**5. Neurodegenerative diseases with deficient DNA repair**

DNA repair processes in mammalian cells normally involve extensive chromatin remodeling. This remodeling involves epigenetic modifications of chromatin that are usually transient, but may persist. When a protein necessary for proper DNA repair is mutationally defective, epigenetic alterations that deviate from a normal functional pattern can be introduced. In a number of neurodegenerative diseases, such epigenetic alterations appear to significantly underlie the disease phenotype.

We describe below four neurodegenerative diseases, ataxia telangiectasia, Huntington's disease, Aicardi-Goutières syndrome and Cockayne syndrome that have inherited genetic deficiencies due to mutations in genes necessary for DNA repair. We briefly summarize for each disease, the notable neurodegenerative features of the disease, the DNA repair processes that are defective, and the accompanying epigenetic alterations that likely have a role in the etiology of the disease. On the basis of the evidence reviewed, it appears that the proper functioning of the nervous system depends on DNA repair processes that not only restore damaged DNA sequence information, but also promote normal gene expression through the maintenance of an appropriate pattern of epigenetic markers.

#### **5.1 Ataxia telangiectasia (AT)**

AT is a multisystem disease characterized by neurodegeneration in the central nervous system. Certain regions of the brain including the cerebellum, are adversely affected in AT resulting in difficulty with movement and coordination. There is also an association with microcephaly. AT is inherited as an autosomal recessive trait, and is caused by mutation of the gene *AT mutated* (*ATM*) that encodes a serine/threonine protein kinase. The wild-type ATM protein has a key role in the DNA damage response. ATM is part of a molecular complex that signals the presence of oxidative DNA damage, including double-strand breaks, and facilitates subsequent repair [45]. *The Role of DNA Repair and the Epigenetic Markers Left after Repair in Neurologic Functions… DOI: http://dx.doi.org/10.5772/intechopen.83477* 

ATM protein is employed in chromatin remodeling and in epigenetic alterations that are required for repairing DNA double-strand breaks [45]. ATM mutation causes defects in epigenetic regulation that likely contribute to the rapid postnatal degeneration of the cerebellum that underlies the progressive ataxia observed in AT [45]. AT is associated with histone acetylation alterations, including significant decreases in histone H3 and H4 acetylation [46]. ATM regulates neuron specific epigenetic alterations involving histone deacetylase-4 [45]. In ATM mutant neurons, misallocation of histone deacetylase-4 represses transcription of genes important in neuronal function and synaptic maintenance [45].

#### **5.2 Huntington's disease (HD)**

HD typically occurs in midlife. The symptoms include progressive movement disorder, cognitive dysfunction and psychiatric impairment. HD is inherited in an autosomal dominant manner. HD results from an unstable expansion of CAG repeat sequences in exon 1 of the *huntingtin* gene (*HTT*). Several lines of evidence link the HTT protein to repair of DNA damage [47]. HTT is a scaffolding protein that directly participates in oxidative DNA damage repair [48]. The ATM protein recruits HTT to sites of DNA damage. HTT co-localizes with, and acts as a scaffold for, proteins of the DNA damage response pathway in response to oxidative stress. The fibroblasts of HD patients with expanded CAG repeats have deficient oxidative damage repair [48].

Impaired DNA repair in HD also appears to cause deleterious epigenetic alterations that are linked to transcriptional dysregulation. Individuals with HD experience accelerated epigenetic aging of the brain, particularly in the frontal lobe, cingulate gyrus and the parietal lobe. This process is associated with substantial changes in brain DNA methylation levels [49]. Also post-translational modifications of histone proteins are significantly altered in HD patients as well as in HD cellular and animal models [50].

