Amyotrophic Lateral Sclerosis - Mechanisms and Therapeutics in The Epigenomic Era

REVIEWS
Amyotrophic lateral sclerosis: mechanisms

and therapeutics in the epigenomic era
Ximena Paez-Colasante, Claudia Figueroa-Romero, Stacey A. Sakowski, Stephen A. Goutman
and Eva L. Feldman
Abstract | Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of the motor
neurons, which results in weakness and atrophy of voluntary skeletal muscles. Treatments do not modify
the disease trajectory effectively, and only modestly improve survival. A complex interaction between genes,
environmental exposure and impaired molecular pathways contributes to pathology in patients with ALS.
Epigenetic mechanisms control the hereditary and reversible regulation of gene expression without altering
the basic genetic code. Aberrant epigenetic patterns—including abnormal microRNA (miRNA) biogenesis
and function, DNA modifications, histone remodeling, and RNA editing—are acquired throughout life and are
influenced by environmental factors. Thus, understanding the molecular processes that lead to epigenetic
dysregulation in patients with ALS might facilitate the discovery of novel therapeutic targets and biomarkers
that could reduce diagnostic delay. These achievements could prove crucial for successful disease
modification in patients with ALS. We review the latest findings regarding the role of miRNA modifications
and other epigenetic mechanisms in ALS, and discuss their potential as therapeutic targets.
Paez-Colasante, X. et al. Nat. Rev. Neurol. 11, 266–279 (2015); published online 21 April 2015, doi:10.1038/nrneurol.2015.57
Introduction
Amyotrophic lateral sclerosis (ALS) is a neurodegen- sporadic ALS has been linked to many environmental
erative disorder that affects motor neurons in the brain, factors, including heavy metal toxicity and exposure to
brainstem and spinal cord, resulting in progressive pesticides and fertilizers.19 A simple view would predict
weakness and atrophy of voluntary skeletal muscles.1 that genetic variation and environmental exposure con-
Treatments are modestly effective at best, and the major- tribute to ALS, yet it is also likely that environmental
ity of patients die within 3–5 years of diagnosis—often exposures influence epigenetic mechanisms—which
from respiratory failure2–7—although survival outside reversibly regulate gene expression—to promote the onset
this timeframe can vary with certain clinical presenta- and progression of ALS.20
tions.8 The disease exhibits phenotypic heterogeneity,5,6,9 Unravelling the complex mechanisms behind the
and although ALS was once considered a single disorder, pathogenesis of ALS is crucial for the development of
newer theories suggest that different disorders might novel diagnostic and therapeutic approaches, and years
share a common final phenotype.10 As many as 50% of of evidence support the existence of unique, pathologi-
patients with ALS also have cognitive impairment, with cal, epigenetic processes in ALS. Here, we briefly review
a subset of patients (~15%) exhibiting characteristics of what is known about the underlying mechanisms and
frontotemporal dementia (FTD), frontotemporal lobar genetic causes of ALS, and then discuss the environmen-
Department of degeneration (FTLD), and progressive social, behav- tal influences and epigenetic basis of this disease. We also
Neurology, University of ioural and/or language dysfunction.11–13 Several ALS- discuss how the main epigenetic mechanisms interact
Michigan, 1500 East
Medical Centre Drive, related mutations, discussed below, are also common in in neurodegeneration, providing insight into potential
1914 Taubman Centre FTD, leading FTD and ALS to be considered part of a therapeutic strategies.
SPC 5316, Ann Arbor,
MI 48109, USA (X.P.‑C.,
continuum of neurodegenerative disorders.11,14,15
C.F.‑R., S.A.G., E.L.F.). The majority of ALS is sporadic with unknown Proposed disease mechanisms in ALS
The A. Alfred Taubman aetiology, whereas ~15% is familial with dominant The pathogenetic mechanisms proposed for ALS involve
Medical Research
Institute, University of inheritance.16,17 Sporadic and familial ALS are clini- a plethora of alterations to the motor neuron microenvi-
Michigan, 109 Zina cally indistinguishable and share several pathogenetic ronment, including accumulation of protein aggregates,
Pitcher Place, 5017
A. Alfred Taubman
pathways.18 A number of ALS-related genes are known; defective RNA processing, oxidative stress, glutamate
Biomedical Science however, genetic mutations do not solely account for excitotoxicity, glial dysfunction, neuroinflammation,
Research Building, neurodegeneration, and they cannot explain the exist- apoptosis, mitochondrial dysfunction, fragmenta-
Ann Arbor, MI 48109,
USA (S.A.S.). ence of the large number of idiopathic cases. Interestingly, tion of the Golgi apparatus, and metal imbalances.21,22
At very early disease stages, many molecular changes
Correspondence to:
E.L.F. Competing interests in the neuromuscular junction precede motor neuron
efeldman@umich.edu The authors declare no competing interests. death.23 The presence of intraneuronal aggregates of
266 | MAY 2015 | VOLUME 11 www.nature.com/nrneurol

© 2015 Macmillan Publishers Limited. All rights reserved
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SOD1 has prion-like properties, with an ability to seques-

