated DSB repair that is catalyzed by the recombinase RAD51, via resolving RNA–DNA hybrids at DSBs [26]. Further biochemical investigations are required to dissect the mechanistic underpinnings of this process. It should be noted that cycling cells face an incessant threat of genomic instability via replication-transcription collisions, wherein the replication and transcription machineries could engage in head-on collisions [104]. This could directly lead to formation of DSBs [76]. Importantly, SETX associates with DNA replication forks and promotes their progression across RNA pol II transcribed regions, a function that appears to be independent of its transcription termination role [105].

Telomeres, which comprise repetitive DNA sequences, cap the ends of each chromosome, and their attrition leads to cellular senescence. Telomerase reverse

**48**

*2.3.4 Telomere maintenance*

Defects in RNA metabolism factors have been associated with multiple motor neuron diseases including ALS and SMA [113, 114]. Despite having distinctive pathological manifestations and clinical onsets, such motor neuropathies could stem from related underlying mechanisms pertaining to RNA homeostasis. In this chapter, we reviewed how defective RNA metabolic pathways could ensue genomic instability, an emerging mechanism in neurodegenerative diseases. Cotranscriptional R-loops are crucial for regulating gene expression in both dividing and postmitotic neurons; however, when not timely resolved, it will lead to genomic instability via generation of DNA strand breaks. While replication-transcription conflicts are the major source of R-loop-induced DSBs in dividing cells that need repair via HR or NHEJ, how R-loops trigger DSBs in postmitotic neurons remains to be investigated. Moreover, small RNAs at DSBs could impede repair and must be cleared nucleolytically or via unwinding of RNA–DNA hybrids. Genetic and cell-based studies of human SETX, together with biochemical characterization of its yeast ortholog Sen1, have suggested that SETX protects genomic stability via resolution of R-loops, assisting replication fork progression across transcribing genomic regions and promoting HR at DSBs. In the light of recent findings on implication of genomic instability in neurodegenerative diseases, as reviewed in [115–118], in-depth studies are required to precisely delineate role of SETX in ALS4. Thus, it is apposite to test if ALS4 gain-of-function mutations affect SETX activities pertaining to DSB repair.

Treatment of ALS4 or other JALS is currently limited to physical and occupational therapies to promote mobility and independence. While an FDA-approved glutamate inhibitor drug, riluzole, that slows down symptoms and prolongs survival is used clinically to treat ALS, there are currently no specific treatment for juvenile ALS diseases. Further studies are obligatory to recognize SETX as a therapeutic target for treatment of ALS4.

#### **Acknowledgements**

Dr. Dutta has been supported by a Brown-Coxe postdoctoral fellowship from Yale University School of Medicine and a Jeane B. Kempner postdoctoral fellowship from the University of Texas Medical Branch, Galveston. The laboratories of Robert Hromas and Patrick Sung have been funded by research grants from the US National Institutes of Health. Patrick Sung is a CPRIT Scholar in Cancer Research and the holder of the Robert A. Welch Distinguished Chair in Chemistry.

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Arijit Dutta1 , Robert Hromas2 and Patrick Sung1 \*

1 Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

2 Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

\*Address all correspondence to: sungp@uthscsa.edu

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**51**

*Senataxin: A Putative RNA: DNA Helicase Mutated in ALS4—Emerging Mechanisms…*

[8] Bennett CL, Dastidar SG, Ling SC, Malik B, Ashe T, Wadhwa M, et al. Senataxin mutations elicit motor neuron degeneration phenotypes and yield TDP-43 mislocalization in ALS4 mice and human patients. Acta Neuropathologica. 2018;**136**:425-443

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#### **References**

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The authors declare no conflict of interest.

**Conflict of interest**

National Institutes of Health. Patrick Sung is a CPRIT Scholar in Cancer Research

and the holder of the Robert A. Welch Distinguished Chair in Chemistry.

**50**

**Author details**

, Robert Hromas2

provided the original work is properly cited.

Antonio, San Antonio, TX, USA

Science Center at San Antonio, San Antonio, TX, USA

\*Address all correspondence to: sungp@uthscsa.edu

and Patrick Sung1

2 Department of Medicine, University of Texas Health Science Center at San

1 Department of Biochemistry and Structural Biology, University of Texas Health

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*

Arijit Dutta1

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*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

irradiated neurons: in vivo. Radiation

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[91] Wang X, Michaelis EK. Selective neuronal vulnerability to oxidative stress in the brain. Frontiers in Aging

[92] Madabhushi R, Gao F, Pfenning AR,

response genes. Cell. 2015;**161**:1592-1605

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[94] Roda RH, Rinaldi C, Singh R, Schindler AB, Blackstone C. Ataxia with

Pan L, Yamakawa S, Seo J, et al. Activity-induced DNA breaks govern the expression of neuronal early-

Neuroscience. 2014;**8**:131

Neuroscience. 2010;**2**:12

2018;**46**:8326-8346

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RNA biogenesis factors cooperate to prevent RNA:DNA hybrids from generating genome instability. Molecular Cell. 2011;**44**:978-988

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[81] Becherel OJ, Yeo AJ, Stellati A, Heng EY, Luff J, Suraweera AM, et al. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing. PLoS Genetics.

[82] Tran PLT, Pohl TJ, Chen CF, Chan A, Pott S, Zakian VA. PIF1 family

DNA helicases suppress R-loop mediated genome instability at tRNA genes. Nature Communications.

Kristiansen MS, Gromak N. RNA/ DNA hybrid Interactome identifies DXH9 as a molecular player in

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[85] Price GB, Modak SP, Makinodan T.

Age-associated changes in the DNA of mouse tissue. Science.

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[83] Cristini A, Groh M,

2018;**23**:1891-1905

2015;**60**:351-361

1971;**171**:917-920

2019;**29**:428-445

2013;**9**:e1003435

2017;**8**:15025

**56**

[95] Storici F, Bebenek K, Kunkel TA, Gordenin DA, Resnick MA. RNAtemplated DNA repair. Nature. 2007;**447**:338-341

[96] Keskin H, Shen Y, Huang F, Patel M, Yang T, Ashley K, et al. Transcript-RNAtemplated DNA recombination and repair. Nature. 2014;**515**:436-439

[97] Wei W, Ba Z, Gao M, Wu Y, Ma Y, Amiard S, et al. A role for small RNAs in DNA double-strand break repair. Cell. 2012;**149**:101-112

[98] Wang Q, Goldstein M. Small RNAs recruit chromatin-modifying enzymes MMSET and Tip60 to reconfigure damaged DNA upon double-Strand break and facilitate repair. Cancer Research. 2016;**76**:1904-1915

[99] Gao M, Wei W, Li MM, Wu YS, Ba Z, Jin KX, et al. Ago2 facilitates Rad51 recruitment and DNA doublestrand break repair by homologous recombination. Cell Research. 2014;**24**:532-541

[100] Ohle C, Tesorero R, Schermann G, Dobrev N, Sinning I, Fischer T. Transient RNA-DNA hybrids are required for efficient double-strand break repair. Cell. 2016;**167**:1001-1013

[101] Chakraborty A, Tapryal N, Venkova T, Horikoshi N, Pandita RK, Sarker AH, et al. Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes. Nature Communications. 2016;**7**:13049

[102] Francia S, Michelini F, Saxena A, Tang D, de Hoon M, Anelli V, et al. Site-specific DICER and DROSHA

RNA products control the DNA-damage response. Nature. 2012;**488**:231-235

[103] Francia S, Cabrini M, Matti V, Oldani A, d'Adda di Fagagna F. DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors. Journal of Cell Science. 2016;**129**:1468-1476

[104] Hamperl S, Cimprich KA. Conflict resolution in the genome: How transcription and replication make it work. Cell. 2016;**167**:1455-1467

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[106] Eitan E, Hutchison ER, Mattson MP. Telomere shortening in neurological disorders: An abundance of unanswered questions. Trends in Neurosciences. 2014;**37**:256-263

[107] De Felice B, Annunziata A, Fiorentino G, Manfellotto F, D'Alessandro R, Marino R, et al. Telomerase expression in amyotrophic lateral sclerosis (ALS) patients. Journal of Human Genetics. 2014;**59**:555-561

[108] Cheng A, Shin-ya K, Wan R, Tang SC, Miura T, Tang H, et al. Telomere protection mechanisms change during neurogenesis and neuronal maturation: Newly generated neurons are hypersensitive to telomere and DNA damage. The Journal of Neuroscience. 2007;**27**:3722-3733

[109] Eitan E, Tichon A, Gazit A, Gitler D, Slavin S, Priel E. Novel telomerase-increasing compound in mouse brain delays the onset of amyotrophic lateral sclerosis. EMBO Molecular Medicine. 2012;**4**:313-329

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[115] Madabhushi R, Pan L, Tsai LH. DNA damage and its links to neurodegeneration. Neuron. 2014;**83**:266-282

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[117] Penndorf D, Witte OW, Kretz A. DNA plasticity and damage in amyotrophic lateral sclerosis. Neural Regeneration Research. 2018;**13**:173-180 [118] Wang H, Hegde ML. New mechanisms of DNA repair defects in fused in sarcoma-associated neurodegeneration: Stage set for DNA repair-based therapeutics? Journal of Experimental Neuroscience. 2019;**13**:1179069519856358

[119] Yeo AJ, Becherel OJ, Luff JE, Graham ME, Richard D, Lavin MF. Senataxin controls meiotic silencing through ATR activation and chromatin remodeling. Cell Discovery. 2015;**1**:15025

[120] Xue B, Dunbrack RL, Williams RW, Dunker AK, Uversky VN. PONDR-FIT: A meta-predictor of intrinsically disordered amino acids. Biochimica et Biophysica Acta. 2010;**1804**:996-1010

**59**

**Chapter 4**

**Abstract**

death in motor neurons.

