RNA Dysregulation in Amyotrophic Lateral Sclerosis

1Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder of motor function. It is characterized by the selective degeneration of the lower and upper motor neurons. Among the symptoms of this disease are progressive muscle weakness and paralysis, swallowing difficulties and breathing impairment due to respiratory muscle weakness that ultimately causes death, usually within 2–5 years following clinical diagnosis. Though most cases of ALS are sporadic, some families (10%) demonstrate a clinically indistinguishable form of ALS with clear Mendelian inheritance and high penetrance. Treatments to slow the progression of ALS to date remains riluzole and edaravone but they are only modestly effective. However, in the past couple years, there has been a real encouragement in witnessing potentially efficacious treatments, such as Masitinib and Pimozide claiming to demonstrate clinical benefit. Furthermore, RNA-targeted therapies are currently intensively being evaluated as potential strategies for treating this ALS. There is indeed hope to have new and potentially more effective treatment options available for ALS in the near future.

2Mutations in over more than 20 genes contribute to the etiology of ALS (Table 1). Amongst these genes, the major established causal ALS genes are SOD1 (Cu-Zn superoxide dismutase 1), TARDBP (transactive response DNA Binding protein 43kDa), FUS (fused in sarcoma) and hexanucleotide expansion repeat in Chromosome 9 Open Reading Frame 72 (C9ORF72). These genetic discoveries have led to the development of animal models that permitted the identification of key pathobiological insights. Currently, RNA dysregulation appears to be a major contributor to ALS pathogenesis. Indeed, TDP-43 and FUS are deeply involved in RNA processing such as transcription, alternative splicing and microRNA (miRNA) biogenesis. Mutations in C9ORF72, lead to a toxic mRNA gain of function through RNA foci formation, and the subsequent sequestration in stress granules and altered activity of RNA-binding proteins. In addition to the major ALS genes, other ALS genes including ataxin-2 (ATXN2), TATA-box binding protein associated factor 15 (TAF15), heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), heterogeneous nuclear ribonucleoprotein A2 B1 (hnRNPA2 B1), matrin 3 (MATR3), Ewing’s sarcoma breakpoint region 1 (EWSR1), T-cell-restricted intracellular antigen-1 (TIA1), senataxin (SETX) and angiogenin (ANG), play critical role in RNA processing (Table 1).

3In this review, we focus on the four major ALS-associated genes (SOD1, TARDBP, FUS, and C9orf72) and present how they play critical roles in various RNA pathways. We particularly highlight recent developments on the dysregulation of RNA pathways (Figure 1) as a major contributor to ALS pathogenesis and discuss the potential of RNA-targeted therapies for ALS.

4A major advance in our understanding of cellular mechanisms in ALS came from the identification of causative mutations in the TARDBP gene. This gene encodes for the evolutionarily conserved RNA/DNA binding protein, TDP-43. It is a protein that is normally nuclear, however, in cases of TARDBP mutations, it is mislocalized to the cytoplasm and forms aggregates. It is found in the pathological aggregates in motor neurons in the majority of cases of ALS. It is believed that TDP-43 aggregation leads to a gain of toxicity and its nuclear depletion results to a loss of function of TDP-43. Indeed, several studies have demonstrated that either overexpression or knockdown of TDP-43 causes neurodegeneration and ALS phenotypes. For instance, the expression of the mutant TDP-43A315T in the C. elegans’ GABAergic motor neurons results in age-dependent motility defects and neurodegeneration. In drosophila, overexpression of TDP-43 in motor neurons was found to cause cytoplasmic accumulation of TDP-43 aggregates, neuromuscular junction (NMJ) morphological defects and cell death. Similarly, the loss of TDP-43 reduced locomotion and lifespan. Implications of TDP-43 loss and toxic gain-of-function in impaired motility, neurodegeneration and survival were further confirmed in higher model systems such as the zebrafish and mice. Altogether, these reports strongly suggest that alterations in the level of TDP-43 are detrimental to neuronal function and survival.