Key points
ter wild-type proteins and seed their aggregation or
■■ The pathogenesis of amyotrophic lateral sclerosis (ALS) is mediated by genetic promote misfolding.42 Additionally, mutations in SOD1
variation, environmental exposure and epigenetic regulation cause misfolding of the nascent SOD1 polypeptide,
■■ Epigenetic mechanisms might explain how gene expression and function
which—in association with other molecules—can lead to
are controlled, and how gene–gene and gene–environmental interactions are
mediated the formation of aggregates, especially during oxidative
■■ DNA methylation, histone remodeling, RNA editing, and microRNA (miRNA) stress.43 Interestingly, expression of mutant SOD1 within
modifications are epigenetic mechanisms that are dysregulated in ALS models motor neurons modulates disease onset, though it is not
and in patients always sufficient to prompt disease.44
■■ miRNAs modulate many physiological processes through an intricate network
of fine-tuning robustness and complexity of the transcriptome and proteome TARDBP and FUS
■■ miRNA pathway disruptions could be a cause or consequence of altered
TAR DNA-binding protein 43 (TDP‑43) and RNA-
RNA and protein metabolism, the inflammatory response, cytotoxicity and/or
binding protein FUS (previously known as fused in
neuromuscular junction impairments, all of which underlie ALS pathology
■■ miRNAs have great potential as novel biomarkers and therapeutic targets for sarcoma or translocated in liposarcoma) are members of
ALS and other neurodegenerative diseases the heterogeneous nuclear ribonucleoprotein (hnRNP)
family. TDP‑43 and FUS are both nuclear RNA binding
proteins that shuttle between the nucleus and cytoplasm,
neurofilaments,24 which can also include peripherin25 or and are involved in transcription, splicing and messenger
occur as hyaline conglomerate inclusions,26 are evident RNP transport.28,30,33,45 Over 40 different missense muta-
in patients with ALS. In particular, spinal motor neurons tions in TARDBP, which encodes the TDP‑43 protein,
show a selective suppression of low molecular weight have been linked to familial ALS.29,45 Most of these muta-
neurofilament light polypeptide (NF‑L) mRNA.27 Also, tions occur in the unstructured, glycine-rich C‑terminal
changes in neurotransmitter release are common to both domain, which binds to hnRNP A/B complexes;46 these
ALS and FTD, as are alterations in synaptic function, C‑terminal regions are commonly observed in ALS and
and the presence of ubiquitinated protein aggregates FTD inclusions.30,47,48 ALS-associated mutations in FUS
that form cytoplasmic inclusion bodies in neurons.28–32 occur within a linkage region on chromosome 16 and
Moreover, disruptions to RNA-processing protein net- affect the protein C‑terminus.49,50 FUS mutations account
works in ALS and other neurodegenerative diseases for ~5% of familial ALS,51,52 and most patients with FUS-
prompt abnormal RNA regulation and exon spli associated ALS have FUS-immunoreactive cytoplasmic
cing.33 Thus, ALS pathogenesis is complex, with several inclusions but the typical phosphorylated TDP‑43-
contributing factors. positive inclusions are absent.49 Mutations in FUS can
also share common pathways with mutant TDP‑43 that
Genetics lead to neurodegeneration,45 or can yield axonal defects
Although the majority of ALS cases are sporadic, in the via interaction and aggregation with survival of motor
past 2 decades, mutations identified in several genes have neuron protein (SMN).53 SMN is required for splice
provided insight into potential disease mechanisms, and osome assembly, and reduced levels of SMN underlie
presented opportunities for disease modelling. Here, the childhood neuromuscular disease spinal muscular
we will briefly introduce some of the most commonly atrophy,54 which has similar features to ALS.
studied mutations, as well as some recently identified, In ALS, TDP‑43 and FUS can interact with other
that are relevant to our current understanding of ALS proteins that regulate RNA metabolism. Under patho-
pathogenesis; more-detailed descriptions of the genetics logical conditions, such as cellular stress, these associ
of ALS can be found elsewhere.5,34 ations between mutant TDP‑43, FUS and mRNAs can
lead to abnormal phosphorylation, ubiquitination and
SOD1 aggregation of proteins, as well as aberrations in their
Dominant mutations in the superoxide dismutase 1 normal cellular functions, 47,48 and the formation of
(SOD1) gene account for one-fifth of familial ALS stress granules.55,56
cases, 35,36 and in vitro and in vivo models based on Stress granules are composed of repressed translation
SOD1 mutations are widely used to investigate disease complexes, where mRNA-binding proteins consolidate
mechanisms. SOD1 protein is an antioxidant found in stalled mRNA. These classically transient structures
the cytosol and mitochondrial intermembrane space, enable a cell to adapt to a stressful response; however,
and converts superoxide radicals to hydrogen peroxide. in neurodegenerative diseases they can become patho-
Over 170 SOD1 missense mutations are known, which logically persistant.48 It is feasible that aggregated RNA-
yield a toxic gain of (unknown) function.37 Although binding proteins become sequestered in stress granules,
reductions in enzymatic dismutase activity were origi- which in turn leads to the formation of cytoplasmic
nally thought to promote oxidative stress and excitotoxi inclusions. TDP‑43 is a principal component of the hall-
city in motor neurons,38,39 degeneration might rather be mark ubiquitinated inclusions seen in many patients with
caused by noxious mutant SOD1 protein aggregation ALS,32,57 and several investigator groups have observed
followed by aberrant association with mitochondria.39,40 co-localization between TDP‑43 and stress granule
SOD1 aggregates are recruited to spinal mitochondria, markers in tissues from patients with ALS or FTD.30,57,58
and crosslinked onto integral membrane components.41 Notably, in most patients with familial ALS, TARDBP
NATURE REVIEWS | NEUROLOGY VOLUME 11 | MAY 2015 | 267

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is not mutated, yet TDP‑43 aggregates may be present Mutations in senataxin (SETX), which codes for a
(except in cases caused by SOD1 or FUS mutations), DNA/RNA helicase involved in RNA processing, have
which disrupt RNA processing and could be one trigger been observed in patients with ALS and patients with pro-
of degeneration.29 gressive motor neuropathy.75 ALS and/or FTD phenotypes
have also been associated with mutations in the non-
C9orf72 structured N‑terminal region or in the α helix of the
The most common genetic cause of ALS and FTD is coiled-coil-helix-coiled-coil-helix-domain-containing
linked to chromosome 9p21, where a founder haplotype 10 (CHCHD10) gene, which encodes a protein located in
occurs in the so-called ALS–FTD locus—which includes the mitochondrial intermembrane space.76,77 Additional
the chromosome 9 open reading frame 72 (C9orf72) associated genes include GRN—one of the major causes
gene—and is associated with autosomal dominant of FTLD with TDP‑43 pathology 78—ANG,79 CHMP2B,80
inheritance.59 In the affected haplotype, expansions in PFN1,81 OPTN, VCP 80,82 and others.5,34 Notably, many of
a large noncoding hexanucleotide (GGGGCC) repeat these genes are also involved in RNA processing, as are
in the first intron of C9orf72 can reach hundreds of TARDBP, FUS, and C9orf72, and are mutated in other
copies in patients presenting an FTD/ALS phenotype neurodegenerative diseases. These observations cat-
and TDP‑43 pathology, as well as in presymptomatic egorically link ALS to a group of pathologies that exhibit
carriers.59,60 Interestingly, although FTD and ALS are altered RNA processing and protein homeostasis.11
related disorders and can sometimes be detected within
the same family harbouring mutations in the ALS–FTD Environmental factors
locus (9p21), ALS is rarely accompanied by FTD in Although there is a clear genetic predisposition to ALS,
SOD1, TARDBP or FUS mutations.60–63 other pathogenetic factors are likely to be environmen-
The exact mechanism by which the C9orf72 repeat tally determined,5,83 and recently, a multistep model
expansion triggers disease is under active investiga- was proposed that suggests a series of events underlie
tion, and a number of possible explanations currently ALS initiation.84 Moreover, sporadic ALS is linked to
exist. First, the repeat expansion could potentially alter toxic exposures, diet, inflammatory cytokines, acquired
the expression of the mutant allele: a decrease in mRNA gene mutations, and other factors—some of which act
expression of C9orf72 transcripts has been observed in synergistically to foster neurodegeneration. Many of
patients with ALS and/or FTD,60,64 providing evidence these factors are involved in molecular m echanisms that
for a loss of function. A more recent study, however, mediate epigenetic changes.
only detected a trend towards decreased expression.65 Lead is the most studied metal in patients with ALS,
Alternatively, accumulation of repeat RNA transcripts and though a link between exposure to lead and ALS
within nuclear foci in the brain and spinal cord of remains inconclusive, multiple studies have reported
patients with ALS and/or FTD has also been observed.60 elevated levels of lead in several different tissues taken
These repeat transcripts undergo unconventional, from patients with ALS.85–89 Furthermore, a recent meta-
bidirect ional repeat associated non-ATG (or ‘RAN’) analysis estimated a 5% increase in the risk of develop-
translation,66,67 thus generating simple peptides that accu- ing ALS after previous lead exposure.90 Accumulation of
mulate into distinct foci in patients with ALS and FTD mercury—a neurotoxin that inhibits SOD1 activity—in
related to C9orf72.66,68 Two of these peptides affect tran- motor neurons damages the cytoskeletal components
scription and translation, interfering with both mRNA and impairs axonal transport, causing phenot ypes
splicing and the biogenesis of ribosomal RNA.69 These similar to ALS.91 However, a direct correlation with the
later observations suggest the repeat expansion leads to disease has not been established.
neurodegeneration via a g ain-of-function mechanism.65 High levels of aluminium are also associated with
the development of symptoms similar to motor neuron
Other mutations disease. Regionally increased incidences of ALS have
Several other genes have also been linked to ALS aetiol- been recorded in a western Pacific region known for
ogy. Mutations in ubiquilin 2 (UBQLN2), which encodes excessive amounts of aluminium in the soil and drink-
a protein that targets abnormal proteins for degrada- ing water. 92 Also, mice fed diets high in aluminium
tion, yield X‑linked FTD or ALS with TDP‑43-positive develop ALS-like characteristics.93 Although the litera-
neuropathology.70 Mutant forms of proteins with a role in ture indicates that metal exposures alone are insufficient
RNA and DNA binding, such as the protein encoded by to explain ALS pathogenesis, the ubiquitous nature of
matrin 3 (MATR3), have also been associated with ALS.71 neurot oxic environmental metals demands further
The RNA-binding proteins hnRNPA1 and hnRNPA2B1 investigation through large-scale epidemiological studies
have a prion-like domain that influences mRNA metabo- with rigorous designs.91
lism and transport,72 and can interact directly with the The risk of ALS has also been linked with exposure to
C‑terminal region of TDP‑43.46,72,73 Mutant forms of fertilizers, insecticides and herbicides.94–96 Occupational
these proteins have been reported in one patient with and home exposure to pesticides and fertilizers tend to
dominantly inherited ALS,74 and in animal models, the be higher in patients with ALS than in controls matched
mutant hnRNPA1 and hnRNPA2 were found to be prone for age and sex,97 and these exposures are also associ-
to fibrillization and incorporation into stress granules ated with increased risk for developing Parkinson
and cytoplasmic inclusions.74 disease (PD).98,99

