Novel Therapeutics and Challenges

**115**

**Chapter 7**

**Abstract**

noncoding RNA

**1. Introduction**

intervention.

**Highlights**

RNA Metabolism and

Lateral Sclerosis

*and Cristina Cereda*

Therapeutics in Amyotrophic

*Orietta Pansarasa, Stella Gagliardi, Daisy Sproviero* 

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder characterized by the selective death of upper and lowers motor neurons in spinal cord, brain stem, and motor cortex, which leads to paralysis and death within 2–3 years of onset. Deeply sequencing technologies, to simultaneously analyze the transcriptional expression of thousands of genes, offered new possibilities to focus on ALS pathogenesis and, most notably, to find new potential targets for novel treatments. The present book chapter illustrates recent advances in transcriptomic studies in animal models and human samples and in new molecular targets related to ALS pathogenesis and disease progression. Additionally, new insights into the involvement of altered transcriptional profiles of noncoding RNAs (microRNA and lncRNA) and ALS-associated ribosomal binding proteins have been investigated, to understand the functional consequences of extensive RNA dysregulation in ALS. Attention has been also turned on how transcrip-

tome alterations could highlight new molecular targets for drug development.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder (ND) that affects the human motor system, that is, the lower and upper

• Understanding RNA-binding protein functions and identifying target RNA regulatory networks is

• miRNAs are strongly linked to the development of ALS and are indicated as new potential biomarkers.

• lncRNAs have been recently indicated to play important roles in CNS in health and disease such as ALS.

• miRNA-based therapeutics as well as deregulated AS are considered important areas for therapeutic

**Keywords:** ALS, RNA metabolism, transcriptomics, gene expression,

• Aberrant RNA metabolism is one of the major contributors to ALS pathogenesis.

crucial to deepen ALS knowledge and to develop new therapeutics.

#### **Chapter 7**

## RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis

*Orietta Pansarasa, Stella Gagliardi, Daisy Sproviero and Cristina Cereda*

#### **Abstract**

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disorder characterized by the selective death of upper and lowers motor neurons in spinal cord, brain stem, and motor cortex, which leads to paralysis and death within 2–3 years of onset. Deeply sequencing technologies, to simultaneously analyze the transcriptional expression of thousands of genes, offered new possibilities to focus on ALS pathogenesis and, most notably, to find new potential targets for novel treatments. The present book chapter illustrates recent advances in transcriptomic studies in animal models and human samples and in new molecular targets related to ALS pathogenesis and disease progression. Additionally, new insights into the involvement of altered transcriptional profiles of noncoding RNAs (microRNA and lncRNA) and ALS-associated ribosomal binding proteins have been investigated, to understand the functional consequences of extensive RNA dysregulation in ALS. Attention has been also turned on how transcriptome alterations could highlight new molecular targets for drug development.

**Keywords:** ALS, RNA metabolism, transcriptomics, gene expression, noncoding RNA

#### **1. Introduction**

#### **Highlights**


Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder (ND) that affects the human motor system, that is, the lower and upper

motor neurons (MNs). Among the symptoms of ALS, there are progressive muscle weakness and paralysis, swallowing difficulties, and breathing impairment due to respiratory muscle weakness that finally causes death, within 2–5 years following clinical diagnosis [1]. Now, also extramotor systems are involved in ALS, thus providing new insight into the pathogenesis of the disease. So far, no effective therapy is available for ALS: Rilutek (riluzole) and Radicava (edaravone) are the only two drugs approved by the Food and Drug Administration for ALS treatment. Unfortunately their effect in slowing disease progression is very modest [2]. The majority of ALS cases, named as sporadic (sALS), has no a family history; a fraction of cases (about 5–10%) are considered familial (fALS) [3], because of mutations in genes involved in a wide range of cellular functions. 60–70% of fALS and 10% of sporadic ALS (sALS) cases can be ascribed to mutations in SOD1, TARDBP, FUS, VCP, C9ORF72, and OPTN [4]; further rare genetic variants have also been identified, MATR3, HNRNPA1, HNRNPA2/B1, EWSR1, TAF15, ANG, UBQLN2, VAPB, TBK1, SQSTM1, PFN1, TUBA4A, KIF5A, ANXA11, and CHCHD10 [5]. Although an in-depth understanding of the mechanisms underlying ALS has yet to be reached, a growing interest was addressed to the impairment of RNA metabolism as one of the major contributor to ALS pathogenesis. This concept is reinforced by the discovery of genetic mutations in FUS and in TARDBP genes coding for RNA binding proteins (RBPs), which play a multifaceted role in transcription and in maintaining RNA metabolism. Recent studies have reported that a substantial portion of the genome is actively transcribed as noncoding RNA molecules. These noncoding RNAs are fundamental key actors in the regulation of biological processes and function as a "fine switch" of gene expression. It is now recognized that dysregulations in the noncoding RNAs gene expression is a putative mechanism in several neurological disorders, including ALS. Moreover, noncoding RNAs are emerging as new potential biomarkers contributing to an early disease diagnosis and treatment follow-up. To date, miRNA have been one the main focus of most ALS studies. miRNAs are differentially expressed in several tissues (CSF, plasma and serum) in ALS patients compared to healthy controls.

In this chapter, we will focus on the involvement of altered transcriptional profiles of microRNAs (miRNAs) and long noncoding RNA (lncRNA) as well as on ALS-related RNA binding proteins. We also review biomarkers and potential therapeutic strategies based on the manipulation of noncoding RNAs.

#### **2. Dysfunctions in RNA metabolism and RNA-binding protein**

It is broadly recognized that an aberrant RNA metabolism may contribute to RNA toxicity, which is due to the accumulation of toxic RNAs and to the dysfunction of RBPs [6].

Messenger RNAs (mRNAs) are subjected to several processing steps including splicing, polyadenylation, editing, transport, translation, and turnover. All these processes are extremely dynamic and require the involvement of RBPs to coordinate both co- and posttranscriptional processing of transcripts. Understanding RBPs functions and identifying their target RNA regulatory networks are crucial to deepen the knowledge in NDs and to promptly develop new therapeutics.

Nussbacher and colleagues by a genome-wide approach, have shed a new light on how RBPs may affect the fate of their targets [7]. Considering the great impact of RBPs on the expression, splicing, and translation of multiple RNA targets, also little changes in their expression and/or activity have amplified effects. Moreover, an altered interaction between RBPs and their targets can induce serious pathological phenotypes, even if the exact mechanism is not clear. Briefly, we focus on RBPs,

**117**

homeostasis in ALS MNs.

*RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis*

TARDBP and FUS, and SOD1 and C9orf72 to highlight recent progresses on their

**TDP-43** is a heterogeneous nuclear RBP of 414 amino acids that contains two RNA recognition motifs (RRM1-2), a glycine rich domain in the C-terminus and nuclear localization and export signals (NLS and NES) [8, 9]. TDP-43 is crucial in RNA processing, that is, RNA splicing, transcription, transport, stability, as well as miRNA production [10]. TDP-43 binds to more than 6000 RNA targets in the brain [11, 12]. TDP-43 binds to mRNA and regulates the expression of other proteins: FUS, Tau, ATXN2 CHMP2B, VAPB, and progranulin, all involved in ALS and in other NDs [12, 13]. Polymenidou and colleagues using an RNA-seq approach, demonstrated the involvement of TDP-43 in the regulation of the expression of 239 mRNAs, many of those encoding synaptic proteins including neurexin NRXN1-3, neuroligin NLGN1-2, Homer2, microtubule-associated protein 1B (MAP1B), GABA receptors subunits (GABRA2, GABRA3), AMPA receptor subunits (GRIA3, GRIA4), syntaxin 1B, and calcium channels [11, 14–17]. Together these data suggest the involvement of TDP-43 in neuronal morphology, synaptic transmission, and neuronal plasticity likely through the regulation of RNA processing of synaptic genes [14]. TDP-43 is also a splicing regulator which decreases its own expression level by binding to the 3′-untranslated (UTR) region of its own pre-mRNA [18]. Moreover, its depletion or overexpression can influence the alternative splicing of specific targets genes, which are altered in ALS [11, 13, 19]. In 2012, Kawahara and Mieda-Sato also demonstrated the involvement of TDP-43 in miRNA biogenesis. TDP-43 helps the production of the precursor miRNAs (pre-miRNAs) through the interaction of the Drosha complex and the binding to the primary miRNAs (pri-miRNAs) [20]. An increased expression of miR-633 and a decreased expression of the let-7b miRNA have been observed when TDP-43 is downregulated [21]. Moreover, TDP-43 binds to lncRNAs, including the nuclearenriched autosomal transcript 1 (NEAT1) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) [13]. Until now, the exact role of this interaction is unclear; however both NEAT1 and MALAT1 levels are enhanced in patients with frontotemporal lobar degeneration (FTLD) and ALS [12, 22]. Nishimoto and coauthors identified paraspeckles, that is, membraneless nuclear bodies, with high levels of NEAT1 and TDP-43 in MNs of patients in the early stage of the disease [22], thus interfering with TDP-43-mediated RNA processing and disrupting RNA

**FUS** is an RBP of 526 amino acids mainly located in the nuclei. It is composed by an RNA recognition motif, a SYGQ (serine, tyrosine, glycine, and glutamine) rich region, several RGG (arginine, glycine, and glycine)-repeat regions, a C2C2 zinc finger motif, and a nuclear localization signal (NLS) [23]. Similar to TDP-43, FUS has a key role in RNA processing. It is involved in transcriptional regulation, mRNA splicing, and miRNA production. FUS co-modulates certain transcription factors, including NF-kB, SPI1, and Runbox transcription factor (RUNX) [24, 25]. Genome-wide approaches have evidenced more than 5000 human RNA targets for FUS [26]. Considering that FUS is part of the hnRNP complex, it is crucial for the splicing mechanism [23], and it may affect the splicing mechanism of more than 900 mRNAs [26]. Among these, FUS may regulate the alternative splicing of genes related to cytoskeletal organization, axonal growth, and guidance such as the microtubule-associated protein tau (MAPT) [27], Netrin G1 (NTNG1) [28], neuronal cell adhesion molecule (NRCAM), and the actin-binding LIM (ABLIM1) [29]. Like TDP-43, FUS also binds to different mRNAs of ALS-related genes, VCP, VAPB, ubiquilin-2, and OPTN, thus modulating their expression [12, 26]. Furthermore, FUS is involved in the biogenesis of miRNA by recruiting Drosha to pri-miRNAs at their transcription sites and supports the biogenesis of a subset of

*DOI: http://dx.doi.org/10.5772/intechopen.90704*

involvement in RNA dysregulation.

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

motor neurons (MNs). Among the symptoms of ALS, there are progressive muscle weakness and paralysis, swallowing difficulties, and breathing impairment due to respiratory muscle weakness that finally causes death, within 2–5 years following clinical diagnosis [1]. Now, also extramotor systems are involved in ALS, thus providing new insight into the pathogenesis of the disease. So far, no effective therapy is available for ALS: Rilutek (riluzole) and Radicava (edaravone) are the only two drugs approved by the Food and Drug Administration for ALS treatment. Unfortunately their effect in slowing disease progression is very modest [2]. The majority of ALS cases, named as sporadic (sALS), has no a family history; a fraction of cases (about 5–10%) are considered familial (fALS) [3], because of mutations in genes involved in a wide range of cellular functions. 60–70% of fALS and 10% of sporadic ALS (sALS) cases can be ascribed to mutations in SOD1, TARDBP, FUS, VCP, C9ORF72, and OPTN [4]; further rare genetic variants have also been identified, MATR3, HNRNPA1, HNRNPA2/B1, EWSR1, TAF15, ANG, UBQLN2, VAPB, TBK1, SQSTM1, PFN1, TUBA4A, KIF5A, ANXA11, and CHCHD10 [5]. Although an in-depth understanding of the mechanisms underlying ALS has yet to be reached, a growing interest was addressed to the impairment of RNA metabolism as one of the major contributor to ALS pathogenesis. This concept is reinforced by the discovery of genetic mutations in FUS and in TARDBP genes coding for RNA binding proteins (RBPs), which play a multifaceted role in transcription and in maintaining RNA metabolism. Recent studies have reported that a substantial portion of the genome is actively transcribed as noncoding RNA molecules. These noncoding RNAs are fundamental key actors in the regulation of biological processes and function as a "fine switch" of gene expression. It is now recognized that dysregulations in the noncoding RNAs gene expression is a putative mechanism in several neurological disorders, including ALS. Moreover, noncoding RNAs are emerging as new potential biomarkers contributing to an early disease diagnosis and treatment follow-up. To date, miRNA have been one the main focus of most ALS studies. miRNAs are differentially expressed in several tissues (CSF, plasma and serum) in ALS patients

In this chapter, we will focus on the involvement of altered transcriptional profiles of microRNAs (miRNAs) and long noncoding RNA (lncRNA) as well as on ALS-related RNA binding proteins. We also review biomarkers and potential

It is broadly recognized that an aberrant RNA metabolism may contribute to RNA toxicity, which is due to the accumulation of toxic RNAs and to the dysfunc-

Messenger RNAs (mRNAs) are subjected to several processing steps including splicing, polyadenylation, editing, transport, translation, and turnover. All these processes are extremely dynamic and require the involvement of RBPs to coordinate both co- and posttranscriptional processing of transcripts. Understanding RBPs functions and identifying their target RNA regulatory networks are crucial to deepen the knowledge in NDs and to promptly develop new therapeutics.

Nussbacher and colleagues by a genome-wide approach, have shed a new light on how RBPs may affect the fate of their targets [7]. Considering the great impact of RBPs on the expression, splicing, and translation of multiple RNA targets, also little changes in their expression and/or activity have amplified effects. Moreover, an altered interaction between RBPs and their targets can induce serious pathological phenotypes, even if the exact mechanism is not clear. Briefly, we focus on RBPs,

therapeutic strategies based on the manipulation of noncoding RNAs.

**2. Dysfunctions in RNA metabolism and RNA-binding protein**

**116**

compared to healthy controls.

tion of RBPs [6].

TARDBP and FUS, and SOD1 and C9orf72 to highlight recent progresses on their involvement in RNA dysregulation.