Pathological Interaction between

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the degeneration of cortical and spinal cord motor neurons. Several mechanisms have been implicated in the pathogenesis of the disease, including mitochondrial dysfunction, oxidative stress, and genome instability. Recently, a combined role between impaired DNA repair and subsequent mitochondrial dysfunction has emerged as a novel pathological interaction in neurodegeneration. This is exemplified by mutations in the RNA-/DNA-binding proteins FUS and TDP-43 as well as superoxide dismutase 1 (SOD1) gene, all related to familial ALS. In this regard, evidence supports either downregulation or impaired recruitment of DNA repair enzymes in both nuclear and mitochondrial genomes. In addition, evidence also suggests a complex metabolic dysregulation as a critical component in the promotion of the disease. This chapter aims to integrate the molecular mechanisms of this pathological interplay and the possible role in cytosolic protein aggregation and cell

**Keywords:** DNA repair, neurodegeneration, mitochondria, motor neuron

ALS has been traditionally classified as sporadic or familial (fALS). Specifically, four genes account for up to 70% of all cases of fALS: *C9orf72*, *TARDBP* (encoding TDP-43), *SOD1*, and *FUS*. Despite the Mendelian inheritance pattern, familial forms of ALS are usually characterized by incomplete penetrance, whereas genetic pleiotropy or oligogenic inheritance is suggested in individuals with sporadic disease [1]. Several hallmarks of neurodegeneration are shared in both familial and sporadic ALS, such as protein aggregation, defective autophagy, impaired DNA damage response (DDR), and mitochondrial dysfunction [1–3]. As ALS is a complex disease, it is plausible to think that regardless of the etiology, there is an interplay between these hallmarks in the pathogenesis and pathological progression. Considering the late age of onset and the neurological tropism, which is the exclusive neurological affectation, it is possible to highlight some aspects of the nervous system that are useful to understand the molecular pathology of the disease. First, due to its high rate of oxygen consumption and metabolic activity, the nervous system is more susceptible to DNA damage [4]. Single-strand breaks (SSBs) are considered the most common damage to DNA in cells [5], mainly derived

**1. Genome instability in the context of ALS pathogenesis**

DNA Repair and Mitochondrial

Dysfunction in ALS

*Luis Bermúdez-Guzmán and Alejandro Leal*

#### **Chapter 4**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

[118] Wang H, Hegde ML. New mechanisms of DNA repair defects in fused in sarcoma-associated neurodegeneration: Stage set for DNA repair-based therapeutics? Journal of Experimental Neuroscience. 2019;**13**:1179069519856358

[119] Yeo AJ, Becherel OJ, Luff JE, Graham ME, Richard D, Lavin MF. Senataxin controls meiotic silencing

chromatin remodeling. Cell Discovery.

[120] Xue B, Dunbrack RL, Williams RW, Dunker AK, Uversky VN. PONDR-FIT: A meta-predictor of intrinsically disordered amino acids. Biochimica et Biophysica Acta. 2010;**1804**:996-1010

through ATR activation and

2015;**1**:15025

[110] Zhu H, Fu W, Mattson MP. The catalytic subunit of telomerase protects

peptide-induced apoptosis. Journal of Neurochemistry. 2000;**75**:117-124

[111] De Amicis A, Piane M, Ferrari F, Fanciulli M, Delia D, Chessa L. Role of senataxin in DNA damage and telomeric stability. DNA Repair (Amst).

[112] Foley NM, Hughes GM, Huang Z, Clarke M, Jebb D, Whelan CV, et al. Growing old, yet staying young: The role of telomeres in bats' exceptional

[113] Grohmann K, Schuelke M, Diers A, Hoffmann K, Lucke B, Adams C, et al. Mutations in the gene encoding immunoglobulin mu-binding protein 2 cause spinal muscular atrophy with respiratory distress type 1. Nature

longevity. Science Advances.

Genetics. 2001;**29**:75-77

Journal of Neurochemistry.

DNA damage and its links to neurodegeneration. Neuron.

[116] Konopka A, Atkin JD. The emerging role of DNA damage in the pathogenesis of the C9orf72 repeat expansion in amyotrophic lateral sclerosis. International Journal of Molecular Sciences. 2018;**19**:E3137

[117] Penndorf D, Witte OW, Kretz A. DNA plasticity and damage in amyotrophic lateral sclerosis. Neural Regeneration Research. 2018;**13**:173-180

[115] Madabhushi R, Pan L, Tsai LH.

2017;**141**:12-30

2014;**83**:266-282

[114] Gama-Carvalho M, L Garcia-Vaquero M, R Pinto F, Besse F, Weis J, Voigt A, Schulz JB, De Las Rivas J. Linking amyotrophic lateral sclerosis and spinal muscular atrophy through RNA-transcriptome homeostasis: A genomics perspective.

neurons against amyloid beta-

2011;**10**:199-209

2018;**4**:eaao0926

**58**

## Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS

*Luis Bermúdez-Guzmán and Alejandro Leal*

#### **Abstract**

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the degeneration of cortical and spinal cord motor neurons. Several mechanisms have been implicated in the pathogenesis of the disease, including mitochondrial dysfunction, oxidative stress, and genome instability. Recently, a combined role between impaired DNA repair and subsequent mitochondrial dysfunction has emerged as a novel pathological interaction in neurodegeneration. This is exemplified by mutations in the RNA-/DNA-binding proteins FUS and TDP-43 as well as superoxide dismutase 1 (SOD1) gene, all related to familial ALS. In this regard, evidence supports either downregulation or impaired recruitment of DNA repair enzymes in both nuclear and mitochondrial genomes. In addition, evidence also suggests a complex metabolic dysregulation as a critical component in the promotion of the disease. This chapter aims to integrate the molecular mechanisms of this pathological interplay and the possible role in cytosolic protein aggregation and cell death in motor neurons.

**Keywords:** DNA repair, neurodegeneration, mitochondria, motor neuron

#### **1. Genome instability in the context of ALS pathogenesis**

ALS has been traditionally classified as sporadic or familial (fALS). Specifically, four genes account for up to 70% of all cases of fALS: *C9orf72*, *TARDBP* (encoding TDP-43), *SOD1*, and *FUS*. Despite the Mendelian inheritance pattern, familial forms of ALS are usually characterized by incomplete penetrance, whereas genetic pleiotropy or oligogenic inheritance is suggested in individuals with sporadic disease [1]. Several hallmarks of neurodegeneration are shared in both familial and sporadic ALS, such as protein aggregation, defective autophagy, impaired DNA damage response (DDR), and mitochondrial dysfunction [1–3]. As ALS is a complex disease, it is plausible to think that regardless of the etiology, there is an interplay between these hallmarks in the pathogenesis and pathological progression.

Considering the late age of onset and the neurological tropism, which is the exclusive neurological affectation, it is possible to highlight some aspects of the nervous system that are useful to understand the molecular pathology of the disease. First, due to its high rate of oxygen consumption and metabolic activity, the nervous system is more susceptible to DNA damage [4]. Single-strand breaks (SSBs) are considered the most common damage to DNA in cells [5], mainly derived from reactive oxygen species [6]. Even physiological brain activity can also cause DNA double-strand breaks (DSBs) in neurons [7]. However, as neurons do not divide, homologous recombination (HR) is not available for DNA repair. In addition, to maintain normal functioning, neurons must ensure to keep mitochondrial homeostasis. Thus, there are different areas with high demands for ATP like synaptic terminals, active growth cones, or axonal branches that contain more mitochondria than other cellular domains [8, 9]. This dependency on mitochondrial homeostasis makes neurons more susceptible and more likely to be affected by mutations in genes regulating mitochondrial dynamics. For example, mutations in MFN2 and GDAP1 (which regulate mitochondrial fusion and fission, respectively) are related to Charcot-Marie-Tooth [10, 11], a peripheral neuropathy that resembles some of the clinical manifestations of ALS, especially in motor neurons. Thus, a disruption in normal nuclear and mitochondrial DNA (mtDNA) repair would affect motor neurons in a progressive way. Supporting this idea, several works have reported mtDNA mutations in presymptomatic animal models of ALS [12]. In fact, similar to CMT, mitochondrial axonal transportation has also been implicated in ALS. The SOD1-G93A mice showed alterations in the axonal traffic of mitochondria at a presymptomatic stage, specifically in motor neurons [13].

Interestingly, recent studies demonstrated that loss-of-function mutations in *KIF5A* are also a cause of ALS [14, 15]. *KIF5A* gene encodes for a specific motor protein implicated in the axonal transport of mitochondria. Although it was previously related with CMT and spastic paraplegia [16, 17], the new variants associated with ALS are primarily located at the C-terminal cargo-binding tail domain. Notably, patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases.

A second aspect behind neurons' susceptibilities is their high rate of transcription. As they depend on the available DNA repair machinery, genomic stability is crucial for the maintenance of homeostasis. Transcription-driven DNA damage can arise from several sources, like the collapse of transcription machinery and subsequent DSBs, damage to genomic segments that have been opened up for transcription, and the formation of unnatural R-loop structures [18–21]. In the case of ALS cells, deficient DNA repair was reported more than three decades ago [22]. It seems that the DDR system in ALS loses adaptive capacity in dependency on the underlying genetic mutation [23]. The next chapter will address the role of the main genes involved in fALS in the generation of genomic instability as a hallmark of neurodegeneration.

#### **2. Role of ALS-related genes in DDR**

The hexanucleotide GGGGCC repeat expansion in *C9ORF72* is one of the most common genetic causes of both ALS and frontotemporal dementia (FTD). It was recently demonstrated that these expansions could cause defective DNA repair, manifested by higher levels of DSBs and impaired DDR [24]. Defective ATMmediated DNA repair was found as a consequence of p62 accumulation, which impairs H2A ubiquitylation and perturbs ATM signaling. Another study reported that c-H2AX, p-ATM, 53BP1, and PARP1 were upregulated in C9orf72 patient spinal cords [25]. However, the mechanism behind the damage to the genome was not addressed. Authors suggest that R-loops, as well as the expression of dipeptide repeat proteins, are more likely to be the cause. Consistent with this notion, elevated levels of DNA damage markers γH2AX, ATR, GADD45, and p53 were present in motor neurons differentiated from iPSC lines from C9orf72-mutant ALS patients in response to oxidative stress [26].

**61**

mice [35].

*Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS*

and severe loss of mitochondrial DNA were demonstrated [27].

repairing spontaneously arising DNA damage [18].

