Single-cell dissection of the human motor and prefrontal cortices in ALS and FTLD.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) share many clinical, pathological, and genetic features, but a detailed understanding of their associated transcriptional alterations across vulnerable cortical cell types is lacking. Here, we report a high-resolution, comparative single-cell molecular atlas of the human primary motor and dorsolateral prefrontal cortices and their transcriptional alterations in sporadic and familial ALS and FTLD. By integrating transcriptional and genetic information, we identify known and previously unidentified vulnerable populations in cortical layer 5 and show that ALS- and FTLD-implicated motor and spindle neurons possess a virtually indistinguishable molecular identity. We implicate potential disease mechanisms affecting these cell types as well as non-neuronal drivers of pathogenesis. Finally, we show that neuron loss in cortical layer 5 tracks more closely with transcriptional identity rather than cellular morphology and extends beyond previously reported vulnerable cell types.

Correction to: ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD.

The original article was published erroneously without mentioning the support of the U.S.

ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD.

The hexanucleotide repeat expansion GGGGCC (G4C2)n in the C9orf72 gene is the most common genetic abnormality associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent findings suggest that dysfunction of nuclear-cytoplasmic trafficking could affect the transport of RNA binding proteins in C9orf72 ALS/FTD. Here, we provide evidence that the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is mislocalized in C9orf72 repeat expansion mediated ALS/FTD. ADAR2 is responsible for adenosine (A) to inosine (I) editing of double-stranded RNA, and its function has been shown to be essential for survival. Here we show the mislocalization of ADAR2 in human induced pluripotent stem cell-derived motor neurons (hiPSC-MNs) from C9orf72 patients, in mice expressing (G4C2)149, and in C9orf72 ALS/FTD patient postmortem tissue. As a consequence of this mislocalization we observe alterations in RNA editing in our model systems and across multiple brain regions. Analysis of editing at 408,580 known RNA editing sites indicates that there are vast RNA A to I editing aberrations in C9orf72-mediated ALS/FTD. These RNA editing aberrations are found in many cellular pathways, such as the ALS pathway and the crucial EIF2 signaling pathway. Our findings suggest that the mislocalization of ADAR2 in C9orf72 mediated ALS/FTD is responsible for the alteration of RNA processing events that may impact vast cellular functions, including the integrated stress response (ISR) and protein translation.

Deleterious mutations in the essential mRNA metabolism factor, hGle1, in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons. Causative mutations in the global RNA-processing proteins TDP-43 and FUS among others, as well as their aggregation in ALS patients, have identified defects in RNA metabolism as an important feature in this disease. Lethal congenital contracture syndrome 1 and lethal arthrogryposis with anterior horn cell disease are autosomal recessive fetal motor neuron diseases that are caused by mutations in another global RNA-processing protein, hGle1. In this study, we carried out the first screening of GLE1 in ALS patients (173 familial and 760 sporadic) and identified 2 deleterious mutations (1 splice site and 1 nonsense mutation) and 1 missense mutation. Functional analysis of the deleterious mutants revealed them to be unable to rescue motor neuron pathology in zebrafish morphants lacking Gle1. Furthermore, in HeLa cells, both mutations caused a depletion of hGle1 at the nuclear pore where it carries out an essential role in nuclear export of mRNA. These results suggest a haploinsufficiency mechanism and point to a causative role for GLE1 mutations in ALS patients. This further supports the involvement of global defects in RNA metabolism in ALS.

Conserved DNA methylation combined with differential frontal cortex and cerebellar expression distinguishes C9orf72-associated and sporadic ALS, and implicates SERPINA1 in disease.