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Box 1 | What is epigenetics?

mechanisms, however, could be driven by the same long-
term environmental influences that contribute to ALS
Epigenetics (‘above genetics’) was first described by the British developmental
risk, suggesting that the accumulation of some epigenetic
biologist and geneticist Conrad H. Waddington (1905–1975) to explain the
interplay between genetics and external forces during development that define
patterns throughout life drives the onset and progres-
the phenotype of an organism.199 The use of the term epigenetics has been sion of ALS. The reversible nature of epigenetic marks
controversial, as the boundaries of the definition have been ambiguous from also makes this mechanism an attractive pharmacologi-
the very beginning. cal target for ALS therapy development. In the following
After Waddington first described epigenetics, two distinct interpretations of the sections, we discuss what is known regarding the contri-
concept emerged. One described epigenetics as the study of “mitotically and/ bution of some of the major epigenetic mechanisms to
or meiotically heritable changes in gene function that cannot be explained by ALS pathogenesis. These findings are also summarized
changes in DNA sequence.”200 Central requirements of this definition are that DNA in Table 1.
methylation and histone modifications are the sole mechanisms that maintain the
epigenome,200,201 and that phenotypic changes resulting from epigenetic memory
DNA methylation
are carried on by transgenerational inheritance.
DNA methylation involves the post-transcriptional
The second interpretation is more global, and more akin to Waddington’s
covalent addition of a methyl group to cytosine residues
definition, describing epigenetics as “the structural adaptation of chromosomal
in DNA, leading to 5‑methylcytosine (5mC) forma-
regions so as to register, signal, or perpetuate altered activity states.”106 This
definition embraces the idea that environmental cues—including behaviour, tion.109 This addition modifies transcriptional protein–
stress, ageing, and exposure to toxins—will be received and maintained by DNA interactions by changing chromatin structure
epigenetic mechanisms, such as DNA methylation, histone modifications, and and transcription rates. 110 The DNA-(cytosine‑5)-
non-coding RNAs, to regulate gene expression.106,201,202 methyltransferase (DNMT) enzymes DNMT1, DNMT3a
In this Review, we use the second definition to account for the phenotypic and DNMT3b catalyse the covalent transfer of methyl
plasticity seen in sporadic cases of late-onset neurological disorders, including groups from S‑adenosyl-methionine to the 5' carbon
amyotrophic lateral sclerosis, which could result from environmental and position of a substrate cytosine residue. 109 Reversal
behaviour-driven epigenetic alterations. of DNA methylation is a complex process and not
fully understood.
Several groups have detected associations between DNA methylation commonly occurs in genomic
cigarette smoking and an elevated risk of ALS, as well regulatory regions, such as at promoter element CpG
as reduced survival. One case–control study identi- dinucleotides,107 which are clustered in CpG islands or
fied trends for a threefold higher risk of ALS in current CpG island shores.111 In promoter regions, 5mC modifi-
smokers compared with nonsmokers,100 and two other cations serve as docking sites for chromatin-remodeling
studies also found that smoking is independently associ proteins, and are associated with negative regulation of
ated with an increased risk of ALS.101,102 However, a gene expression107 through interference with transcrip-
meta-analysis detected only a moderate association of tion factor binding or by providing a binding site for
ALS with current smokers,103 a prospective cohort study transcriptional repressors.112 By contrast, intragenic 5mC
found an increased risk for ALS and worse prognosis modifications facilitate gene expression.21,113,114
only in female smokers, 104 and a case–control study The establishment and maintenance of DNA methy
found no correlation at all.19,97 Inconsistencies in results lation patterns during brain development, learning
are probably due to limitations in experimental design, and memory follow complex processes involving the
small sample sizes and the use of relatives and/or friends interaction between DMNTs and methyl-CpG binding
as controls; therefore, additional studies are required. domain proteins (MBDs). 115 In addition, the ten–
Finally, the physical exertion and trauma, especially eleven translocation (TET; also known as methylcyto-
head injuries, experienced by war veterans and athletes sine dioxygenase) family of enzymes oxidizes 5mC to
could be associated with increased risk of ALS.19,23,105 generate an alternatively modified cytosine residue,
5‑hydroxymethylcytosine (5hmC), which inhibits MBD
Epigenetics protein binding and opposes 5mC function.107 Thus,
Epigenetics represents a potential convergence between DNA methylation is a powerful molecular strategy to
genetic predisposition and environmental exposures. regulate gene expression, and it might further serve as
Although controversy has surrounded the definition an epigenetic biomarker and therapeutic target because
of epigenetics (Box 1), in this Review we identify epi- methylation is stable, easily detectable and reversible.
genetics as “the structural adaptation of chromosomal Supporting a role for reversible DNA methylation in
regions so as to register, signal, or perpetuate altered ALS, DNMT3A is present in the brains and spinal cords
activity states.”106 Epigenetic mechanisms include DNA of patients with ALS, and its overexpression prompts cell
methylation, histone remodelling, RNA editing, and death in motor-neuron-like cells in vitro.109 Blood and
noncoding RNAs such as microRNAs (miRNAs). They neural tissue from patients with ALS also exhibit enhanced
coordinate neural development, plasticity and ageing— global DNA 5mC and 5hmC,21,109,116 and sites of gene
and are essential for cellular homeostasis—by responding methylation in brain and spinal cord tissue from patients
to stimuli beyond the inherited genetic blueprint.107,108 with ALS have been identified.21,117 For example, genome-
Epigenetic programming through previous experi- wide methylation arrays have revealed hypomethylation
ence might also allow the brain to adapt to a chroni- of the ALS-related gene OPTN.21 Additionally, loss of
cally stressful environment. Dysregulation of epigenetic function of the protein arginine N‑methyltransferase 1