**3. Mitochondrial dysfunction in ALS**

In a model of C9orf72-induced ALS, the overexpression of SETX or depletion of p62 was capable of reducing γH2AX foci in MRC5 cells. In fact, the combined overexpression of SETX and depletion of p62 further reduced DSB levels compared to levels observed by SETX overexpression or p62 depletion alone [24]. Interestingly, mutations in SETX cause both ataxia with oculomotor apraxia type 2 (AOA2) (autosomal recessive) and amyotrophic lateral sclerosis 4 (ALS4) (autosomal dominant). The N-terminal protein interaction and C-terminal RNA/DNA helicase domains are conserved in the *Saccharomyces cerevisiae* SETX homolog Sen1p. In a mutant model of Sen1p in *S. cerevisiae,* alterations in redox state, unfolded protein response, TOR,

Other ALS-related genes can cause DNA damage. The cytoplasmic inclusions of TDP-43 and FUS seen in ALS can be derived from a toxic gain of function, which, in turn, triggers motor neuron cell death [18]. As the majority of ALS-causing mutations are located within the C-terminus of FUS, it has been suggested that these mutations impair the DNA pairing function of FUS, compromising genome stability [28]. Moreover, depletion of either FUS or TDP-43 in cells treated with α-amanitin (an RNA Pol II inhibitor) led to higher DNA strand breaks [18]. It also was demonstrated that after UV damage-induced transcription arrest, FUS localizes at genomic sites where active transcription has been arrested by DNA damage, and it does so together with BRCA1 [18]. TDP-43 is also able to localize at sites of transcription-associated DNA damage, given its colocalization with γH2AX and phosphorylated RPA. Interestingly, TDP-43 also colocalized with γH2AX and phosphorylated RPA in undamaged cells, consistent with its role in preventing or

FUS and TDP-43 are also required to maintain R-loop homeostasis since their depletion leads to R-loop-mediated genomic instability [29]. It is speculated that RNA processing factors prevent R-loop by binding to the nascent RNA transcript and blocking R-loop formation. In this regard, it has been shown that FUS and TDP-43 colocalize with active RNA polymerase II at sites of DNA damage along with BRCA1, either to prevent or repair R-loop-associated DNA damage, normally considered an indicator of defective transcription and/or RNA processing [18].

The space-time location of mitochondria is vital for the metabolic requirements of the nervous system, especially in the case of motor neurons. As neurons cannot rely on glycolysis, mitochondria are crucial to meet the high energy demands mainly by oxidative phosphorylation (OXPHOS). Several aspects of mitochondrial dynamics have been involved in the pathogenesis of ALS. For example, ATP generation was markedly decreased in neuronal cells of mutant SOD1-G93A mice [30, 31]. Moreover, cytosolic ATP levels were significantly reduced in neuroblastoma cells expressing mutant SOD1-G37R and in rotenone-treated SOD1-G93A neuronal cells [32]. Impairment of intracellular Ca2+ homeostasis is also considered as a hallmark of mitochondrial dysfunction in neurodegeneration. In this regard, an imbalance in Ca2+ dynamics has been reported in cells expressing both mutant SOD1-G93A and SOD1-G37R [33, 34] and in motor neurons from mutant SOD1-G93A transgenic

Mitochondrial fusion and fission are also key processes to preserve organelle functioning and cellular homeostasis. Dynamin-related protein 1 (Drp1) influences cell survival and apoptosis by mediating the mitochondrial fission process in mammals [36]. An excessive mitochondrial fragmentation and dysfunction were reported in patient-derived fibroblasts and cultured motor neurons of several

*DOI: http://dx.doi.org/10.5772/intechopen.90217*

#### *Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS DOI: http://dx.doi.org/10.5772/intechopen.90217*

In a model of C9orf72-induced ALS, the overexpression of SETX or depletion of p62 was capable of reducing γH2AX foci in MRC5 cells. In fact, the combined overexpression of SETX and depletion of p62 further reduced DSB levels compared to levels observed by SETX overexpression or p62 depletion alone [24]. Interestingly, mutations in SETX cause both ataxia with oculomotor apraxia type 2 (AOA2) (autosomal recessive) and amyotrophic lateral sclerosis 4 (ALS4) (autosomal dominant). The N-terminal protein interaction and C-terminal RNA/DNA helicase domains are conserved in the *Saccharomyces cerevisiae* SETX homolog Sen1p. In a mutant model of Sen1p in *S. cerevisiae,* alterations in redox state, unfolded protein response, TOR, and severe loss of mitochondrial DNA were demonstrated [27].

Other ALS-related genes can cause DNA damage. The cytoplasmic inclusions of TDP-43 and FUS seen in ALS can be derived from a toxic gain of function, which, in turn, triggers motor neuron cell death [18]. As the majority of ALS-causing mutations are located within the C-terminus of FUS, it has been suggested that these mutations impair the DNA pairing function of FUS, compromising genome stability [28]. Moreover, depletion of either FUS or TDP-43 in cells treated with α-amanitin (an RNA Pol II inhibitor) led to higher DNA strand breaks [18]. It also was demonstrated that after UV damage-induced transcription arrest, FUS localizes at genomic sites where active transcription has been arrested by DNA damage, and it does so together with BRCA1 [18]. TDP-43 is also able to localize at sites of transcription-associated DNA damage, given its colocalization with γH2AX and phosphorylated RPA. Interestingly, TDP-43 also colocalized with γH2AX and phosphorylated RPA in undamaged cells, consistent with its role in preventing or repairing spontaneously arising DNA damage [18].

FUS and TDP-43 are also required to maintain R-loop homeostasis since their depletion leads to R-loop-mediated genomic instability [29]. It is speculated that RNA processing factors prevent R-loop by binding to the nascent RNA transcript and blocking R-loop formation. In this regard, it has been shown that FUS and TDP-43 colocalize with active RNA polymerase II at sites of DNA damage along with BRCA1, either to prevent or repair R-loop-associated DNA damage, normally considered an indicator of defective transcription and/or RNA processing [18].

#### **3. Mitochondrial dysfunction in ALS**

The space-time location of mitochondria is vital for the metabolic requirements of the nervous system, especially in the case of motor neurons. As neurons cannot rely on glycolysis, mitochondria are crucial to meet the high energy demands mainly by oxidative phosphorylation (OXPHOS). Several aspects of mitochondrial dynamics have been involved in the pathogenesis of ALS. For example, ATP generation was markedly decreased in neuronal cells of mutant SOD1-G93A mice [30, 31]. Moreover, cytosolic ATP levels were significantly reduced in neuroblastoma cells expressing mutant SOD1-G37R and in rotenone-treated SOD1-G93A neuronal cells [32]. Impairment of intracellular Ca2+ homeostasis is also considered as a hallmark of mitochondrial dysfunction in neurodegeneration. In this regard, an imbalance in Ca2+ dynamics has been reported in cells expressing both mutant SOD1-G93A and SOD1-G37R [33, 34] and in motor neurons from mutant SOD1-G93A transgenic mice [35].

Mitochondrial fusion and fission are also key processes to preserve organelle functioning and cellular homeostasis. Dynamin-related protein 1 (Drp1) influences cell survival and apoptosis by mediating the mitochondrial fission process in mammals [36]. An excessive mitochondrial fragmentation and dysfunction were reported in patient-derived fibroblasts and cultured motor neurons of several

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

presymptomatic stage, specifically in motor neurons [13].

tive to typical ALS cases.

neurodegeneration.

**2. Role of ALS-related genes in DDR**

in response to oxidative stress [26].

from reactive oxygen species [6]. Even physiological brain activity can also cause DNA double-strand breaks (DSBs) in neurons [7]. However, as neurons do not divide, homologous recombination (HR) is not available for DNA repair. In addition, to maintain normal functioning, neurons must ensure to keep mitochondrial homeostasis. Thus, there are different areas with high demands for ATP like synaptic terminals, active growth cones, or axonal branches that contain more mitochondria than other cellular domains [8, 9]. This dependency on mitochondrial homeostasis makes neurons more susceptible and more likely to be affected by mutations in genes regulating mitochondrial dynamics. For example, mutations in MFN2 and GDAP1 (which regulate mitochondrial fusion and fission, respectively) are related to Charcot-Marie-Tooth [10, 11], a peripheral neuropathy that resembles some of the clinical manifestations of ALS, especially in motor neurons. Thus, a disruption in normal nuclear and mitochondrial DNA (mtDNA) repair would affect motor neurons in a progressive way. Supporting this idea, several works have reported mtDNA mutations in presymptomatic animal models of ALS [12]. In fact, similar to CMT, mitochondrial axonal transportation has also been implicated in ALS. The SOD1-G93A mice showed alterations in the axonal traffic of mitochondria at a

Interestingly, recent studies demonstrated that loss-of-function mutations in *KIF5A* are also a cause of ALS [14, 15]. *KIF5A* gene encodes for a specific motor protein implicated in the axonal transport of mitochondria. Although it was previously related with CMT and spastic paraplegia [16, 17], the new variants associated with ALS are primarily located at the C-terminal cargo-binding tail domain. Notably, patients harboring loss-of-function mutations displayed an extended survival rela-

A second aspect behind neurons' susceptibilities is their high rate of transcription. As they depend on the available DNA repair machinery, genomic stability is crucial for the maintenance of homeostasis. Transcription-driven DNA damage can arise from several sources, like the collapse of transcription machinery and subsequent DSBs, damage to genomic segments that have been opened up for transcription, and the formation of unnatural R-loop structures [18–21]. In the case of ALS cells, deficient DNA repair was reported more than three decades ago [22]. It seems that the DDR system in ALS loses adaptive capacity in dependency on the underlying genetic mutation [23]. The next chapter will address the role of the main genes involved in fALS in the generation of genomic instability as a hallmark of

The hexanucleotide GGGGCC repeat expansion in *C9ORF72* is one of the most common genetic causes of both ALS and frontotemporal dementia (FTD). It was recently demonstrated that these expansions could cause defective DNA repair, manifested by higher levels of DSBs and impaired DDR [24]. Defective ATMmediated DNA repair was found as a consequence of p62 accumulation, which impairs H2A ubiquitylation and perturbs ATM signaling. Another study reported that c-H2AX, p-ATM, 53BP1, and PARP1 were upregulated in C9orf72 patient spinal cords [25]. However, the mechanism behind the damage to the genome was not addressed. Authors suggest that R-loops, as well as the expression of dipeptide repeat proteins, are more likely to be the cause. Consistent with this notion, elevated levels of DNA damage markers γH2AX, ATR, GADD45, and p53 were present in motor neurons differentiated from iPSC lines from C9orf72-mutant ALS patients

**60**

familial forms of ALS expressing SOD1 mutant [37]. In the same article, authors demonstrated that inhibition of Drp1/Fis1 interaction led to a significant reduction in ROS levels and improved mitochondrial function and structure. Mutations in SOD1, TARDBP, or FUS are related to protein aggregation. In this regard, aggregated proteins can also contribute to mitochondrial dysfunction. Moreover, ATP decrease and ROS increase are the main features of mitochondrial damage. While ROS can increase Drp1 leading to sustained mitochondrial fission and fragmentation, a decrease in ATP levels can impair autophagy and proteasomal degradation. This can worsen protein aggregates and trigger ER stress [37, 38].