**TDP-43** is a heterogeneous nuclear RBP of 414 amino acids that contains two RNA recognition motifs (RRM1-2), a glycine rich domain in the C-terminus and nuclear localization and export signals (NLS and NES) [8, 9]. TDP-43 is crucial in RNA processing, that is, RNA splicing, transcription, transport, stability, as well as miRNA production [10]. TDP-43 binds to more than 6000 RNA targets in the brain [11, 12]. TDP-43 binds to mRNA and regulates the expression of other proteins: FUS, Tau, ATXN2 CHMP2B, VAPB, and progranulin, all involved in ALS and in other NDs [12, 13]. Polymenidou and colleagues using an RNA-seq approach, demonstrated the involvement of TDP-43 in the regulation of the expression of 239 mRNAs, many of those encoding synaptic proteins including neurexin NRXN1-3, neuroligin NLGN1-2, Homer2, microtubule-associated protein 1B (MAP1B), GABA receptors subunits (GABRA2, GABRA3), AMPA receptor subunits (GRIA3, GRIA4), syntaxin 1B, and calcium channels [11, 14–17]. Together these data suggest the involvement of TDP-43 in neuronal morphology, synaptic transmission, and neuronal plasticity likely through the regulation of RNA processing of synaptic genes [14]. TDP-43 is also a splicing regulator which decreases its own expression level by binding to the 3′-untranslated (UTR) region of its own pre-mRNA [18]. Moreover, its depletion or overexpression can influence the alternative splicing of specific targets genes, which are altered in ALS [11, 13, 19]. In 2012, Kawahara and Mieda-Sato also demonstrated the involvement of TDP-43 in miRNA biogenesis. TDP-43 helps the production of the precursor miRNAs (pre-miRNAs) through the interaction of the Drosha complex and the binding to the primary miRNAs (pri-miRNAs) [20]. An increased expression of miR-633 and a decreased expression of the let-7b miRNA have been observed when TDP-43 is downregulated [21]. Moreover, TDP-43 binds to lncRNAs, including the nuclearenriched autosomal transcript 1 (NEAT1) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) [13]. Until now, the exact role of this interaction is unclear; however both NEAT1 and MALAT1 levels are enhanced in patients with frontotemporal lobar degeneration (FTLD) and ALS [12, 22]. Nishimoto and coauthors identified paraspeckles, that is, membraneless nuclear bodies, with high levels of NEAT1 and TDP-43 in MNs of patients in the early stage of the disease [22], thus interfering with TDP-43-mediated RNA processing and disrupting RNA homeostasis in ALS MNs.

**FUS** is an RBP of 526 amino acids mainly located in the nuclei. It is composed by an RNA recognition motif, a SYGQ (serine, tyrosine, glycine, and glutamine) rich region, several RGG (arginine, glycine, and glycine)-repeat regions, a C2C2 zinc finger motif, and a nuclear localization signal (NLS) [23]. Similar to TDP-43, FUS has a key role in RNA processing. It is involved in transcriptional regulation, mRNA splicing, and miRNA production. FUS co-modulates certain transcription factors, including NF-kB, SPI1, and Runbox transcription factor (RUNX) [24, 25]. Genome-wide approaches have evidenced more than 5000 human RNA targets for FUS [26]. Considering that FUS is part of the hnRNP complex, it is crucial for the splicing mechanism [23], and it may affect the splicing mechanism of more than 900 mRNAs [26]. Among these, FUS may regulate the alternative splicing of genes related to cytoskeletal organization, axonal growth, and guidance such as the microtubule-associated protein tau (MAPT) [27], Netrin G1 (NTNG1) [28], neuronal cell adhesion molecule (NRCAM), and the actin-binding LIM (ABLIM1) [29]. Like TDP-43, FUS also binds to different mRNAs of ALS-related genes, VCP, VAPB, ubiquilin-2, and OPTN, thus modulating their expression [12, 26]. Furthermore, FUS is involved in the biogenesis of miRNA by recruiting Drosha to pri-miRNAs at their transcription sites and supports the biogenesis of a subset of

miRNAs [30]. However, if FUS directly regulates the function of mature miRNAs remains to be understand. Finally, FUS is crucial for the regulation of NEAT 1 levels and paraspeckle formation. FUS nuclear deficiency, its loss of nuclear function, as well as its aggregation might cause sequestration of paraspeckle components into pathological inclusions. Thus, the interaction between FUS and NEAT 1 is involved in the development of neuronal dysfunction in ALS [31].

**SOD1** is not an RBP; however, several authors demonstrated that mutant SOD1 has a role in RNA metabolism regulation [32, 33]. These authors reported that mutant SOD1 can bind mRNA species, that is, vascular endothelial growth factor (VEGF) and neurofilament light chain (NFL), and alter their expression, stabilization, and function [32, 33]. Mutant SOD1, by the direct bind to the 3' UTR of VEGF mRNA, promotes the sequestration of other RBPs such as TIA-1-related protein (TIAR) and Hu antigen R (HuR) into insoluble aggregates. This, in turn, determines the impairment of HuR function and interferes with the HuR neuroprotective effect during stress responses [32]. Chen and colleagues further demonstrated that, through the modification of neurofilament (NF) stoichiometry, mutant SOD1 destabilizes NFL mRNA. Consequently, NFs aggregate in MNs and are considered a hallmark of ALS disease [33]. NFL mRNA stability could also be regulated by a common interaction between SOD1 and TDP-43 [34]. The exact mechanism is not completely understood; however, it is hypothesized that mutant SOD1 removes TDP-43 from the NFL mRNA, thus disturbing NFL mRNA metabolism and promoting the formation of aggregates.

In 2011, the large GGGGCC hexanucleotide repeat expansion of **C9orf72** has been recognized as a new cause of ALS [35, 36], accounting for about 50% of fALS and 5–10% of sALS [37]. The C9orf72 repeat expansion is transcribed in both the sense and antisense directions and causes the accumulations of repeat containing RNA foci [38]. RNA foci formation allows the recruitment of RBPs and alter RNA metabolism [39]. Mori and co-authors observed that RNA foci can sequester the RBP hnRNP-A3 and can suppress its RNA processing function. Notably, RNA foci are also able to sequester nuclear proteins such as TDP-43 and FUS, thus affecting the expression of their RNA targets, mainly involved in RNA metabolism, stress response, and nuclear transport. Moreover, RNA-Seq data unveil new candidate genes, that is, genes involved in synaptic transmission, protein targeting, and cell–cell signaling; however future validation are required [40]. Moreover, poly-PR and poly-GR can alter the splicing patterns of specific RNAs. The poly-PR causes the exon skipping in RAN and PTX3 RNA [41]. Finally, C9orf72 repeats can interfere with transcription or splicing of C9orf72 transcripts and can disrupt the C9orf72 promoter activity [42, 43].

#### **3. Dysfunctions in RNA metabolism and miRNA**

miRNAs are short noncoding RNAs, approximately 18–25 nucleotides long, that play a key role in the regulation of gene expression in many fundamental cellular processes and, posttranscriptionally, at the translation levels of target mRNA transcripts [44, 45]. A high number of protein-coding genes have been demonstrated to be regulated by miRNA through base-pairing interactions within the UTR of the targeted mRNAs [46, 47]. Alongside their gene silencing functions, miRNAs can also induce upregulation of their targets [48]. An accurate regulatory pathway is fundamental to control and maintain the physiological processes of cells. However, when abnormalities occur, as in diseases, a complex dysregulation of the miRNA expression takes place. In this paragraph, we will focus on miRNAs which are linked to the development of ALS and miRNA with a potential role as biomarkers.

One of the most interesting miRNAs involved in ALS is **miRNA206**. miRNA206 is skeletal muscle-specific, regulates myogenesis, and promotes the formation of

**119**

*RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis*

new neuromuscular junctions [49, 50]. Generally, the miRNA206 is overexpressed in muscle fibers and in serum of ALS patients [50, 51]. Pegoraro and co-authors associated the high levels of miRNA206 to the remodeling of the muscle, that is, atrophy, hypertrophy, and/or reinnervation of some fibers [51]. de Andrade et al. evidenced that miRNA206 increases early in the disease course and then decreases, thus suggesting its role during muscle loss [50]. miRNAs might also have a protective role in ALS; higher levels of miRNA206 were indeed observed in slow progressors, that is, in long-term ALS patients [52]. Thanks to the possibility to detect miRNA206 in accessible samples like serum and the correlation between miRNA206 levels and disease characteristics, miRNA206 could be indicated as a potential biomarker for ALS [53]. Other three miRNAs, miRNA133a/b, miRNA 1, and miRNA 27a, are indicated as muscle-specific. **miRNA133** is higher in serum and muscle of ALS patients, and it is also higher in spinal ALS compared to bulbar ALS [51, 54]. An upregulation of **miRNA27a** was observed in CD14+ CD16− monocytes, in muscle fibers, and in CSF of ALS patients [51, 55], while a downregulation was reported in serum samples [54]. **miRNA338-3p** is another miRNA frequently upregulated. An increase of more than twofold was reported in leukocytes of sALS patients [56, 57]. Moreover, De Felice and co-authors showed an increase in miRNA338-3p in serum, CSF, and spinal cord of sALS patients. The evidence that it can be easily obtainable in body fluids suggested the possibility that miRNA338-3p might be a suitable biomarker for ALS. The inflammatory **miRNA146a** is overexpressed in CD14+ CD16− monocytes, CSF, spinal cord, and muscle fibers [55, 58]. miRNA146a can also interact with NFL mRNA 3′UTR, according to low mRNA levels observed in spinal neurons of sALS [58]. Tasca and co-authors, on the other hand, identified a reduction of miRNA146a in serum of ALS patients, both bulbar and spinal [54]. Tasca et al. and Pegoraro et al. found a downregulation in serum, muscle fibers, and leukocytes of sALS of the inflammatory **miRNA149/149\***. Also **miRNA221** seems to contribute to ALS development by acting on muscle growth and/or atrophy and inflammation, through the NF-kB pathway [53, 54]. **miRNA155** was evaluated in CD14+ CD16− monocytes [55] and spinal cords of ALS patients, and it increases both in fALS and sALS [59]. Two other miRNAs targeting TGFβ1, **miRNA21,** and **miRNA106b** were upregulated in CD14+ CD16− monocytes [55], and, at least for miRNA21, an upregulation was reported in muscle samples [50] in ALS patients even if its role in the pathology has yet to be fully explained. The same authors identified an inverse correlation between **miRNA424** levels and disease progression, thus suggesting miRNA 424 as a potential biomarker [50] (**Figure 1**). The ALS genes, TDP-43 and FUS, play a role in miRNA biogenesis [60]. Mutations in TARDBP result in differential expression of **miRNA9**, **miRNA132**, **miRNA143**, **miRNA558** [61], and **let7** families [53], and differences between CSF and serum levels were observed. For instance, miRNA9, a brain-specific miRNA highly conserved during evolution is 2–3 times more elevated in CSF with respect to serum [62]. Differences are reported also for the presence of mutations. In induced pluripotent stem cell (iPSC)-derived neuron obtained from patients carrying the TARDBP p.A90V and the M337 V mutation, miRNA9 and pri-miRNA9-2 levels were lower when compared to controls [61]). Likewise, miRNA9 also decreases in lumbar motor neurons of sALS and SOD1 A4V mutated patients [63]. Moreover, a correlation between these miRNAs and disease duration and site of onset was identified. Specifically, **miRNA 143-3p** levels increase in later-collected samples, and the increase becomes significant in lower limb-onset patients [53] (**Figure 2**). Morlando and co-authors reported that, upon FUS depletion, the expression of **miRNA9**, **miRNA132**, **miRNA143**, **miRNA125,** and **miRNA192** is altered [30]. The involvement of these miRNAs in motor neuron development and maintenance, axonal growth, and synaptic transmission accounts for their contribution to the ALS

*DOI: http://dx.doi.org/10.5772/intechopen.90704*

#### *RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.90704*

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

in the development of neuronal dysfunction in ALS [31].

**3. Dysfunctions in RNA metabolism and miRNA**

miRNAs are short noncoding RNAs, approximately 18–25 nucleotides long, that play a key role in the regulation of gene expression in many fundamental cellular processes and, posttranscriptionally, at the translation levels of target mRNA transcripts [44, 45]. A high number of protein-coding genes have been demonstrated to be regulated by miRNA through base-pairing interactions within the UTR of the targeted mRNAs [46, 47]. Alongside their gene silencing functions, miRNAs can also induce upregulation of their targets [48]. An accurate regulatory pathway is fundamental to control and maintain the physiological processes of cells. However, when abnormalities occur, as in diseases, a complex dysregulation of the miRNA expression takes place. In this paragraph, we will focus on miRNAs which are linked

One of the most interesting miRNAs involved in ALS is **miRNA206**. miRNA206 is skeletal muscle-specific, regulates myogenesis, and promotes the formation of

to the development of ALS and miRNA with a potential role as biomarkers.

miRNAs [30]. However, if FUS directly regulates the function of mature miRNAs remains to be understand. Finally, FUS is crucial for the regulation of NEAT 1 levels and paraspeckle formation. FUS nuclear deficiency, its loss of nuclear function, as well as its aggregation might cause sequestration of paraspeckle components into pathological inclusions. Thus, the interaction between FUS and NEAT 1 is involved

**SOD1** is not an RBP; however, several authors demonstrated that mutant SOD1 has a role in RNA metabolism regulation [32, 33]. These authors reported that mutant SOD1 can bind mRNA species, that is, vascular endothelial growth factor (VEGF) and neurofilament light chain (NFL), and alter their expression, stabilization, and function [32, 33]. Mutant SOD1, by the direct bind to the 3' UTR of VEGF mRNA, promotes the sequestration of other RBPs such as TIA-1-related protein (TIAR) and Hu antigen R (HuR) into insoluble aggregates. This, in turn, determines the impairment of HuR function and interferes with the HuR neuroprotective effect during stress responses [32]. Chen and colleagues further demonstrated that, through the modification of neurofilament (NF) stoichiometry, mutant SOD1 destabilizes NFL mRNA. Consequently, NFs aggregate in MNs and are considered a hallmark of ALS disease [33]. NFL mRNA stability could also be regulated by a common interaction between SOD1 and TDP-43 [34]. The exact mechanism is not completely understood; however, it is hypothesized that mutant SOD1 removes TDP-43 from the NFL mRNA, thus disturbing NFL mRNA metabolism and promoting the formation of aggregates. In 2011, the large GGGGCC hexanucleotide repeat expansion of **C9orf72** has been recognized as a new cause of ALS [35, 36], accounting for about 50% of fALS and 5–10% of sALS [37]. The C9orf72 repeat expansion is transcribed in both the sense and antisense directions and causes the accumulations of repeat containing RNA foci [38]. RNA foci formation allows the recruitment of RBPs and alter RNA metabolism [39]. Mori and co-authors observed that RNA foci can sequester the RBP hnRNP-A3 and can suppress its RNA processing function. Notably, RNA foci are also able to sequester nuclear proteins such as TDP-43 and FUS, thus affecting the expression of their RNA targets, mainly involved in RNA metabolism, stress response, and nuclear transport. Moreover, RNA-Seq data unveil new candidate genes, that is, genes involved in synaptic transmission, protein targeting, and cell–cell signaling; however future validation are required [40]. Moreover, poly-PR and poly-GR can alter the splicing patterns of specific RNAs. The poly-PR causes the exon skipping in RAN and PTX3 RNA [41]. Finally, C9orf72 repeats can interfere with transcription or splicing of C9orf72 transcripts and can disrupt the C9orf72 promoter activity [42, 43].