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Table 1 | Epigenetic mechanisms and findings in ALS

Player Epigenetic mechanism Importance to ALS Reference(s)
DNMTs Methylation In vitro overexpression of Dnmt3a prompts cell death Chestnut et al. (2011)109
in motor-neuron-like cells
DNMTs upregulated in brain and spinal cords of patients
with ALS
OPTN Methylation Hypomethylated in patients with ALS Figueroa-Romero et al. (2012)21
5mC and Methylation and Blood and neural tissue from patients with ALS and ALS Figueroa-Romero et al. (2012)21
5hmC hydroxymethylation models exhibit enhanced global 5mC and 5hmC Chestnut et al. (2011)109
Tremolizzo et al. (2013)87
PRMT1 Methylation and histone Redistribution of FUS to the cytoplasm yields loss- Tibshirani et al. (2014)118
modification of-function in PRMT1, altering the histone code
C9orf72 Methylation and histone C9orf72 expansion CpG islands are hypermethylated Xi et al. (2013, 2014)64,91
methylation in tissue from patients with ALS Belzil et al. (2014)120
Altered histone methylation pattern causes reduced
brain C9orf72 mRNA expression in ALS and FTD
C9orf72 expansion-repeat carriers
HAT Histone modifications A mutation in SS18L1 ablates the interaction with HAT Teyssou et al. (2014)131
machinery and modifies HAT catalytic subunit in ELP3, Simpson et al. (2009)132
which is important for motor neuron axonal homeostasis
HDAC Histone modifications Forced expression of HDAC3 causes degeneration of rat Bardai et al. (2011)128
neurons and HT22 cultured hippocampal cells
AMPA RNA editing Reductions in ADAR2 promote an abnormal AMPA Kwak et al. (2010)133
receptor variant associated with ALS, and causes mislocalization Al-Chalabi et al. (2013)20
and aggregation of TDP‑43 Hideyama et al. (2012)134
Yamashita et al. (2014)136
miRNAs Posttranscriptional Dysregulation is observed in several tissues from See Table 2 for specific
silencing mechanisms patients with ALS and ALS models; the majority of references
dysregulated miRNAs are downregulated
Abbreviations: 5hmC, 5‑hydroxymethylcytosine; 5mC, 5‑methylcytosine; ADAR2, adenosine deaminase RNA-specific 2; ALS, amyotrophic lateral sclerosis; AMPA,
alpha-amino‑3-hydroxy‑5-methyl‑4-isoxazolepropionate; C9orf72, chromosome 9 open reading frame 72; DNMT, DNA-(cytosine‑5)-methyltransferase; ELP3, RNA
polymerase II elongator complex protein 3; HAT, histone acetyltranferase; HDAC, histone deacetylase; miRNA, microRNA; OPTN, optineurin; PRMT1, protein
arginine N‑methyltransferase 1; SS18L1, synovial sarcoma translocation gene on chromosome 18 like 1; TDP‑43, TAR DNA-binding protein 43.
(PRMT1)—which is associated with FUS redistribution Histone modifications and chromatin remodelling
from the nucleus to the cytoplasm in patients with ALS Post-translational modifications of H3 and H4 histone
—leads to changes in the histone code.118 tails via acetylation and methylation allow chromatin to
Epigenetic mechanisms could also be important in dynamically change from a highly packaged, transcrip-
C9orf72-related ALS, because the hexanucleotide repeat tionally inactive conformation (heterochromatin) to a
expansion is associated with hypermethylation of CpG transcriptionally active state (euchromatin).107,125 Whereas
islands. In one study, global hypermethylation of the acetylation directly changes chromatin structure, histone
C9orf72 CpG island was associated with the presence of methylation—as with DNA methylation—serves as a
the repeat expansion in tissue samples from patients with docking site for transcriptional-regulation proteins. The
ALS.64 In another study, methylation levels were signifi acetylation of histone tails by the histone acetyltrans-
cantly higher in blood from patients with FTLD who ferases promotes transcription, whereas removal of acetyl
carried C9orf72 expansions relative to noncarriers.119,120 groups by histone deacetylases (HDACs) represses gene
Interestingly, C9orf72 promoter hypermethylation is expression.126 Methylation of histone tails is catalysed by
hypothesized to prevent abnormalities associated with methyltransferases; monomethylation is associated with
the hexanucleotide repeat expansion. In patient-derived euchromatization, and dimethylation and trimethylation
lymphoblast cell lines, promoter hypermethylation led to are associated with heterochromatization.127
transcriptional silencing of C9orf72, whereas demethy HDAC overexpression can be detrimental to the CNS,
lation reversed these protective properties.121 However, and reduced histone acetylation is a common observation
it is possible that the reduction in expression of C9orf72 in models of neurodegenerative disease.125 Forced expres-
could lead to disease through haploinsufficiency. sion of HDAC3 causes degeneration of rat neurons and
Notably, alterations in DNA methylomes are present in cultured HT22 hippocampal cells,128 and a postmortem
other human nucleotide repeat expansion and neuro- analysis reported an increase in HDAC2 mRNA and a
degenerative diseases, including spinocerebellar ataxia reduction in HDAC11 mRNA in spinal cord and brain
type 1, Friedreich ataxia, fragile X syndrome, myotonic tissue from patients with ALS.129 Conversely, a more
dystrophy, PD and Alzheimer disease (AD). 64,122–124 recent study using two SOD1 mouse models found an
Together, these findings support the premise that altered increase in HDAC11 mRNA,130 although these results are
DNA methylation contributes to neurodegeneration in yet to be corroborated by subsequent studies. Similarly,
patients with ALS. a single point mutation in SS18L1, a component of the