5TDP-43 contains two RNA recognition motifs (RRM1-2), a glycine rich domain in the C-terminus and nuclear localization and export signals (NLS and NES). TDP-43 plays a major role in multiple steps of RNA processing such as splicing, RNA stability and mRNA transport. For instance, TDP43 has been shown to bind to mRNA and regulate the expression of other proteins implicated in ALS and other neurodegenerative diseases such as FUS, Tau, ATXN 2 and progranulin. This suggests that TDP-43 may be a central component in the pathogenesis of several neurodegenerative conditions. By RNA-seq analysis, reported that TDP-43 is required for regulating the expression of 239 mRNAs, many of those encoding synaptic proteins. Several independent studies have corroborated that TDP-43 plays an important role in regulating genes involved in synaptic formation and function and in the regulation of neurotransmitter processes. Examples of such genes are neurexin (NRXN1-3), neuroligin (NLGN1-2), scaffolding protein Homer2, microtubule-associated protein 1B (MAP1B), GABA receptors subunits (GABRA2, GABRA3), AMPA receptor subunits (GRIA3, GRIA4), syntaxin 1B, and calcium channel cacophony. The development of TDP-43 animal models has offered the opportunity to explore synaptic alterations in ALS and continuous efforts are being made to identify compounds that can facilitate synaptic transmission in ALS. reported that expression of mutant TARDPG348C mRNA in zebrafish resulted in impaired synaptic transmission, reduced frequency of miniature endplate currents (mEPCs) and reduced quantal transmission. Remarkably, they also demonstrated that all these synaptic dysfunction features in their zebrafish TARDBP mutant were stabilized by chronic treatment the L-type calcium channel agonists. In drosophila neurons, TDP-43 depletion was shown to reduce dendritic branching as well as synaptic formation. Overexpression or knocking down TDP-43 in cultured mammalian neurons also led to reduced dendritic branching. In TDP-43A315T mice, showed that expression of mutant TDP-43 alters dendritic spine development, spine morphology and neuronal synaptic transmission. Collectively, these independent studies on several model systems, suggest that TDP-43 may play an important role in neuronal morphology, synaptic transmission and neuronal plasticity likely via regulation of RNA processing of various synaptic genes.

6TDP-43 is also known to act as a splicing regulator to reduce its own expression level by binding to the 3′ UTR of its own pre-mRNA. Additionally, it functions as a splicing factor whose depletion or overexpression can affect the alternative splicing of specific targets. Indeed, the alternative splicing of several genes were reported to be altered in human CNS tissues from TDP-43 ALS cases. For instance, the level of the polymerase delta interacting protein 3 (POLDIP3) variant-2 mRNA (lacking exon 3) was significantly increased in the CNS of ALS patients with ALS, while that of variant-1 mRNA remained unchanged. This was consistent with findings that TDP-43 directly regulates the inclusion of exon 3 of POLDIP3 and that depletion of TDP-43 in cell culture models increased variant-2 mRNA. TDP-43 has also been shown to regulate splicing of the cystic fibrosis transmembrane regulator (CFTR) gene and controls exon skipping by within the pre-mRNA. Importantly, it controls the alternative splicing of apolipoprotein AII (APOAII) and survival of motor neuron (SMN) transcripts. Specifically, TDP-43 was shown to enhance the inclusion of exon 7 during the maturation of human SMN2 pre-mRNA, which results to an increase in full-length SMN2 mRNA level in neurons. Furthermore, recently TDP-43 was shown to bind to HNRNPA1 pre-mRNA to modulate its alternative splicing. TDP-43 depletion resulted in exon7B inclusion, culminating in a longer hnRNAP A1B isoform that is aggregation-prone and cytotoxic. Collectively, these studies demonstrated that loss of TDP-43 results to alterations in alternative splicing of many genes and some of which, for example HNRNPA1, can contribute to cellular vulnerability. It would be interesting further to investigate the contribution of the alteration of splicing of these genes (POLDIP3, CFTR, APOAII, SMN2, HNRNPA1) to the pathogenesis of ALS.

7TDP-43 is actively transported along axons and co-localizes with other well-known transport RNA binding proteins close to synaptic terminals. It was reported that TDP-43 mutations impair mRNA transport function in vivo and in vitro. In addition to a role in mRNA transport, TDP-43 also acts as a regulator of mRNA stability. It was shown to directly interacts with the 3′ UTR of neurofilament light chain (NFL) mRNA to stabilize it and associates with futsch/MAP1B mRNA in Drosophila to regulates its localization and translation. Particularly, TDP-43 was found to interact with 14-3-3 protein subunits to modulate the stability of the NFL mRNA. Abnormal regulation of NFL mRNA has been observed in ALS patients and disruption of NFL mRNA stoichiometry leads to motor neuron death and symptoms of ALS in animal models. It is, thus, very likely that TDP-43 mutations may cause motor neuron degeneration by interfering with RNA processing of NFL mRNA.

8Other important identified targets regulated by TDP-43 at mRNA level that may play a role in disease are G3BP and TBC1D1. G3BP is an essential component of stress granules, which are cytoplasmic non-membrane organelles that store translationally arrested mRNAs that accumulate during cellular stress. Stress granules consists of polyadenylated mRNAs, translation initiation factors (e.g., eIF3, eIF4E, and eIF4G), small ribosomal subunits and a numerous RNA-binding proteins. TDP-43 is recruited to stress granules in cellular models upon exposure to different stressors. Importantly, cytosolic TDP-43 mutants are more efficiently recruited to stress granules upon cellular stress compared to nuclear wild-type TDP-43. Prolonged stress is thought to promote sequestration of TDP-43 and their mRNA targets in stress granules; thereby inhibiting translation and potentially contributing to ALS progression.