More than two decades ago, Kong and Xu reported that in mice expressing SOD1-G93A, the onset of the disease involved a decline of muscle strength and a transient explosive increase in vacuoles derived from degenerating mitochondria. Interestingly, this damage did not involve motor neuron death at the time of onset. Based on their results, the authors suggested that SOD1-mutant toxicity was mediated by damage to mitochondria in motor neurons [39]. The early findings of mitochondrial dysfunction were also confirmed by Magrané and colleagues, who studied the mitochondrial transport within the sciatic nerve in SOD1-G93A and TDP-43-A315T mice. Defects of retrograde mitochondrial transport were detected at 45 days of age in both mice, before the onset of symptoms [40]. In the SOD1-G93A mice, mitochondrial morphological abnormalities were apparent at day 15 of age, thus preceding transport abnormalities. Conversely, in TDP-43-A315T mice, morphological abnormalities appeared after the onset of transport defects. Interestingly, the authors reported neither morphological nor mitochondrial distribution changes in sensory neurons [40].

Another study evaluated the role of SOD1 mutations in mitochondrial homeostasis, in spinal cord sections from early symptomatic 10-month mice. Authors showed that mutant SOD1 binding to mitochondria disrupts normal distribution and morphology as an early pathogenic feature. In their work, mitochondria from SOD G85R/G37R mice became progressively smaller and rounder and are unevenly distributed along axons [41]. As in SOD1 mutant experiments, oxidative stress and DNA damage were increased in iPSC-derived C9ORF72 motor neurons in an age-dependent manner [26]. Poly (GR) preferentially binds to mitochondrial ribosomal proteins and compromised mitochondrial function, which could lead to increased oxidative stress. Reducing oxidative stress partially rescued DNA damage in C9ORF72 motor neurons cells [26].

The role of mutant FUS and TDP-43 has also been evaluated in mitochondrial dysfunction. In cultured neurons expressing FUS-R495X, it was revealed that mutant FUS controlled the translation of genes that were associated with mitochondria function, which resulted in a significant reduction of mitochondrial size [42]. Specifically, protein levels of KIF5B, DNM1L, and CSDE1 were significantly reduced in R495X-expressing neurons. FUS is also able to translocate and accumulate inside mitochondria. Following this approach, it was revealed that FUS could interact with mitochondrial chaperonin HSP60 and this interaction mediates FUS localization to the organelle, leading to damage [43]. This damage derives from reduced mitochondrial membrane potential and increased production of ROS. Authors also reported elevated HSP60 expression in two of three cases of FTLD-FUS patients' brain samples. Curiously, mutations in the *HSPD1* gene which encodes for HSP60 protein have also been found in patients with spastic paraplegia type 13 [44]. Another work revealed that mitochondrial impairment is a critical early event in FUS proteinopathy. When mutant FUS-P525L accumulated inside mitochondria, it interacted with the mitochondrial ATP synthase catalytic subunit ATP5B and reduced mitochondrial ATP synthesis. Importantly, FUS accumulation also induced the mitochondrial unfolded protein response (UPTmt), making the damage even worse [45].

**63**

future studies.

**4.1 The role of SIRT1-PARP1 axis**

*Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS*

in ER-mitochondria associations and mitochondrial Ca2+ levels.

closely related to the selective involvement of motor neurons in ALS.

**4. Nuclear-mitochondrial interplay and genomic integrity in ALS**

Poly (ADP-ribose) polymerase 1 (PARP1) is a NAD+-dependent protein. It is able to posttranslationally modify itself and other proteins involved in multiple DDR. PARP1 is also crucial for the stabilization of DNA replication forks and chromatin remodeling [55]. Deletion of *Parp1* gene in *Xrcc1* knockout mice is able to prevent neurodegeneration, indicating that the overactivation of PARP1 is a neurotoxic

In the ALS but not control brains, levels of a common mitochondrial DNA deletion mutation (mt DNA4977) were an average of more than 30-fold in Brodmann area 4 [52]. In addition, the oxidative damage derived from 8-oxoG was accumulated in the mitochondria of motor neurons in ALS, and according to previous findings, OGG1 did not repair the damage efficiently, driving the loss of motor neurons in ALS [53]. Likewise, motor neurons in G93A-mSOD1 transgenic mice undergo slow degeneration characterized by mitochondrial swelling and formation of DNA single-strand breaks prior to double-strand breaks occurring in both nuclear and mitochondrial DNA [54]. These cells accumulated mitochondria from the axon terminals and generated higher levels of ROS/RNS. Despite experimental evidence demonstrating mtDNA damage, more research is required to elucidate the mechanisms that lead to mitochondrial damage in ALS. The mechanisms that can link the DNA repair deficiency induced by errors in ALS-associated proteins and the subsequent mitochondrial dysfunction in terms of genetic integrity are not yet understood. We believe that this is an issue that should be taken into account for

Similarly, mutant TDP-43 accumulates in the mitochondria of neurons in subjects with ALS or frontotemporal dementia (FTD). Wild-type and mutant TDP-43 preferentially bind to mitochondria-transcribed mRNAs encoding respiratory complex I subunits ND3/ND6, causing complex I disassembly [46]. The suppression of TDP-43 mitochondrial localization abolished mitochondrial dysfunction and neuronal loss and improved the phenotypes of transgenic mutant TDP-43 mice. Mutant FUS can also alter mitochondrial-endoplasmic reticulum (ER) communication. Stoica and colleagues showed that FUS disrupts the VAPB-PTPIP51 complex, impairing ER-mitochondria association. As a consequence, there was a perturbation of Ca2+ uptake by mitochondria and impaired ATP production [47]. Remarkably, inhibition of glycogen synthase kinase-3b (GSK-3b) corrected FUS-induced defects

Mitochondrial DNA damage is also important in the context of neurodegenerative diseases [48, 49]. Maintaining mitochondrial DNA stability is critical for cellular function, especially in the context of the nervous system. Several pathways have been implicated in mtDNA repair, but base excision repair (BER) is the predominant one [50]. Oxoguanine glycosylase 1 (OGG1) is an enzyme that plays a major role in the mitochondrial BER pathway, removing 8-hydroxy-2-deoxyguanosine (8-OHdG). In the spinal cord of SOD1 mutant transgenic mice, the presence of 8-OHdG was progressively accumulated in the ventral horn neurons from early and presymptomatic stage (25 weeks) [51]. This was a suggestion that an oxidative damage to mitochondrial DNA was affecting spinal motor neurons at a very early stage of the disease. Early and selective impairment of DNA repair enzymes in mitochondria was also reported in presymptomatic transgenic mice carrying a mutant SOD1 gene [51]. Notably, the selective expression of these enzymes in spinal neurons is

*DOI: http://dx.doi.org/10.5772/intechopen.90217*

#### *Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS DOI: http://dx.doi.org/10.5772/intechopen.90217*

Similarly, mutant TDP-43 accumulates in the mitochondria of neurons in subjects with ALS or frontotemporal dementia (FTD). Wild-type and mutant TDP-43 preferentially bind to mitochondria-transcribed mRNAs encoding respiratory complex I subunits ND3/ND6, causing complex I disassembly [46]. The suppression of TDP-43 mitochondrial localization abolished mitochondrial dysfunction and neuronal loss and improved the phenotypes of transgenic mutant TDP-43 mice. Mutant FUS can also alter mitochondrial-endoplasmic reticulum (ER) communication. Stoica and colleagues showed that FUS disrupts the VAPB-PTPIP51 complex, impairing ER-mitochondria association. As a consequence, there was a perturbation of Ca2+ uptake by mitochondria and impaired ATP production [47]. Remarkably, inhibition of glycogen synthase kinase-3b (GSK-3b) corrected FUS-induced defects in ER-mitochondria associations and mitochondrial Ca2+ levels.

Mitochondrial DNA damage is also important in the context of neurodegenerative diseases [48, 49]. Maintaining mitochondrial DNA stability is critical for cellular function, especially in the context of the nervous system. Several pathways have been implicated in mtDNA repair, but base excision repair (BER) is the predominant one [50]. Oxoguanine glycosylase 1 (OGG1) is an enzyme that plays a major role in the mitochondrial BER pathway, removing 8-hydroxy-2-deoxyguanosine (8-OHdG). In the spinal cord of SOD1 mutant transgenic mice, the presence of 8-OHdG was progressively accumulated in the ventral horn neurons from early and presymptomatic stage (25 weeks) [51]. This was a suggestion that an oxidative damage to mitochondrial DNA was affecting spinal motor neurons at a very early stage of the disease. Early and selective impairment of DNA repair enzymes in mitochondria was also reported in presymptomatic transgenic mice carrying a mutant SOD1 gene [51]. Notably, the selective expression of these enzymes in spinal neurons is closely related to the selective involvement of motor neurons in ALS.

In the ALS but not control brains, levels of a common mitochondrial DNA deletion mutation (mt DNA4977) were an average of more than 30-fold in Brodmann area 4 [52]. In addition, the oxidative damage derived from 8-oxoG was accumulated in the mitochondria of motor neurons in ALS, and according to previous findings, OGG1 did not repair the damage efficiently, driving the loss of motor neurons in ALS [53]. Likewise, motor neurons in G93A-mSOD1 transgenic mice undergo slow degeneration characterized by mitochondrial swelling and formation of DNA single-strand breaks prior to double-strand breaks occurring in both nuclear and mitochondrial DNA [54]. These cells accumulated mitochondria from the axon terminals and generated higher levels of ROS/RNS. Despite experimental evidence demonstrating mtDNA damage, more research is required to elucidate the mechanisms that lead to mitochondrial damage in ALS. The mechanisms that can link the DNA repair deficiency induced by errors in ALS-associated proteins and the subsequent mitochondrial dysfunction in terms of genetic integrity are not yet understood. We believe that this is an issue that should be taken into account for future studies.