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chromatin-remodelling complex, ablates the interaction evolutionarily conserved miR‑124 affects neuronal differ-
with histone acetyltransferase machinery 131 and modi- entiation of mesenchymal stem cells,148 and miR‑9 exhibits
fies the histone acetyltransferase catalytic subunit in the abundant neuron-specific expression.149,150
RNA polymerase II elongator complex protein 3 (ELP3), miRNAs undergo a detailed maturation process that
which regulates histone acetylation and methylation and occurs in the nucleus and cytoplasm. In the canonical bio-
is important for motor neuron axonal homeostasis.132 genesis pathway (Figure 1a), primary miRNA transcripts
C9orf72 repeat-expansion carriers with ALS and/or FTD (pri-miRNAs) are transcribed in the nucleus151 and pro-
also exhibit reduced brain C9orf72 mRNA expression, cessed by Drosha (also called ribonuclease 3) to produce
owing to an altered histone methylation pattern featuring precursor miRNAs (pre-miRNAs).152–154 Pre-miRNAs
trimethylation at four lysine residues.120 This methylation are then exported to the cytoplasm and processed by
pattern alone might be sufficient to produce neuropatho- endoribonuclease Dicer to become mature double-
logical alterations. Thus, DNA methylation and histone stranded short miRNAs.155,156 Next, argonaute (AGO) pro-
modifications might be important therapeutic targets. teins associate with miRNAs to prompt the separation of
the double-stranded molecule and form the RNA-induced
RNA editing silencing complex (RISC).157 The miRNAs then guide the
RNA editing enzymatically changes nucleotide sequences RISC complex toward the 3' untranslated region of specific
so that they differ from those encoded by the original mRNA targets157 to epigenetically control gene expression
genomic DNA. The most common change in patients by either translational repression or transcript degrada-
with ALS is the deamination of adenosine to inosine. tion. In mammals, only a single member of the AGO
This modification might underlie the excitotoxic effects family, AGO2, harbours the catalytic activity required for
of variants of the AMPA (alpha-amino‑3-hydroxy‑ mRNA cleavage.158 Cleavage is initiated in the context of
5-methyl‑4-isoxazolepropionate) receptor in ALS,133 adequate miRNA and target complementarity; however,
whereby reductions in the enzyme adenosine deamin complementarity is limited for most animal miRNAs and
ase RNA-specific 2 (ADAR2; also known as double- only a short ‘seed sequence’ of 2‑8 nucleotides at the 5'
stranded RNA-specific editase 1) limits the conversion end is essential for determining specificity.159Alternatively,
of adenosine to inosine at the GluA2 (Glu/Arg) site of mammalian miRNA biogenesis can also occur via non-
the AMPA receptor pore-lining domain, and promote an canonical pathways.160 In the mirtron pathway, miRNA
abnormal AMPA variant in ALS.20,134 The consequences synthesis does not require processing by Drosha,161,162 and
of altered RNA editing in this case are further supported direct pre-miRNA loading onto AGO2 does not require
by the observation that motor neurons in patients Dicer.163 Some nuclear miRNAs also target noncoding
with ALS are particularly susceptible to RNA-editing RNAs via AGO2-mediated cleavage, in which circular
deficiencies, 135 as ADAR2-deficient motor neurons noncoding antisense RNA stabilizes a sense transcript,
undergo slow death in conditional ADAR2 knockout whereas destabilization of the antisense transcript occurs
mice. Furthermore, this enzymatic deficiency causes by nuclear miRNA.164
mislocalization and aggregation of TDP‑43.136 These For the vast majority of diseases, differentially
defects can be reversed in conditional ADAR2-knockout expressed miRNAs have been found between diseased
mice with injection of AAV9-ADAR2, which enhances and normal tissue, and miRNAs are usually subject to
the expression of exogenous ADAR2 in the CNS, and change during the pathogenetic course. 146 Thus, the
prevents progressive motor dysfunction.137 These results miRNA-mediated alteration of biological pathways could
suggest that delivery of ADAR2 via gene therapy also explain, at least partly, complex diseases like ALS. In
correct defects in patients with ALS. neurodegenerative diseases, the implications of miRNA
dysregulation have not been fully elucidated, and it is
miRNA crucial to determine whether dysregulation occurs via
miRNAs are evolutionarily conserved noncoding RNAs, transcriptional or post-transcriptional mechanisms, or
about 22 nucleotides long, that can individually regu- both. Notably, the dysfunction of components in miRNA
late several hundred targets via RNA-dependent post- biogenesis can have severe consequences. Loss of Dicer1
transcriptional silencing mechanisms.138–142 Conversely, induces symptoms similar to spinal muscular atrophy
mRNA transcripts can be regulated by many miRNAs.139 in rodent motor neurons 165 and also contributes to
These noncoding molecules can be intergenic or intra- amyloid-β accumulation and dopamine loss.166 Similarly,
genic, and some miRNAs can be cotranscribed as poly decreases in TDP‑43 can impact miRNA processing via
cistronic clusters.138,143 The human genome harbours at interactions with Drosha-containing protein complexes
least 1,881 precursor miRNAs capable of modulating (Figure 1b).45 In addition to forming a complex with
many physiological processes144—including neuronal DGCR8, Drosha can interact with several polypeptides—
homeostasis and cell differentiation145—by intricate fine- including TDP‑43, AGO2, and some nuclear primary
tuning of the transcriptome and proteome.143,146 miRNAs miRNAs or cytoplasmic pre-miRNAs—to establish an
are also hypothesized to contribute to genetic networks alternative complex with limited processing function.167
controlled by feed-forward loops,146 and they are tempo- Whereas miRNAs are generally stable, long-lived mol-
rally and differentially expressed among different tissues; ecules, high complementarity between miRNAs and
for example, the miRNA miR‑138 is restricted to the CNS their targets can further lead to trimming and destabi-
whereas its precursor is ubiquitously expressed,147 the lization of miRNAs.168 An accelerated turnover rate in