**118**

new neuromuscular junctions [49, 50]. Generally, the miRNA206 is overexpressed in muscle fibers and in serum of ALS patients [50, 51]. Pegoraro and co-authors associated the high levels of miRNA206 to the remodeling of the muscle, that is, atrophy, hypertrophy, and/or reinnervation of some fibers [51]. de Andrade et al. evidenced that miRNA206 increases early in the disease course and then decreases, thus suggesting its role during muscle loss [50]. miRNAs might also have a protective role in ALS; higher levels of miRNA206 were indeed observed in slow progressors, that is, in long-term ALS patients [52]. Thanks to the possibility to detect miRNA206 in accessible samples like serum and the correlation between miRNA206 levels and disease characteristics, miRNA206 could be indicated as a potential biomarker for ALS [53]. Other three miRNAs, miRNA133a/b, miRNA 1, and miRNA 27a, are indicated as muscle-specific. **miRNA133** is higher in serum and muscle of ALS patients, and it is also higher in spinal ALS compared to bulbar ALS [51, 54]. An upregulation of **miRNA27a** was observed in CD14+ CD16− monocytes, in muscle fibers, and in CSF of ALS patients [51, 55], while a downregulation was reported in serum samples [54]. **miRNA338-3p** is another miRNA frequently upregulated. An increase of more than twofold was reported in leukocytes of sALS patients [56, 57]. Moreover, De Felice and co-authors showed an increase in miRNA338-3p in serum, CSF, and spinal cord of sALS patients. The evidence that it can be easily obtainable in body fluids suggested the possibility that miRNA338-3p might be a suitable biomarker for ALS. The inflammatory **miRNA146a** is overexpressed in CD14+ CD16− monocytes, CSF, spinal cord, and muscle fibers [55, 58]. miRNA146a can also interact with NFL mRNA 3′UTR, according to low mRNA levels observed in spinal neurons of sALS [58]. Tasca and co-authors, on the other hand, identified a reduction of miRNA146a in serum of ALS patients, both bulbar and spinal [54]. Tasca et al. and Pegoraro et al. found a downregulation in serum, muscle fibers, and leukocytes of sALS of the inflammatory **miRNA149/149\***. Also **miRNA221** seems to contribute to ALS development by acting on muscle growth and/or atrophy and inflammation, through the NF-kB pathway [53, 54]. **miRNA155** was evaluated in CD14+ CD16− monocytes [55] and spinal cords of ALS patients, and it increases both in fALS and sALS [59]. Two other miRNAs targeting TGFβ1, **miRNA21,** and **miRNA106b** were upregulated in CD14+ CD16− monocytes [55], and, at least for miRNA21, an upregulation was reported in muscle samples [50] in ALS patients even if its role in the pathology has yet to be fully explained. The same authors identified an inverse correlation between **miRNA424** levels and disease progression, thus suggesting miRNA 424 as a potential biomarker [50] (**Figure 1**). The ALS genes, TDP-43 and FUS, play a role in miRNA biogenesis [60]. Mutations in TARDBP result in differential expression of **miRNA9**, **miRNA132**, **miRNA143**, **miRNA558** [61], and **let7** families [53], and differences between CSF and serum levels were observed. For instance, miRNA9, a brain-specific miRNA highly conserved during evolution is 2–3 times more elevated in CSF with respect to serum [62]. Differences are reported also for the presence of mutations. In induced pluripotent stem cell (iPSC)-derived neuron obtained from patients carrying the TARDBP p.A90V and the M337 V mutation, miRNA9 and pri-miRNA9-2 levels were lower when compared to controls [61]). Likewise, miRNA9 also decreases in lumbar motor neurons of sALS and SOD1 A4V mutated patients [63]. Moreover, a correlation between these miRNAs and disease duration and site of onset was identified. Specifically, **miRNA 143-3p** levels increase in later-collected samples, and the increase becomes significant in lower limb-onset patients [53] (**Figure 2**).

Morlando and co-authors reported that, upon FUS depletion, the expression of **miRNA9**, **miRNA132**, **miRNA143**, **miRNA125,** and **miRNA192** is altered [30]. The involvement of these miRNAs in motor neuron development and maintenance, axonal growth, and synaptic transmission accounts for their contribution to the ALS

#### *Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

#### **Figure 1.**

*Representation of the key miRNAs involved in amyotrophic lateral sclerosis sporadic patients (sALS).*

#### **Figure 2.**

*Representation of the key miRNAs involved in amyotrophic lateral sclerosis mutated patients.*

pathological phenotype [64, 65]. In motor neurons progenitors derived from human ALS iPSCs, Rizzuti et al. observed that **miRNA34a** and **miRNA504**, involved in vesicle regulation and cell survival, were dysregulated [66]. Also **miRNA1825** is downregulated in CNS of sALS and fALS patients, thus inducing depolymerization and degradation of tubulin alpha-4A (TUBA4A), which is encoded by the known ALS gene [67].

Taken together, these studies significantly contribute to evidence the importance of miRNAs, also as biomarkers for ALS. Despite these evidences, several issues need to be addressed mainly on the utility of miRNAs to serve as accurate and fast biomarkers for an early ALS diagnosis.

#### **4. Dysfunctions in RNA metabolism and lncRNA**

Long noncoding RNAs (lncRNAs) are transcripts, greater than 200 nucleotides in length, with no protein-coding potential which are found in sense or antisense

**121**

*RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis*

orientation to protein-coding genes or within intergenic regions. lncRNAs control the gene expression through different mechanisms, that is, epigenetic modulation through chromatin remodeling, activation or repression of transcription, posttranscriptional modifications of mRNA, and regulation of protein activity by acting as scaffold to recruit RBPs and/or drive RBPs to DNA. Moreover, they can compete for and disrupt protein-binding interactions or sponge miRNAs away from their mRNA targets [68]. Recently, lncRNAs have been indicated to play important roles in the CNS in health and disease such as ALS. Nishimoto and colleagues first identify a relation between **NEAT1** and ALS pathogeneses [22]. The NEAT1 gene produces two transcripts, NEAT1\_1 and NEAT1\_2; NEAT1\_2 expression is very low in the adult nervous system, and it is the only one that forms paraspeckles [69]. Specifically, NEAT1 acts as a scaffold for paraspeckles thus enhancing their de novo formation in spinal motor neurons in a cohort of sALS patients [22]. Paraspeckles function through the retention of specific RNAs; the regulation of gene expression by sequestration of transcription factors; and the modulation of miRNA biogenesis and mitochondrial function [70]. Paraspeckles are enriched in pathological proteins for ALS and are indicated as a hallmark of the disease [71]. Moreover, paraspeckle proteins, including TDP-43 and FUS, are related to ALS and FTD. The increase in paraspeckle formation in ALS could be triggered, at least in part, by the nuclear depletion of TDP-43. TDP-43 binds NEAT1, and, in turn, its downregulation stimulates NEAT1\_2 accumulation and paraspeckle association in cultured cells [71]. Regarding FUS, in the spinal cord of FUS mutated ALS patients, Shelkovnikova and co-authors reported the presence of pathological aggregation of NONO, a core paraspeckle protein [31]. This evidence allows to speculate that, considering that FUS and NONO are both required to set up paraspeckles, the formation of paraspeckles is disrupted in FUS mutated ALS patients. Also aberrant nuclear RNA foci formed by the expanded C9ORF72 repeats sequester paraspeckle proteins including TDP-43 [72]. **MALAT1** is abundantly expressed and evolutionarily conserved lncRNA. It is one of the first lncRNAs associated with human disease, and it is involved in alternative splicing, epigenetic modification of gene expression, synapse formation, and myogenesis. In NDs MALAT1 is significantly increased in FTLD patients, where it recruits splicing factors to nuclear speckles and affects phosphorylation of SR proteins37 [13]. Some lncRNA transcripts have been associated to FUS, among these the **lncRNA CCND1** which binds to the FUS consensus sequence GGUG [73]. Data form Wang and colleagues suggested that FUS is a specific repressor of CCND1, which is downregulated in response to DNA damage signals. Until now the lncRNA CCND1 has not been described in relation to ALS; but taken together, these observations point out that this lncRNA could be, at least partly, responsible in ALS

Together with the lncRNA an increasing interest was addressed to the antisense (AS) noncoding transcripts. They are generated from the strand opposite the sense strand [74]. AS lncRNAs act by regulating chromatin, by controlling DNA methylation and/or histones modification, or by removing repressors. They promote sense transcription by recruiting transcription factors, they also regulate the half-life of their sense partners, and, in turn, they regulate gene expression [74]. About 70% of the human genome creates antisense transcripts with a great physiological and pathological significance. Ataxin 2 (ATXN2) is a coding gene related to ALS because of the association between the length of ATXN2 repeat expansion and the disease risk of ALS [75]. In 2016, Li and co-authors described the **ATXN2-AS** [76]. ATXN2-AS with its CUG repeat expansion is neurotoxic and may contribute to ALS pathogenesis. The CUG transcript toxicity is related to the structure formed by the repeats; that is, the stems of hairpin structures act with sponge-like features,

sequester RBPs, and induce alterations of the RNA metabolism [77].

*DOI: http://dx.doi.org/10.5772/intechopen.90704*

and other neurodegenerative diseases.

#### *RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.90704*

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

pathological phenotype [64, 65]. In motor neurons progenitors derived from human ALS iPSCs, Rizzuti et al. observed that **miRNA34a** and **miRNA504**, involved in vesicle regulation and cell survival, were dysregulated [66]. Also **miRNA1825** is downregulated in CNS of sALS and fALS patients, thus inducing depolymerization and degradation of tubulin alpha-4A (TUBA4A), which is encoded by the known ALS gene [67]. Taken together, these studies significantly contribute to evidence the importance

*Representation of the key miRNAs involved in amyotrophic lateral sclerosis mutated patients.*

*Representation of the key miRNAs involved in amyotrophic lateral sclerosis sporadic patients (sALS).*

of miRNAs, also as biomarkers for ALS. Despite these evidences, several issues need to be addressed mainly on the utility of miRNAs to serve as accurate and fast

Long noncoding RNAs (lncRNAs) are transcripts, greater than 200 nucleotides in length, with no protein-coding potential which are found in sense or antisense

biomarkers for an early ALS diagnosis.

**4. Dysfunctions in RNA metabolism and lncRNA**

**120**

**Figure 1.**

**Figure 2.**

orientation to protein-coding genes or within intergenic regions. lncRNAs control the gene expression through different mechanisms, that is, epigenetic modulation through chromatin remodeling, activation or repression of transcription, posttranscriptional modifications of mRNA, and regulation of protein activity by acting as scaffold to recruit RBPs and/or drive RBPs to DNA. Moreover, they can compete for and disrupt protein-binding interactions or sponge miRNAs away from their mRNA targets [68]. Recently, lncRNAs have been indicated to play important roles in the CNS in health and disease such as ALS. Nishimoto and colleagues first identify a relation between **NEAT1** and ALS pathogeneses [22]. The NEAT1 gene produces two transcripts, NEAT1\_1 and NEAT1\_2; NEAT1\_2 expression is very low in the adult nervous system, and it is the only one that forms paraspeckles [69]. Specifically, NEAT1 acts as a scaffold for paraspeckles thus enhancing their de novo formation in spinal motor neurons in a cohort of sALS patients [22]. Paraspeckles function through the retention of specific RNAs; the regulation of gene expression by sequestration of transcription factors; and the modulation of miRNA biogenesis and mitochondrial function [70]. Paraspeckles are enriched in pathological proteins for ALS and are indicated as a hallmark of the disease [71]. Moreover, paraspeckle proteins, including TDP-43 and FUS, are related to ALS and FTD. The increase in paraspeckle formation in ALS could be triggered, at least in part, by the nuclear depletion of TDP-43. TDP-43 binds NEAT1, and, in turn, its downregulation stimulates NEAT1\_2 accumulation and paraspeckle association in cultured cells [71]. Regarding FUS, in the spinal cord of FUS mutated ALS patients, Shelkovnikova and co-authors reported the presence of pathological aggregation of NONO, a core paraspeckle protein [31]. This evidence allows to speculate that, considering that FUS and NONO are both required to set up paraspeckles, the formation of paraspeckles is disrupted in FUS mutated ALS patients. Also aberrant nuclear RNA foci formed by the expanded C9ORF72 repeats sequester paraspeckle proteins including TDP-43 [72]. **MALAT1** is abundantly expressed and evolutionarily conserved lncRNA. It is one of the first lncRNAs associated with human disease, and it is involved in alternative splicing, epigenetic modification of gene expression, synapse formation, and myogenesis. In NDs MALAT1 is significantly increased in FTLD patients, where it recruits splicing factors to nuclear speckles and affects phosphorylation of SR proteins37 [13]. Some lncRNA transcripts have been associated to FUS, among these the **lncRNA CCND1** which binds to the FUS consensus sequence GGUG [73]. Data form Wang and colleagues suggested that FUS is a specific repressor of CCND1, which is downregulated in response to DNA damage signals. Until now the lncRNA CCND1 has not been described in relation to ALS; but taken together, these observations point out that this lncRNA could be, at least partly, responsible in ALS and other neurodegenerative diseases.