We previously found C9orf72-associated (c9ALS) and sporadic amyotrophic lateral sclerosis (sALS) brain transcriptomes comprise thousands of defects, among which, some are likely key contributors to ALS pathogenesis. We have now generated complementary methylome data and combine these two data sets to perform a comprehensive "multi-omic" analysis to clarify the molecular mechanisms initiating RNA misregulation in ALS. We found that c9ALS and sALS patients have generally distinct but overlapping methylome profiles, and that the c9ALS- and sALS-affected genes and pathways have similar biological functions, indicating conserved pathobiology in disease. Our results strongly implicate SERPINA1 in both C9orf72 repeat expansion carriers and non-carriers, where expression levels are greatly increased in both patient groups across the frontal cortex and cerebellum. SERPINA1 expression is particularly pronounced in C9orf72 repeat expansion carriers for both brain regions, where SERPINA1 levels are strictly down regulated across most human tissues, including the brain, except liver and blood, and are not measurable in E18 mouse brain. The altered biological networks we identified contain critical molecular players known to contribute to ALS pathology, which also interact with SERPINA1. Our comprehensive combined methylation and transcription study identifies new genes and highlights that direct genetic and epigenetic changes contribute to c9ALS and sALS pathogenesis.

ERBB4 mutations that disrupt the neuregulin-ErbB4 pathway cause amyotrophic lateral sclerosis type 19.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by the degeneration of motor neurons and typically results in death within 3-5 years from onset. Familial ALS (FALS) comprises 5%-10% of ALS cases, and the identification of genes associated with FALS is indispensable to elucidating the molecular pathogenesis. We identified a Japanese family affected by late-onset, autosomal-dominant ALS in which mutations in genes known to be associated with FALS were excluded. A whole- genome sequencing and parametric linkage analysis under the assumption of an autosomal-dominant mode of inheritance with incomplete penetrance revealed the mutation c.2780G>A (p. Arg927Gln) in ERBB4. An extensive mutational analysis revealed the same mutation in a Canadian individual with familial ALS and a de novo mutation, c.3823C>T (p. Arg1275Trp), in a Japanese simplex case. These amino acid substitutions involve amino acids highly conserved among species, are predicted as probably damaging, and are located within a tyrosine kinase domain (p. Arg927Gln) or a C-terminal domain (p. Arg1275Trp), both of which mediate essential functions of ErbB4 as a receptor tyrosine kinase. Functional analysis revealed that these mutations led to a reduced autophosphorylation of ErbB4 upon neuregulin-1 (NRG-1) stimulation. Clinical presentations of the individuals with mutations were characterized by the involvement of both upper and lower motor neurons, a lack of obvious cognitive dysfunction, and relatively slow progression. This study indicates that disruption of the neuregulin-ErbB4 pathway is involved in the pathogenesis of ALS and potentially paves the way for the development of innovative therapeutic strategies such using NRGs or their agonists to upregulate ErbB4 functions.

ALS and FTD: an epigenetic perspective.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two fatal neurodegenerative diseases seen in comorbidity in up to 50 % of cases. Despite tremendous efforts over the last two decades, no biomarkers or effective therapeutics have been identified to prevent, decelerate, or stop neuronal death in patients. While the identification of multiple mutations in more than two dozen genes elucidated the involvement of several mechanisms in the pathogenesis of both diseases, identifying the hexanucleotide repeat expansion in C9orf72, the most common genetic abnormality in ALS and FTD, opened the door to the discovery of several novel pathogenic biological routes, including chromatin remodeling and transcriptome alteration. Epigenetic processes regulate DNA replication and repair, RNA transcription, and chromatin conformation, which in turn further dictate transcriptional regulation and protein translation. Transcriptional and post-transcriptional epigenetic regulation is mediated by enzymes and chromatin-modifying complexes that control DNA methylation, histone modifications, and RNA editing. While the alteration of DNA methylation and histone modification has recently been reported in ALS and FTD, the assessment of epigenetic involvement in both diseases is still at an early stage, and the involvement of multiple epigenetic players still needs to be evaluated. As the epigenome serves as a way to alter genetic information not only during aging, but also following environmental signals, epigenetic mechanisms might play a central role in initiating ALS and FTD, especially for sporadic cases. Here, we provide a review of what is currently known about altered epigenetic processes in both ALS and FTD and discuss potential therapeutic strategies targeting epigenetic mechanisms. As approximately 85 % of ALS and FTD cases are still genetically unexplained, epigenetic therapeutics explored for other diseases might represent a profitable direction for the field.