#### **4. Nuclear-mitochondrial interplay and genomic integrity in ALS**

#### **4.1 The role of SIRT1-PARP1 axis**

Poly (ADP-ribose) polymerase 1 (PARP1) is a NAD+-dependent protein. It is able to posttranslationally modify itself and other proteins involved in multiple DDR. PARP1 is also crucial for the stabilization of DNA replication forks and chromatin remodeling [55]. Deletion of *Parp1* gene in *Xrcc1* knockout mice is able to prevent neurodegeneration, indicating that the overactivation of PARP1 is a neurotoxic

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

This can worsen protein aggregates and trigger ER stress [37, 38].

bution changes in sensory neurons [40].

in C9ORF72 motor neurons cells [26].

familial forms of ALS expressing SOD1 mutant [37]. In the same article, authors demonstrated that inhibition of Drp1/Fis1 interaction led to a significant reduction in ROS levels and improved mitochondrial function and structure. Mutations in SOD1, TARDBP, or FUS are related to protein aggregation. In this regard, aggregated proteins can also contribute to mitochondrial dysfunction. Moreover, ATP decrease and ROS increase are the main features of mitochondrial damage. While ROS can increase Drp1 leading to sustained mitochondrial fission and fragmentation, a decrease in ATP levels can impair autophagy and proteasomal degradation.

More than two decades ago, Kong and Xu reported that in mice expressing SOD1-G93A, the onset of the disease involved a decline of muscle strength and a transient explosive increase in vacuoles derived from degenerating mitochondria. Interestingly, this damage did not involve motor neuron death at the time of onset. Based on their results, the authors suggested that SOD1-mutant toxicity was mediated by damage to mitochondria in motor neurons [39]. The early findings of mitochondrial dysfunction were also confirmed by Magrané and colleagues, who studied the mitochondrial transport within the sciatic nerve in SOD1-G93A and TDP-43-A315T mice. Defects of retrograde mitochondrial transport were detected at 45 days of age in both mice, before the onset of symptoms [40]. In the SOD1-G93A mice, mitochondrial morphological abnormalities were apparent at day 15 of age, thus preceding transport abnormalities. Conversely, in TDP-43-A315T mice, morphological abnormalities appeared after the onset of transport defects. Interestingly, the authors reported neither morphological nor mitochondrial distri-

Another study evaluated the role of SOD1 mutations in mitochondrial homeostasis, in spinal cord sections from early symptomatic 10-month mice. Authors showed that mutant SOD1 binding to mitochondria disrupts normal distribution and morphology as an early pathogenic feature. In their work, mitochondria from SOD G85R/G37R mice became progressively smaller and rounder and are unevenly distributed along axons [41]. As in SOD1 mutant experiments, oxidative stress and DNA damage were increased in iPSC-derived C9ORF72 motor neurons in an age-dependent manner [26]. Poly (GR) preferentially binds to mitochondrial ribosomal proteins and compromised mitochondrial function, which could lead to increased oxidative stress. Reducing oxidative stress partially rescued DNA damage

The role of mutant FUS and TDP-43 has also been evaluated in mitochondrial dysfunction. In cultured neurons expressing FUS-R495X, it was revealed that mutant FUS controlled the translation of genes that were associated with mitochondria function, which resulted in a significant reduction of mitochondrial size [42]. Specifically, protein levels of KIF5B, DNM1L, and CSDE1 were significantly reduced in R495X-expressing neurons. FUS is also able to translocate and accumulate inside mitochondria. Following this approach, it was revealed that FUS could interact with mitochondrial chaperonin HSP60 and this interaction mediates FUS localization to the organelle, leading to damage [43]. This damage derives from reduced mitochondrial membrane potential and increased production of ROS. Authors also reported elevated HSP60 expression in two of three cases of FTLD-FUS patients' brain samples. Curiously, mutations in the *HSPD1* gene which encodes for HSP60 protein have also been found in patients with spastic paraplegia type 13 [44]. Another work revealed that mitochondrial impairment is a critical early event in FUS proteinopathy. When mutant FUS-P525L accumulated inside mitochondria, it interacted with the mitochondrial ATP synthase catalytic subunit ATP5B and reduced mitochondrial ATP synthesis. Importantly, FUS accumulation also induced the mitochondrial unfolded protein response (UPTmt), making the damage even worse [45].

**62**

consequence of defective DNA repair [56]. Recently, it was demonstrated that FUS can bind directly to PAR, the polymer made by PARP1 [57]. Authors revealed that FUS is recruited to chromosomal sites of oxidative DNA damage and that this recruitment is reduced by the FUS-R521G mutation. Additionally, when cells lack PARP1, FUS failed to accumulate at sites of UVA laser-induced damage. Moreover, under oxidative stress, PARP1 activity increases, leading to an accumulation of ADP-ribose polymers and NAD+ depletion. This condition induces a metabolic and energetic crisis driving cell death [58]. As PARP1 hyperactivation and toxicity have been implicated in diseases like Alzheimer's, Parkinson's, and Huntington's, a predominant role in ALS is also plausible [59].

Sirtuins are also NAD-dependent proteins. SIRT1 is known as a nuclear and cytoplasmic deacetylase, which is predominantly expressed in neurons in the context of the nervous system [60]. Notably, PARP1 consumes NAD+, reducing SIRT1 activity by increasing PARylation of DNA and proteins involved in DDR. Thus, SIRT1 activity appears to be impeded by PARP1 hyperactivation [61]. Interestingly, NAD+ replenishment shows a reactivation of SIRT1 and prevented neurodegeneration [62]. Thus, compromised DNA repair leads to PARP1 hyperactivation, resulting in the depletion of NAD+ levels, inhibiting SIRTs (**Figure 1**). Notably, the Km of SIRT1 for NAD+ is higher than that of PARP1, so when NAD+ levels become so low following cell stress or senescence, SIRT1 no longer has the chance to regulate the activity of PARP1 [61]. Cantó and colleagues demonstrated that deletion of *Parp1* gene increased NAD+ levels and SIRT1 activity in brown adipose tissue and muscle. *Parp1* −/− mice presented a higher mitochondrial content, resulting in increased energy expenditure and protection against metabolic disease. As previously reported, authors showed that pharmacologic inhibition of PARP in vitro and in vivo increased NAD+, leading to SIRT1 expression and enhanced oxidative metabolism [63].

SIRT1 is vital in the maintenance of metabolic homeostasis [64]. SIRT1 can activate the peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) through deacetylation [65]. PGC-1a can regulate genes of the ROS defense system. It has been shown that PGC-1a can protect WT neural cells from oxidative stress by increasing the expression of ROS-detoxifying genes including SOD1/SOD2 and UCP2 [66]. SIRT1 may also deacetylate PGC-1α to induce mitochondrial biogenesis by increasing mitochondrial gene expression via nuclear respiratory factor 1 (NRF-1) and nuclear-encoded mitochondrial transcription factor A (TFAM) [67]. Interestingly, it was revealed that PGC-1 and SIRT1 are also present inside the mitochondria and are in close proximity to mtDNA [68]. In addition, an unexpected role for serotonin (5-HT) was also recently demonstrated. Fanibunda and colleagues showed that in rodent cortical neurons, serotonin is a regulator of mitochondrial biogenesis, via 5-HT2A receptor-mediated recruitment of the SIRT1-PGC-1α axis [69].

SIRT1 can form a protein complex with the Forkhead transcription factor FOXO3 and promote its deacetylation. SIRT1 induces several effects mediated by FOXO3 like cell cycle arrest and transcription of DNA repair target genes [70]. Cantó and colleagues demonstrated that AMPK enhances SIRT1 activity by increasing cellular NAD+ levels [71]. This resulted in the deacetylation and modulation of the activity of downstream SIRT1 targets including PGC-11α and FOXO1/FOXO3a transcription factors. Thus, SIRT1 could promote both mitochondrial biogenesis in conditions of energy deficiency in disease or induce the clearance of damaged mitochondria [72].

In XPA-deficient cells, a model of xeroderma pigmentosum, the DNA repair deficiency leads to PARP1 activation, and the attenuated NAD+/SIRT1/PGC-1α axis resulted in defective mitophagy. The condition could be rescued either by

**65**

**Figure 1.**

*Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS*

PARP1 inhibition or by supplementation with NAD+ [73]. In a different work, Scheibye-Knudsen and colleagues showed that hyperactivation of PARP1 drives SIRT1 depression in Cockayne syndrome (CS). Similar to previous findings, NAD+ replenishment rescued the phenotype in CS, but in this case, they also demonstrated that diet can also increase SIRT1 activity [62]. Following the same line, it was revealed that NAD+ levels are reduced in aged mice and *Caenorhabditis elegans* [74]. Genetic or pharmacological restoration of NAD+ prevented age-associated metabolic decline and promoted longevity in worms. These effects were associated with deacetylase sir-2.1 levels, the homolog of SIRT1. Indeed, hyperacetylation of the SIRT1 substrate PGC-1α resulted from low levels of NAD+ in aged mice [74]. Supporting this approach, it was demonstrated that the intracellular nicotinamide phosphoribosyltransferase (iNAMPT) is essential to projection neuron function and viability. The iNAMPT is the rate-limiting enzyme of the mammalian NAD+ biosynthesis. When eliminating *Nampt* in adult mice, the resulting phenotype