Together with the lncRNA an increasing interest was addressed to the antisense (AS) noncoding transcripts. They are generated from the strand opposite the sense strand [74]. AS lncRNAs act by regulating chromatin, by controlling DNA methylation and/or histones modification, or by removing repressors. They promote sense transcription by recruiting transcription factors, they also regulate the half-life of their sense partners, and, in turn, they regulate gene expression [74]. About 70% of the human genome creates antisense transcripts with a great physiological and pathological significance. Ataxin 2 (ATXN2) is a coding gene related to ALS because of the association between the length of ATXN2 repeat expansion and the disease risk of ALS [75]. In 2016, Li and co-authors described the **ATXN2-AS** [76]. ATXN2-AS with its CUG repeat expansion is neurotoxic and may contribute to ALS pathogenesis. The CUG transcript toxicity is related to the structure formed by the repeats; that is, the stems of hairpin structures act with sponge-like features, sequester RBPs, and induce alterations of the RNA metabolism [77].


#### **Table 1.**

*List of lncRNAs related to ALS. In square bracket the relative references.*

Thanks to the deep sequencing technologies which allow high-throughput massive RNA sequencing, a wide characterization of the transcriptome profile of cell populations and tissues is now available. Three different massive transcriptome profiles have been published in different tissues (spinal cord, monocytes, and peripheral blood mononuclear cells) of ALS patients, and matched controls reported a deregulation in expressed genes [78, 79] and in lncRNAs [80]. Differences in transcriptome profiles (coding and lncRNAs) were observed in PBMCs of unmutated sALS patients, SOD1, TARDBP, and FUS mutated ALS patients and healthy controls [80]. Specifically, the authors reported a remarkable AS deregulation of genes involved in the transcription regulation pathway such as **ZEB1-AS** and **ZBTB11-AS** in sALS patients. ZEB1 acts as a repressor or an activator of the transcription, that is, it may repress histone organization or activate chromatin regulators [81]. As regards ZBTB11-AS, it decreases in sALS patients compared to healthy controls. ZBTB11-AS is annotated as AS of Zinc finger and BTB domain-containing protein 11 (ZBTB11) gene, and it is reported to be a negative regulator of cell cycle; however its exact role has yet to be defined [82]. Moreover, Gagliardi and co-authors evidenced **UBXN7-AS**, **ATG10-AS,** and **ADORA2A-AS** in sALS patients, all related to NDs [83–85]. Specifically, the regulation of UBXN7, an ubiquitin protein bound by VCP a known ALS protein, through its AS controlled the ubiquitination in ALS disease. ATG10 is involved in the autophagy pathway, while ADORA2A is involved in Huntington's disease and Parkinson's disease in relation to defects in DNA methylation [84, 86] (**Table 1**).

#### **5. Therapeutics**

In the era of noncoding RNA, understanding the involvement of dysregulated miRNAs and of their targets in ALS disease is crucial to identify new pathways contributing to neurodegeneration that also offer novel opportunities for targeted intervention. miRNA-based therapeutics take advantages of two different approaches. The first involves the use of an anti-miRNA, that is, chemically modified antisense RNA, to decrease miRNA. Thus, miRNA duplex is not active and counteracts the negative regulatory effects of miRNA. This approach was first used to deliver the anti-miR-155 to the SOD1G93A transgenic mice via ventricular osmotic pumps; after this treatment the mortality was successfully delayed [59]. The second therapeutic approach using miRNA involves miRNA mimics, that is, small RNA molecules resembling miRNA precursors, that are reintroduced into cells exhibiting downregulation thus re-starting the key-related pathways [87]. Biomedical and nanoparticle engineering has begun to develop tools allowing

**123**

**6. Conclusion**

*RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis*

the exon or including alternatively spliced exons [89].

cokinetics [90].

for this specific targeting. These second-generation miRNA-based therapeutics offer the potential for a greater delivery cargo to the tissue site while reducing RNA-mediated toxicity. Overall, the continued development of innovative RNA modifications and delivery items such as nanoparticles will aid in the development

Deregulated AS is considered an important area for therapeutic intervention. Particularly, gene therapy is an encouraging pharmacological approach for patients with diseases of genetic origins. This therapy is principally based on antisense oligonucleotides (ASOs), spliceosome-mediated RNA trans-splicing (SMaRT), or small interfering RNAs (siRNAs) [88]. ASOs, that is, synthetic single-stranded nucleic acids, bind the pre-mRNA intron/exon junctions and control the splicing through their action on enhancers or repressor sequences, thus determining the skipping of

In ALS, one of the first ASO-based clinical trials was designed to silence SOD1. The intrathecal administration of this ASO pass with good results the phase I testing. Now a phase Ib/IIa trial is in process to assess safety, tolerability, and pharma-

Among the ALS-related genes, C9orf72 is one of the best candidates for ASOs therapy. Early testing of ASO-based therapeutics for C9orf72 was performed on iPSC-derived neurons and fibroblasts [91]. Specifically, ASOs were designed to target the repeat expansion or within N-terminal regions of the mRNA transcript to destroy the transcript or to prevent the interaction between the repeat expansion and the RBPs, determining a decrease in RNA foci and dipeptide proteins and recovering the normal gene expression [91]. Other studies investigated the effects of ASO on the oligonucleotide backbone, sugar, and heterocycles to promote delivery, potency, and stability to target FUS. These studies evidenced that the affinities of nucleic acid binding domains depend on chemical changes and that the interaction between ASO and protein affects the localization of ASOs themselves [92]. These data strongly indicate that ASO-based therapy could be central in treating ALSrelated genes, although there is great attention on the relation between the therapeutic outcomes and the stage of disease progression and on the time of intervention. Also many novel lncRNAs have been discovered, and the potential to become therapeutic targets is gradually increasing. Considering that lncRNAs function as decoys, regulators of translation, and scaffolds directing chromatin-modifying enzymes to specific genomic loci, they are an attractive class of therapeutic targets. The relation between HOTAIR in breast cancer [93] and MALAT1 in metastatic lung cancer [94] is a remarkable example of this association. Therefore, there is enthusiasm about the possibility to develop therapeutic tools to modulate mis-regulated lncRNAs in diseases. Although lncRNAs represent appealing pharmacological and therapeutic targets, inhibiting lncRNAs in vivo remains a challenge. A possible approach could be the use of small molecules that disrupt the complex lncRNA-chromatin that alter the epigenetic state of the target cells. All these delivery efforts, along with further elucidation of lncRNA regulatory mechanisms, will ultimately lead to the development of effective therapeutic strategies that target lncRNAs in vivo.

The impairment in RNA regulation and processing is crucial in ALS pathogenesis. Defects at different steps of RNA processing alter both cellular function and survival; thus RNA metabolism can be an essential target for therapeutic intervention for ALS and for other NDs. The application of RNA-based therapies to modulate gene and protein expression is an interesting therapeutic strategy:

of future RNA-based therapeutics for a broader range of chronic disease.

*DOI: http://dx.doi.org/10.5772/intechopen.90704*

#### *RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.90704*

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

ATXN2-AS [76] NEAT1

ZEB1-AS [80] MALAT1

*List of lncRNAs related to ALS. In square bracket the relative references.*

ZBTB11-AS [80] UBXN7-AS [80] ATG10-AS [80] ADORA2A-AS [80]

**Table 1.**

**sALS TARDBP mutation FUS mutation**

[22, 69–71]

[13]

lncCCND1 [73]

in relation to defects in DNA methylation [84, 86] (**Table 1**).

Thanks to the deep sequencing technologies which allow high-throughput massive RNA sequencing, a wide characterization of the transcriptome profile of cell populations and tissues is now available. Three different massive transcriptome profiles have been published in different tissues (spinal cord, monocytes, and peripheral blood mononuclear cells) of ALS patients, and matched controls reported a deregulation in expressed genes [78, 79] and in lncRNAs [80]. Differences in transcriptome profiles (coding and lncRNAs) were observed in PBMCs of unmutated sALS patients, SOD1, TARDBP, and FUS mutated ALS patients and healthy controls [80]. Specifically, the authors reported a remarkable AS deregulation of genes involved in the transcription regulation pathway such as **ZEB1-AS** and **ZBTB11-AS** in sALS patients. ZEB1 acts as a repressor or an activator of the transcription, that is, it may repress histone organization or activate chromatin regulators [81]. As regards ZBTB11-AS, it decreases in sALS patients compared to healthy controls. ZBTB11-AS is annotated as AS of Zinc finger and BTB domain-containing protein 11 (ZBTB11) gene, and it is reported to be a negative regulator of cell cycle; however its exact role has yet to be defined [82]. Moreover, Gagliardi and co-authors evidenced **UBXN7-AS**, **ATG10-AS,** and **ADORA2A-AS** in sALS patients, all related to NDs [83–85]. Specifically, the regulation of UBXN7, an ubiquitin protein bound by VCP a known ALS protein, through its AS controlled the ubiquitination in ALS disease. ATG10 is involved in the autophagy pathway, while ADORA2A is involved in Huntington's disease and Parkinson's disease

In the era of noncoding RNA, understanding the involvement of dysregulated miRNAs and of their targets in ALS disease is crucial to identify new pathways contributing to neurodegeneration that also offer novel opportunities for targeted intervention. miRNA-based therapeutics take advantages of two different approaches. The first involves the use of an anti-miRNA, that is, chemically modified antisense RNA, to decrease miRNA. Thus, miRNA duplex is not active and counteracts the negative regulatory effects of miRNA. This approach was first used to deliver the anti-miR-155 to the SOD1G93A transgenic mice via ventricular osmotic pumps; after this treatment the mortality was successfully delayed [59]. The second therapeutic approach using miRNA involves miRNA mimics, that is, small RNA molecules resembling miRNA precursors, that are reintroduced into cells exhibiting downregulation thus re-starting the key-related pathways [87]. Biomedical and nanoparticle engineering has begun to develop tools allowing

**122**

**5. Therapeutics**

for this specific targeting. These second-generation miRNA-based therapeutics offer the potential for a greater delivery cargo to the tissue site while reducing RNA-mediated toxicity. Overall, the continued development of innovative RNA modifications and delivery items such as nanoparticles will aid in the development of future RNA-based therapeutics for a broader range of chronic disease.

Deregulated AS is considered an important area for therapeutic intervention. Particularly, gene therapy is an encouraging pharmacological approach for patients with diseases of genetic origins. This therapy is principally based on antisense oligonucleotides (ASOs), spliceosome-mediated RNA trans-splicing (SMaRT), or small interfering RNAs (siRNAs) [88]. ASOs, that is, synthetic single-stranded nucleic acids, bind the pre-mRNA intron/exon junctions and control the splicing through their action on enhancers or repressor sequences, thus determining the skipping of the exon or including alternatively spliced exons [89].

In ALS, one of the first ASO-based clinical trials was designed to silence SOD1. The intrathecal administration of this ASO pass with good results the phase I testing. Now a phase Ib/IIa trial is in process to assess safety, tolerability, and pharmacokinetics [90].

Among the ALS-related genes, C9orf72 is one of the best candidates for ASOs therapy. Early testing of ASO-based therapeutics for C9orf72 was performed on iPSC-derived neurons and fibroblasts [91]. Specifically, ASOs were designed to target the repeat expansion or within N-terminal regions of the mRNA transcript to destroy the transcript or to prevent the interaction between the repeat expansion and the RBPs, determining a decrease in RNA foci and dipeptide proteins and recovering the normal gene expression [91]. Other studies investigated the effects of ASO on the oligonucleotide backbone, sugar, and heterocycles to promote delivery, potency, and stability to target FUS. These studies evidenced that the affinities of nucleic acid binding domains depend on chemical changes and that the interaction between ASO and protein affects the localization of ASOs themselves [92]. These data strongly indicate that ASO-based therapy could be central in treating ALSrelated genes, although there is great attention on the relation between the therapeutic outcomes and the stage of disease progression and on the time of intervention.

Also many novel lncRNAs have been discovered, and the potential to become therapeutic targets is gradually increasing. Considering that lncRNAs function as decoys, regulators of translation, and scaffolds directing chromatin-modifying enzymes to specific genomic loci, they are an attractive class of therapeutic targets. The relation between HOTAIR in breast cancer [93] and MALAT1 in metastatic lung cancer [94] is a remarkable example of this association. Therefore, there is enthusiasm about the possibility to develop therapeutic tools to modulate mis-regulated lncRNAs in diseases. Although lncRNAs represent appealing pharmacological and therapeutic targets, inhibiting lncRNAs in vivo remains a challenge. A possible approach could be the use of small molecules that disrupt the complex lncRNA-chromatin that alter the epigenetic state of the target cells. All these delivery efforts, along with further elucidation of lncRNA regulatory mechanisms, will ultimately lead to the development of effective therapeutic strategies that target lncRNAs in vivo.

#### **6. Conclusion**

The impairment in RNA regulation and processing is crucial in ALS pathogenesis. Defects at different steps of RNA processing alter both cellular function and survival; thus RNA metabolism can be an essential target for therapeutic intervention for ALS and for other NDs. The application of RNA-based therapies to modulate gene and protein expression is an interesting therapeutic strategy:

the preclinical application of RNA-based therapies targeting SOD1 and C9orf72 mutations are promising and pave the way to apply similar approaches for FUS and TDP-43 mutations. In conclusion, RNA-based therapies could be recommended for the future treatment of ALS.

### **Funding**

Authors acknowledge the economic support of the Fondazione Regionale per la Ricerca Biomedica (FRRB): TRANS\_ALS [2015-0023]; Finanziamento 5x1000 2016; Italian Ministry of Health GR-2016-02361552.