Exome sequencing identifies FUS mutations as a cause of essential tremor.

Essential tremor (ET) is a common neurodegenerative disorder that is characterized by a postural or motion tremor. Despite a strong genetic basis, a gene with rare pathogenic mutations that cause ET has not yet been reported. We used exome sequencing to implement a simple approach to control for misdiagnosis of ET, as well as phenocopies involving sporadic and senile ET cases. We studied a large ET-affected family and identified a FUS p.Gln290(∗) mutation as the cause of ET in this family. Further screening of 270 ET cases identified two additional rare missense FUS variants. Functional considerations suggest that the pathogenic effects of ET-specific FUS mutations are different from the effects observed when FUS is mutated in amyotrophic lateral sclerosis cases; we have shown that the ET FUS nonsense mutation is degraded by the nonsense-mediated-decay pathway, whereas amyotrophic lateral sclerosis FUS mutant transcripts are not.

Mechanisms of toxicity in C9FTLD/ALS.

A hexanucleotide repeat expansion within a non-coding region of the C9ORF72 gene is the most common mutation causative of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Elucidating how this bidirectionally transcribed G4C2·C4G2 expanded repeat causes "C9FTLD/ALS" has since become an important goal of the field. Likely pathogenic mechanisms include toxicity induced by repeat-containing RNAs, and loss of C9orf72 function due to epigenetic changes resulting in decreased C9ORF72 mRNA expression. With regards to the former, sense and antisense transcripts of the expanded repeat aberrantly interact with various RNA-binding proteins and form discrete nuclear structures, termed RNA foci. These foci have the capacity to sequester select RNA-binding proteins, thereby impairing their function. (G4C2)exp and (C4G2)exp transcripts also succumb to an alternative fate: repeat-associated non-ATG (RAN) translation. This unconventional mode of translation, which occurs in the absence of an initiating codon, results in the abnormal production of poly(GA), poly(GP), poly(GR), poly(PR) and poly(PA) peptides, collectively referred to as C9RAN proteins. C9RAN proteins form neuronal inclusions throughout the central nervous system of C9FTLD/ALS patients and may contribute to disease pathogenesis. This review aims to summarize the important findings from studies examining mechanisms of disease in C9FTLD/ALS, and will also highlight some of the many questions in need of further investigation.

Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress.

The occurrence of repeat-associated non-ATG (RAN) translation, an atypical form of translation of expanded repeats that results in the synthesis of homopolymeric expansion proteins, is becoming more widely appreciated among microsatellite expansion disorders. Such disorders include amyotrophic lateral sclerosis and frontotemporal dementia caused by a hexanucleotide repeat expansion in the C9ORF72 gene (c9FTD/ALS). We and others have recently shown that this bidirectionally transcribed repeat is RAN translated, and the "c9RAN proteins" thusly produced form neuronal inclusions throughout the central nervous system of c9FTD/ALS patients. Nonetheless, the potential contribution of c9RAN proteins to disease pathogenesis remains poorly understood. In the present study, we demonstrate that poly(GA) c9RAN proteins are neurotoxic and may be implicated in the neurodegenerative processes of c9FTD/ALS. Specifically, we show that expression of poly(GA) proteins in cultured cells and primary neurons leads to the formation of soluble and insoluble high molecular weight species, as well as inclusions composed of filaments similar to those observed in c9FTD/ALS brain tissues. The expression of poly(GA) proteins is accompanied by caspase-3 activation, impaired neurite outgrowth, inhibition of proteasome activity, and evidence of endoplasmic reticulum (ER) stress. Of importance, ER stress inhibitors, salubrinal and TUDCA, provide protection against poly(GA)-induced toxicity. Taken together, our data provide compelling evidence towards establishing RAN translation as a pathogenic mechanism of c9FTD/ALS, and suggest that targeting the ER using small molecules may be a promising therapeutic approach for these devastating diseases.

