Epigenetics of neurodegenerative diseases

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a motor neuron disease that involves neurogeneration. All skeletal muscles in the body are controlled by motor neurons that communicate signals from the brain to the muscle through a neuromuscular junction. When the motor neurons degenerate, the muscles no longer receive signals from the brain and begin to waste away. ALS is characterized by stiff muscles, muscle twitching, and progressive muscle weakness from muscle wasting. The parts of the body affected by early symptoms of ALS depend on which motor neurons in the body are damaged first, usually the limbs. As the disease progresses most patients are unable to walk or use their arms and eventually develop difficulty speaking, swallowing and breathing. Most patients retain cognitive function and sensory neurons are generally unaffected. Patients are often diagnosed after the age of 40 and the median survival time from onset to death is around 3–4 years. In the final stages, patients can lose voluntary control of eye muscles and often die of respiratory failure or pneumonia as a result of degeneration of the motor neurons and muscles required for breathing. Currently there is no cure for ALS, only treatments that may prolong life.

To date, multiple genes and proteins have been implicated in ALS. One of the common themes between many of these genes and their causative mutations is the presence of protein aggregates in motor neurons.[42] Other common molecular features in ALS patients are altered RNA metabolism[43] and general histone hypoacetylation.[44]

Chromossome 21

SOD1

The SOD1 gene on chromosome 21 that codes for the superoxide dismutase protein is associated with 2% of cases and is believed to be transmitted in an autosomal dominant manner.[45] Many different mutations in SOD1 have been documented in ALS patients with varying degrees of progressiveness. SOD1 protein is responsible for destroying naturally occurring, but harmful superoxide radicals produced by the mitochondria. Most of the SOD1 mutations associated with ALS are gain-of-function mutations in which the protein retains its enzymatic activity, but aggregate in motor neurons causing toxicity.[46][47] Normal SOD protein is also implicated in other cases of ALS due to potentially cellular stress.[48] An ALS mouse model through gain-of-function mutations in SOD1 has been developed.[49]

c9orf72

A gene called c9orf72 was found to have a hexanucleotide repeat in the non-coding region of the gene in association with ALS and ALS-FTD.[50] These hexanucleotide repeats may be present in up 40% of familial ALS cases and 10% of sporadic cases. C9orf72 likely functions as a guanine exchange factor for a small GTPase, but this is likely not related to the underlying cause of ALS.[51] The hexanucleotide repeats are likely causing cellular toxicity after they are spliced out of the c9orf72 mRNA transcripts and accumulate in the nuclei of affected cells.[50]

UBQLN2

The UBQLN2 gene encodes the protein ubiquilin 2 which is responsible for controlling the degradation of ubiquitinated proteins in the cell. Mutations in UBQLN2 interfere with protein degradation resulting in neurodegeneration through abnormal protein aggregation.[52] This form of ALS is X chromosome-linked and dominantly inherited and can also be associated with dementia.

ALS patients and mouse models show general histone hypoacetylation that can ultimately trigger apoptosis of cells.[53] In experiments with mice, HDAC inhibtors counteract this hypoacetylation, reactivate aberrantly down-regulated genes, and counteract apoptosis initiation.[12][54] Furthermore, HDAC inhibitors are known to prevent SOD1 protein aggregates in vitro.[55]

Sodium phenylbutyrate

Sodium phenylbutyrate treatment in a SOD1 mouse model of ALS showed improved motor performance and coordination, decreased neural atrophy and neural loss, and increased weight gain.[8][9] Release of pro-apoptotic factors was also abrogated as well as a general increase in histone acetylation.[54] A human trial using phenylbuturate in ALS patients showed some increase in histone acetylation, but the study did not report whether ALS symptoms improved with treatment.[10]

Valproic scid

Valproic acid in mice studies restored histone acetylation levels, increased levels of pro-survival factors, and mice showed improved motor performance.[56] However, while the drug delayed the onset of ALS, it did not increase lifespan or prevent denervation.[57] Human trials of valproic acid in ALS patients did not improve survival or slow progression.[11]

Trichostatin A

Trichostatin A trials in mouse ALS models restored histone acetylation in spinal neurons, decreased axon demyelination, and increased survival of mice.[12]

Alpha motor neurons are derived from the basal plate (basal lamina).