### **Abbreviations**


**125**

*RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis*

NEAT1 nuclear-enriched autosomal transcript 1

NONO non-POU domain-containing octamer-binding protein

*DOI: http://dx.doi.org/10.5772/intechopen.90704*

ND neurodegenerative disorder

NF-kB nuclear factor kappa B subunit 1 NFL neurofilament light chain

NRCAM neuronal cell adhesion molecule

sALS sporadic amyotrophic lateral sclerosis

SMaRT spliceosome-mediated RNA trans-splicing

SYGQ N-terminal serine-tyrosine-glycine-glutamine TAF15 TATA-box binding protein-associated factor 15

TIAR TIA1 cytotoxic granule-associated RNA binding protein like 1

VAPB vesicle-associated membrane protein-associated protein B/C

NES nuclear export signals

NLS nuclear localization

RBP RNA-binding proteins RRM1-2 RNA recognition motifs 1-2 RUNX Runbox transcription factor

siRNAs small interfering RNAs

SOD1 superoxide dismutase 1 SPI1 Spi-1 proto-oncogene SQSTM1 sequestosome 1

TARDBP TAR DNA-binding protein TBK1 TANK-binding kinase 1

TUBA4A tubulin alpha 4a UBQLN2 ubiquilin 1

UBXN7 UBX domain protein 7 UTR 3′-untranslated

VCP valosin-containing protein

VEGF vascular endothelial growth factor

ZBTB11 zinc finger and BTB domain-containing 11 ZEB1 zinc finger E-box-binding homeobox 1

TGF-β1 transforming growth factor-beta

miRNAs microRNAs MN motor neuron

NLGN1-2 neuroligin

NRXN1-3 neurexin NTNG1 netrin G1 OPTN optineurin PFN1 profilin 1 PTX3 pentraxin 3 *RNA Metabolism and Therapeutics in Amyotrophic Lateral Sclerosis DOI: http://dx.doi.org/10.5772/intechopen.90704*

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

the future treatment of ALS.

Italian Ministry of Health GR-2016-02361552.

ABLIM1 actin-binding LIM ADORA2A adenosine A2a receptor ALS amyotrophic lateral sclerosis

ASOs antisense oligonucleotides ATG10 autophagy related 10

CNS central nervous system CSF cerebrospinal fluid

EWSR1 EWS RNA binding protein 1

tor subunits

tor subunits

iPSC induced pluripotent stem cell KIF5A kinesin family member 5A lncRNA long noncoding RNA lncRNAs long noncoding RNAs

MAP1B microtubule-associated protein 1B MAPT microtubule-associated protein tau

Homer2 homer scaffold protein 2 HOTAIR HOX transcript antisense RNA

HuR Hu antigen R

MATR3 matrin 3

C9ORF72 chromosome 9 open reading frame 72

CHMP2B charged multivesicular body protein 2B

fALS familial amyotrophic lateral sclerosis FTLD frontotemporal lobar degeneration

FUS fused in sarcoma/translocated in liposarcoma

HNRNPA1 heterogeneous nuclear ribonucleoprotein A1 HNRNPA2/B1 heterogeneous nuclear ribonucleoprotein A2/B1

CHCHD10 coiled-coil-helix-coiled-coil-helix domain containing 10

GABRA2 gamma-aminobutyric acid type A receptor alpha2 subunit GABRA3 gamma-aminobutyric acid type A receptor alpha3 subunit GRIA3 glutamate ionotropic receptor AMPA type subunit 3AMPA recep-

MALAT1 metastasis-associated lung adenocarcinoma transcript 1

GRIA4 glutamate ionotropic receptor AMPA type subunit 4AMPA recep-

ANG angiogenin ANXA11 annexin A11 AS antisense

ATXN2 ataxin 2

CCND1 cyclin D1

**Funding**

**Abbreviations**

the preclinical application of RNA-based therapies targeting SOD1 and C9orf72 mutations are promising and pave the way to apply similar approaches for FUS and TDP-43 mutations. In conclusion, RNA-based therapies could be recommended for

Authors acknowledge the economic support of the Fondazione Regionale per la Ricerca Biomedica (FRRB): TRANS\_ALS [2015-0023]; Finanziamento 5x1000 2016;

**124**


*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

#### **Author details**

Orietta Pansarasa\*, Stella Gagliardi, Daisy Sproviero and Cristina Cereda Genomic and Post-Genomic Center, IRCCS Mondino Foundation, Pavia, Italy

\*Address all correspondence to: orietta.pansarasa@mondino.it

© 2019 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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**Author details**

Orietta Pansarasa\*, Stella Gagliardi, Daisy Sproviero and Cristina Cereda Genomic and Post-Genomic Center, IRCCS Mondino Foundation, Pavia, Italy

© 2019 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,

\*Address all correspondence to: orietta.pansarasa@mondino.it

provided the original work is properly cited.

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**132**

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**135**

**Chapter 8**

**Abstract**

**1. Introduction**

phenomenon [3].

Targeting Axonal Transport: A

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

New Therapeutic Avenue for ALS

Motor neurons have an extreme polarized morphology and heavily rely on efficient cargo transport along axons to maintain their neuronal connections and connections with muscles. Axonal transport deficits have been observed in almost all model systems of ALS. More and more studies have confirmed the close genetic and mechanistic linkage between axonal transport deficits with ALS pathogenesis. Moreover, several therapeutic approaches have been developed to target axonal transport deficits in ALS and showed promising effects in disease models. In this concise chapter, we summarize some major discoveries of axonal transport deficits in ALS pathogenesis and some related therapeutic strategies. We propose that targeting axonal transport may provide a potential therapeutic avenue for ALS.

The unique morphological feature of neuronal cells compared with other cell types is their extreme polarity and the incredibly long axons [1]. Although the soma size ranges from 5 μm to 100 µm, axons can be up to 1 m long [1]. Axons keep the efficient communication between soma and axonal terminals [1]. This axonal communication is especially important to motor neuron as they not only need connections with each other but also far reach to muscles in order to control proper muscle contractions [2]. As for ALS, axonal transport defects are one of the most prevalent reported phenotypes from different model systems [2]. In addition, the classical "dying-back" hypothesis could explain the sequence of events during motor neuron degeneration in ALS [3]. The idea is that motor neurons lose their connection with muscle fibers and that the axon retracts back towards the soma, which ultimately results in cell death [3]. This theory is supported by the observation that motor neuron pathology begins at the terminal part of the axon and proceeds in a "dyingback" pattern [3]. In addition, the longest and largest neurites with the highest metabolic demand seem to be the most susceptible to this "dying-back"

Mechanistically, axonal transport process relies on three main elements: cargoes, microtubules, and motor proteins and their adaptors [1]. Axonal transport is tracked on microtubules which are polymers of α-tubulin and β-tubulins [1]. Microtubules determine the neuronal polarity with "plus" end at the axonal distal part and "minus" end at the soma part [1]. This polarity allows the directionality of the axonal transport [1]. Kinesin (mainly responsible for anterograde axonal transport, from soma to axonal terminal) and dynein (mainly responsible for retrograde

**Keywords:** ALS, axonal transport, pathogenesis, therapeutics

#### **Chapter 8**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

[93] Han B, Peng X, Cheng D, Zhu Y, Du J, Li J, et al. Delphinidin suppresses breast carcinogenesis through the HOTAIR/microRNA-34a axis. Cancer Science. 2019;**110**(10):3089-3097. DOI:

[94] Gomes CP, Nóbrega-Pereira S, Domingues-Silva B, Rebelo K, Alves-Vale C, Marinho SP, et al. An antisense transcript mediates MALAT1 response in human breast cancer. BMC Cancer. 2019;**19**(1):771. DOI: 10.1186/

10.1111/cas.14133

s12885-019-5962-0

**134**

## Targeting Axonal Transport: A New Therapeutic Avenue for ALS

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

#### **Abstract**

Motor neurons have an extreme polarized morphology and heavily rely on efficient cargo transport along axons to maintain their neuronal connections and connections with muscles. Axonal transport deficits have been observed in almost all model systems of ALS. More and more studies have confirmed the close genetic and mechanistic linkage between axonal transport deficits with ALS pathogenesis. Moreover, several therapeutic approaches have been developed to target axonal transport deficits in ALS and showed promising effects in disease models. In this concise chapter, we summarize some major discoveries of axonal transport deficits in ALS pathogenesis and some related therapeutic strategies. We propose that targeting axonal transport may provide a potential therapeutic avenue for ALS.

**Keywords:** ALS, axonal transport, pathogenesis, therapeutics

#### **1. Introduction**

The unique morphological feature of neuronal cells compared with other cell types is their extreme polarity and the incredibly long axons [1]. Although the soma size ranges from 5 μm to 100 µm, axons can be up to 1 m long [1]. Axons keep the efficient communication between soma and axonal terminals [1]. This axonal communication is especially important to motor neuron as they not only need connections with each other but also far reach to muscles in order to control proper muscle contractions [2]. As for ALS, axonal transport defects are one of the most prevalent reported phenotypes from different model systems [2]. In addition, the classical "dying-back" hypothesis could explain the sequence of events during motor neuron degeneration in ALS [3]. The idea is that motor neurons lose their connection with muscle fibers and that the axon retracts back towards the soma, which ultimately results in cell death [3]. This theory is supported by the observation that motor neuron pathology begins at the terminal part of the axon and proceeds in a "dyingback" pattern [3]. In addition, the longest and largest neurites with the highest metabolic demand seem to be the most susceptible to this "dying-back" phenomenon [3].

Mechanistically, axonal transport process relies on three main elements: cargoes, microtubules, and motor proteins and their adaptors [1]. Axonal transport is tracked on microtubules which are polymers of α-tubulin and β-tubulins [1]. Microtubules determine the neuronal polarity with "plus" end at the axonal distal part and "minus" end at the soma part [1]. This polarity allows the directionality of the axonal transport [1]. Kinesin (mainly responsible for anterograde axonal transport, from soma to axonal terminal) and dynein (mainly responsible for retrograde axonal transport, from axonal terminal to soma) are two types of ATP-dependent motor proteins responsible for carrying cargoes along the microtubules [1]. Adaptor proteins are responsible for the connection between motor proteins and cargoes [1]. Axonal transport maintains the efficient supply of cargoes including proteins, RNAs, lipids, and organelles from soma to terminals and is responsible to clear or recycle some misfolded proteins or aggregates under cell stress [1]. It is known that several ALS genes can directly cause axonal transport defects, and axonal transport also actively interacts with other major ALS pathological changes. Gene therapies and compounds that target axonal transport have shown beneficial effects in ALS animal models, and some have already been tested in clinical trials in the context of other diseases. Therefore, a better understanding of transport mechanisms and its role in the disease will open up a new therapeutic avenue for ALS.

#### **2. Axonal transport defects in ALS pathogenesis**

#### **2.1 ALS mutations directly interfere with the axonal transport machinery**

With the fact that about 10% of ALS cases are considered as "familial ALS" with clear genetic factors involved [4], several ALS gene mutations have been shown which could directly interfere with different aspects of the axonal transport machinery and eventually cause axonal transport defects.

ALS genes that are directly linked to kinesin- and dynein-mediated axonal transport have been uncovered by the discovery of ALS mutations in kinesin family member 5A (KIF5A) and dynactin subunit 1 (DCTN1) [5]. As a member of the kinesin family, KIF5A is mainly expressed in neuronal cells. In the year 2018, two independent large-scale genome-wide association and exome sequencing studies have found that mutations in KIF5A cause ALS [5, 6]. Most of the mutations are loss-of-function mutations that localized in the C-terminal region of the protein where cargoes bind. KIF5A mutations cause altered ATP activity and the dysfunction of kinesin-1 that eventually interfere with the anterograde transport of cargoes along the microtubules [6]. Moreover, for one of the most common ALS-causing genes called fused in sarcoma (FUS) it has been reported that its protein product functions as DNA-/RNA-binding protein that can bind or regulate mRNAs of several other motor proteins including KIF5C, KIF1B, and KIF3A [7], which are actively involved in regulating mitochondrial transport in neurons. Dynactin is a multi-subunit protein that binds and activates dynein by forming a dynein-dynactin motor complex that conveys cargoes in a retrograde transport [8]. The DCTN1 gene encodes dynactin subunit 1, which is responsible for binding microtubules and motor proteins. Heterozygous missense mutations in the DCTN1 gene have been suggested as risk factors for ALS in both sporadic and familial ALS patients [9].

The direct influence from ALS genes to microtubules has been described by the tubulin alpha 4a (TUBA4A) gene and spastic paraplegia 11 (SPG11) gene [10]. Exome-wide variant burden analysis revealed that mutations in TUBA4A associate with both sporadic and familial ALS cases [11, 12]. TUBA4A encodes the tubulin alpha 4A protein. Tubulins are the basic constituents of microtubules [11]. Mutations in TUBA4A destabilize the microtubule network, diminishing its re-polymerization capability that eventually disrupts the transport process [11]. Mutations in the SPG11 gene are the cause of autosomal recessive juvenile-onset ALS. Most of the mutations are loss-of-function mutations [10]. SPG11 encodes a protein called spatacsin, which co-localizes with the cytoskeleton in neurons [13]. Knockdown of SPG11 in mice showed a decreased acetylation level of α-tubulins [13]. With the fact that acetylation of α-tubulin facilitates the stabilization of the

**137**

axonal transport in ALS.