9Mutations in FUS are detected in 4–5% of familial ALS patients as well as in sporadic ALS. FUS is an RNA/DNA-binding protein of 526 amino acids, consisting of an RNA-recognition motif, a SYGQ (serine, tyrosine, glycine and glutamine)-rich region, several RGG (arginine, glycine and glycine)-repeat regions, a C2C2 zinc finger motif and a nuclear localization signal (NLS). C-terminal ALS FUS mutations disrupt the NLS region and the nuclear import of FUS; resulting in cytoplasmic accumulation.

10Similarly to TDP-43, FUS plays multiple roles in RNA processing by directly binding to RNA. Using CLIP-based methods, several groups have identified thousands of RNA targets bound by FUS in various cell lines, and brain tissues. Interestingly, FUS was identified in spliceosomal complexes and interacting with several key splicing factors (such as hnRNP A1, YB-1) as well as with the U1 snRNP. FUS regulates splicing events for neuronal maintenance and survival. Given that FUS plays an essential role in splicing regulation, the consequence of its loss of function in ALS on RNA splicing has been immensely investigated. For instance, showed by mass spectrometric analysis that minor spliceosome components are highly enriched among the FUS-interacting proteins. They further reported that FUS interacts with the minor spliceosome and directly regulates the removal of minor introns. Moreover, the FUSP525L ALS mutation, which destroys the NLS and results in cytoplasmic retention of FUS, inhibits splicing of minor introns and causes mislocalization of the minor spliceosome components U11 and U12 snRNA to the cytoplasm and inhibits splicing of minor introns. Loss of function of FUS led to splicing changes in more than 300 genes mice brains and importantly a vast majority minor intron containing mRNAs was altered. Corroborating the results with mouse brain, many minor intron-containing genes were found to be downregulated in FUS-depleted SH-SY5Y cells. FUS depletion has been shown to affect minor intron containing genes that are important for neurogenesis (PPP2R2C), dendritic development (ACTL6B) and action potential transmission in skeletal muscles (SCN8A and SCN4A) and may contribute to ALS pathogenesis. FUS has also been shown to regulate alternative splicing of genes related to cytoskeletal organization, axonal growth and guidance such as the microtubule-associated protein tau (MAPT), Netrin G1 (NTNG1), neuronal cell adhesion molecule (NRCAM) and the actin-binding LIM (ABLIM1). For example, FUS knockdown has been shown to promote inclusion of exon 10 in the MAPT/tau protein and to significantly cause shortened axon length and growth cone enlargement. Loss of function of FUS altered MAPT/tau isoform expression and likely disturbed cytoskeletal function impairing axonal growth and maintenance. Interestingly, axon retraction and denervation are early events in ALS. Disruption of cytoskeleton function may thus play an important role in neurodegeneration in ALS.

11Besides its functions in splicing, FUS has been proposed to regulate transcription by RNA polymerase II (RNAP2), RNA polymerase III (RNAP3) or cyclin D1. For instance, transcriptomic analyses showed that knockdown of FUS results in differential expression several genes including many mRNAs encoding proteins important for neuronal function. Transcriptome changes have also been observed in human motoneurons obtained from FUS mutant induced pluripotent stem cells (IPSCs) and transgenic FUS knockin mice. Alterations in the expression of several genes involved in pathways related to cell adhesion, apoptosis, synaptogenesis and other neurodegenerative diseases were reported in these FUS models. Among these genes TAF15, which is mutated in some case of ALS, has been found to be upregulated in several ALS FUS models including human mutant IPSC derived motoneurons, FUS knockout and knockin mouse. However, it remains to be determined whether TAF15 upregulation upon FUS loss- or toxic gain- of function contributes to ALS pathogenesis.

12FUS is also incorporated into stress granules under cellular stress conditions. Sequestration of FUS and its protein partners into these cytoplasmic organelles appears to contribute to ALS pathogenesis. An example of such a protein partner is Pur-alpha, which co-localizes with mutant FUS and becomes trapped in stress granules in stress conditions, as reported in ALS patient cells carrying FUS mutations. It has been shown that FUS physically interacts with Pur-alpha. In vivo expression of Pur-alpha in Drosophila significantly exacerbates the neurodegeneration caused by mutated FUS. Conversely, showed that the downregulation of Pur-alpha in neurons expressing mutated FUS significantly improves fly climbing activity. It was subsequently demonstrated that overexpression Pur-alpha inhibits cytoplasmic mislocalization of mutant FUS and promotes neuroprotection. However, the function of Pur-alpha in regulating ALS pathogenesis remains elusive.

13Unlike TDP43 and FUS, SOD1 does not contain RNA-binding motifs, however, several reports have demonstrated a potential role of mutant SOD1 in regulating RNA metabolism. Particularly, mutant SOD1 can bind mRNA species such as vascular endothelial growth factor (VEGF) and NFL and negatively affects their expression, stabilization and function. More precisely, mutant SOD1 can directly bind to specific adenylate- and uridylate-rich stability elements (AREs) located in the 3′ UTR of transcripts of VEGF and NFL. It is believed that such a gain of abnormal protein–RNA interactions can be caused by SOD1 misfolding that results in the exposure of polypeptide portions with the ability to bind nucleic acids.