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by mutations in the SMN1 gene.[58] Symptoms vary greatly with each subset of SMA and the stage of the disease. General symptoms include overall muscle weakness and poor muscle tone including extremities and respiratory muscles leading to difficulty walking, breathing, and feeding. Depending on the type of SMA, the disease can present itself from infancy through adulthood. As SMN protein generally promotes the survival of motor neurons, mutations in SMN1 results in slow degeneration motor neurons leading to progressive system-wide muscle wasting. Specifically, over time, decreased levels of SMN protein results in gradual death of the alpha motor neurons in the anterior horn of the spinal cord and brain. Muscles depend on connections to motor neurons and the central nervous system to stimulate muscle maintenance and therefore degeneration of motor neurons and subsequent denervation of muscles lead to loss of muscle control and muscle atrophy. The muscles of the lower extremities are often affected first followed by upper extremities and sometime the muscles of respiration and mastication. In general, proximal muscle are always affected more than distal muscle.

Spinal muscular atrophy is linked to genetic mutations in the SMN1 (Survival of Motor Neuron 1) gene. The SMN protein is widely expressed in neurons and serves many functions within neurons including spliceosome construction, mRNA axon transport, neurite outgrowth during development, and neuromuscular junction formation. The causal function loss in SMA is currently unknown.

SMN1 is located in a telomeric region of human chromosome 5 and also contains SMN2 in a centromeric region. SMN1 and SMN2 are nearly identical except for a single nucleotide change in SMN2 resulting in an alternative splicing site where intron 6 meets exon 8. This single base pair change leads to only 10-20% of SMN2 transcripts resulting in fully functional SMN protein and 80-90% of transcripts leading to a truncated protein that is rapidly degraded. Most SMA patients have 2 or more copies of the SMN2 gene with more copies resulting a decrease in disease severity.[59] Most SMA patients have either point mutations or a deletion in exon 7 often leading to a protein product similar to the truncated and degraded version of the SMN2 protein. In SMA patients this small amount of functional SMN2 protein product allows for the some neurons to survive.

Although SMA is not caused by an epigenetic mechanism, therapeutic drugs that target epigenetic marks may provide SMA patients with some relief, halting or even reversing the progression of the disease. As SMA patients with higher copy numbers of the SMN2 gene have less severe symptoms, researchers predicted that epigenetic drugs that increased SMN2 mRNA expression would increase the amount of functional SMN protein in neurons leading to a reduction in SMA symptoms. Histone deacetylase (HDAC) inhibitors are the main compounds that have been tested to increase SMN2 mRNA expression. Inhibiting HDACs would allow for hyperacetylation of the SMN2 gene loci theoretically resulting in an increase in SMN2 expression.[40] Many of these HDAC inhibitors (HDACi) are first tested in mouse models of SMA created through a variety of mutations in the mouse SMN1 gene. If the mice show improvement and the drug does not cause very many side effects or toxicity, the drug may be used in human clinical trials. Human trials with all of the below HDAC inhibitors are extremely variable and often impacted by the patient's exact SMA subtype.

Alzheimer’s disease (AD) is the most prevalent form of dementia among the elderly. The disease is characterized behaviorally by chronic and progressive decline in cognitive function, beginning with short term memory loss, and neurologically by buildup of misfolded tau protein and associated neurofibrillary tangles, and by amyloid-beta senile plaques amyloid-beta senile plaques. Several genetic factors have been identified as contributing to AD, including mutations to the amyloid precursor protein (APP) and presenilins 1 and 2 genes, and familial inheritance of apolipoprotein E allele epsilon 4. In addition to these common factors, there are a number of other genes that have shown altered expression in Alzheimer's disease, some of which are associated with epigenetic factors.