RNA-mediated toxicity in neurodegenerative disease.

Cellular viability depends upon the well-orchestrated functions carried out by numerous protein-coding and non-coding RNAs, as well as RNA-binding proteins. During the last decade, it has become increasingly evident that abnormalities in RNA processing represent a common feature among many neurodegenerative diseases. In "RNAopathies", which include diseases caused by non-coding repeat expansions, RNAs exert toxicity via diverse mechanisms: RNA foci formation, bidirectional transcription, and the production of toxic RNAs and proteins by repeat associated non-ATG translation. The mechanisms of toxicity in "RNA-binding proteinopathies", diseases in which RNA-binding proteins like TDP-43 and FUS play a prominent role, have yet to be fully elucidated. Nonetheless, both loss of function of the RNA binding protein, and a toxic gain of function resulting from its aggregation, are thought to be involved in disease pathogenesis. As part of the special issue on RNA and Splicing Regulation in Neurodegeneration, this review intends to explore the diverse RNA-related mechanisms contributing to neurodegeneration, with a special emphasis on findings emerging from animal models.

Reduced C9orf72 gene expression in c9FTD/ALS is caused by histone trimethylation, an epigenetic event detectable in blood.

Individuals carrying (GGGGCC) expanded repeats in the C9orf72 gene represent a significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Elucidating how these expanded repeats cause "c9FTD/ALS" has since become an important goal of the field. Toward this end, we sought to investigate whether epigenetic changes are responsible for the decrease in C9orf72 expression levels observed in c9FTD/ALS patients. We obtained brain tissue from ten c9FTD/ALS individuals, nine FTD/ALS cases without a C9orf72 repeat expansion, and nine disease control participants, and generated fibroblastoid cell lines from seven C9orf72 expanded repeat carriers and seven participants carrying normal alleles. Chromatin immunoprecipitation using antibodies for histone H3 and H4 trimethylated at lysines 9 (H3K9), 27 (H3K27), 79 (H3K79), and 20 (H4K20) revealed that these trimethylated residues bind strongly to C9orf72 expanded repeats in brain tissue, but not to non-pathogenic repeats. Our finding that C9orf72 mRNA levels are reduced in the frontal cortices and cerebella of c9FTD/ALS patients is consistent with trimethylation of these histone residues, an event known to repress gene expression. Moreover, treating repeat carrier-derived fibroblasts with 5-aza-2-deoxycytidine, a DNA and histone demethylating agent, not only decreased C9orf72 binding to trimethylated histone residues, but also increased C9orf72 mRNA expression. Our results provide compelling evidence that trimethylation of lysine residues within histones H3 and H4 is a novel mechanism involved in reducing C9orf72 mRNA expression in expanded repeat carriers. Of importance, we show that mutant C9orf72 binding to trimethylated H3K9 and H3K27 is detectable in blood of c9FTD/ALS patients. Confirming these exciting results using blood from a larger cohort of patients may establish this novel epigenetic event as a biomarker for c9FTD/ALS.

Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation.

A repeat expansion in the C9orf72 (C9) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we investigate single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem motor and frontal cortices from C9-ALS, C9-FTD, and control donors. C9-ALS donors present pervasive alterations of gene expression with concordant changes in chromatin accessibility and histone modifications. The greatest alterations occur in upper and deep layer excitatory neurons, as well as in astrocytes. In neurons, the changes imply an increase in proteostasis, metabolism, and protein expression pathways, alongside a decrease in neuronal function. In astrocytes, the alterations suggest activation and structural remodeling. Conversely, C9-FTD donors have fewer high-quality neuronal nuclei in the frontal cortex and numerous gene expression changes in glial cells. These findings highlight a context-dependent molecular disruption in C9-ALS and C9-FTD, indicating unique effects across cell types, brain regions, and diseases.

A mutation in the RNF170 gene causes autosomal dominant sensory ataxia.