*Dotted lines illustrate pathological interactions.*

*Nuclear-mitochondrial interplay in the context of ALS. (A) Activation of PARP1 upon DNA damage facilitates DNA repair but inhibits SIRT1 due to NAD+ depletion. FUS binds directly to PAR, the polymer made by PARP1 in chromosomal sites of oxidative DNA damage. Both TDP-43 and FUS localize at genomic sites where active transcription has been arrested by DNA damage and also maintain R-loop homeostasis. SETX is also necessary for R-loop repair. In addition, FUS interacts with HDAC1 and enhances its activity in both physiological conditions and DNA damage. (B) SIRT1 positively regulates DDR. SIRT is able to deacetylase HDAC1, ATM, WRN, TDG, XPA, and KU70. (C) SIRT1 also regulates mitochondrial homeostasis through AMPK, FOXOs, and PGC1α. These enzymes regulate mitochondrial biogenesis and oxidative stress, transcribing proteins such as UCP2 and SODs. (D) Accumulation of misfolded proteins leads to the aberrant p62 expression, disrupting normal mitophagy. Likewise, defective ATM-mediated DNA repair is a consequence of p62 accumulation and RNF168 inhibition. C9orf72 can also cause defective DNA repair and p62 accumulation with the subsequent impaired mitophagy. (E) ALS-related mitochondrial dysfunction leads to mitochondrial fragmentation through DRP1 overexpression, decreased ATP production, higher ROS levels, mtDNA damage, decrease in OXPHOS, and Ca+2 imbalance. FUS can accumulate inside the mitochondria, leading to impaired ATP production and increased ROS levels. C9ORF72 can also generate oxidative stress and mtDNA damage in motor neurons. TDP-43 preferentially bind to mitochondria-transcribed mRNAs, causing complex I disassembly. SOD1 mutations can decrease cytosolic ATP levels, impair intracellular Ca2+ homeostasis, generate excessive mitochondrial fragmentation, and deregulate DDR. The final effect of this cascade is derived from mitochondria-ER stress, consequent misfolded proteins, and further protein aggregation.* 

*DOI: http://dx.doi.org/10.5772/intechopen.90217*

*Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS DOI: http://dx.doi.org/10.5772/intechopen.90217*

#### **Figure 1.**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

predominant role in ALS is also plausible [59].

consequence of defective DNA repair [56]. Recently, it was demonstrated that FUS can bind directly to PAR, the polymer made by PARP1 [57]. Authors revealed that FUS is recruited to chromosomal sites of oxidative DNA damage and that this recruitment is reduced by the FUS-R521G mutation. Additionally, when cells lack PARP1, FUS failed to accumulate at sites of UVA laser-induced damage. Moreover, under oxidative stress, PARP1 activity increases, leading to an accumulation of ADP-ribose polymers and NAD+ depletion. This condition induces a metabolic and energetic crisis driving cell death [58]. As PARP1 hyperactivation and toxicity have been implicated in diseases like Alzheimer's, Parkinson's, and Huntington's, a

Sirtuins are also NAD-dependent proteins. SIRT1 is known as a nuclear and cytoplasmic deacetylase, which is predominantly expressed in neurons in the context of the nervous system [60]. Notably, PARP1 consumes NAD+, reducing SIRT1 activity by increasing PARylation of DNA and proteins involved in DDR. Thus, SIRT1 activity appears to be impeded by PARP1 hyperactivation [61]. Interestingly, NAD+ replenishment shows a reactivation of SIRT1 and prevented neurodegeneration [62]. Thus, compromised DNA repair leads to PARP1 hyperactivation, resulting in the depletion of NAD+ levels, inhibiting SIRTs (**Figure 1**). Notably, the Km of SIRT1 for NAD+ is higher than that of PARP1, so when NAD+ levels become so low following cell stress or senescence, SIRT1 no longer has the chance to regulate the activity of PARP1 [61]. Cantó and colleagues demonstrated that deletion of *Parp1* gene increased NAD+ levels and SIRT1 activity in brown adipose tissue and muscle. *Parp1* −/− mice presented a higher mitochondrial content, resulting in increased energy expenditure and protection against metabolic disease. As previously reported, authors showed that pharmacologic inhibition of PARP in vitro and in vivo increased NAD+, leading to SIRT1 expression and enhanced oxidative

SIRT1 is vital in the maintenance of metabolic homeostasis [64]. SIRT1 can activate the peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) through deacetylation [65]. PGC-1a can regulate genes of the ROS defense system. It has been shown that PGC-1a can protect WT neural cells from oxidative stress by increasing the expression of ROS-detoxifying genes including SOD1/SOD2 and UCP2 [66]. SIRT1 may also deacetylate PGC-1α to induce mitochondrial biogenesis by increasing mitochondrial gene expression via nuclear respiratory factor 1 (NRF-1) and nuclear-encoded mitochondrial transcription factor A (TFAM) [67]. Interestingly, it was revealed that PGC-1 and SIRT1 are also present inside the mitochondria and are in close proximity to mtDNA [68]. In addition, an unexpected role for serotonin (5-HT) was also recently demonstrated. Fanibunda and colleagues showed that in rodent cortical neurons, serotonin is a regulator of mitochondrial biogenesis, via 5-HT2A receptor-mediated recruitment

SIRT1 can form a protein complex with the Forkhead transcription factor FOXO3 and promote its deacetylation. SIRT1 induces several effects mediated by FOXO3 like cell cycle arrest and transcription of DNA repair target genes [70]. Cantó and colleagues demonstrated that AMPK enhances SIRT1 activity by increasing cellular NAD+ levels [71]. This resulted in the deacetylation and modulation of the activity of downstream SIRT1 targets including PGC-11α and FOXO1/FOXO3a transcription factors. Thus, SIRT1 could promote both mitochondrial biogenesis in conditions of energy deficiency in disease or induce the clearance of damaged

In XPA-deficient cells, a model of xeroderma pigmentosum, the DNA repair deficiency leads to PARP1 activation, and the attenuated NAD+/SIRT1/PGC-1α axis resulted in defective mitophagy. The condition could be rescued either by

**64**

metabolism [63].

of the SIRT1-PGC-1α axis [69].

mitochondria [72].

*Nuclear-mitochondrial interplay in the context of ALS. (A) Activation of PARP1 upon DNA damage facilitates DNA repair but inhibits SIRT1 due to NAD+ depletion. FUS binds directly to PAR, the polymer made by PARP1 in chromosomal sites of oxidative DNA damage. Both TDP-43 and FUS localize at genomic sites where active transcription has been arrested by DNA damage and also maintain R-loop homeostasis. SETX is also necessary for R-loop repair. In addition, FUS interacts with HDAC1 and enhances its activity in both physiological conditions and DNA damage. (B) SIRT1 positively regulates DDR. SIRT is able to deacetylase HDAC1, ATM, WRN, TDG, XPA, and KU70. (C) SIRT1 also regulates mitochondrial homeostasis through AMPK, FOXOs, and PGC1α. These enzymes regulate mitochondrial biogenesis and oxidative stress, transcribing proteins such as UCP2 and SODs. (D) Accumulation of misfolded proteins leads to the aberrant p62 expression, disrupting normal mitophagy. Likewise, defective ATM-mediated DNA repair is a consequence of p62 accumulation and RNF168 inhibition. C9orf72 can also cause defective DNA repair and p62 accumulation with the subsequent impaired mitophagy. (E) ALS-related mitochondrial dysfunction leads to mitochondrial fragmentation through DRP1 overexpression, decreased ATP production, higher ROS levels, mtDNA damage, decrease in OXPHOS, and Ca+2 imbalance. FUS can accumulate inside the mitochondria, leading to impaired ATP production and increased ROS levels. C9ORF72 can also generate oxidative stress and mtDNA damage in motor neurons. TDP-43 preferentially bind to mitochondria-transcribed mRNAs, causing complex I disassembly. SOD1 mutations can decrease cytosolic ATP levels, impair intracellular Ca2+ homeostasis, generate excessive mitochondrial fragmentation, and deregulate DDR. The final effect of this cascade is derived from mitochondria-ER stress, consequent misfolded proteins, and further protein aggregation. Dotted lines illustrate pathological interactions.*

PARP1 inhibition or by supplementation with NAD+ [73]. In a different work, Scheibye-Knudsen and colleagues showed that hyperactivation of PARP1 drives SIRT1 depression in Cockayne syndrome (CS). Similar to previous findings, NAD+ replenishment rescued the phenotype in CS, but in this case, they also demonstrated that diet can also increase SIRT1 activity [62]. Following the same line, it was revealed that NAD+ levels are reduced in aged mice and *Caenorhabditis elegans* [74]. Genetic or pharmacological restoration of NAD+ prevented age-associated metabolic decline and promoted longevity in worms. These effects were associated with deacetylase sir-2.1 levels, the homolog of SIRT1. Indeed, hyperacetylation of the SIRT1 substrate PGC-1α resulted from low levels of NAD+ in aged mice [74]. Supporting this approach, it was demonstrated that the intracellular nicotinamide phosphoribosyltransferase (iNAMPT) is essential to projection neuron function and viability. The iNAMPT is the rate-limiting enzyme of the mammalian NAD+ biosynthesis. When eliminating *Nampt* in adult mice, the resulting phenotype

resembled ALS: motor neuron degeneration, hypothermia, motor dysfunction and paralysis, reduced general motor activity, and anxiety-like behaviors. Remarkably, nicotinamide mononucleotide (NMN), a NAD+ precursor, improved health span, restored motor function, and extended lifespan [75]. Since the authors also found that iNAMPT protein levels were significantly reduced in the spinal cord of ALS patients, the NAD+/SIRT1 axis represents an outstanding therapeutic opportunity.

For a long time, the beneficial effect of resveratrol has been controversial due to inconsistent results and tissue-dependent effects. Price and colleagues showed that a moderate dose of resveratrol is able to stimulate the AMPK activity, increase NAD+ levels, enhance mitochondrial biogenesis, and improve mitochondrial function. All of these functions were entirely dependent upon SIRT1 in skeletal muscle [76]. Interestingly, a tenfold higher dose of resveratrol activated AMPK in a SIRT1-independent manner, although improvements in mitochondrial function were SIRT1 dependent [76]. Finally, it was demonstrated that resveratrol-mediated SIRT1 activation protects against p25 and mutant SOD1-G93A neurotoxicity both in vitro and in vivo [77].

#### **4.2 The role of SIRT in DDR**

Beyond its role in metabolism, SIRT1 is also important in DNA repair. In postmitotic neurons, SIRT1 was rapidly recruited to DSBs where it showed a synergistic relationship with ATM, stimulating its autophosphorylation and stabilizing ATM at DSBs' sites [78]. Additionally, authors reported that after DSBs' induction, SIRT1 also bound HDAC1, a neuroprotective histone deacetylase, stimulating its enzymatic activity. This activity is necessary for the nonhomologous end joining (NHEJ) pathway [78]. Likewise, upon exposure to radiation, SIRT1 can enhance DNA repair capacity and deacetylation of repair protein Ku70, involved in NHEJ as well [79].