**mechanisms**

*Targeting Axonal Transport: A New Therapeutic Avenue for ALS*

microtubules and the binding of motor proteins to the microtubules, silencing SPG11 causes axon outgrowth defects and retrograde axonal retraction in cortical neurons of mice [13]. This is in line with the reduced axon plasticity in human iPSC-derived neurons from patients carrying SPG11 mutations [13]. These studies highlighted the importance of spatacsin in axon maintenance due to insufficient

**2.2 Interplay between axonal transport defects with other ALS pathogenic** 

Based on the genetic discoveries of ALS, intensive studies have proposed several major pathogenic mechanisms that contribute to motor neuron degeneration in ALS. Together with axonal transport defects, other dysfunctional mechanisms such as mitochondrial dysfunction, endoplasmic reticulum (ER) stress, neuroinflammation, excitotoxicity, and abnormal DNA/RNA metabolism have been identified [4]. Although it is still under debate which mechanism plays the vital role in initiating the motor neuron degenerative process, axonal transport defects have been reported to interplay with other mechanisms involved in disease progression. With the most polarized morphological structure, motor neurons demand high-energy supply to maintain their normal function in controlling muscle

Except influencing motor proteins and microtubules, ALS genes can also interfere with axonal transport by affecting the cargoes. As the cargoes can participate into different mechanisms, the interplay between axonal transport and other ALS pathological mechanisms is also linked via cargo-specific transport. Rab proteins are a group of small GTPases that belong to the Ras superfamily [14]. Rabs are responsible for proper vesicle sorting, fission, docking, fusion, and transporting spatially and temporally by switching from an inactive guanosine 5'-diphosphate (GDP)-bound state to an active guanosine 5'-triphosphate [14]. Several Rabs have been reported to play a crucial role in driving neuronal transport in the central nervous system [14]. Mutations in the ALS2 gene cause juvenile-onset ALS. The protein product of the ALS2 gene specifically binds to Rab5 and functions as a guanine nucleotide exchange factor (GEF) for Rab5. The vacuolar protein sorting 9 (VPS9) domain of alsin mediates the activation of Rab5 through endosome and guanine-nucleotide exchanging reaction [14]. Most of the ALS2 mutations are loss-of-function mutations that cause the loss of the VPS9 domain and eventually fail in Rab5 activation [14]. Subsequently, Rab5-dependent endosome and AMPA receptor trafficking are hampered in neuronal culture [15, 16]. The reduction of GluR2-containing AMPA receptors at the synaptic surface in ALS2 knockout neurons results in vulnerability to glutamate toxicity [16]. In addition, more than 20 mutations in the OPTN gene have been described being the causative mutations of ALS [17]. Optineurin, the protein product of OPTN, regulates vesicle trafficking by forming a complex with myosin VI and Rab8 [17]. Myosins are a superfamily of motor proteins that move cargoes along microtubules, while Rab8 is a marker of recycling endosomes. The formation of the complex is Optineurin dependent [17]. This complex localizes at the Golgi apparatus and in cytoplasmic vesicles. It mediates Golgi organization, post-Golgi trafficking, exocytosis, and the basolateral delivery of membrane proteins [17]. In line with this, impaired axonal vesicle transport has been observed in a zebrafish model with optineurin loss. Furthermore, chromosome 9 open reading frame 72 (C9orf72), the most common genetic cause of ALS, is a GEF for Rab8 and is also associated with Rab1 [18, 19]. The knockdown of C9orf72 affects cellular trafficking from the cell membrane to Golgi. Overall, Rab-related transport processes are a good example of the cargo-induced impaired

*DOI: http://dx.doi.org/10.5772/intechopen.91963*

transport and cargo trafficking [13].

#### *Targeting Axonal Transport: A New Therapeutic Avenue for ALS DOI: http://dx.doi.org/10.5772/intechopen.91963*

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

role in the disease will open up a new therapeutic avenue for ALS.

**2.1 ALS mutations directly interfere with the axonal transport machinery**

With the fact that about 10% of ALS cases are considered as "familial ALS" with clear genetic factors involved [4], several ALS gene mutations have been shown which could directly interfere with different aspects of the axonal transport

ALS genes that are directly linked to kinesin- and dynein-mediated axonal transport have been uncovered by the discovery of ALS mutations in kinesin family member 5A (KIF5A) and dynactin subunit 1 (DCTN1) [5]. As a member of the kinesin family, KIF5A is mainly expressed in neuronal cells. In the year 2018, two independent large-scale genome-wide association and exome sequencing studies have found that mutations in KIF5A cause ALS [5, 6]. Most of the mutations are loss-of-function mutations that localized in the C-terminal region of the protein where cargoes bind. KIF5A mutations cause altered ATP activity and the dysfunction of kinesin-1 that eventually interfere with the anterograde transport of cargoes along the microtubules [6]. Moreover, for one of the most common ALS-causing genes called fused in sarcoma (FUS) it has been reported that its protein product functions as DNA-/RNA-binding protein that can bind or regulate mRNAs of several other motor proteins including KIF5C, KIF1B, and KIF3A [7], which are actively involved in regulating mitochondrial transport in neurons. Dynactin is a multi-subunit protein that binds and activates dynein by forming a dynein-dynactin motor complex that conveys cargoes in a retrograde transport [8]. The DCTN1 gene encodes dynactin subunit 1, which is responsible for binding microtubules and motor proteins. Heterozygous missense mutations in the DCTN1 gene have been suggested as risk factors for ALS in both sporadic and familial ALS patients [9]. The direct influence from ALS genes to microtubules has been described by the tubulin alpha 4a (TUBA4A) gene and spastic paraplegia 11 (SPG11) gene [10]. Exome-wide variant burden analysis revealed that mutations in TUBA4A associate with both sporadic and familial ALS cases [11, 12]. TUBA4A encodes the tubulin alpha 4A protein. Tubulins are the basic constituents of microtubules [11]. Mutations in TUBA4A destabilize the microtubule network, diminishing its re-polymerization capability that eventually disrupts the transport process [11]. Mutations in the SPG11 gene are the cause of autosomal recessive juvenile-onset ALS. Most of the mutations are loss-of-function mutations [10]. SPG11 encodes a protein called spatacsin, which co-localizes with the cytoskeleton in neurons [13]. Knockdown of SPG11 in mice showed a decreased acetylation level of α-tubulins [13]. With the fact that acetylation of α-tubulin facilitates the stabilization of the

**2. Axonal transport defects in ALS pathogenesis**

machinery and eventually cause axonal transport defects.

axonal transport, from axonal terminal to soma) are two types of ATP-dependent motor proteins responsible for carrying cargoes along the microtubules [1]. Adaptor proteins are responsible for the connection between motor proteins and cargoes [1]. Axonal transport maintains the efficient supply of cargoes including proteins, RNAs, lipids, and organelles from soma to terminals and is responsible to clear or recycle some misfolded proteins or aggregates under cell stress [1]. It is known that several ALS genes can directly cause axonal transport defects, and axonal transport also actively interacts with other major ALS pathological changes. Gene therapies and compounds that target axonal transport have shown beneficial effects in ALS animal models, and some have already been tested in clinical trials in the context of other diseases. Therefore, a better understanding of transport mechanisms and its

**136**

microtubules and the binding of motor proteins to the microtubules, silencing SPG11 causes axon outgrowth defects and retrograde axonal retraction in cortical neurons of mice [13]. This is in line with the reduced axon plasticity in human iPSC-derived neurons from patients carrying SPG11 mutations [13]. These studies highlighted the importance of spatacsin in axon maintenance due to insufficient transport and cargo trafficking [13].

Except influencing motor proteins and microtubules, ALS genes can also interfere with axonal transport by affecting the cargoes. As the cargoes can participate into different mechanisms, the interplay between axonal transport and other ALS pathological mechanisms is also linked via cargo-specific transport. Rab proteins are a group of small GTPases that belong to the Ras superfamily [14]. Rabs are responsible for proper vesicle sorting, fission, docking, fusion, and transporting spatially and temporally by switching from an inactive guanosine 5'-diphosphate (GDP)-bound state to an active guanosine 5'-triphosphate [14]. Several Rabs have been reported to play a crucial role in driving neuronal transport in the central nervous system [14]. Mutations in the ALS2 gene cause juvenile-onset ALS. The protein product of the ALS2 gene specifically binds to Rab5 and functions as a guanine nucleotide exchange factor (GEF) for Rab5. The vacuolar protein sorting 9 (VPS9) domain of alsin mediates the activation of Rab5 through endosome and guanine-nucleotide exchanging reaction [14]. Most of the ALS2 mutations are loss-of-function mutations that cause the loss of the VPS9 domain and eventually fail in Rab5 activation [14]. Subsequently, Rab5-dependent endosome and AMPA receptor trafficking are hampered in neuronal culture [15, 16]. The reduction of GluR2-containing AMPA receptors at the synaptic surface in ALS2 knockout neurons results in vulnerability to glutamate toxicity [16]. In addition, more than 20 mutations in the OPTN gene have been described being the causative mutations of ALS [17]. Optineurin, the protein product of OPTN, regulates vesicle trafficking by forming a complex with myosin VI and Rab8 [17]. Myosins are a superfamily of motor proteins that move cargoes along microtubules, while Rab8 is a marker of recycling endosomes. The formation of the complex is Optineurin dependent [17]. This complex localizes at the Golgi apparatus and in cytoplasmic vesicles. It mediates Golgi organization, post-Golgi trafficking, exocytosis, and the basolateral delivery of membrane proteins [17]. In line with this, impaired axonal vesicle transport has been observed in a zebrafish model with optineurin loss. Furthermore, chromosome 9 open reading frame 72 (C9orf72), the most common genetic cause of ALS, is a GEF for Rab8 and is also associated with Rab1 [18, 19]. The knockdown of C9orf72 affects cellular trafficking from the cell membrane to Golgi. Overall, Rab-related transport processes are a good example of the cargo-induced impaired axonal transport in ALS.

#### **2.2 Interplay between axonal transport defects with other ALS pathogenic mechanisms**

Based on the genetic discoveries of ALS, intensive studies have proposed several major pathogenic mechanisms that contribute to motor neuron degeneration in ALS. Together with axonal transport defects, other dysfunctional mechanisms such as mitochondrial dysfunction, endoplasmic reticulum (ER) stress, neuroinflammation, excitotoxicity, and abnormal DNA/RNA metabolism have been identified [4]. Although it is still under debate which mechanism plays the vital role in initiating the motor neuron degenerative process, axonal transport defects have been reported to interplay with other mechanisms involved in disease progression.

With the most polarized morphological structure, motor neurons demand high-energy supply to maintain their normal function in controlling muscle

contractions [20]. Mitochondria play a pivotal role as the energy supply center in cells. Abnormal mitochondrial morphology and function are observed in postmortem tissues from ALS patients as well as in different ALS animal models [21]. Mitochondrial transport is important to clear the damaged mitochondria and maintain sufficient energy supply from the soma side to the distal side in motor neurons [21]. In line with this, fast motor neurons that have the highest needs of ATP, are more severely affected in ALS compared to slow motor neurons [22]. Therefore, the interplay between mitochondria quality control and transport is crucial for motor neuron survival. Mitochondrial movement along microtubules is controlled by a large complex containing kinesin, dynein, mitochondrial Rho (Miro), and milton [23]. Milton is an adaptor protein that connects Miro and motor proteins. Miro is a GTPase localized to the outer membrane of mitochondria. Miro is regulated by PINK/Parkin, which are genetic modifiers of FUS-induced neurodegeneration [24]. Axonal transport defects have been observed in ALS patient-derived motor neurons and FUS transgenic flies. Decreased expression of either PINK or Parkin is beneficial for reversing the locomotive defects and enhancing the survival of FUS transgenic flies [24]. In addition, overexpression of FUS also increased the ubiquitination of the Miro1 protein [24]. We have observed that mitochondria-associated ER membranes (MAM) are significantly decreased in motor neurons carrying FUS mutation [49]. As Miro1 tends to localize at MAM sites, the decrease of MAM might cause mitochondrial axonal transport reduction due to reduced Miro1-linkage [49]. Similarly, mitochondrial transport defects have also been reported for other ALS genes including vesicle-associated membrane protein-associated protein B/C (VAPB) and C9orf72 genes both in primary cultured neurons and transgenic flies [25]. ALS mutant VAPB interferes with anterograde mitochondrial axonal transport through disrupting Ca2+ homeostasis and affecting the amounts of tubulin associated with the Miro1/kinesin-1 complex [25, 26]. The C9orf72 repeat expansion can cause a severe disruption of mitochondrial transport but only a slight inhibition of vesicle transport. Although the exact mechanisms are not clear yet, evidence showed that the toxicity comes from the DPRs translated from the hexanucleotide repeats present in C9orf72 [26].

ER stress is a widely observed pathological change in different ALS models [27]. The ER is responsible for protein synthesis and quality control. Misfolded proteins are one of the earliest finding in models based on SOD1 mutations and later on in other ALS subtypes [27]. In normal conditions, the ER can identify the misfolded protein and trigger the unfolded protein response (UPR) to clear these proteins [27]. While under ER stress, the misfolded proteins will be accumulated without a proper UPR process [27]. It has been suggested that the ER stress can also cause an axonopathy and an irregular microtubule distribution [28]. This has been highlighted by the discovery that mutations in two major genes called PDLA1 and PDLA3 that code ER chaperons are linked with ALS [28]. The ER chaperones or protein disulfide isomerases (PDIs) play a pivotal role in the UPR process [27]. Expression of mutant PDIs in motor neurons affects dendrite outgrowth and causes motor defects in mice [26, 28].

TDP-43 aggregation has been identified as the most prevalent clinical pathological hallmark of ALS patients. In addition, the coding gene TARDBP is an ALS-causing gene. TDP-43 aggregation also widely occurs in other familial as well as sporadic ALS patients. Axonal transport defects have been observed in different model systems of ALS with TDP-43 aggregation [29, 30]. TDP-43 mutations impair mRNA transport in transgenic Drosophila, primary cultured mouse cortical neurons and stem cell-derived motor neurons from ALS patients [29, 30]. Impaired anterograde axonal transport of microtubule plus tip proteins has been

**139**

*Targeting Axonal Transport: A New Therapeutic Avenue for ALS*

with the microtubule-based transport machinery [32].

observed in primary cultured rat cortical neurons [29]. It has been suggested that cytoplasmic TDP-43 aggregation impairs the cytoskeletal integrity and results in transport deficits [29, 30]. While another study has shown that age-dependent organelle transport defects in iPSC-derived motor neurons from ALS patients carrying TARDBP mutations is independent from TDP-43 aggregation [31], thus, a clear interplay between TDP-43 aggregation and axonal transport still needs to be clarified. Recently, we found that arginine-rich dipeptide repeat proteins (DPRs), which are the pathological translational products from C9orf72 repeat expansion, can cause axonal transport defects in human stem cell-derived motor neurons [32]. We found that several components of the axonal transport machinery interact with arginine-rich DPRs both in vitro and in vivo. It has been proposed that argininerich DPRs might directly affect axonal transport through an inhibitory interaction

DNA damage has been recently proposed as an early pathological change in ALS patients [33]. With the fact that TDP-43 and FUS are DNA-/RNA-binding proteins, the mutations in these genes cause insufficient DNA damage repair and result in motor neuron degeneration [33]. Both DNA damage and distal axonal transport defects have been observed in ALS patients carrying FUS mutations [34, 35]. It has been proposed that DNA damage might play a role in axonal transport defects [34]. When DNA damage is induced in cultured motor neurons, axonal transport defects occur thereafter [34]. In line with this, improving the DNA damage repair process by inhibiting poly(ADP-ribose) glycohydrolase (PARG) shows a rescue of axonal transport defects [34]. Although the exact underling mechanism is not clear yet, it is very likely that DNA damage might induce universal transcriptional changes of

some genes that produce proteins that are involved in axonal transport.