#### **5.3 Aicardi-Goutières syndrome (AGS)**

 AGS is characterized by early onset, often in early infancy. Features of AGS include neurological dysfunction, psychomotor retardation, seizures, and microcephaly [51]. AGS is an inherited disease and most cases are inherited in an autosomal recessive pattern. AGS arises from mutations in genes encoding proteins TREX 1 (AGS1), RNase H2 (AGS2, 3 and 4) and SAMHD1 (AGS5) [51]. The incorporation of ribonucleotide triphosphates (rNTPs) into DNA is perhaps the most common type of endogenous DNA damage encountered in proliferating cells [52]. Removal of rNTPs incorporated into DNA is referred to as rNTP excision repair. Key players in rNTP excision repair are TREX1 and RNase H2 [52]. RNase H2 is the predominant nuclear enzyme to hydrolyze the RNA strand of RNA/DNA hybrids [53].

*TREX1*, *RNASEH2* and *SAMHD1* mutations in AGS cells cause common molecular abnormalities including increased levels of RNA:DNA hybrid species and genomewide DNA hypomethylation, a substantial epigenetic perturbance [51]. AGS2 and AGS4 mutant cells display about a 20% reduction in genomic methylation levels overall, and this reduction is spread along the length of entire chromosomes impacting nearly all compartments including genic, intergenic unique and repeat regions [51].

#### **5.4. Cockayne syndrome (CS)**

Due to impaired neurological development, individuals with CS are characteristically mentally retarded and have microcephaly. CS is caused by mutations in the *CSA* and *CSB* genes. CS is inherited as an autosomal recessive trait. Transcription of DNA can be inhibited by DNA damage, and restoration of transcription requires removal

of blocking damages by a sub-pathway of nucleotide excision repair that specifically removes transcription-blocking DNA damages. This sub-pathway is referred to as transcription-coupled DNA repair (TCR). In mammals, TCR depends on the CSA and CSB proteins. More than 70% of CS syndrome patients have a mutation in the *CSB* gene. CSA and CSB proteins regulate recruitment of chromatin remodeling and repair factors to stalled RNA polymerase at sites of DNA damage [54, 55].

 Among the proteins recruited by CSA and CSB are nucleotide excision repair proteins and histone acetyltransferase, an enzyme that catalyzes chromatin remodeling and epigenetic alteration [54]. CSB can slide histones along DNA and histone chaperone proteins that accept and donate histones can greatly facilitate this process [55]. Nucleosome remodeling by CSB is important for TCR, and inability to efficiently mobilize nucleosomes appears to contribute to the underlying mechanism of CS [55]. The chromatin remodeling activity of CSB appears to create an epigenetic landscape that permits more efficient DNA repair or facilitates transcription resumption after repair is completed [56].

### **5.5 Perspective on the role of DNA repair in neurodegeneration**

The neurodegenerative diseases AT, HD, AGS and CS are due to mutation in genes that encode proteins employed in DNA repair. Inadequate DNA repair can lead directly to cell death and neuron depletion that may be reflected in microcephaly, as is seen in AT, AGS and CS. The defects in DNA repair also cause disruptions in the pattern of epigenetic alteration required for normal neuronal function. These epigenetic alterations likely underlie characteristic features of the disease phenotype. Thus it appears that important functions of the nervous system, including those involved in various aspects of cognition and motor function, depend on the role of intact DNA repair processes in maintaining normal patterns of epigenetic markers.

#### **5.6 Other neurodegenerative diseases deficient in DNA repair**

In addition to the four neurodegenerative diseases discussed above, there is also evidence for defective DNA repair in the neurodegenerative diseases amyotrophic lateral sclerosis [57], fragile X syndrome [58], Friedrich's ataxia [59], spinocerebellar ataxia type 1 [60], trichothiodystrophy [61], and xeroderma pigmentosum [62].

 Amyotrophic lateral sclerosis is causally linked to mutations in the gene *FUS* [57]. ALS patients with *FUS* mutations have increased neuronal DNA damage. FUS protein functions in the DNA damage response including recruitment to doublestrand breaks and homologous recombinational DNA repair. FUS protein also directly interacts with histone deacetylase 1 in response to DNA damage, and this interaction is necessary for efficient DNA repair [57].