SIRT1 also interacts with WRN both in vitro and in vivo, and this interaction is enhanced after DNA damage [80]. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Moreover, the MRE11- RAD50-NBS1 (MRN) is a conserved nuclease complex that exhibits properties of a DNA damage sensor in the context of DSBs. SIRT1 can associate with the MRN complex and maintains NBS1 in a hypoacetylated state following ionizing radiation [81]. SIRT1 is also important for the base excision repair (BER) pathway. It was shown that SIRT1 deacetylates APE1 in vitro and in vivo following genotoxic insults [82]. Knockdown of SIRT1 increases cellular abasic DNA content, sensitizing cells to death induced by genotoxic stress. Interestingly, the activation of SIRT1 with resveratrol promoted binding of APE1 to XRCC1 [82]. Thymine DNA glycosylase (TDG) is an essential multifunctional enzyme also involved in BER. SIRT1 was shown to interact with TDG and enhance its glycosylase activity due to deacetylation [83]. Nucleotide excision repair (NER) is another important pathway in DNA repair. NER removes DNA damage induced by ultraviolet light (UV). Xeroderma pigmentosum group A (XPA) is a critical protein for this process. SIRT1 interacts with XPA, and the interaction is enhanced after UV irradiation [84]. In fact, downregulation of SIRT1 sensitized cells to UV irradiation.

#### **4.3 The role of FUS in DDR**

FUS is important for both NHEJ- and HR-mediated DSB repair in neurons. As SIRT1, FUS interacts with HDAC1 and enhances its activity in both physiological conditions and DNA damage in cortical neurons [85]. The same was demonstrated in human-induced pluripotent stem cells (hiPSCs) and hiPSC-derived motor neurons. The degree of FUS mislocalization correlated well with the clinical severity

**67**

*Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS*

of the underlying ALS, with a higher accumulation of DNA damage in postmitotic mutated FUS motor neurons than in dividing hiPSCs [86]. Although FUS has been associated with multiple DNA repair pathways including DSB repair, quantification of the level of DNA SSBs vs. DSBs in FUS knockdown (KD) and knockout (KO) cells by comet analysis revealed that most unrepaired DNA strand breaks that

FUS protects the genome by facilitating PARP1-dependent recruitment of XRCC1/DNA ligase IIIα (LigIII) to oxidized genome sites and activating LigIII via direct interaction [88]. Cells presenting mutant FUS showed a significantly reduced association between this DNA repair protein complex, SSBs' accumulation, and ROS levels in motor neurons. LigIII was demonstrated to be an essential enzyme for mtDNA integrity but dispensable for nuclear DNA repair [89]. LigI was critical for nuclear DNA repair in a cooperative manner with LigIII, but inactivation of LigIII resulted in mtDNA loss, mitochondrial dysfunction, and incapacitating ataxia in the mouse nervous system. However, as neurons do not divide, it is possible that LigIII is equally important for nuclear DNA repair. Following the idea that mtDNA integrity is vital in postmitotic tissues, the authors also found that inactivation of LigIII in cardiac muscle resulted in mitochondrial dysfunction and defective heartpump function leading to heart failure [89]. More research is needed to elucidate the interaction of ALS-associated proteins with both nuclear and mitochondrial

**4.4 Cross talk between auto-/mitophagy and DNA repair in ALS**

Autophagy is a crucial process to eliminate misfolded proteins and organelles. Mitophagy is a special subroutine of autophagy, involved in the maintenance of mitochondrial homeostasis. An important protein regulating this process is p62. The molecular mechanism of p62-mediated mitochondrial defective clearance was demonstrated by Geisler and colleagues [90]. Knockdown of p62 significantly diminished mitochondria recognition by the autophagy machinery and its subsequent elimination [91]. Notably, accumulation of misfolded proteins leads to the aberrant p62 expression, which influences the balance of mitophagy, worsens mitochondrial dysfunction, and induces more protein aggregates [92]. This can explain the vulnerability of non-cycling cells to proteinopathies in the context of

Autophagy is also important to maintain functional DNA repair by preventing p62 accumulation. In the context of ALS, where p62 accumulates, RNF168 mediated H2A ubiquitylation (U) is perturbed, leading to impaired DSBs' repair and genomic instability [29]. Wang and colleagues reported that p62/SQSTM1, which accumulates in autophagy-defective cells, directly binds to and inhibits nuclear RNF168, which is crucial for histone H2A ubiquitination and DNA damage responses. As a result, DNA repair proteins such as BRCA1, RAP80, and Rad51 cannot be recruited to the sites of DNA double-strand breaks (DSBs), which impairs DSBs' repair [97]. The interaction between mitochondrial dysfunction, defective mitophagy, and protein aggregation should be the subject of further investigation. This could generate new therapeutic avenues in the context of neurodegenerative

Despite the clear role of mitochondria in neurodegeneration, the application of translational medicine in this field remains to be addressed. The lack of new

*DOI: http://dx.doi.org/10.5772/intechopen.90217*

DNA repair enzymes (**Figure 1**).

mitochondrial dysfunction [93–96].

diseases that occur with proteinopathies.

**5. Therapeutic challenges and opportunities**

accumulated after the loss of FUS were SSBs [87].

#### *Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS DOI: http://dx.doi.org/10.5772/intechopen.90217*

of the underlying ALS, with a higher accumulation of DNA damage in postmitotic mutated FUS motor neurons than in dividing hiPSCs [86]. Although FUS has been associated with multiple DNA repair pathways including DSB repair, quantification of the level of DNA SSBs vs. DSBs in FUS knockdown (KD) and knockout (KO) cells by comet analysis revealed that most unrepaired DNA strand breaks that accumulated after the loss of FUS were SSBs [87].

FUS protects the genome by facilitating PARP1-dependent recruitment of XRCC1/DNA ligase IIIα (LigIII) to oxidized genome sites and activating LigIII via direct interaction [88]. Cells presenting mutant FUS showed a significantly reduced association between this DNA repair protein complex, SSBs' accumulation, and ROS levels in motor neurons. LigIII was demonstrated to be an essential enzyme for mtDNA integrity but dispensable for nuclear DNA repair [89]. LigI was critical for nuclear DNA repair in a cooperative manner with LigIII, but inactivation of LigIII resulted in mtDNA loss, mitochondrial dysfunction, and incapacitating ataxia in the mouse nervous system. However, as neurons do not divide, it is possible that LigIII is equally important for nuclear DNA repair. Following the idea that mtDNA integrity is vital in postmitotic tissues, the authors also found that inactivation of LigIII in cardiac muscle resulted in mitochondrial dysfunction and defective heartpump function leading to heart failure [89]. More research is needed to elucidate the interaction of ALS-associated proteins with both nuclear and mitochondrial DNA repair enzymes (**Figure 1**).

#### **4.4 Cross talk between auto-/mitophagy and DNA repair in ALS**

Autophagy is a crucial process to eliminate misfolded proteins and organelles. Mitophagy is a special subroutine of autophagy, involved in the maintenance of mitochondrial homeostasis. An important protein regulating this process is p62. The molecular mechanism of p62-mediated mitochondrial defective clearance was demonstrated by Geisler and colleagues [90]. Knockdown of p62 significantly diminished mitochondria recognition by the autophagy machinery and its subsequent elimination [91]. Notably, accumulation of misfolded proteins leads to the aberrant p62 expression, which influences the balance of mitophagy, worsens mitochondrial dysfunction, and induces more protein aggregates [92]. This can explain the vulnerability of non-cycling cells to proteinopathies in the context of mitochondrial dysfunction [93–96].

Autophagy is also important to maintain functional DNA repair by preventing p62 accumulation. In the context of ALS, where p62 accumulates, RNF168 mediated H2A ubiquitylation (U) is perturbed, leading to impaired DSBs' repair and genomic instability [29]. Wang and colleagues reported that p62/SQSTM1, which accumulates in autophagy-defective cells, directly binds to and inhibits nuclear RNF168, which is crucial for histone H2A ubiquitination and DNA damage responses. As a result, DNA repair proteins such as BRCA1, RAP80, and Rad51 cannot be recruited to the sites of DNA double-strand breaks (DSBs), which impairs DSBs' repair [97]. The interaction between mitochondrial dysfunction, defective mitophagy, and protein aggregation should be the subject of further investigation. This could generate new therapeutic avenues in the context of neurodegenerative diseases that occur with proteinopathies.

#### **5. Therapeutic challenges and opportunities**

Despite the clear role of mitochondria in neurodegeneration, the application of translational medicine in this field remains to be addressed. The lack of new

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

in vitro and in vivo [77].

**4.2 The role of SIRT in DDR**

regulation of SIRT1 sensitized cells to UV irradiation.

**4.3 The role of FUS in DDR**

resembled ALS: motor neuron degeneration, hypothermia, motor dysfunction and paralysis, reduced general motor activity, and anxiety-like behaviors. Remarkably, nicotinamide mononucleotide (NMN), a NAD+ precursor, improved health span, restored motor function, and extended lifespan [75]. Since the authors also found that iNAMPT protein levels were significantly reduced in the spinal cord of ALS patients, the NAD+/SIRT1 axis represents an outstanding therapeutic opportunity. For a long time, the beneficial effect of resveratrol has been controversial due to inconsistent results and tissue-dependent effects. Price and colleagues showed that a moderate dose of resveratrol is able to stimulate the AMPK activity, increase NAD+ levels, enhance mitochondrial biogenesis, and improve mitochondrial function. All of these functions were entirely dependent upon SIRT1 in skeletal muscle [76]. Interestingly, a tenfold higher dose of resveratrol activated AMPK in a SIRT1-independent manner, although improvements in mitochondrial function were SIRT1 dependent [76]. Finally, it was demonstrated that resveratrol-mediated SIRT1 activation protects against p25 and mutant SOD1-G93A neurotoxicity both