Brain-derived neurotrophic factor

ncRNA

ncRNA that is encoded antisense from an intron within the beta-amyloid cleaving enzyme gene, BACE1, is involved in AD.[5] This ncRNA, BACE1-AS (for antisense), which overlaps exon 6 of BACE1, is involved in increasing the stability of the BACE1 mRNA transcript. As that gene's name suggests, BACE1 is an enzymatic protein that cleaves the Amyloid Precursor Protein into the insoluble amyloid beta form, which then aggregates into senile plaques. With increased stability of BACE1 mRNA resulting from BACE1-AS, more BACE1 mRNA is available for translation into BACE1 protein.

miRNA

factors have not consistently been shown to play a role in progression of AD. miRNAs are involved in post-transcriptional gene silencing via inhibiting translation or involvement in RNAi pathways. Some studies have shown upregulation of miRNA-146a, which differentially regulates neuroimmune-related Interleukin-1R associated kinases IRAK1 and IRAK2 expression, in human AD brain, while other studies have shown upregulation or downregulation of miRNA-9 in brain.[61]

DNA methylation

In Alzheimer’s disease cases, global DNA hypomethylation and gene-specific hypermethylation has been observed, though findings have varied between studies, especially in studies of human brains. Hypothetically, global hypomethylation should be associated with global increases in transcription, since CpG islands are most prevalent in gene promoters; gene-specific hypermethylation, however, would indicate that these hypermethylated genes are repressed by the methylation marks. Generally, repressive hypermethylation of genes related to learning and memory has been observed in conjunction with derepressive hypomethylation of neuroinflammatory genes and genes related to pathological expression of Alzheimer's disease. Reduced methylation has been found in the long-term memory-associated temporal neocortex neurons in monozygotic twins with Alzheimer’s disease compared to the healthy twin.[62] Global hypomethylation of CpG dinucleotides has also been observed in hippocampus[63] and in entorhinal cortex layer II[64] of human AD patients, both of which are susceptible to AD pathology. These results, found by probing with immunoassays, have been challenged by studies that interrogate DNA sequence by bisulfite sequencing, a CpG transformation technique which is sensitive to CpG methylation status, in which global hypomethylation has been observed.[65][66]

COX-2

At the individual gene level, hypomethylation and thus derepression of COX-2 occurs, inhibition of which reduces inflammation and pain, and hypermethylation of BDNF, a neurotrophic factor important for long-term memory.[66] Expression of CREB, an activity-dependent transcription factor involved in regulating BDNF among many other genes, has also been shown to be hypermethylated, and thus repressed, in AD brains, further reducing BDNF transcription.[66] Furthermore, synaptophysin (SYP), the major synaptic vesicle protein-encoding gene, has been shown to be hypermethylated and thus repressed, and transcription factor NF-κB, which is involved in immune signaling, has been shown to be hypomethylated and thus derepressed.[66] Taken together, these results have elucidated a role for dysregulation of genes involved in learning and memory and synaptic transmission, as well as with immune response.

Hypomethylation

has been observed in promoters of presenilin 1,[67] GSK3beta, which phosphorylates tau protein,[68] and BACE1,[69] an enzyme that cleaves APP into the amyloid-beta form, which in turn aggregates into insoluble senile plaques. Repressive hypermethylation caused by amyloid-beta has been observed at the promoter of NEP, the gene for neprilysin, which is the major amyloid-beta clearing enzyme in the brain.[70] This repression of NEP could result in a feed-forward buildup of senile plaques; combined with the observed increase in AD brains of BACE1-AS and corresponding increases in BACE1 protein and amyloid beta,[5] multiple levels of epigenetic regulation may be involved in controlling amyloid-beta formation, clearance or aggregation, and senile plaque deposition. There may be some effect of age in levels of DNA methylation at specific gene promoters, as one study found greater levels of methylation at APP promoters in AD patients up to 70 years old, but lower levels of methylation in patients greater than 70 years old.[71] Studies on differential DNA methylation in human AD brains remain largely inconclusive possibly owing to the high degree of variability between individuals and to the numerous combinations of factors that may lead to AD.