REVIEWS
a ◀ Figure 1 | Disruption of miRNA biogenesis and function by

cytoplasmic protein aggregates. a | In the canonical miRNA
Cytoplasm
biogenesis, a primary transcript is encoded in the nucleus
2
by specific miRNA genes via RNA polymerase II/III (1).
Dicer
Further processing by the RNase III family member
1 enzymatic complex Drosha and DGCR8 produces the
RNA Drosha
polymerase
DGCR8 70-nucleotide stem loop pre-miRNAs that are transported
TDP-43 Exportin 5
II/III to the cytoplasm by exportin 5. Pre-miRNAs are
5'-cap Poly-A tail consequently cleaved at the stem loop by Dicer to generate
miRNA gene Primary
or intron miRNA Pre-miRNA Pre-miRNA ~22-nucleotide double-stranded mature miRNAs (2). AGO2
protein then binds to the 2‑nucleotide overhang of the
Mature
Nucleus 3 miRNA mature miRNAs to initiate their incorporation into the RISC
RISC and trigger strand separation (3). The single-stranded
TNRC6
4 RISC TNRC6 miRNA guides the complex toward specific mRNA targets,
Translational AGO2
repression AGO2 inducing mRNA silencing and repressing protein production
either by blocking translation or by transcript degradation
(4). Inclusion of TNRC6 in RISCs induces deadenylation,
Target mRNA
decapping, and decay of mRNAs. b | As another component
of the Drosha/DGCR8 miRNA-processing complex, loss of
b TDP‑43 induces alterations in TDP‑43-binding miRNAs,
Cytoplasm while c | TDP‑43 co-localization with stress granules might
sequester miRNAs, thus inhibiting their negative regulatory
Dicer function on nascent mRNAs. Abbreviations: AGO2,
argonaute 2; DGCR8, microprocessor complex subunit
RNA Drosha DGCR8; miRNA, microRNA; RISC, RNA-induced silencing
polymerase DGCR8
II/III TDP-43 Exportin 5 complex; TDP-43, TAR DNA binding protein 43; TNRC6,
5'-cap Poly-A tail trinucleotide repeat-containing gene 6A protein.
miRNA gene Primary
or intron miRNA
TNRC6 CSF, indicating that changes in serum do not necessarily
Nucleus AGO2 RISC reflect CSF miRNA levels.169 Moreover, strongly regu-
lated miRNAs were identified in lymphoblast cell lines
Translational from patients with familial ALS. Specifically, both strands
activation Target
Ribosomes mRNA of miR‑143 were downregulated, whereas miR‑558-3p
was unaffected. Suppression of miR‑132-5p/3p and
miR‑574-5p/3p was also evident in all patients except
those with SOD1 mutations (that is, those with mutations
c in TARDBP, FUS, or C9orf72). Furthermore, miR‑663a
Cytoplasm and miR‑9-5p were exclusively downregulated in patients
with FUS mutations, and let-7b was altered in both FUS
Dicer
and C9orf72 mutation carriers.169 Overall, the prevalence
Drosha
of mostly downregulated TDP‑43-binding miRNAs sug-
RNA
polymerase
DGCR8
Exportin 5 gests that a general defect in RNA metabolism may be
II/III TDP-43 TDP-43
5'-cap Poly-A tail aggregates present in patients with ALS.
miRNA gene Primary miRNA alterations that impact the proposed patho-
or intron miRNA Pre-miRNA Pre-miRNA
genetic mechanisms of ALS have also been observed.
Nucleus A disease-specific, more than twofold upregulation of
RISC
TNRC6
miR‑338-3p is present in blood leukocytes, CSF, serum
Translational AGO2 Stress and spinal cords of patients with sporadic ALS rela-
activation granules
Ribosomes tive to controls, and in situ hybridization verified that
Target
miR‑338-3p accumulates in lumbar dorsal horn grey
mRNA matter.170 Interestingly, miR‑338-3p is also enriched in
distal axons where it modulates mitochondrial function
and is involved in apoptosis, neurodegeneration and
Nature Reviews | Neurology
neuronal miRNAs suggests a major role for this pathway glutamate clearance, presumably via interactions with
in neuronal physiology.168 targets such as the the glutamate transporter SLC1A2
Several studies have examined miRNAs in ALS models and apoptosis-associated tyrosine kinase.170 Expression of
and in patient biopsy samples. In one study, the expres- excitatory amino acid transporter 2 (EAAT2) is indirectly
sion of certain miRNAs previously shown to bind to increased by neuronal miRNAs, such as miR‑124a, and
TDP‑43 in vitro was altered in cerebrospinal fluid (CSF) EAAT2 packaged in exosomes is internalized by astro-
and serum from patients with sporadic ALS.169 This cytes.171 Decreases in miR‑124a are seen in the spinal
study further determined that some miRNAs are nor- cord of mutant SOD1 mice, suggesting that dysregulated
mally found in serum, whereas others, such as miR‑9-5p, glutamate transport pathways probably contribute to the
miR‑132-5p, and miR‑558-3p, are more abundant in exacerbation of ALS pathology.171

REVIEWS
Increased expression of miR‑23a, miR‑29b, miR‑206, Converging pathways

and miR‑455 has been observed in skeletal muscle Given the highly tuned epigenetic regulation of gene
samples from patients with ALS.172 miR‑23a suppresses expression, neuronal homeostasis is most likely orches-
the activity of the peroxisome proliferator-activated trated by complex molecular networks directed by
receptor‑γ co-activator (PGC)‑1α, a protein involved dynamic crosstalk between multiple epigenetic mecha-
in mitochondrial biogenesis and function, which might nisms.177 Therefore, alterations to epigenetic players or
indicate that specific miRNAs represent therapeutic pathways can cause deleterious downstream effects. For
targets.172 Similarly, miR‑206 is increased in skeletal example, miRNA function goes beyond that of RNA
muscles of patients with ALS and in symptomatic mutant interference by directly interacting with gene promot-
SOD1 mice, possibly owing to the effects of denerva- ers, thereby driving epigenetic regulation of DNA methy
tion.172,173 The loss of miR‑206 accelerates disease pro- lation and histone modification. The control of miRNAs
gression in mice, probably because of its involvement in is similar to those of protein-coding genes with double-
skeletal muscle development, synaptic plasticity, neuro- negative feedback loops.178 At least one identified miRNA
muscular junction regeneration, and nerve to muscle directs chromatin remodelling of a promoter region,
communication. 173 Finally, one group examined the and synthetically produced miRNA-like molecules can
expression of numerous miRNAs in spinal cords from similarly cause gene silencing.179 Acceleration of ALS
patients with ALS, and found that downregulations disease progression is also associated with HDAC4: ele-
accounted for the vast majority of significant differ- vated levels were found in muscle biopsy samples from
ences in miRNA expression.174 Two additional studies a patient with ALS who had poor functional outcome
from the same group determined that downregulation and low capacity for reinnervation.180 miR‑206 regulates
of miR‑b1336 and miR‑b2403, which normally stabilize HDAC4 expression and is likewise upregulated in muscle
NF‑L, leads to the loss of NF‑L mRNA and subsequent samples from patients with ALS; however, expression did
neuromuscular junction pathology.27 This study also not correlate with reinnervation.180
revealed that miR‑146a*, miR‑524-5p, and miR‑582-3p, Mutations in ALS-related genes can also have a modu-
which have miRNA recognition elements within the latory effect on miRNAs. FUS mutations—particularly
human NF‑L mRNA 3' untranslated region, are also FUS Gly48Ala, which causes increased FUS protein
dysregulated in the spinal cords of patients with ALS.174 expression—are found in the 3' untranslated region
Inflammation and the immune system are also and are complemented by miR‑141 and miR‑200a, thus
implicated in ALS pathology and disease progression: linking FUS and these miRNAs by a feed-forward regula-
microglial activation, T‑cell-independent macrophage tory loop.181 TDP‑43 and FUS also interact, functioning
activation, monocyte recruitment to diseased tissue, in a biochemical complex to modulate HDAC6 mRNA
and dysregulation of immune-related genes are all production, whereas SOD1 acts independently of this
present in patients with ALS.21,175,176 Moreover, unique complex.182 Moreover, a polymorphism in the GRN 3'
pro-inflammatory miRNA and gene profiles are present untranslated region hinders miR‑659 binding, resulting
in the blood of patients with ALS and in microglia and in decreased expression of progranulin and a higher risk
LY6C hi peripheral monocytes of mutant SOD1 mice, of FTD–ALS spectrum disorders.183 miR‑29b can also
including increased expression of miR‑27a, miR‑155, directly regulate progranulin levels, thus supporting the
miR‑146a, miR‑451, miR‑223, miR‑142-5p, let-7a/b, induction of miR‑29 as a potential therapeutic strategy to
and miR‑532-3p, among others.175 The TGF‑β1-targeting increase proper protein translation.184 Moreover, TDP‑43
miRNAs miR‑21 and miR‑106b are upregulated in knockdown in human cultured cells alters miRNAs that
patients with ALS.175 miR‑155, which reduces TGF‑β1 bind TDP‑43, including downregulation of let-7b and
production, promotes macrophage inflammatory upregulation of miR‑663.185 Also binding to TDP‑43
responses, and increases pro-inflammatory cytokine in the nucleus are primary miR‑132 (highly enriched
secretion, is also upregulated in the spinal cords and in neurons), primary miR‑143, primary miR‑558, and
peripheral blood cells of mutant SOD1 mice and patients primary miR‑574.167 These findings support the con-
with ALS.159 Furthermore, upregulation of additional tention that TDP‑43 is a nuclear Drosha-associated
immune-related miRNAs, including miR‑22, miR‑125b, protein with post-transcriptional involvement in miRNA
miR‑146b, and miR‑365, are present within activated biogenesis (Figure 1b).
microglia in the brains of SOD1-Gly93Ala mice.176 The Although it is unclear how the activity of mature
pro-inflammatory and anti-inflammatory cytokine miRNAs is regulated in the cytoplasm, we hypothesize
interleukin 6 (IL‑6) is normally negatively regulated by that the dysregulation of key miRNAs could potentially
miR‑365. In microglia of patients with ALS, miRNA‑365 be caused by their sequestration within protein aggre-
is upregulated, which is accompanied by a correspond- gates, namely TDP‑43 aggregates formed through cellu-
ing significant reduction in IL‑6. On the other hand, lar stress, or overexpression of or mutations in TARDBP
miR‑125b targets signal transducer and activator of tran- (Figure 1c). Prior evidence shows that TDP‑43 nega-
scription 3 (STAT3), and downregulation of these two tively regulates the miR‑1 family (miR‑1 and miR‑206)
signalling pathways favours pro-inflammatory signals by limiting their bioavailability to RNA-induced silenc-
by increasing expression of tumour necrosis factor ing complexes. Additionally, overexpression of TDP‑43
(TNF).176 Table 2 summarizes the miRNAs with known increases levels of HDAC4, a miR‑1-family target, and
importance to ALS pathology. this dysregulation of HDAC4 is correlated with ALS