14Binding of mutant SOD1 to the 3′ UTR of the VEGF mRNA results in the sequestration of other ribonucleoproteins such as TIAR and HuR into insoluble aggregates. These interactions, which are specific to mutant SOD1, result in decline levels of VEGF mRNA, impairment of HuR function and ultimately hampering their neuroprotective actions during stress responses.

15In motor neuron-like NSC34 cell lines expressing mutant SOD1 (G37R or G93A), the level of NFL mRNA is significantly reduced. Reduction in NFL mRNA levels has also been reported in G93A transgenic mice and human spinal motor neurons from SOD1-ALS cases. It is proposed that destabilization NFL mRNA by mutant SOD1, result to altered stoichiometry of neurofilament (NF) subunits and subsequent NF aggregation in motor neurons. NF inclusion in the soma and proximal axons of spinal motor neurons is a hallmark of ALS pathology. In IPSC-derived model of ALS, a reduction of NFL mRNA level has been reported to result in NF aggregation and neurite degeneration. Altogether, these studies support a pathogenic role for dysregulation of RNA processing in SOD1-related ALS.

16Interestingly, SOD1 has been shown to interact with TDP-43 to modulate NFL mRNA stability. As mentioned above, TDP-43 was found to directly interact with the 3′ UTR of NFL mRNA to stabilize it. Altogether, these studies suggest that SOD1 and TDP-43 may act in a possible common action in regulating specific RNA stability. In the case of NFL mRNA, it would be interesting to investigate whether mutant SOD1 dislodges TDP-43 from the NFL mRNA in a manner that would affect its mRNA metabolism and potentially making NF prone to form aggregates.

17Furthermore, there have been several transcriptome investigations in SOD1 human samples, motor neuron-like NSC34 cell culture model and transgenic animals including mice, rat and drosophila. These studies have reported dysregulation of genes involved in pathways related to the neuroinflammatory and immune response, oxidative stress, mitochondria, lipid metabolism, synapse and neurodevelopment. However, in these studies it is not clear whether SOD1 directly or indirectly impact the regulation of the differentially expressed genes. In a recent elegant study,, compared transcriptome changes in SOD1 and TDP-43 models. They found that most genes that were altered in the SOD1G93A model were not dysregulated in the TDP-43A315T model, and vice versa. There were, however, a few genes whose expressions were altered in both ALS models. These findings are consistent with the ALS pathology, which is distinguishable between the ALS-related SOD1 phenotype and the TDP-43 phenotype. Although different cellular pathways are likely activated by SOD1 versus TDP-43, it is very plausible that they ultimately convergence onto common targets to result in similar motor neuron toxicity and ALS phenotype.

18In 2011, a large GGGGCC hexanucleotide repeat expansion in the first intron or promoter region of the C9orf72 gene has been discovered as a new cause of ALS. C9orf72 repeat expansion mutations account for about 50% of familial ALS and 5–10% of sporadic ALS. It remains a topic of debate whether the repeat expansion in C9orf72 causes neurodegeneration primarily through a toxic gain of function, loss of function, or both. The C9orf72 repeat expansion is transcribed in both the sense and antisense directions and leads to accumulations of repeat-containing RNA foci in patient tissues. The formation of RNA foci facilitates the recruitment of RNA-binding proteins, causes their mislocalization and interferes with their normal functions. Indeed, RNA foci may bind RNA binding proteins and alter RNA metabolism. For example, and showed that RNA foci can sequester hnRNP-A3 and repress its RNA processing function. Aborted transcripts containing the repeat can also disrupt nucleolar function. Importantly, these foci can sequester nuclear proteins such as TDP-43 and FUS, impacting expression of the their RNA targets and culminating in a range of RNA misprocessing events. Other RNA binding proteins binding to RNA foci include hnRNP A1, hnRNP-H, ADARB2, Pur-α, ASF/SF2, ALYREF and nucleolin. Antisense oligonucleotides (ASOs) targeting the C9orf72 repeat expansion suppress RNA foci formation, attenuate sequestration of specific RNA-binding proteins and reverse gene expression alterations in C9orf72 ALS motor neurons derived from IPSCs.

19Additionally, simple dipeptide repeats (poly-GA, poly-GP, poly-GR, poly-PA, and poly-PR) can be generated by repeat-associated non-ATG-dependent (RAN) translation of both the sense and antisense strands that have a variety of toxic effects. Poly-PR and poly-GR can alter the splicing patterns of specific RNAs. For example, poly-PR has been shown to cause exon-skipping in RAN and PTX3 RNA. Dipeptides repeat proteins have also been found to be toxic by creating aggregates sequestrating cytoplasmic proteins. Poly-GR dipeptide co-localizes with several ribosomal subunits and with a transcription factor elF3η. This suggests a ribosomal dysfunction, which implies a defect in RNA translation. In line with these findings, a recent report demonstrated that poly-PR co-localizes with the nucleolar protein, nucleophosmin, and reduces the expression of several ribosomal RNA. further showed that the reduction in the expression of ribosomal RNA results in neuronal cell death and this could be rescued by overexpression of an accelerator of ribosome biogenesis, Myc. RNA sequencing reveals that more than 6,000 genes are up or down regulated in mice that express the dipeptide construct in the brain. Other findings show that poly-PR dipeptide binds nuclear pores channels blocking the import and export of molecules. The dipeptide actually binds the nucleoporin proteins Nup54 and Nup98 that rim the central channel of the pore. The accumulation of poly-PR dipeptide at the nuclear pore was found to correlate with defect in nuclear transport of RNA and protein, which is consistent with previous findings.