Histone marks

Acetylation of lysine residues on histone tails is typically associated with transcriptional activation, whereas deacetylation is associated with transcriptional repression. There are few studies investigating specific histone marks in AD. These studies have elucidated a decrease in acetylation of lysines 18 and 23 on N-terminal tails of histone 3 (H3K18 and H3K23, respectively)[72] and increases in HDAC2 in AD brains[73] - both marks related to transcriptional repression. Age-related cognitive decline has been associated with deregulation of H4K12 acetylation, a cognitive effect that was restored in mice by induction of this mark.[74]

Treatment for prevention or management of Alzheimer's disease has proven troublesome since the disease is chronic and progressive, and many epigenetic drugs act globally and not in a gene-specific manner. As with other potential treatments to prevent or ameliorate symptoms of AD, these therapies do not work to cure, but only ameliorate symptoms of the disease temporarily, underscoring the chronic, progressive nature of AD, and the variability of methylation in AD brains.

Folate and other B vitamins

B vitamins are involved in the metabolic pathway that leads to SAM production. SAM is the donor of the methyl group utilized by DNA methyltransferases (DNMTs) to methylate CpGs. Using animal models, Fuso et al. have demonstrated restoration of methylation at previously hypomethylated promoters of presenilin 1, BACE1 and APP[75] - a hypothetically stable epigenetic modification that should repress those genes and slow the progression of AD. Dietary SAM supplementation has also been shown to reduce oxidative stress and delay buildup of neurological hallmarks of AD such as amyloid beta and phosphorylated tau protein in transgenic AD mice.

AZA

Khan and colleagues have demonstrated a potential role for neuroglobinin attenuating amyloid-related neurotoxicity.[76] 5-aza-2' deoxycitidine (AZA, or decitabine), a DNMT inhibitor, has shown some evidence for regulating neuroglobin expression, though this finding has not been tested in AD models.[77]

Histone-directed treatments

Though studies of histone marks in AD brains are few in number, several studies have looked at the effects of HDACis in treatment of Alzheimer’s disease. Class I and II HDAC inhibitors such as trichostatin A, vorinostat, and sodium butyrate, and Class III HDACis, such as nicotinamide, have been effective at treating symptoms in animal models of AD. While promising as a therapeutic in animal models, studies on the long-term efficacy of HDACis and human trials have yet to be conducted.

Sodium butyrate

Sodium butyrate is a class I and II HDACi and has been shown to recover learning and memory after 4 weeks,[13] decrease phosphorylated tau protein, and restore dendritic spine density in the hippocampus of AD transgenic mice.[14] Histone acetylation resulting from diffuse sodium butyrate application is especially prevalent in the hippocampus, and genes involved in learning and memory showed increased acetylation in AD mice treated with this drug.[15]

Trichostatin A

Trichostatin A is also a class I and II HDACi that rescues fear learning in a fear conditioning paradigm in transgenic AD mice to wild type levels via acetylation on histone 4 lysine tails.[17]

Vorinostat

Vorinostat is a class I and II HDACi that has been shown to be especially effective at inhibiting HDAC2 and restoring memory functions in non-AD models of learning deficits.[78] One study showed vorinostat is effective at reversing contextual memory deficits in transgenic AD mice.[16]

This is a transverse section of the striatum from a structural MR image. The striatum, in red, includes the caudate nucleus (top), the putamen (right), and, when including the term 'corpus' striatum, the globus pallidus (lower left).

Huntington’s disease (HD) is an inherited disorder that causes progressive degeneration of neurons within the cerebral cortex and striatum of the brain[79] resulting in loss of motor functions (involuntary muscle contractions), decline in cognitive ability (eventually resulting in dementia), and changes in behavior.[6]

Huntington’s is caused by an autosomal dominant mutation expanding the number of glutamine codon repeats (CAG) within the Huntingtin gene (Htt).[79] The Htt gene encodes for the huntingtin protein which plays a role in normal development but its exact function remains unknown.[80] The length of this CAG repeat correlates with the age-of-onset of the disease. The average person without Huntington’s has less than 36 CAG repeats present within the Htt gene. When this repeat length exceeds 36, the onset of neuronal degradation and the physical symptoms of Huntington’s can range from as early as 5 years of age (CAG repeat > 70) to as late as 80 years of age (CAG repeat < 39).[81]

