Understanding the relationship between cerebellum and the frontal-cortex region of C9orf72-related amyotrophic lateral sclerosis: A comparative analysis of genetic features.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive and fatal neurodegenerative diseases for which at present no cure is available. Despite the extensive research the progress from diagnosis to prognosis in ALS and frontotemporal dementia (FTD) has been slow which represents suboptimal understanding of disease pathophysiological processes. In recent studies, several genes have been associated with the ALS and FTD diseases such as SOD1, TDP43, and TBK1, whereas the hexanucleotide GGGGCC repeat expansion (HRE) in C9orf72 gene is a most frequent cause of ALS and FTD, that has changed the understanding of these diseases. METHODS: The goal of this study was to identify and spatially determine differential gene expression signature differences between cerebellum and frontal cortex in C9orf72-associated ALS (C9-ALS), to study the network properties of these differentially expressed genes, and to identify miRNAs targeting the common differentially expressed genes in both the tissues. This study thus highlights underlying differential cell susceptibilities to the disease mechanisms in C9-ALS and suggesting therapeutic target selection in C9-ALS. RESULTS: In this manuscript, we have identified that the genes involved in neuron development, protein localization and transcription are mostly enriched in cerebellum of C9-ALS patients, while the UPR-related genes are enriched in the frontal cortex. Of note, UPR pathway genes were mostly dysregulated both in the C9-ALS cerebellum and frontal cortex. Overall, the data presented here show that defects in normal RNA processing and the UPR pathway are the pathological hallmarks of C9-ALS. Interestingly, the cerebellum showed more strong transcriptome changes than the frontal cortex. CONCLUSION: Interestingly, the cerebellum region showed more significant transcriptomic changes as compared to the frontal cortex region suggesting its active participation in the disease process. This nuanced understanding may offer valuable insights for the development of targeted therapeutic strategies aimed at mitigating disease progression in C9-ALS.

KCNJ2 inhibition mitigates mechanical injury in a human brain organoid model of traumatic brain injury.

Traumatic brain injury (TBI) strongly correlates with neurodegenerative disease. However, it remains unclear which neurodegenerative mechanisms are intrinsic to the brain and which strategies most potently mitigate these processes. We developed a high-intensity ultrasound platform to inflict mechanical injury to induced pluripotent stem cell (iPSC)-derived cortical organoids. Mechanically injured organoids elicit classic hallmarks of TBI, including neuronal death, tau phosphorylation, and TDP-43 nuclear egress. We found that deep-layer neurons were particularly vulnerable to injury and that TDP-43 proteinopathy promotes cell death. Injured organoids derived from C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) patients displayed exacerbated TDP-43 dysfunction. Using genome-wide CRISPR interference screening, we identified a mechanosensory channel, KCNJ2, whose inhibition potently mitigated neurodegenerative processes in vitro and in vivo, including in C9ORF72 ALS/FTD organoids. Thus, targeting KCNJ2 may reduce acute neuronal death after brain injury, and we present a scalable, genetically flexible cerebral organoid model that may enable the identification of additional modifiers of mechanical stress.

C9ORF72 patient-derived endothelial cells drive blood-brain barrier disruption and contribute to neurotoxicity.

The blood-brain barrier (BBB) serves as a highly intricate and dynamic interface connecting the brain and the bloodstream, playing a vital role in maintaining brain homeostasis. BBB dysfunction has been associated with multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS); however, the role of the BBB in neurodegeneration is understudied. We developed an ALS patient-derived model of the BBB by using cells derived from 5 patient donors carrying C9ORF72 mutations. Brain microvascular endothelial-like cells (BMEC-like cells) derived from C9ORF72-ALS patients showed altered gene expression, compromised barrier integrity, and increased P-glycoprotein transporter activity. In addition, mitochondrial metabolic tests demonstrated that C9ORF72-ALS BMECs display a significant decrease in basal glycolysis accompanied by increased basal and ATP-linked respiration. Moreover, our study reveals that C9-ALS derived astrocytes can further affect BMECs function and affect the expression of the glucose transporter Glut-1. Finally, C9ORF72 patient-derived BMECs form leaky barriers through a cell-autonomous mechanism and have neurotoxic properties towards motor neurons.

Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

Abundant transcriptomic alterations in the human cerebellum of patients with a C9orf72 repeat expansion.