20The last proposed mechanism involved in ALS pathogenesis is a haploinsufficiency due to the expansion of repetition leading to a decreased transcription of the gene and consequently to a decrease of its translation. Studies have demonstrated that C9orf72 expansion repeat can interfere with transcription or splicing of C9orf72 transcripts. It has also been proposed that the C9orf72 expansion repeat could disrupt the C9orf72 promoter activity thereby reducing its expression. Several studies have demonstrated alterations in the C9orf72 ALS transcriptome. Interestingly, a recent article reported an increased expression of the calcium-permeable GluA1 AMPA receptor subunit in motoneurons derived from IPSC of patients with C9orf72 mutations. This alteration in AMPA receptor composition led to an enhanced motoneuron vulnerability to AMPA-induced excitotoxicity. It remains to be determined whether the increased expression of GluA1 AMPA subunit is related to reduced levels of C9orf72, RNA foci and/or dipeptide repeats.

21C9orf72 has also been showed to be involved in the generation of stress granules and sequestering other RNA binding proteins that are involved in nucleo-cytoplasmic transport. It has been found that stress granules observed in C9orf72 mutants co-localizes with Ran GAP; which is known to activate Ran GTPase. This GTPase in involved in nucleo-cytoplasmic transport. It has also been published that expressing Ran GAP rescues the age-related motor defects in flies expressing the GGGGCC repeats. Very recently, it has also been reported that one of the dipeptide generated by the expansion has a role in formation of these stress granules. Moreover, importins and exportins are sequestered in stress granules; which also implies that protein transport in altered.

22These toxic gain- or loss-of function mechanisms are thought to be all involved in synergy in ALS pathogenesis and it can be summed up that that altered RNA processing plays a key role in C9orf72-mediated toxicity through two ways. The first is altered processing of the expanded C9orf72 transcript itself, in terms of altered transcription, splicing defects, nuclear aggregation and non-conventional translation. The second involves downstream and indirect changes in RNA processing of other transcripts. A thorough understanding of RNA metabolism dysregulation could definitely bring a major enlightenment on how C9orf72 mutation leads to ALS and provide insights on therapeutic targets.

23Multiple mechanisms control the proper levels of RNA and subsequent protein expression; among these are microRNAs (miRNAs). They are endogenous small non-coding RNAs (approximately 22 nucleotides in length) that are initially transcribed by the RNA polymerase II as primary miRNA (pri-miRNAs) transcripts. These pri-miRNAs are processed into precursor miRNAs (pre-miRNAs) by the nuclear ribonuclease III (RNase III), DROSHA, and the double-stranded RNA-binding protein, DGCR8, which anchors DROSHA to the pri-miRNA transcript. Pre-miRNA is then exported into the cytoplasm by exportin-5, where it is processed into a mature miRNA by the DICER enzyme. The mature miRNA is then incorporated with a ribonucleoprotein (RNP) complex with argonaute (AGO) proteins to form the RNA-induced silencing complex (RISC), which mediates inhibition of translation and/or mRNA degradation of targeted transcripts that are complementary to the miRNA. The recognition of mRNAs by miRNAs occurs through base-pairing interactions within the 3′-untranslated region (UTR) of the targeted mRNAs. Besides their well-known gene silencing functions, miRNAs can also induce up-regulation of their targets.

24MiRNAs play important roles in several biological processes such as cell proliferation, cell differentiation, apoptosis, and patterning of the nervous system. Interestingly, several miRNAs have been particularly shown to be essential for motor neuron development and survival (see review, ). For example, in developing chick, it was demonstrated that the activation of the miRNA miR9 is necessary to suppress the expression of the transcription factor onecut1, which in turn helps to drive differentiation of neural progenitor cells into spinal motor neurons. It is believed that several miRNAs work in concert to establish motor neuron identity. Indeed, in addition to miR9, other miRNAs such as miR-128, miR-196, miR-375 have been shown to play a role in motor neuron differentiation and localization. Loss of DICER function within progenitor cells results in aberrant motor neuron development while its loss in motor neuron leads to progressive motor neuron degeneration. Furthermore, miRNAs are important players for NMJ function, synaptic plasticity and for maintaining cytoskeletal integrity (see review, ).