Beyond its role in metabolism, SIRT1 is also important in DNA repair. In postmitotic neurons, SIRT1 was rapidly recruited to DSBs where it showed a synergistic relationship with ATM, stimulating its autophosphorylation and stabilizing ATM at DSBs' sites [78]. Additionally, authors reported that after DSBs' induction, SIRT1 also bound HDAC1, a neuroprotective histone deacetylase, stimulating its enzymatic activity. This activity is necessary for the nonhomologous end joining (NHEJ) pathway [78]. Likewise, upon exposure to radiation, SIRT1 can enhance DNA repair capacity and deacetylation of repair protein Ku70, involved in NHEJ as well [79]. SIRT1 also interacts with WRN both in vitro and in vivo, and this interaction is enhanced after DNA damage [80]. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Moreover, the MRE11- RAD50-NBS1 (MRN) is a conserved nuclease complex that exhibits properties of a DNA damage sensor in the context of DSBs. SIRT1 can associate with the MRN complex and maintains NBS1 in a hypoacetylated state following ionizing radiation [81]. SIRT1 is also important for the base excision repair (BER) pathway. It was shown that SIRT1 deacetylates APE1 in vitro and in vivo following genotoxic insults [82]. Knockdown of SIRT1 increases cellular abasic DNA content, sensitizing cells to death induced by genotoxic stress. Interestingly, the activation of SIRT1 with resveratrol promoted binding of APE1 to XRCC1 [82]. Thymine DNA glycosylase (TDG) is an essential multifunctional enzyme also involved in BER. SIRT1 was shown to interact with TDG and enhance its glycosylase activity due to deacetylation [83]. Nucleotide excision repair (NER) is another important pathway in DNA repair. NER removes DNA damage induced by ultraviolet light (UV). Xeroderma pigmentosum group A (XPA) is a critical protein for this process. SIRT1 interacts with XPA, and the interaction is enhanced after UV irradiation [84]. In fact, down-

FUS is important for both NHEJ- and HR-mediated DSB repair in neurons. As SIRT1, FUS interacts with HDAC1 and enhances its activity in both physiological conditions and DNA damage in cortical neurons [85]. The same was demonstrated in human-induced pluripotent stem cells (hiPSCs) and hiPSC-derived motor neurons. The degree of FUS mislocalization correlated well with the clinical severity

**66**

therapeutic options and the limitations of current genetic editing techniques make it difficult to have a clear path for future research. Due to the failure in targeting specific aspects of mitochondria, mitochondrial transplantation presents a new paradigm of therapeutic intervention that may benefit neuronal survival and regeneration in ALS and similar diseases. The clinical application of mitochondrial transplantation was extensively reviewed by Chang and colleagues already [98].

The importance of this approach has been demonstrated in different lines of research. For example, it was shown that astrocytes in mice release functional mitochondria that enter neurons and amplify cell survival signals after stroke [99]. Cardiomyocytes share several similitudes with neurons, including the postmitotic state, the high transcription rate, the similar metabolic profile, and the underlying dependence on mitochondrial homeostasis. Following myocardial ischemia and reperfusion (IR), several alterations have been demonstrated in mitochondrial structure and function. Mitochondrial transplantation has been proposed as a strong opportunity for the treatment of IR [100]. Masuzawa and colleagues showed in a model of ischemia-reperfusion injury that transplantation of autologously derived mitochondria immediately prior to reperfusion ameliorated the damage. By using New Zealand white rabbits, they demonstrated a complete recovery and showed that the transplanted mitochondria enhanced oxygen consumption, highenergy phosphate synthesis, and enhanced post-infarct cardiac function [101].

In a model of Parkinson's disease induced by the respiratory chain inhibitor MPTP, Shi and colleagues demonstrated in mice that intravenous injection of mitochondria prevented the progression of the disease. Specifically, the authors reported increased activity of the electron transport chain, decreased ROS levels, and prevention of cell apoptosis and necrosis [102]. Other approaches have been addressed following this so-called mitochondrial medicine approach in ALS, such as ketogenic diet, antioxidants, and metabolic targeting drugs [103]. Based on the evidence provided until this point, mitochondrial transplantation should be validated as a therapeutic option in ALS. The safety and efficacy, delivery strategies, the periodicity of the transplant, and the capture by the cells of interest should be rigorously analyzed experimentally. Thus, if its effectiveness is proven in animal models, hopefully, clinical trials can be performed soon.

#### **6. Conclusions**

The DNA repair deficiency is a hallmark that combines at the pathological level with mitochondrial dysfunction, which highlights the special dependence of neurons on genomic integrity and metabolic stability. This highlights what we called the pathological tropism of the nervous system. At this point, it is clear that whatever the etiology behind ALS and motor neuron degeneration, mitochondrial dysfunction is present in all fALS and sporadic ALS patients. The fact that mitochondrial damage appears in the presymptomatic stage of the disease raises an opportunity for targeting mitochondria and hopefully makes disease progression slower or even stops it. Mitochondrial transplantation represents a promising avenue for the following years, as well as NAD+ repletion therapy and mitophagy-inducing agents.

#### **Acknowledgements**

We acknowledge the support from the University of Costa Rica (project 111- B2–372). We also thank Gabriel Jiménez Huezo for the graphic design of the picture in this chapter.

**69**

*Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS*

*DOI: http://dx.doi.org/10.5772/intechopen.90217*

The authors declare no conflict of interests.

ALS amyotrophic lateral sclerosis

DDR DNA damage response DSBs double-strand breaks

ROS reactive oxygen species RNS reactive nitrogen species NHEJ nonhomologous end joining

BER base excision repair

SSBs single-strand breaks HR homologous recombination

mtDNA mitochondrial DNA

53BP1 p53-binding protein 1

alpha

BRCA1 breast cancer 1

NMN nicotinamide mononucleotide RNF168 RING-type E3 ubiquitin transferase

AMPK MP-activated protein kinase FOXOs forkhead box O proteins

FTD frontotemporal dementia ER endoplasmic reticulum GSK-3b glycogen synthase kinase-3b

KIF5A kinesin heavy chain isoform 5A ATM ataxia telangiectasia mutated

UPRmt mitochondrial unfolded protein response

MFN2 mitofusin 2

FUS fused in sarcoma HDAC1 histone deacetylase 1

SETX senataxin

SIRT Sirtuin 1

fALS familial amyotrophic lateral sclerosis

NAD+ nicotinamide adenine dinucleotide

HiPSCs human-induced pluripotent stem cells PARP1 poly (ADP-ribose) polymerase 1 XRCC1 X-ray repair cross-complementing 1 TDP-43 TAR DNA-binding protein 43 SOD1 superoxide dismutase 1

C9orf72 chromosome 9 open reading frame 72

GDAP1 ganglioside-induced differentiation-associated protein 1

PGC-1α peroxisome proliferator-activated receptor gamma coactivator-1

iNAMPT intracellular nicotinamide phosphoribosyltransferase

MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

**Conflict of interest**

**Nomenclature**

*Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS DOI: http://dx.doi.org/10.5772/intechopen.90217*

#### **Conflict of interest**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

models, hopefully, clinical trials can be performed soon.

The DNA repair deficiency is a hallmark that combines at the pathological level with mitochondrial dysfunction, which highlights the special dependence of neurons on genomic integrity and metabolic stability. This highlights what we called the pathological tropism of the nervous system. At this point, it is clear that whatever the etiology behind ALS and motor neuron degeneration, mitochondrial dysfunction is present in all fALS and sporadic ALS patients. The fact that mitochondrial damage appears in the presymptomatic stage of the disease raises an opportunity for targeting mitochondria and hopefully makes disease progression slower or even stops it. Mitochondrial transplantation represents a promising avenue for the following years, as well as NAD+ repletion therapy and mitophagy-inducing agents.

We acknowledge the support from the University of Costa Rica (project 111- B2–372). We also thank Gabriel Jiménez Huezo for the graphic design of the picture

therapeutic options and the limitations of current genetic editing techniques make it difficult to have a clear path for future research. Due to the failure in targeting specific aspects of mitochondria, mitochondrial transplantation presents a new paradigm of therapeutic intervention that may benefit neuronal survival and regeneration in ALS and similar diseases. The clinical application of mitochondrial transplantation was extensively reviewed by Chang and colleagues already [98]. The importance of this approach has been demonstrated in different lines of research. For example, it was shown that astrocytes in mice release functional mitochondria that enter neurons and amplify cell survival signals after stroke [99]. Cardiomyocytes share several similitudes with neurons, including the postmitotic state, the high transcription rate, the similar metabolic profile, and the underlying dependence on mitochondrial homeostasis. Following myocardial ischemia and reperfusion (IR), several alterations have been demonstrated in mitochondrial structure and function. Mitochondrial transplantation has been proposed as a strong opportunity for the treatment of IR [100]. Masuzawa and colleagues showed in a model of ischemia-reperfusion injury that transplantation of autologously derived mitochondria immediately prior to reperfusion ameliorated the damage. By using New Zealand white rabbits, they demonstrated a complete recovery and showed that the transplanted mitochondria enhanced oxygen consumption, highenergy phosphate synthesis, and enhanced post-infarct cardiac function [101]. In a model of Parkinson's disease induced by the respiratory chain inhibitor MPTP, Shi and colleagues demonstrated in mice that intravenous injection of mitochondria prevented the progression of the disease. Specifically, the authors reported increased activity of the electron transport chain, decreased ROS levels, and prevention of cell apoptosis and necrosis [102]. Other approaches have been addressed following this so-called mitochondrial medicine approach in ALS, such as ketogenic diet, antioxidants, and metabolic targeting drugs [103]. Based on the evidence provided until this point, mitochondrial transplantation should be validated as a therapeutic option in ALS. The safety and efficacy, delivery strategies, the periodicity of the transplant, and the capture by the cells of interest should be rigorously analyzed experimentally. Thus, if its effectiveness is proven in animal

**68**

**6. Conclusions**

**Acknowledgements**

in this chapter.

The authors declare no conflict of interests.

#### **Nomenclature**


#### **Author details**

Luis Bermúdez-Guzmán1 and Alejandro Leal1,2\*

1 Section of Genetics and Biotechnology, School of Biology, Universidad de Costa Rica, San José, Costa Rica

2 Neuroscience Research Center, Universidad de Costa Rica, San José, Costa Rica

\*Address all correspondence to: alejandro.leal@ucr.ac.cr

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**71**

*Pathological Interaction between DNA Repair and Mitochondrial Dysfunction in ALS*

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**70**

**Author details**

Luis Bermúdez-Guzmán1

Rica, San José, Costa Rica

and Alejandro Leal1,2\*

\*Address all correspondence to: alejandro.leal@ucr.ac.cr

provided the original work is properly cited.

1 Section of Genetics and Biotechnology, School of Biology, Universidad de Costa

2 Neuroscience Research Center, Universidad de Costa Rica, San José, Costa Rica

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

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Section 2

Emerging Etiopathology and

Implications

**79**

### Section 2