This CAG expansion results in mRNA downregulation of specific genes, decreased histone acetylation, and increased histone methylation.[82][83] The exact mechanism of how this repeat causes gene dysregulation is unknown, but epigenome modification may play a role. For early-onset Huntington's (ages 5–15), both transgenic mice and mouse striatal cell lines show brain specific histone H3 hypoacetylation and decreased histone association at specific downregulated genes within the striatum (namely Bdnf, Cnr1, Drd2 - dopamine 2 receptor, and Penk1 - preproenkephalin).[84] For both late- and early-onset Huntington’s, the H3 and H4 core histones associated with these downregulated genes in Htt mutants have hypoacetylation (decreased acetylation) compared to wild-type Htt.[83][84] This hypoacetylation is sufficient to cause tighter chromatin packing and mRNA downregulation.[83]

Along with H3 hypoacetylation, both human patients and mice with the mutant Htt have increased levels of histone H3 lysine 9 trimethylation.[82] This increase in H3-K9 trimethylation is linked to an increased expression of the methyltransferase ESET/SETDB1 (ERG-associated protein with SET domain (ESET)), which targets and trimethylates H3-K9 residues.[82] It’s proposed that this hypermethylation may account for the onset of specific gene repression in Htt mutants.[82]

Huntington patients and both mouse and Drosophila models show histone H3 and H4 hypoacetylation. There are currently no treatments for the disease but numerous HDAC inhibitors have been tested and shown to reverse the certain symptoms caused by the Htt mutation.

Lewy bodies

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra by causes unknown. Several genes and environmental factors (e.g. pesticide exposure) may play a role in onset of PD. Hallmarks include mutations to the alpha-synuclein gene, SNCA, as well as PARK2, PINK1, UCHL1, DJ1, and LRRK2 genes, and fibrillar accumulation of Lewy bodies from misfolded alpha-synuclein. Symptoms are most noticeably manifested in disorders of movement, including shaking, rigidity, deficits in making controlled movements, and slow and difficult walking. The late stages of the disease result in dementia and depression. Levodopa and dopaminergic therapy may ameliorate symptoms, though there is no treatment to halt progression of the disease.

ncRNA

Reductions of miR-133b correlated to decreased numbers of dopaminergic neurons in the midbrain of PD patients.[87] miR-132, meanwhile, is negatively correlated with dopaminergic neuron differentiation in the midbrain.[88] miR-7 and miR-153 act to reduce alpha-synuclein levels (a hallmark of PD) but are reduced in PD brain.[89]

DNA methylation

Neurons of PD patients show hypomethylation of tumor necrosis factor (TNF) alpha encoding sequence, overexpression of which leads to apoptosis of neurons.[90] Cerebrospinal fluid of PD patients also shows elevated TNF alpha.[91] Research indicates there may be a link between DNA methylation and SNCA expression.[92][93] Furthermore, human and mouse models have shown reduction of nuclear DNMT1 levels in PD subjects, resulting in hypomethylated states associated with transcriptional repression.[94]

Histone marks

alpha-synuclein, the protein encoded by SNCA, can associate with histones and prevent their acetylation in concert with the HDACs HDAC1 and Sirt2.[25][95] Furthermore, it has been demonstrated that alpha-synuclein binds histone 3 and inhibits its acetylation in Drosophila.[25] Dopamine depletion in Parkinson’s disease is associated with repressive histone modifications, including reduced H3K4me3, and lower levels of H3 and H4 lysine acetylation after levodopa therapy (a common treatment of PD).

Epigenetic treatments tested in models of PD are few, though some promising research has been conducted. Most treatments investigated thus far are directed at histone modifications and analysis of their roles in mediating alpha-synuclein expression and activity. Pesticides and paraquat increase histone acetylation, producing neurotoxic effects similar to those seen in PD, such as apoptosis of dopaminergic cells.[96] Despite this, treatment with HDACis[97] seems to have a neuroprotective effect.