25The ALS genes, TDP-43 and FUS, were identified in a protein complex with RNAse III DORSHA and shown to play a role in miRNA biogenesis. TDP-43, in particular was shown to associate with proteins involved in the cytoplasmic cleavage of pre-miRNA mediated by the DICER enzyme. It is thus to no surprise that dysregulation of miRNAs has been observed in ALS. Indeed, mutations in TARDBP result in differential expression of miRNAs – miR-9, miR-132, miR-143, and miR-558. Interestingly, the expression of several of these miRNAs (miR-9, miR-132, miR-143) and including others (such as miR-125, miR-192) are altered upon FUS depletion. MiR-9 expression is also found to be upregulation in mutant SOD1 mice. These dysregulated miRNAs are essential for motor neuron development and maintenance, axonal growth and synaptic transmission. Thus, these miRNA alterations likely contribute to the pathological phenotype observed in ALS.

26Additionally, depletion of TDP-43 in cell culture systems has also been shown to change the total miRNA expression profile. A similar observation was recently observed in motoneurons progenitors derived from human ALS IPSCs. Particularly, it was reported that 15 miRNAs were dysregulated including disease-relevant miR-34a and miR504, which are known to be, implicated synaptic vesicle regulation and cell survival. Additionally, another important miRNA, namely microRNA-1825, was found to be downregulated in CNS of both sporadic and familial ALS patients. Interestingly, reduced levels of microRNA-1825 was demonstrated to cause a translational upregulation of tubulin-folding cofactor b (TBCB) which consequently to depolymerization and degradation of tubulin alpha-4A (TUBA4A), which is encoded by a known ALS gene.

27In several repeats diseases such as myotonic dystrophy, fragile X tremor and ataxia syndrome, toxic RNA from expansion repeats cause widespread RNA splicing abnormalities, degeneration of affected tissues and alter miRNA processing. Since its discovery, C9orf72 GGGGCC expansion repeat was also questioned as a disruptor of miRNA processing. Recently, the DROSHA protein was found to be mislocalized in dipeptide repeat protein-aggregates in frontal cortex and cerebellum C9orf72 ALS/FTLD patients. An involvement of the miRNA pathway in motor neuron impairment in ALS is evident and further investigations on miRNAs dysregulation in ALS pathogenesis could eventually lead to the identification of therapeutic targets.

28Our understanding of RNA biology has expanded tremendously over the past decades, resulting in new approaches to engage RNA as a therapeutic target. More precisely, RNA-targeted therapeutics have been developed to mediate the reduction or expression of a given target RNA by employing mechanisms such as RNA cleaving, modulation of RNA splicing, inhibition of mRNA translation into protein, inhibition of miRNA binding sites, increasing translation by targeting upstream open reading frames and disruption of RNA structures regulating RNA stability. Therapeutics that directly target RNAs are promising for a broad spectrum of disorders, including the neurodegenerative diseases and are currently under evaluation as potential strategies for treating ALS. The RNA therapeutics approaches include RNA interference (RNAi) and ASOs (Figure 2), both bind to their target nucleic acid via Watson-Crick base pairing and cause degradation of or inactivate the targeted mRNA. Recently, application of innovative drug discovery approaches has showed that targeting RNA with bioactive small molecules is achievable. A few researchers including us are currently exploiting such a new type of RNA-targeted therapeutics to search for RNA-targeted small molecules as C9orf72 ALS therapeutics.

29RNAi is an endogenous cellular mechanism to regulate mRNA. It operates sequence specifically and post-transcriptionally via the RISC. Methods of mediating the RNAi effects are via small interfering RNA (siRNA), short hairpin RNA (shRNA), and artificial miRNA. These approaches can help to reduce the expression of mutant (toxic) gene and can provide significant therapeutic benefit in treating ALS and other neurodegenerative disease implicating aberrant accumulation of misfolded proteins.

30The challenge of using siRNA for treating ALS is that it has to be designed to have the specificity and ability to reduce the aberrant mutant protein while leaving wild-type protein intact. Attempts were made to design siRNA, which could recognize just a single nucleotide alternation to selectively suppress mutant SOD1 (particularly G93A) expression leaving wild-type SOD1 intact. The design of siRNA G93A.1 and G93A.2 by were found to successfully suppress the expression of approximately 90% of mutant SOD1 G93A. Importantly, both siRNA had virtually little or no effect on wild-type SOD1 expression. To achieve long-term expression of siRNA in cells, the use of viral delivery system has proved powerful to provide a continuous delivery and expression of shRNA in sufficient quantities. Indeed, diverse viral vectors have been studied such as adeno-associated virus (AAV), lentivirus (LV), and rabies-glycoprotein-pseudotyped lentivirus (RGP-LV). Recombinant AAVs are currently the choice of RNAi treatment vehicle for neurological diseases because they are non-pathogenic and safe. Several studies have aimed at engineering AAV serotypes with better cell-type and tissue specificities and an improved immune-evasion potential. AAV9 and AAVrh10 serotypes have been shown to cross the blood–brain barrier and efficiently transduce cells in the CNS, with widespread and sustained transgene expression in the spinal cord and brain even after just a single injection. Importantly, they can efficiently target neurons and astrocytes, making them the most applicable delivery systems for treating ALS.