The most prominent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a repeat expansion in the gene C9orf72. Importantly, the transcriptomic consequences of the C9orf72 repeat expansion remain largely unclear. Here, we used short-read RNA sequencing (RNAseq) to profile the cerebellar transcriptome, detecting alterations in patients with a C9orf72 repeat expansion. We focused on the cerebellum, since key C9orf72-related pathologies are abundant in this neuroanatomical region, yet TDP-43 pathology and neuronal loss are minimal. Consistent with previous work, we showed a reduction in the expression of the C9orf72 gene and an elevation in homeobox genes, when comparing patients with the expansion to both patients without the C9orf72 repeat expansion and control subjects. Interestingly, we identified more than 1000 alternative splicing events, including 4 in genes previously associated with ALS and/or FTLD. We also found an increase of cryptic splicing in C9orf72 patients compared to patients without the expansion and controls. Furthermore, we demonstrated that the expression level of select RNA-binding proteins is associated with cryptic splice junction inclusion. Overall, this study explores the presence of widespread transcriptomic changes in the cerebellum, a region not confounded by severe neurodegeneration, in post-mortem tissue from C9orf72 patients.

Generation of induced pluripotent stem cells (CSSi017-A)(12862) from an ALS patient carrying a repeat expansion in the C9orf72 gene.

Genetic expansions of the hexanucleotide repeats (GGGGCC) in the C9orf72 gene appear in approximately 40% of patients with familial ALS and 7% of patients with sporadic ALS in the European population, making this mutation one of the most prevalent genetic mutations in ALS. Here, we generated a human induced pluripotent stem cell (hiPSC) line from the dermal fibroblasts of a patient carrying a 56-repeat expansion in an ALS disease-causing allele of C9orf72. These iPSCs showed stable amplification in vitro with normal karyotype and high expression of pluripotent markers and differentiated spontaneously in vivo into three germ layers.

Glucose hypometabolism prompts RAN translation and exacerbates C9orf72-related ALS/FTD phenotypes.

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, but its role in disease pathogenesis is unknown. Here, we show alterations in glucose metabolic pathways and ATP levels in the brains of asymptomatic C9-BAC mice. We find that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also show that one of the arginine-rich DPRs (PR) could directly contribute to glucose metabolism and metabolic stress by inhibiting glucose uptake in neurons. Our findings provide a potential mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and suggest a feedforward loop model with potential opportunities for therapeutic intervention.

LINC complex alterations are a key feature of sporadic and familial ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that primarily affects motor neurons, leading to progressive muscle weakness and loss of voluntary muscle control. While the exact cause of ALS is not fully understood, emerging research suggests that dysfunction of the nuclear envelope (NE) may contribute to disease pathogenesis and progression. The NE plays a role in ALS through several mechanisms, including nuclear pore defects, nucleocytoplasmic transport impairment, accumulation of mislocalized proteins, and nuclear morphology abnormalities. The LINC complex is the second biggest multi-protein complex in the NE and consists of the SUN1/2 proteins spanning the inner nuclear membrane and Nesprin proteins embedded in the outer membrane. The LINC complex, by interacting with both the nuclear lamina and the cytoskeleton, transmits mechanical forces to the nucleus regulating its morphology and functional homeostasis. In this study we show extensive alterations to the LINC complex in motor and cortical iPSC-derived neurons and spinal cord organoids carrying the ALS causative mutation in the C9ORF72 gene (C9). Importantly, we show that such alterations are present in vivo in a cohort of sporadic ALS and C9-ALS postmortem spinal cord and motor cortex specimens. We also found that LINC complex disruption strongly correlated with nuclear morphological alterations occurring in ALS neurons, independently of TDP43 mislocalization. Altogether, our data establish morphological and functional alterations to the LINC complex as important events in ALS pathogenic cascade, making this pathway a possible target for both biomarker and therapy development.

Neuromuscular organoids model spinal neuromuscular pathologies in C9orf72 amyotrophic lateral sclerosis.

Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Due to the lack of trunk neuromuscular organoids (NMOs) from ALS patients' induced pluripotent stem cells (iPSCs), an organoid system was missing to model the trunk spinal neuromuscular neurodegeneration. With the C9orf72 ALS patient-derived iPSCs and isogenic controls, we used an NMO system containing trunk spinal cord neural and peripheral muscular tissues to show that the ALS NMOs could model peripheral defects in ALS, including contraction weakness, neural denervation, and loss of Schwann cells. The neurons and astrocytes in ALS NMOs manifested the RNA foci and dipeptide repeat proteins. Acute treatment with the unfolded protein response inhibitor GSK2606414 increased the glutamatergic muscular contraction 2-fold and reduced the dipeptide repeat protein aggregation and autophagy. This study provides an organoid system for spinal neuromuscular pathologies in ALS and its application for drug testing.

DDX3X overexpression decreases dipeptide repeat proteins in a mouse model of C9ORF72-ALS/FTD.