Autosomal dominant sensory ataxia is a rare genetic condition that results in a progressive ataxia that is caused by degeneration of the posterior columns of the spinal cord. To date only two families have been clinically ascertained with this condition, both from Maritime Canada. We previously mapped both families to chromosome 8p12-8q12 and have now screened the majority of annotated protein-coding genes in the shared haplotype region by direct DNA sequencing. We have identified a putative pathogenic mutation in the gene encoding ring-finger protein RNF170, a potential ubiquitin ligase. This mutation is a rare non-synonymous change in a well-conserved residue and is predicted to be pathogenic by SIFT, PolyPhen, PANTHER and Align-GVD. Microinjection of wild-type or mutant orthologous messenger RNAs into zebrafish (Danio rerio) embryos confirmed that the mutation dominantly disrupts normal embryonic development. Together these results suggest that the mutation in RNF170 is causal for the sensory ataxia in these families.

Identification of a FUS splicing mutation in a large family with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by the degeneration of upper and lower motor neurons. Genetic studies have led, thus far, to the identification of 12 loci and 9 genes for familial ALS (FALS). Although the distribution and impact of superoxide dismutase 1 mutations has been extensively examined for over a decade, the recently identified FALS-associated FUS gene has been less studied. Therefore, we set out to screen our collection of FALS cases for FUS mutations. All 15 exons of FUS were amplified and sequenced in 154 unrelated FALS cases and 475 ethnically matched healthy individuals. One substitution located in the acceptor splice site of intron 14 was identified in all affected members of a large family, causing the skipping of the last 13 amino acids of the protein and the translation of 7 novel amino acids, resulting from the new translation of a part of the 3' untranslated region. Our study identified a new splicing mutation in the highly conserved C-terminal of the FUS protein. Thus far most FUS mutations are missenses, and our findings, combined with those of others, confirm the importance of the C-terminal portion of the protein, adding additional support for FUS mutations having a critical role in ALS.

Identification of novel FUS mutations in sporadic cases of amyotrophic lateral sclerosis.

Mutations in the FUS gene have been recently associated with amyotrophic lateral sclerosis (ALS). While most of the variants have been identified in patients with a family history of the disease, a few mutations were also found in sporadic patients. Considering this, we wanted to evaluate the frequency of mutations in the coding region of the FUS gene in a sporadic ALS (SALS) cohort compared to a control population. We tested 475 SALS cases of European origin and 475 matched controls for coding variations in the 15 exons of the FUS gene. Rare novel variants were identified in a total of five SALS patients: one missense, one deletion, one frameshift, and one nonsense substitution. Two of the four variants are located in the carboxy terminal of the protein where the previously reported variants were mostly clustered. In conclusion, FUS gene mutations are rare in SALS, with four new FUS variants identified in five different SALS cases. These findings will help evaluate the proportion of FUS variations in the SALS population, and to better understand its contributing role to ALS pathology.

An Epigenetic Spin to ALS and FTD.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two devastating and lethal neurodegenerative diseases seen comorbidly in up to 15% of patients. Despite several decades of research, no effective treatment or disease-modifying strategies have been developed. We now understand more than before about the genetics and biology behind ALS and FTD, but the genetic etiology for the majority of patients is still unknown and the phenotypic variability observed across patients, even those carrying the same mutation, is enigmatic. Additionally, susceptibility factors leading to neuronal vulnerability in specific central nervous system regions involved in disease are yet to be identified. As the inherited but dynamic epigenome acts as a cell-specific interface between the inherited fixed genome and both cell-intrinsic mechanisms and environmental input, adaptive epigenetic changes might contribute to the ALS/FTD aspects we still struggle to comprehend. This chapter summarizes our current understanding of basic epigenetic mechanisms, how they relate to ALS and FTD, and their potential as therapeutic targets. A clear understanding of the biological mechanisms driving these two currently incurable diseases is urgent-well-needed therapeutic strategies need to be developed soon. Disease-specific epigenetic changes have already been observed in patients and these might be central to this endeavor.