Fragile X syndrome is a common form of inherited mental retardation. The fragile X mental retardation protein FMRP is a chromatin-binding protein that functions in the DNA damage response, likely in DNA repair [58]. Fragile X syndrome is caused by loss of expression of the *FMR1* gene, most often due to an expansion of a CGG repeat in the first exon of *FMR1*. The repeat expansion results in abnormal methylation of the promoter region which leads to transcriptional silencing of the *FMR1* gene [63].

 Friedreich ataxia, a progressive neurodegenerative disease, is caused by deficient frataxin protein resulting from downregulation of the *FXN* gene. Frataxin is employed in the repair of DNA double-strand breaks [59]. Most individuals with Friedrich ataxia have a homozygous mutation consisting of a GAA trinucleotide repeat expansion within the first intron of the FXN gene. This expansion itself may lead to downregulation of the *FXN* gene. In addition, there is a repressive

#### *The Role of DNA Repair and the Epigenetic Markers Left after Repair in Neurologic Functions… DOI: http://dx.doi.org/10.5772/intechopen.83477*

heterochromatin effect around the *FXN* gene caused by the expanded GAA repeats, consisting of high levels histone methylation of H3K9 and H3K27 [64].

 Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease characterized by progressive motor incoordination. SCA1 results from mutation in the *ATXN1* gene that leads to a pathogenic glutamine-repeat expansion in the protein ataxin-1 (ATXN1). The multifunctional protein TERA/VCP/p97 acts in DNA damage repair. Glutamine-repeat expansion mutant proteins such as mutant ATXN1 impair accumulation and function of TERA/VCP/p67 leading to an increase in unrepaired DNA double-strand breaks [60]. Also mutant *ATXN1* represses gene transcription by decreasing histone acetylation [65].

 Trichothiodystrophy (TTD) is an autosomal recessive disorder with a range of clinical neurodevelopmental features and often photosensitivity. All photosensitive TTD individuals have a mutation in the *XPB*, *XPD* or *TTDA* genes that encode subunits of the dual functional repair/transcription factor IIH (TFIIH) [61]. These individuals deficient for TFIIH are defective in nucleotide excision repair, a process that repairs transcription-blocking DNA damages, including UV induced DNA damages, thus explaining their photosensitivity. Induction of DNA damage in cells with *XPB* or *XPD* mutations that cause TTD results in reduced transient DNA strand breaks that are intermediates during DNA repair [66]. Also methylation of histone H3 (H3K9Me3) was reduced in an evaluated model promoter region [66].

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. XP has characteristic neurological manifestations, but the most prominent feature of the condition is sensitivity to sunlight resulting in a high predisposition to UV-induced skin cancer. Seven different complementation groups (genes) *XPA*, *XPB*, *XPC*, *XPD*, *XPE*, *XPF* and *XPG* encode proteins employed in nucleotide excision repair, a process that repairs bulky DNA damages including damages caused by UV-light [62]. XPF and XPG proteins are endonucleases that also trigger chromatin looping and DNA demethylation that promote accurate expression of activated genes [67].

### **6. Mental activity is associated with DNA damage and repair in the brain**

 An easy type of DNA damage to measure is the double-strand break. When a double-strand break occurs there is a rapid effect on particular histones near the break. A variant histone, H2AX, is sometimes present in histone cores, and it constitutes about 2–25% of the H2A histones in mammalian chromatin [68]. After a double-strand break, H2AX histones near the break are phosphorylated by the kinases ATM, ATR and DNA-PK [69], allowing formation of H2AX phosphorylated on serine 139 near the break. This histone is then designated γH2AX. γH2AX can be detected as soon as 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurs in 1 minute [68]. Chromatin with phosphorylated γH2AX extends to about a million base pairs on each side of a DNA double-strand break [68]. It is easy to detect γH2AX by immunohistochemistry, and these large segments of chromatin with γH2AX are called γH2AX foci.