**3. Therapeutic strategies targeting axonal transport in ALS**

**3.1 Restoring the tracks: approaches to modulate microtubule dynamics**

devised to target the system at different levels.

and to extend the survival of SOD1G93A mice [43].

As summarized above, different molecular mechanisms underlie axonal transport deficits in ALS. Based on these evidences, therapeutic strategies to restore axonal transport deficits have started to emerge. Given the complexity of the axonal transport machinery and its regulatory mechanisms, multiple approaches have been

Microtubule-stabilizing agents are currently in clinical use as chemotherapeutic drugs [36]. Compounds that modulate microtubule stability have shown promising results also in the context of neurodegeneration. Epothilone D, for example, has shown beneficial effects in several models of Alzheimer's disease [37, 38]. Because of these findings, Epothilone D underwent a clinical phase 1 trial investigation in patients with mild Alzheimer's disease (NCT01492374, NCT01966666) [39]. Beneficial effects have been reported also for Parkinson's disease [40] and hereditary spastic paraplegia (HSP) [41]. Epothilone D was shown to protect the soma and distal axon of spinal motor neurons early in the disease course of the SOD1G93A model mouse of ALS [42]. However, this was not associated with improved motor performance or survival [42]. While another microtubule-stabilizing agent, Noscapine, was shown to restore axonal transport, to delay the onset of symptoms

Pharmacological agents that increase the level of microtubule acetylation have been also used to rescue axonal transport deficits. Acetylated microtubules are considered to be stable, long-lived microtubules [44]. Although the

*DOI: http://dx.doi.org/10.5772/intechopen.91963*

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

hexanucleotide repeats present in C9orf72 [26].

motor defects in mice [26, 28].

contractions [20]. Mitochondria play a pivotal role as the energy supply center in cells. Abnormal mitochondrial morphology and function are observed in postmortem tissues from ALS patients as well as in different ALS animal models [21]. Mitochondrial transport is important to clear the damaged mitochondria and maintain sufficient energy supply from the soma side to the distal side in motor neurons [21]. In line with this, fast motor neurons that have the highest needs of ATP, are more severely affected in ALS compared to slow motor neurons [22]. Therefore, the interplay between mitochondria quality control and transport is crucial for motor neuron survival. Mitochondrial movement along microtubules is controlled by a large complex containing kinesin, dynein, mitochondrial Rho (Miro), and milton [23]. Milton is an adaptor protein that connects Miro and motor proteins. Miro is a GTPase localized to the outer

membrane of mitochondria. Miro is regulated by PINK/Parkin, which are genetic modifiers of FUS-induced neurodegeneration [24]. Axonal transport defects have been observed in ALS patient-derived motor neurons and FUS transgenic flies. Decreased expression of either PINK or Parkin is beneficial for reversing the locomotive defects and enhancing the survival of FUS transgenic flies [24]. In addition, overexpression of FUS also increased the ubiquitination of the Miro1 protein [24]. We have observed that mitochondria-associated ER membranes (MAM) are significantly decreased in motor neurons carrying FUS mutation [49]. As Miro1 tends to localize at MAM sites, the decrease of MAM might cause mitochondrial axonal transport reduction due to reduced Miro1-linkage [49]. Similarly, mitochondrial transport defects have also been reported for other ALS genes including vesicle-associated membrane protein-associated protein B/C (VAPB) and C9orf72 genes both in primary cultured neurons and transgenic flies [25]. ALS mutant VAPB interferes with anterograde mitochondrial axonal transport through disrupting Ca2+ homeostasis and affecting the amounts of tubulin associated with the Miro1/kinesin-1 complex [25, 26]. The C9orf72 repeat expansion can cause a severe disruption of mitochondrial transport but only a slight inhibition of vesicle transport. Although the exact mechanisms are not clear yet, evidence showed that the toxicity comes from the DPRs translated from the

ER stress is a widely observed pathological change in different ALS models [27]. The ER is responsible for protein synthesis and quality control. Misfolded proteins are one of the earliest finding in models based on SOD1 mutations and later on in other ALS subtypes [27]. In normal conditions, the ER can identify the misfolded protein and trigger the unfolded protein response (UPR) to clear these proteins [27]. While under ER stress, the misfolded proteins will be accumulated without a proper UPR process [27]. It has been suggested that the ER stress can also cause an axonopathy and an irregular microtubule distribution [28]. This has been highlighted by the discovery that mutations in two major genes called PDLA1 and PDLA3 that code ER chaperons are linked with ALS [28]. The ER chaperones or protein disulfide isomerases (PDIs) play a pivotal role in the UPR process [27]. Expression of mutant PDIs in motor neurons affects dendrite outgrowth and causes

TDP-43 aggregation has been identified as the most prevalent clinical pathological hallmark of ALS patients. In addition, the coding gene TARDBP is an ALS-causing gene. TDP-43 aggregation also widely occurs in other familial as well as sporadic ALS patients. Axonal transport defects have been observed in different model systems of ALS with TDP-43 aggregation [29, 30]. TDP-43 mutations impair mRNA transport in transgenic Drosophila, primary cultured mouse cortical neurons and stem cell-derived motor neurons from ALS patients [29, 30]. Impaired anterograde axonal transport of microtubule plus tip proteins has been

**138**

observed in primary cultured rat cortical neurons [29]. It has been suggested that cytoplasmic TDP-43 aggregation impairs the cytoskeletal integrity and results in transport deficits [29, 30]. While another study has shown that age-dependent organelle transport defects in iPSC-derived motor neurons from ALS patients carrying TARDBP mutations is independent from TDP-43 aggregation [31], thus, a clear interplay between TDP-43 aggregation and axonal transport still needs to be clarified. Recently, we found that arginine-rich dipeptide repeat proteins (DPRs), which are the pathological translational products from C9orf72 repeat expansion, can cause axonal transport defects in human stem cell-derived motor neurons [32]. We found that several components of the axonal transport machinery interact with arginine-rich DPRs both in vitro and in vivo. It has been proposed that argininerich DPRs might directly affect axonal transport through an inhibitory interaction with the microtubule-based transport machinery [32].

DNA damage has been recently proposed as an early pathological change in ALS patients [33]. With the fact that TDP-43 and FUS are DNA-/RNA-binding proteins, the mutations in these genes cause insufficient DNA damage repair and result in motor neuron degeneration [33]. Both DNA damage and distal axonal transport defects have been observed in ALS patients carrying FUS mutations [34, 35]. It has been proposed that DNA damage might play a role in axonal transport defects [34]. When DNA damage is induced in cultured motor neurons, axonal transport defects occur thereafter [34]. In line with this, improving the DNA damage repair process by inhibiting poly(ADP-ribose) glycohydrolase (PARG) shows a rescue of axonal transport defects [34]. Although the exact underling mechanism is not clear yet, it is very likely that DNA damage might induce universal transcriptional changes of some genes that produce proteins that are involved in axonal transport.

### **3. Therapeutic strategies targeting axonal transport in ALS**

As summarized above, different molecular mechanisms underlie axonal transport deficits in ALS. Based on these evidences, therapeutic strategies to restore axonal transport deficits have started to emerge. Given the complexity of the axonal transport machinery and its regulatory mechanisms, multiple approaches have been devised to target the system at different levels.

#### **3.1 Restoring the tracks: approaches to modulate microtubule dynamics**

Microtubule-stabilizing agents are currently in clinical use as chemotherapeutic drugs [36]. Compounds that modulate microtubule stability have shown promising results also in the context of neurodegeneration. Epothilone D, for example, has shown beneficial effects in several models of Alzheimer's disease [37, 38]. Because of these findings, Epothilone D underwent a clinical phase 1 trial investigation in patients with mild Alzheimer's disease (NCT01492374, NCT01966666) [39]. Beneficial effects have been reported also for Parkinson's disease [40] and hereditary spastic paraplegia (HSP) [41]. Epothilone D was shown to protect the soma and distal axon of spinal motor neurons early in the disease course of the SOD1G93A model mouse of ALS [42]. However, this was not associated with improved motor performance or survival [42]. While another microtubule-stabilizing agent, Noscapine, was shown to restore axonal transport, to delay the onset of symptoms and to extend the survival of SOD1G93A mice [43].

Pharmacological agents that increase the level of microtubule acetylation have been also used to rescue axonal transport deficits. Acetylated microtubules are considered to be stable, long-lived microtubules [44]. Although the mechanisms and functional consequences of microtubule acetylation is not fully understood [44], it has been suggested that acetylation of α-tubulin promotes the recruitment of molecular motors kinesin-1 and dynein [45, 46], indicating that boosting the level of microtubule acetylation might positively affect intracellular transport. Strong evidence has shown that inhibition of HDAC6, a major tubulin de-acetylating enzyme, stimulates intracellular transport of different cargoes in several models [45–50].

HDAC6 belongs to the histone deacetylases (HDACs) family, and, unlike the other HDACs, HDAC6 is mainly localized in the cytoplasm where it associates with microtubules and with the dynein-dynactin motor complex containing p150glued [51]. In line with these observations, HDAC6 has been implicated in the regulation of cytoskeletal stability, intracellular transport, and cell motility [51, 52]. The beneficial effect of HDAC6 inhibitors has been shown in a broad variety of neurodegenerative diseases. For example, the inhibition of HDAC6 by trichostatin (TSA) increases microtubule acetylation and rescues axonal transport deficit in primary neurons carrying the LRRK2 mutation, which is the most common genetic causes of Parkinson's disease. In addition, in vivo knockdown of HDAC6 and administration of TSA restore locomotor deficits caused by LRRK2 mutation in a Drosophila model [48]. Inhibition of HDAC6 by TSA or Tubastatin A (TubA) restores the levels of acetylated α-tubulin and corrects the axonal transport defects in a mutant HSPB1-induced Charcot-Marie-Tooth disease (CMT) mouse model [47, 53]. TSA also enhances tubulin acetylation and rescues microtubule-based transport deficits observed in Huntington's disease (HD) mutant cells [46]. However, despite the increased microtubule acetylation, the loss of HDAC6 did not rescue neurodegenerative phenotypes and deficits in motor coordination in a HD mouse model [54]. In contrast, genetic deletion of HDAC6 significantly slows disease progression and extends survival of the mutant SOD1G93A mouse model of ALS [55]. The therapeutic potential of HDAC6 inhibition in ALS has been further investigated in FUS iPSC-derived motor neurons [49]. Both TubA and ACY-738 HDAC6 inhibitors rescue the axonal transport deficit in ALS patient-derived motor neurons. This beneficial effect on intracellular transport was further confirmed using HDAC6 antisense oligonucleotides (ASOs) [49]. Furthermore, HDAC6 inhibition increases the acetylation level of α-tubulin in patient-derived motor neurons [49].

Overall, targeting microtubules might represent an interesting therapeutic target in ALS. In particular, modulating the acetylation levels of α-tubulin might be beneficial in restoring axonal transport deficits observed early in the disease course of ALS. HDAC6 inhibitors have shown promising results in the context of ALS; however additional studies are required. For instance, it has been shown that HDAC6 plays an important role in autophagy by promoting the clearance of protein aggregates [56] including mutant SOD1 [57–59]. In this regard, inhibitors of the deacetylation function of HDAC6 that leave the other functions unhampered need to be further validated in the available ALS disease models as they might represent an interesting therapeutic approach.

#### **3.2 Restoring the motors: the role of kinases**

Several kinases can directly modulate axonal transport through phosphorylation of motors, adapters, and cargoes [60]. Deregulation of axonal transport by protein kinases has been associated to ALS; therefore the possibility of targeting protein kinases has started to emerge as a novel therapeutic avenue.

An abnormal activation of p38 MAP kinase (MAPK) was reported in mutant SOD1 mice [61–63]. It has been shown that active p38 MAPK phosphorylates kinesin-1, leading to impaired translocation of kinesin-1 along axonal microtubules

**141**

*Targeting Axonal Transport: A New Therapeutic Avenue for ALS*

and inhibition of fast axonal transport [62]. The p38 MAPK inhibitor, SB203580, completely inhibits mutant SOD1-induced apoptosis of motor neurons in vitro [61]. In addition, Semapimod, a p38 MAPK inhibitor potentially suitable for clinical purposes, protects motor neurons from degeneration in vivo, although it only mildly extends the survival of SOD1G93A mice [61]. Importantly, a more recent study has shown that p38 MAPK is directly responsible for SOD1G93A-induced axonal transport deficits in motor neurons, further strengthening the link between p38 MAPK and axonal transport [63]. Both genetic and acute pharmacological inhibitions of p38 MAPK rescue axonal transport deficits in motor neurons of SOD1G93A mice both in vivo and in vitro [63]. However, long-term treatment with the p38 MAPKα inhibitor SB239063 (a potentially interesting compound given the ability to cross the blood-brain barrier) has shown significant toxic side effects and, probably because of that, failed to improve axonal transport and muscle function in SOD1G93A mice [63]. Therefore, additional investigation is required to evaluate and optimize

Overactivation of GSK3β has been found in the brain and spinal cord of SOD1G93A mice as well as in spinal cord samples from sporadic ALS patients [64–67]. Inhibition of GSK3β was protective in SOD1G93A transgenic mice in some studies [67, 68], but these findings were not confirmed in later ones [69, 70]. Therefore, at present, the involvement of GSK3β in ALS remains controversial. Aberrant activation of cyclin-dependent kinase 5 (CDK5) has been reported in the spinal cord of mouse models of ALS [71–73]. Hyperactivation of CDK5 mis-regulates transport of several cargoes via the Lis1/Ndel1 complex, which directly regulates dynein activity [72]. Reduction of CDK5 activity in neurons from SOD1G93A mice by roscovitine rescues transport deficits [72]. Similarly, inhibition of CDK5 by overexpression of calpastatin improves motor axon survival, delays disease onset, and increases

Overall, modulating kinase activation seems to be beneficial for the transport defects in ALS; however most of these studies focus on the SOD1G93A mouse model. It remains to be determined whether targeting kinases is beneficial also in the context of other ALS-causing mutations. In addition, many protein kinases have multiple targets and are involved in several cellular processes. Therefore a more detailed understanding of kinase signaling pathways is required to effectively

Axonal transport defects have been strongly linked with ALS pathogenesis. Different therapeutic strategies have been tested in ALS disease models, showing prospective results. However, a deeper understanding of the pathological mechanisms that are responsible for axonal transport deficit is required to properly target axonal transport in ALS. Most of the therapeutic strategies proposed have been mainly tested in the SOD1G93A transgenic mice, and it is still unknown whether they are beneficial in other familial ALS models. In addition, the use of iPSC-derived motor neurons could potentially help to validate whether these compounds might

Other cellular mechanisms have been shown to be altered in ALS. Therefore it is not easy to clarify whether altered axonal transport causes neuronal degeneration or whether neuronal dysfunction, due to other upstream mechanisms, ultimately leads to malfunctioning of axonal transport. However, the evidence that axonal transport is an early identifiable phenotype in several in vivo models suggests that targeting axonal transport needs to be addressed for an effective treatment.