31Several researchers have independently use siRNA or shRNA to silence mutant SOD1 expression in vitro and in vivo. Intramuscular delivery of siRNA targeting mutant SOD1 in SOD1G93A mice delays the onset of motor neuron symptoms and extend their survival. Similarly, SOD1G93A mice treated with injection of AAV encoding shRNA against human SOD1 mRNA (hSOD1) exhibited delayed diseases onset and significantly increased their survival by 23%. The same group later demonstrated the efficacy of this approach in SOD1G93A rats, showing that silencing of hSOD1 expression selectively in the motor cortex also delayed disease onset and prolonged survival. Silencing of SOD1 using an artificial miRNA (miR-SOD1) systemically delivered using the viral vector AAVrh10 in SOD1G93A mice was also found to significantly delayed disease onset, preserved muscle motor functions and extended survival. Interestingly, similar findings were observed in non-human primates treated with AAVrh10-miR-SOD1. These findings suggest that miRNA silencing strategy warrants further investigations and may offer promise for the development for the treatment of SOD1-related ALS.

32The concept of ASOs was first introduced in 1978, when Stephenson and Zamecnik used a chemically modified oligonucleotide, designed to bind to its complementary sequence in a Rous sarcoma virus transcript to inhibit its gene expression and viral replication. ASOs are synthetic single-stranded oligonucleotides that activate the RNAse H, an endonuclease in the nucleus, to degrade the complementary mRNA. They can be designed to specifically target mutant RNAs or mRNA splicing. An ASO therapy based (nusinersen) approach designed to promote exon skipping has proven to be very effective in treating spinal muscular atrophy (SMA) in clinical trials. In late 2016, this antisense drug (marketed as Spinraza) has received FDA approval for the treatment of SMA. This was the first exciting success of ASO therapeutics in neurodegeneration and a significant milestone for ASO therapy, in general. With increased understanding of gain- and loss-of-function mechanisms of genetic forms of ALS, ASOs therapies have also been tested principally tested in SOD1 and C9ORF72 models to target the mutant forms of RNA but not the wild-type.

33The first study using an ASO to target SOD1 showed an effective silencing of SOD1 and reduced mutated SOD1 protein throughout the brain and spinal cord of SOD1G93A rats. Infusion of ASOs complementary to hSOD1 mRNA extended survival in SOD1G93A rats. Given these promising preclinical results, the ASO IONIS-SOD1Rx (ISIS 333611 and BIIB067) has been proposed as a therapeutic strategy for SOD1-link ALS and has been clinical tested. In a phase I testing, intrathecal administration of the ASO IONIS-SOD1Rx was showed to be both practical and safe in SOD1 ALS patients. A phase Ib/IIa trial (NCT02623699) is currently underway to further evaluate safety, tolerability, and pharmacokinetics of IONIS-SOD1Rx. Altogether, the preclinical and clinical tests suggest that ASOs delivered to the CNS represent a feasible treatment for SOD1-related ALS and are safe, however, ASOs are not specific for mutant over wild-type SOD1 and the long-term effects of the reduction of SOD1 need further investigation.

34In addition, silencing of SOD1 can be induced by exon skipping of hSOD1 using ASOs complementary to splicing regulatory elements on the primary transcript. For instance, administrating an exon-2-targeted ASO embedded in a modified U7 small-nuclear RNA and delivered by AAV10, in either newborn or adult (P50) SOD1G93A mice, was shown to increase survival and restore neuromuscular function. These recent findings provide new hope for treatment of ALS and open perspectives for a clinical development.

35Strong evidence supports that the mechanism by which the GGGGCC repeat expansion in C9orf72 causes the diseases is by toxicity of RNAs that they generate. Thus early development of ASO-based therapeutics for C9orf72 ALS focused on reducing gain-of-function toxicity associated with the repeat expansion. Testing of the efficacy of ASO-based therapeutics for C9orf72 was initially performed on clinically relevant human IPSC-derived neurons and fibroblasts. More recently, ASOs were also evaluated in mouse models expressing the expanded C9orf72.

36Antisense oligonucleotides were designed to bind within the GGGGCC repeat expansion or within surrounding N-terminal regions of the C9orf72 mRNA transcript to either degrade the transcript or block the interaction between the repeat expansion and RNA-binding proteins. ASOs effectively reduced RNA foci formation, dipeptide proteins, increased survival from glutamate excitotoxicity and restored normal gene expression markers. These promising findings suggest that ASO-based therapy can be a powerful way for treating C9orf72 ALS. They also provided the basis for the initiation of the first C9orf72 ASO clinical trial that is anticipated to start by the end of 2018.