REVIEWS
Table 2 | Key miRNAs differentially expressed in ALS

miRNA Function Importance to ALS Reference(s)
miR‑206 Human skeletal-muscle-specific; affects Increased in patients with ALS Williams et al. (2009)173
nerve–muscle interactions Absence causes delay in muscle Toivonen et al. (2014)44
reinnervation via lack of a local signalling
miR‑143 Possibly involved in cardiac morphogenesis Downregulated in ALS Freischmidt et al. (2013)169
miR‑365 Inhibitory effect on IL‑6 3' untranslated Negative regulator of IL‑6 in microglia, Parisi et al. (2014)176
region augmented in ALS
miR‑9 Highly involved in brain neuronal Elevated in patients with ALS Zhang et al. (2013)197
development harbouring TARDBP mutations Kye and Goncalves Ido
Modulates axon growth via direct miR‑9-5p is decreased in patients (2014)198
regulation of MAP1B mRNA with FUS mutations Freischmidt et al. (2013)169
miR‑132p Highly enriched in neurons Cytoplasmic TDP‑43 aggregates hinder Kawahara et al. (2012)167
Promotes neuronal outgrowth by reducing the biogenesis of miR‑132p
ρ250GAP levels
miR‑124a Negatively regulates the expression of the Decreased expression in spinal cords Morel et al. (2013)171
glutamate transporters EAAT2 of SOD1 mutant mice
miR‑23a Suppresses PGC‑1α translation in a Elevated in skeletal muscles of patients Russell et al. (2013)172
manner dependent on the 3' untranslated with ALS
region Might explain mitochondrial dysfunction
miR‑29b and miR‑29b has a role in muscle Elevated in skeletal muscle tissues Russell et al. (2013)172
miR‑455 regeneration from patients with ALS
miR‑455 is involved in muscle wasting
miR‑21 and Target TGF‑β1 expression Upregulated in patients with ALS Butovsky et al. (2012)175
miR‑106b Reduce TGF‑β1 levels
miR‑155 Targets TGF‑β1, promotes macrophage Upregulated in spinal cords and blood Koval et al. (2013)159
inflammatory responses, and increases of patients with ALS, and in SOD1- Butovsky et al. (2012)175
pro-inflammatory cytokine secretion mutant mice
Treatment by anti-miR‑155 delays SOD1-
mouse mortality
miR‑365 and Regulates the pro-inflammatory and Downregulated in patients with ALS, Parisi et al. (2013)176
miR‑125b anti-inflammatory cytokine IL‑6 in familial favouring pro-inflammatory signals by
ALS (miR‑365); targets STAT3 (miR‑125b) increasing TNF
miR‑b1336 and Stabilize NF‑L at the neuromuscular Differentially expressed in spinal cords Ishtiaq et al. (2014)27
miR‑b2403 junction of patients with sporadic ALS
miR‑146a*, Target human NF‑L mRNA Dysregulated in spinal cords of patients Campos-Melo et al.
miR‑524-5p, with ALS (2013)174
and miR‑582-3p
Abbreviations: ALS, amyotrophic lateral sclerosis; EAAT2, excitatory amino acid transporter 2; FUS, fused in sarcoma; MAP1B, microtubule-associated protein 1b;
miRNA, microRNA; NF‑L, neurofilament light polypeptide; SOD1, superoxide dismutase; ρ250GAP, Rho GTPase-activating protein 32; STAT3, signal transducer
and activator of transcription 3; TARDBP, TAR DNA binding protein; TDP‑43, TAR DNA-binding protein 43; TGF‑β1, transforming growth factor beta 1; TNF, tumour
necrosis factor.
progression.186 Three members of the miR‑132 cluster mutant protein has proven effective and safe when deliv-
are significantly downregulated in the brains of patients ered to the CSF of patients with SOD1-related familial
with FTD who present with TDP‑43 inclusions,183 and ALS.188 A similar drug that targets the sense strand of
most TDP‑43-binding miRNAs are generally downregu- the C9orf72 hexanucleotide repeat mitigated toxicity by
lated as well.169 Furthermore, FUS C‑terminal mutants suppressing RNA foci formation in vitro65,68 and in vivo189
are mislocalized in the cytoplasm, and are recruited to without reducing RNA levels. These results support
stress granules along with wild-type FUS and other RNA- the development of antisense oligonucleotides to treat
binding proteins to form large aggregates, thus disrupt- C9orf72-linked and SOD1-linked ALS. On the other
ing RNA metabolism.72 Similarly, aggregated TDP‑43 is hand, understanding the contribution to ALS made by
capable of sequestering mRNAs as well as other small dysregulated miRNAs and their targets might help iden-
molecules, such as miRNAs. tify new pathways involved in neurodegeneration, thus
offering novel opportunities for targeted intervention.
Therapeutics There are two approaches to the development of
There is no effective treatment for ALS, and riluzole— miRNA-based therapeutics. miRNA antagonists inhibit
the lone FDA-approved disease-modifying drug—only endogenous miRNAs that have a toxic gain-of-function
modestly slows disease progression.187 However, alter- in diseased tissues, and involve the use of an anti-miR—
native strategies are under investigation. A phase I clini- a chemically modified antisense RNA—to knockdown
cal trial of an antisense oligonucleotide (ISIS 333611) miRNA. The resulting miRNA duplex is not active, thus
that targets SOD1 mRNA to halt the production of the counteracting the miRNA’s negative regulatory effects.