*DOI: http://dx.doi.org/10.5772/intechopen.91963*

the long-term effects of this approach.

survival of SOD1G93A mice [73].

implement this strategy.

**4. Conclusions and perspectives**

also be beneficial for the sporadic ALS cases.

#### *Targeting Axonal Transport: A New Therapeutic Avenue for ALS DOI: http://dx.doi.org/10.5772/intechopen.91963*

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

several models [45–50].

mechanisms and functional consequences of microtubule acetylation is not fully understood [44], it has been suggested that acetylation of α-tubulin promotes the recruitment of molecular motors kinesin-1 and dynein [45, 46], indicating that boosting the level of microtubule acetylation might positively affect intracellular transport. Strong evidence has shown that inhibition of HDAC6, a major tubulin de-acetylating enzyme, stimulates intracellular transport of different cargoes in

HDAC6 belongs to the histone deacetylases (HDACs) family, and, unlike the other HDACs, HDAC6 is mainly localized in the cytoplasm where it associates with microtubules and with the dynein-dynactin motor complex containing p150glued [51]. In line with these observations, HDAC6 has been implicated in the regulation of cytoskeletal stability, intracellular transport, and cell motility [51, 52]. The beneficial effect of HDAC6 inhibitors has been shown in a broad variety of neurodegenerative diseases. For example, the inhibition of HDAC6 by trichostatin (TSA) increases microtubule acetylation and rescues axonal transport deficit in primary neurons carrying the LRRK2 mutation, which is the most common genetic causes of Parkinson's disease. In addition, in vivo knockdown of HDAC6 and administration of TSA restore locomotor deficits caused by LRRK2 mutation in a Drosophila model [48]. Inhibition of HDAC6 by TSA or Tubastatin A (TubA) restores the levels of acetylated α-tubulin and corrects the axonal transport defects in a mutant HSPB1-induced Charcot-Marie-Tooth disease (CMT) mouse model [47, 53]. TSA also enhances tubulin acetylation and rescues microtubule-based transport deficits observed in Huntington's disease (HD) mutant cells [46]. However, despite the increased microtubule acetylation, the loss of HDAC6 did not rescue neurodegenerative phenotypes and deficits in motor coordination in a HD mouse model [54]. In contrast, genetic deletion of HDAC6 significantly slows disease progression and extends survival of the mutant SOD1G93A mouse model of ALS [55]. The therapeutic potential of HDAC6 inhibition in ALS has been further investigated

in FUS iPSC-derived motor neurons [49]. Both TubA and ACY-738 HDAC6

the acetylation level of α-tubulin in patient-derived motor neurons [49].

an interesting therapeutic approach.

**3.2 Restoring the motors: the role of kinases**

kinases has started to emerge as a novel therapeutic avenue.

inhibitors rescue the axonal transport deficit in ALS patient-derived motor neurons. This beneficial effect on intracellular transport was further confirmed using HDAC6 antisense oligonucleotides (ASOs) [49]. Furthermore, HDAC6 inhibition increases

Overall, targeting microtubules might represent an interesting therapeutic target in ALS. In particular, modulating the acetylation levels of α-tubulin might be beneficial in restoring axonal transport deficits observed early in the disease course of ALS. HDAC6 inhibitors have shown promising results in the context of ALS; however additional studies are required. For instance, it has been shown that HDAC6 plays an important role in autophagy by promoting the clearance of protein aggregates [56] including mutant SOD1 [57–59]. In this regard, inhibitors of the deacetylation function of HDAC6 that leave the other functions unhampered need to be further validated in the available ALS disease models as they might represent

Several kinases can directly modulate axonal transport through phosphorylation of motors, adapters, and cargoes [60]. Deregulation of axonal transport by protein kinases has been associated to ALS; therefore the possibility of targeting protein

An abnormal activation of p38 MAP kinase (MAPK) was reported in mutant SOD1 mice [61–63]. It has been shown that active p38 MAPK phosphorylates kinesin-1, leading to impaired translocation of kinesin-1 along axonal microtubules

**140**

and inhibition of fast axonal transport [62]. The p38 MAPK inhibitor, SB203580, completely inhibits mutant SOD1-induced apoptosis of motor neurons in vitro [61]. In addition, Semapimod, a p38 MAPK inhibitor potentially suitable for clinical purposes, protects motor neurons from degeneration in vivo, although it only mildly extends the survival of SOD1G93A mice [61]. Importantly, a more recent study has shown that p38 MAPK is directly responsible for SOD1G93A-induced axonal transport deficits in motor neurons, further strengthening the link between p38 MAPK and axonal transport [63]. Both genetic and acute pharmacological inhibitions of p38 MAPK rescue axonal transport deficits in motor neurons of SOD1G93A mice both in vivo and in vitro [63]. However, long-term treatment with the p38 MAPKα inhibitor SB239063 (a potentially interesting compound given the ability to cross the blood-brain barrier) has shown significant toxic side effects and, probably because of that, failed to improve axonal transport and muscle function in SOD1G93A mice [63]. Therefore, additional investigation is required to evaluate and optimize the long-term effects of this approach.

Overactivation of GSK3β has been found in the brain and spinal cord of SOD1G93A mice as well as in spinal cord samples from sporadic ALS patients [64–67]. Inhibition of GSK3β was protective in SOD1G93A transgenic mice in some studies [67, 68], but these findings were not confirmed in later ones [69, 70]. Therefore, at present, the involvement of GSK3β in ALS remains controversial. Aberrant activation of cyclin-dependent kinase 5 (CDK5) has been reported in the spinal cord of mouse models of ALS [71–73]. Hyperactivation of CDK5 mis-regulates transport of several cargoes via the Lis1/Ndel1 complex, which directly regulates dynein activity [72]. Reduction of CDK5 activity in neurons from SOD1G93A mice by roscovitine rescues transport deficits [72]. Similarly, inhibition of CDK5 by overexpression of calpastatin improves motor axon survival, delays disease onset, and increases survival of SOD1G93A mice [73].

Overall, modulating kinase activation seems to be beneficial for the transport defects in ALS; however most of these studies focus on the SOD1G93A mouse model. It remains to be determined whether targeting kinases is beneficial also in the context of other ALS-causing mutations. In addition, many protein kinases have multiple targets and are involved in several cellular processes. Therefore a more detailed understanding of kinase signaling pathways is required to effectively implement this strategy.

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

Axonal transport defects have been strongly linked with ALS pathogenesis. Different therapeutic strategies have been tested in ALS disease models, showing prospective results. However, a deeper understanding of the pathological mechanisms that are responsible for axonal transport deficit is required to properly target axonal transport in ALS. Most of the therapeutic strategies proposed have been mainly tested in the SOD1G93A transgenic mice, and it is still unknown whether they are beneficial in other familial ALS models. In addition, the use of iPSC-derived motor neurons could potentially help to validate whether these compounds might also be beneficial for the sporadic ALS cases.

Other cellular mechanisms have been shown to be altered in ALS. Therefore it is not easy to clarify whether altered axonal transport causes neuronal degeneration or whether neuronal dysfunction, due to other upstream mechanisms, ultimately leads to malfunctioning of axonal transport. However, the evidence that axonal transport is an early identifiable phenotype in several in vivo models suggests that targeting axonal transport needs to be addressed for an effective treatment.

#### *Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

Relatedly, studies are ongoing to improve the specificity and the ability of drugs to cross the blood–brain barrier. The advent of techniques such as gene therapy and antisense oligonucleotides might speed up the process of effectively targeting axonal transport in patients. The feasibility of gene therapy to ameliorate axonal transport deficits has been already successfully shown in ALS mice [58]. In addition, ASOs that specifically target HDAC6 have been already tested in iPSCderived motor neurons of FUS patients showing positive effects on transport [49]. Therefore, these approaches might represent an interesting area for future research and might help to identify effective therapeutic strategies.

#### **Author details**

Wenting Guo1,2,3, Laura Fumagalli1,2 and Ludo Van Den Bosch1,2\*

1 Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), KU Leuven, University of Leuven, Leuven, Belgium

2 Center for Brain & Disease Research, Laboratory of Neurobiology, VIB, Leuven, Belgium

3 Stem Cell Institute (SCIL), KU Leuven, Leuven, Belgium

\*Address all correspondence to: ludo.vandenbosch@kuleuven.vib.be

© 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.

**143**

brain/awp325

*Targeting Axonal Transport: A New Therapeutic Avenue for ALS*

[11] Smith BN et al. Exome-wide rare variant analysis identifies TUBA4A mutations associated with familial ALS. Neuron. 2014;**84**:324-331

[12] Perrone F et al. Investigating the role of ALS genes CHCHD10 and TUBA4A in Belgian FTD-ALS spectrum patients. Neurobiology of Aging. 2017;**51**:177.e9-177.e16. DOI: 10.1016/j.

[13] Pérez-Brangulí F et al. Dysfunction of spatacsin leads to axonal pathology in SPG11-linked hereditary spastic paraplegia. Human Molecular Genetics.

[14] Mignogna ML, D'Adamo P. Critical importance of RAB proteins for synaptic function. Small GTPases. 2018;**9**:145-157

endosomal motility and degradation by amyotrophic lateral sclerosis 2/alsin.

[16] Lai C et al. Amyotrophic lateral sclerosis 2-deficiency leads to neuronal degeneration in amyotrophic lateral sclerosis through altered AMPA receptor trafficking. The Journal of Neuroscience. 2006;**26**:11798-11806

[17] Toth RP, Atkin JD. Dysfunction of optineurin in amyotrophic lateral sclerosis and glaucoma. Frontiers in

[18] Song W et al. Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity. Nature Medicine. 2011;**17**:377-382. DOI:

[19] Farg MA et al. C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking. Human

Molecular Genetics. 2014;**23**:3579-3595

Immunology. 2018;**9**:1017

10.1038/nm.2313

neurobiolaging.2016.12.008

[15] Lai C et al. Regulation of

Molecular Brain. 2009;**2**:1-12

2014;**23**:4859-4874

*DOI: http://dx.doi.org/10.5772/intechopen.91963*

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[2] De Vos KJ, Hafezparast M. Neurobiology of axonal transport defects in motor neuron diseases: Opportunities for translational research? Neurobiology of Disease.

[3] Dadon-Nachum M, Melamed E, Offen D. The 'dying-back' phenomenon of motor neurons in ALS. Journal of Molecular Neuroscience. 2011;**43**:470-477

[4] Taylor JP, Brown RH, Cleveland DW. Decoding ALS: From genes to mechanism.

[5] Nicolas A et al. Genome-wide analyses identify KIF5A as a novel ALS gene. Neuron. 2018;**97**:1268-1283.e6

[6] Brenner D et al. Hot-spot KIF5A mutations cause familial ALS. Brain.

[7] Burk K, Pasterkamp RJ. Disrupted neuronal trafficking in amyotrophic lateral sclerosis. Acta Neuropathologica.

[8] Konno T et al. DCTN1-related

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neurodegeneration: Perry syndrome and beyond. 2017;**41**:14-24. DOI: 10.1016/j. parkreldis.2017.06.004.DCTN1

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*Targeting Axonal Transport: A New Therapeutic Avenue for ALS DOI: http://dx.doi.org/10.5772/intechopen.91963*

#### **References**

*Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges*

and might help to identify effective therapeutic strategies.

Relatedly, studies are ongoing to improve the specificity and the ability of drugs

to cross the blood–brain barrier. The advent of techniques such as gene therapy and antisense oligonucleotides might speed up the process of effectively targeting axonal transport in patients. The feasibility of gene therapy to ameliorate axonal transport deficits has been already successfully shown in ALS mice [58]. In addition, ASOs that specifically target HDAC6 have been already tested in iPSCderived motor neurons of FUS patients showing positive effects on transport [49]. Therefore, these approaches might represent an interesting area for future research

Wenting Guo1,2,3, Laura Fumagalli1,2 and Ludo Van Den Bosch1,2\*

3 Stem Cell Institute (SCIL), KU Leuven, Leuven, Belgium

provided the original work is properly cited.

Institute (LBI), KU Leuven, University of Leuven, Leuven, Belgium

\*Address all correspondence to: ludo.vandenbosch@kuleuven.vib.be

1 Department of Neurosciences, Experimental Neurology, and Leuven Brain

2 Center for Brain & Disease Research, Laboratory of Neurobiology, VIB, Leuven,

© 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,

**142**

**Author details**

Belgium

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and protein phosphatase

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journal.pone.0020696

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2015;**15**:339-349

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*Edited by Muralidhar L. Hegde*

A flurry of recent research on the role of the RNA/DNA-binding proteins TDP-43 and FUS as well as a dozen other factors (e.g., C9ORF72 and profilin) has led to a new paradigm in our understanding of the pathobiology of the motor neuron disease, Amyotrophic Lateral Sclerosis (ALS). How these factors trigger neuromuscular dysfunction is critical for developing more effective ALS therapeutics. The 'gain-oftoxicity' or 'loss-of-function' of these etiological factors is a key question. Recent studies on the imbalance in genome damage versus repair have opened avenues for potential DNA repair-based therapeutics. This book highlights emerging science in the area of ALS and discusses key approaches and mechanisms essential for developing a cure for ALS.

Published in London, UK © 2020 IntechOpen © bestdesigns / iStock

Amyotrophic Lateral Sclerosis - Recent Advances and Therapeutic Challenges

Amyotrophic Lateral Sclerosis

Recent Advances and Therapeutic Challenges

*Edited by Muralidhar L. Hegde*