37These planned ASOs trials in ALS as well as ongoing trials of ASOs in SMA, Huntington’s disease and Alzheimer’s disease will enhance our understanding of this therapeutic approach. Importantly, positive outcomes from these clinical trials will revolutionize the treatment of genetically mediated neurodegenerative diseases.

38RNAs adopt discrete secondary and tertiary structures and have pivotal roles in biology and diseases. The ALS-associated C9orf72 GGGGCC repeat RNA can stably fold to into a four-stranded structure formed by the stacking of planar tetrads of four guanosine residues, termed G-quadruplex. This G-quadruplex structure can affect various RNA processing including splicing and translation. In particular, the C9or72 repeat RNA G-quadruplexes have been shown to specifically sequester RNA-binding proteins and have toxic functions. GGGGCC repeat RNA sequence can also adopt a hairpin structure in addition to G-quadruplexes. Hairpin is composed of a base-paired stem and a loop and it can affect transcription and alternative splicing. Targeting these RNA structures of the C9or72 repeat is a potential therapeutic strategy.

39Recent developments in technologies and approaches have made the long sought-after goal of developing small-molecule drugs that target RNA possible. Small molecules binding to RNA hairpin or G-quadruplex structure have been identified. This has provided the springboard to initiate the search for small molecules that can specifically target C9orf72 repeat RNA and hinder pathogenic interactions with RNA-binding proteins and/or by interfering with RAN translation (Figure 2C).

40showed that (GGGGCC)8 RNA can adopt a hairpin structure in equilibrium with a quadruplex structure. They designed three compounds targeting mainly the hairpin structure of the (GGGGCC)n RNA and showed that the bioactive small molecule 1a significantly inhibited RAN translation and foci formation in cultured cells (GGGGCC)66 repeat expansion and in patient-derived neurons. However, these small molecules were only tested in vitro on cellular models. Recently, a drug screen study to identify compounds that specifically target the C9orf72 RNA G-quadruplex structure led to the identification of three lead compounds. These compounds were then functionally validated as ALS therapeutics in C9orf72 IPSC-derived neurons and C9orf72 repeat-expressing fruit flies. Interestingly, two of the lead compounds reduced RNA foci formation and the levels of toxic dipeptide repeat proteins in IPSC-derived spinal motor neurons and cortical neurons. The most effective small molecule (DB1273) was then tested in vivo on C9orf72 repeat-expressing fruit flies and was found to significantly reduce dipeptide repeats levels. Furthermore, D1273 improved the survival of the fruit flies. These studies support the further development of small molecules that selectively bind GGGGCC RNA as a therapeutic strategy for C9orf72 ALS and FTLD.

41RNA-targeted therapeutic approaches offer a treatment strategy with greater specificity, improved potency, and decreased toxicity compared to the small molecules against traditional drug targets (signaling proteins). They represent an important way to treat ALS and other neurodegenerative diseases that need to be considered in the near future. However, there are still some concerns and challenges to overcome for ALS therapeutic applications.

42Off-target effects RNAi and ASO remain an important consideration though thorough toxicological and safety research prior to clinical application can diminish some of this concern. The negative charge of siRNA and ASO as well as their size makes it difficult for them to cross the cell membrane. Viral packing is currently widely used to deliver ASO and siRNA into cells. Although, viral vectors are highly efficient as transfer vehicles, immunogenicity of the viral vectors is a major concern. Various other delivery strategies such as nanoparticles, liposomes and aptamers could be more effective and safe. Efforts are also underway to chemically stabilize siRNA, which will avoid the need for viral vectors. RNA foci and dipeptide products are generated from both sense and antisense directions of the C9orf72 transcript. However, ASOs for C9orf72 ALS preferentially target sense strand transcripts. There may be a need to design ASO strategies to target toxic RNA transcribed from both directions in order to adequately treat the C9orf72 ALS. Furthermore, ASO-based therapeutic strategy for C9orf72 ALS only target gain-of-function mechanisms, but loss-of-function mechanisms may also act in synergy to cause pathogenesis in C9orf72 ALS. It is very plausible that an integrated therapeutic approach to inhibit toxic RNA foci/dipeptide repeat protein formation and restore normal levels of C9orf72 may be necessary to fully address the cellular deficits in C9orf72 ALS.

43TDP-43, SOD1, FUS, and C9orf72 mutations are involved at various aspects of RNA processing and many of which are shared. It is becoming clear that impaired RNA regulation and processing is a central feature ALS pathogenesis. Given that defects at multiple steps of RNA processing impair cellular function and survival, RNA metabolism can be considered an essential target for therapeutic intervention for ALS and other neurodegenerative disease such as FTLD. The application of RNA-based therapies to modulation of gene and subsequent protein expression is an attractive therapeutic strategy. The preclinical testing of RNA-based therapies targeting SOD1 and C9orf72 mutations are indeed very promising. Similar studies are yet to be undertaken for FUS and TDP-43 mutations. RNA-based therapies could be considered in the future for the treatment of ALS.

44SP contributed to the idea conception and overall review design. ZB and SP wrote the manuscript.

45The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.