Hexanucleotide repeat expansion in C9ORF72 (C9) is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One of the proposed pathogenic mechanisms is the neurotoxicity arising from dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG (RAN) translation. Therefore, reducing DPR levels emerges as a potential therapeutic strategy for C9ORF72-ALS/FTD. We previously identified an RNA helicase, DEAD-box helicase 3 X-linked (DDX3X), modulates RAN translation. DDX3X overexpression decreases poly-GP accumulation in C9ORF72-ALS/FTD patient-derived induced pluripotent stem cell (iPSC)-differentiated neurons (iPSNs) and reduces the glutamate-induced neurotoxicity. In this study, we examined the in vivo efficacy of DDX3X overexpression using a mouse model. We expressed exogenous DDX3X or GFP in the central nervous system (CNS) of the C9-500 ALS/FTD BAC transgenic or non-transgenic control mice using adeno-associated virus serotype 9 (AAV9). The DPR levels were significantly reduced in the brains of DDX3X-expressing C9-BAC mice compared to the GFP control even twelve months after virus delivery. Additionally, p62 aggregation was also decreased. No neuronal loss or neuroinflammatory response were detected in the DDX3X overexpressing C9-BAC mice. This work demonstrates that DDX3X overexpression effectively reduces DPR levels in vivo without provoking neuroinflammation or neurotoxicity, suggesting the potential of increasing DDX3X expression as a therapeutic strategy for C9ORF72-ALS/FTD.

PolyGR and polyPR knock-in mice reveal a conserved neuroprotective extracellular matrix signature in C9orf72 ALS/FTD neurons.

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-ß1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-ß1 followed by COL6A1. Knockdown of TGF-ß1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-ß1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.

Sulfated disaccharide protects membrane and DNA damages from arginine-rich dipeptide repeats in ALS.

Hexanucleotide repeat expansion in C9ORF72 (C9) is the most prevalent mutation among amyotrophic lateral sclerosis (ALS) patients. The patients carry over ~30 to hundreds or thousands of repeats translated to dipeptide repeats (DPRs) where poly-glycine-arginine (GR) and poly-proline-arginine (PR) are most toxic. The structure-function relationship is still unknown. Here, we examined the minimal neurotoxic repeat number of poly-GR and found that extension of the repeat number led to a loose helical structure disrupting plasma and nuclear membrane. Poly-GR/PR bound to nucleotides and interfered with transcription. We screened and identified a sulfated disaccharide that bound to poly-GR/PR and rescued poly-GR/PR-induced toxicity in neuroblastoma and C9-ALS-iPSC-derived motor neurons. The compound rescued the shortened life span and defective locomotion in poly-GR/PR expressing Drosophila model and improved motor behavior in poly-GR-injected mouse model. Overall, our results reveal structural and toxicity mechanisms for poly-GR/PR and facilitate therapeutic development for C9-ALS.

The C9orf72-SMCR8 complex suppresses primary ciliogenesis as a RAB8A GAP.

Approximately half of the familial cases of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD) are attributed to the abnormal GGGGCC repeat expansion within the first intron of C9orf72, potentializing C9orf72 and its product as the most promising target for ALS therapeutics. Nevertheless, the biological function of C9orf72 remains unclear. Previously, we reported that C9orf72 and its binding partner, SMCR8, form a GTPase-activating protein (GAP) complex, which is proposed to regulate membrane trafficking and autophagy. Hereby, we found that the C9orf72-SMCR8 complex negatively regulates primary ciliogenesis and hedgehog (HH) signaling. Furthermore, the biochemical analysis and cell biology experiments identified C9orf72 as the RAB8A binding subunit and SMCR8 as the GAP subunit within the complex. Further, we discussed the relationship among the C9orf72-SMCR8 complex, primary ciliogenesis, and autophagy.

C9orf72-associated dipeptide protein repeats form A11-positive oligomers in amyotrophic lateral sclerosis and frontotemporal dementia.

Hexanucleotide repeat expansion in C9orf72 is one of the most common causes of amyotrophic lateral sclerosis and frontotemporal dementia. The hexanucleotide expansion, formed by GGGGCC (G4C2) repeats, leads to the production of five dipeptide protein repeats (DPRs) via repeat-associated non-AUG translation. Among the five dipeptide repeats, Gly-Arg, Pro-Arg, and Gly-Ala form neuronal inclusions that contain aggregates of the peptides. Several studies have attempted to model DPR-associated toxicity using various repeat lengths, which suggests a unique conformation that is cytotoxic and is independent of the repeat length. However, the structural characteristics of DPR aggregates have yet to be determined. Increasing evidence suggests that soluble species, such as oligomers, are the main cause of toxicity in proteinopathies, such as Alzheimer's and Parkinson's disease. To investigate the ability of DPRs to aggregate and form toxic oligomers, we adopted a reductionist approach using small dipeptide repeats of 3, 6, and 12. This study shows that DPRs, particularly glycine-arginine and proline-arginine, form oligomers that exhibit distinct dye-binding properties and morphologies. Importantly, we also identified toxic DPR oligomers in amyotrophic lateral sclerosis and frontotemporal dementia postmortem brains that are morphologically similar to those generated recombinantly. This study demonstrates that, similar to soluble oligomers formed by various amyloid proteins, DPR oligomers are toxic, independent of their repeat length.