REVIEWS
Neuromuscular (NaPB) upregulates astrocytic neurotrophin expression

RNA and protein junction Inflammatory in the CNS of AD‑model mice, thus improving cogni-
metabolism structure/function Cytotoxicity response
tion,192 and in SOD1 Gly93Ala mice, riluzole and NaPB
Nerve/ TGFβ1 synergistically improve survival and phenotype. 193
Glutamate
Protein muscle clearance Apoptosis Inflammatory Likewise, a human clinical trial assessing NaPB further
aggregates signalling cytokine
impairment demonstrated that it is safe and tolerable, and does not
impairment secretion
alter blood riluzole levels.194 In addition, certain DNMT
miRNA dysregulation and HDAC inhibitors are already approved by the
FDA for use in cancer,195 and these agents might pos-
ALS pathology sibly reverse other aberrant epigenetic changes in the
CNS as well.196 Thus, the identification of epigenetic
biomarkers for sporadic ALS could elucidate pathologi-
Cell body cal molecular mechanisms, aid in diagnosis, and reveal
therapeutic targets.
Conclusions
Phagocyte The mechanisms leading to ALS are complex, and despite
Axon extensive research into the genetic causes and environ-
mental risk factors, the vulnerability of motor neurons
to neurodegeneration cannot be explained by the indi-
Schwann cell vidual involvement of these factors. The joint genetic
and environm ental contributions involving post-
Skeletal muscle transcriptional changes or epigenetic mechanisms,
Normal motor neuron Early-stage ALS Late-stage ALS however, are probably crucial to understanding ALS
and skeletal muscle
pathogenesis, and establishing a path to treatment.
Figure 2 | Dysregulation of miRNA biogenesis and functionNature
may beReviews
a cause| and/or
Neurology Furthermore, disruptions to miRNA pathways are a likely
consequence of ALS pathogenesis. Impairments in miRNA biogenesis or function
cause and/or consequence of mechanisms that lead to
occur through the dysregulation of various cellular pathways. Specifically, miRNA–
protein complex metabolism is possibly altered by cytoplasmic protein inclusions, ALS pathology (Figure 2), including altered RNA and
transmission of wrongful cues due to disrupted signalling at the neuromuscular protein metabolism, inflammatory responses, cytotoxic-
junction, hampering of cell homeostasis due to cytotoxicity associated with faulty ity, and impaired structure and signalling at the neuro-
glutamate clearance, and an overactive inflammatory response. Conversely, evidence muscular junction. Thus, epigenetics offers new scientific
also suggests that the dysregulation of key miRNAs triggers the alterations in these approaches and tools for uncovering the pathophysio
cellular pathways that cause the downstream effects ending in the neurodegeneration logy of neurodegeneration. Furthermore, the revers-
associated with ALS pathology. Abbreviations: ALS, amyotrophic lateral sclerosis;
ible nature of epigenetic mechanisms positions them as
miRNA, microRNA; TGFβ1, transforming growth factor β1.
attractive targets for therapeutic development. In fact,
some molecules and environmental manipulations have
In one of the first attempts to use this strategy in ALS, successfully reversed certain epigenetic marks in both
delivery of anti-miR‑155 to SOD1 Gly93Ala mice via laboratory and clinical settings, supporting the potential
ventricular osmotic pumps successfully delayed mortal- of therapeutic agents based on epigenetic mechanisms.
ity.159 A pitfall associated with this approach is the poten- Further investigation of proteins involved in miRNA
tial for nonspecific binding to other RNAs. Alternatively, biogenesis and disruption, and identification of targets
the second approach to miRNA therapeutics involves that could restore altered cellular pathways, such as those
miRNA mimics and miRNA replacement therapies, mediated by TDP‑43 and FUS, might also open new
which can reintroduce miRNAs into cells exhibiting therapeutic avenues.
downregulation, thereby reactivating key pathways.190
Notably, injection of artificial miR‑124a oligonucleotides
can counteract the increase in EAAT2 expression associ- Review criteria
ated with decreased miR‑124a that is observed in mutant References were identified from English language
SOD1 murine spinal cords.171 publications in PubMed through January 2015, and
The first miRNA-based therapeutic (currently being from the authors’ collections of scientific literature.
evaluated in phase II clinical trials) is the miR‑122 antag- Combinations of the following key words were used:
onist SPC3649, which targets the hepatitis C virus. So far, “amyotrophic lateral sclerosis”, “ALS”, “frontotemporal
dementia”, “neurodegeneration”, “Alzheimer’s disease”,
this agent has exhibited no adverse effects.191 Although
“Parkinson’s disease”, “pathology”, “neuroinflammation”,
miRNA-targeted therapeutics are still in infancy, con- “protein aggregates”, “disease modifier”, “genetics”,
tinued, rapid technological progress has the potential “C9orf72”, “TDP‑43”, “FUS/TLS”, “epigenetics”, “miRNA”,
to facilitate the translation of these approaches to future “posttranslational modifications”, “DNA modifications”,
human clinical trials. “chromatin remodeling”, “noncoding RNA”, “RNA editing”,
In recent years, therapies that target histone modifica- “motor neuron disease”, “environment”, “exposure”, “risk
tions have been successful in fighting neurodegeneration factor”, “ heavy metals”, “biomarkers”, “therapeutics”.
Publications from the past five years were prioritized.
and cancer. The HDAC inhibitor sodium phenylbutyrate

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interact in genetic models of amyotrophic lateral activation of cAMP-response element-binding Funding support during the preparation of this Review
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Amyotrophic Lateral Sclerosis - Mechanisms and Therapeutics in The Epigenomic Era

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REVIEWS

Amyotrophic lateral sclerosis: mechanisms

266 | MAY 2015 | VOLUME 11  www.nature.com/nrneurol

SOD1 has prion-like properties, with an ability to seques-

NATURE REVIEWS | NEUROLOGY VOLUME 11 | MAY 2015 | 267

268 | MAY 2015 | VOLUME 11  www.nature.com/nrneurol

Box 1 | What is epigenetics?

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Table 1 | Epigenetic mechanisms and findings in ALS

270 | MAY 2015 | VOLUME 11  www.nature.com/nrneurol

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a ◀ Figure 1 | Disruption of miRNA biogenesis and function by

272 | MAY 2015 | VOLUME 11  www.nature.com/nrneurol

Increased expression of miR‑23a, miR‑29b, miR‑206, Converging pathways

NATURE REVIEWS | NEUROLOGY VOLUME 11 | MAY 2015 | 273

Table 2 | Key miRNAs differentially expressed in ALS

274 | MAY 2015 | VOLUME 11  www.nature.com/nrneurol

Neuromuscular (NaPB) upregulates astrocytic neurotrophin expression

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276 | MAY 2015 | VOLUME 11  www.nature.com/nrneurol

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