Learning and new memories occur when mice explore a new, strange environment. This is a low level stimulation. Exploration of a novel environment increased the number of neurons with double-strand breaks in neuronal DNA as measured by γH2AX foci [70]. This occurs in different brain regions but particularly in the dentate gyrus, which is involved in spatial learning and memory. Within 24 hours of break formation, DNA repair occurs with removal of the breaks [70]. When double-strand breaks in this situation were also measured by the comet assay (another simple assay), roughly 30–40% of dentate gyrus nuclei had comet tails indicating double-strand breaks in the nuclear DNA [70].

#### **6.1 Visual stimulation**

 Another neuronal activity also caused double-strand breaks. Exposure of anesthetized mice to visual stimuli activated the primary visual cortex (V1) of the brain. One eye was exposed to visual stimuli for 15 minutes, while the other was shielded from light. One hour after the visual stimulation began, the number of cells with γH2AX foci in the stimulated contralateral V1 was roughly twice as high as that in the unstimulated ipsilateral V1 [70].

#### **6.2 Optogenetic stimulation**

 Optogenetic stimulation of a mouse striatum brain region also caused DNA double-strand breaks [70]. Transgenic mice expressing Cre-recombinase in medium spiny neurons of the dorsomedial striatum were used. The Cre-recombinase gene inserted into DNA of the striatum neurons in these mice provides a topoisomerase I like mechanism to carry out site-specific recombination events. Using this system, a viral vector was infused into the striatum, carrying a genetic segment coding for a light sensitive ChR2 protein. The ChR2 gene frequently recombined into the mouse dorsomedial striatum DNA. A glass fiber was then implanted close to the viral injection site. Two weeks later, awake mice were stimulated by light through the glass fiber. This caused neuronal activity in the dorsomedial striatum, resulting in behavioral ipsiversive rotations in mice (mice turning in a circle). The mice were then terminated and the mouse brains examined. The illuminated striata contained many more cells with γH2AX foci than the non-illuminated contralateral striata [70].

#### **6.3 Non-homologous end joining (NHEJ) repair required for long-term memory retention**

One form of long-term memory, through associative learning, is contextual fear conditioning [71]. This fear conditioning occurs, for instance, when a rodent is placed in a novel environment (a new context) and is then subjected to an electric shock (e.g. a footshock). This produces robust fear learning, shown by a strong fear response, when the rodent is placed in that context again. Contextual fear conditioning occurs very rapidly (can occur with a single event) and has a lasting effect.

Madabhushi et al. [72] subjected wild-type C57BL/6 mice to a training paradigm for contextual fear conditioning, following which they prepared hippocampal lysates and measured γH2AX levels (as a measure of double-strand breaks in DNA). Elevated γH2AX levels were detectable in hippocampal lysates within 15 minutes after exposure to the fear-conditioning paradigm.

NHEJ, which repairs double-strand breaks in DNA, appears to be needed specifically for consolidation of memory into long-term memory. Contextual fear conditioning in mice increased NHEJ repair activity in the hippocampus brain region measured at 10 and 60 minutes after training [73]. The hippocampus is important in forming memories [74].

 When NHEJ repair was active, memories were demonstrated in fear-conditioned mice at 6 and 24 hours after training. Ara-C (cytosine arabinoside) interferes with DNA synthesis. Injecting animals systemically with ara-C 1 hour before exposing them to the conditioning inhibited NHEJ repair [68]. If NHEJ repair was blocked before fear conditioning, memories of fear conditioning were substantially diminished at the 6- and 24-hour time periods tested. Thus it appears that NHEJ repair is required for memory formation. Other cognitive elements were not blocked by ara-C. Mice given ara-C and then subjected to contextual fear conditioning maintained their short-term memory (tested at 30 seconds after training) and exploratory

*The Role of DNA Repair and the Epigenetic Markers Left after Repair in Neurologic Functions… DOI: http://dx.doi.org/10.5772/intechopen.83477* 

behavior in an open field 24 hours after training. Treatment with ara-C also did not cause general malaise, motor in-coordination, sedation, or anxiety.