Cytoplasmic Accumulation of Histones Induced by BET Inhibition Protects Cells from C9orf72 Poly(PR)-Induced Cell Death.

Repeat dipeptides such as poly(proline-arginine) (polyPR) are generated from the hexanucleotide GGGGCC repeat expansions in the C9orf72 gene. These dipeptides are often considered as the genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In the study, fluorescein isothiocyanate (FITC) labeled PR20 is used to investigate PR20-induced cell death. The findings reveal that the cell death induced by PR20 is dependent on its nuclear distribution and can be blocked by a nuclear import inhibitor called importazole. Further investigation reveals that BRD4 inhibitors, such as JQ-1 and I-BET762, restrict cytoplasmic localization of PR20, thereby reducing its cytotoxic effect. Mechanistically, the inhibition of BRD4 leads to an increase in the expression of numerous histones, resulting in the accumulation of histones in the cytoplasm. These cytoplasmic histones associate with PR20 and limit its distribution within the nucleus. Notably, the ectopic expression of histones alone is enough to confer protection to cells treated with PR20. In addition, phenylephrine (PE) induces cellular hypertrophy and cytoplasmic distribution of histone, which also helps protect cells from PR20-induced cell death. The research suggests that temporarily inducing the presence of cytoplasmic histones may alleviate the neurotoxic effects of dipeptide repeat proteins.

Metformin ameliorates mitochondrial damage induced by C9orf72 poly(GR) via upregulating AKT phosphorylation.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating neurodegenerative diseases with no effective cure. GGGGCC repeat expansion in C9orf72 is the most common genetic cause of both ALS and FTD. A key pathological feature of C9orf72 related ALS/FTD is the presence of abnormal dipeptide repeat proteins translated from GGGGCC repeat expansion, including poly Glycine-Arginine (GR). In this study, we observed that (GR)50 conferred significant mitochondria damage and cytotoxicity. Metformin, the most widely used clinical drug, successfully relieved (GR)50 induced mitochondrial damage and inhibited (GR)50 related cytotoxicity. Further research revealed metformin effectively restored mitochondrial function by upregulating AKT phosphorylation in (GR)50 expressed cells. Taken together, our results indicated restoring mitochondrial function with metformin may be a rational therapeutic strategy to reduce poly(GR) toxicity in C9orf72 ALS/FTD patients.

SUN1 facilitates CHMP7 nuclear influx and injury cascades in sporadic amyotrophic lateral sclerosis.

We have recently identified the aberrant nuclear accumulation of the ESCRT-III protein CHMP7 as an initiating event that leads to a significant injury to the nuclear pore complex (NPC) characterized by the reduction of specific nucleoporins from the neuronal NPC in sporadic amyotrophic lateral sclerosis (sALS) and C9orf72 ALS/frontotemporal dementia (FTD)-induced pluripotent stem cell-derived neurons (iPSNs), a phenomenon also observed in post-mortem patient tissues. Importantly, this NPC injury is sufficient to contribute to TDP-43 dysfunction and mislocalization, a common pathological hallmark of neurodegenerative diseases. However, the molecular mechanisms and events that give rise to increased nuclear translocation and/or retention of CHMP7 to initiate this pathophysiological cascade remain largely unknown. Here, using an iPSN model of sALS, we demonstrate that impaired NPC permeability barrier integrity and interactions with the LINC complex protein SUN1 facilitate CHMP7 nuclear localization and the subsequent 'activation' of NPC injury cascades. Collectively, our data provide mechanistic insights in the pathophysiological underpinnings of ALS/FTD and highlight SUN1 as a potent contributor to and modifier of CHMP7-mediated toxicity in sALS pathogenesis.

Pathogenesis underlying hexanucleotide repeat expansions in C9orf72 gene in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder. Mutations in C9orf72 and the resulting hexanucleotide repeat (GGGGCC) expansion (HRE) has been identified as a major cause of familial ALS, accounting for about 40 % of familial and 6 % of sporadic cases of ALS in Western patients. The pathological outcomes of HRE expansion in ALS have been recognized as the results of two mechanisms that include both the toxic gain-of-function and loss-of-function of C9ORF72. The gain of toxicity results from RNA and dipeptide repeats (DPRs). The HRE can be bidirectionally transcribed into RNA foci, which can bind to and disrupt RNA splicing, transport, and translation. The DPRs that include poly-glycine-alanine, poly-glycine-proline, poly-glycine- arginine, poly-proline-alanine, and poly-proline-arginine can induce toxicity by direct binding and sequestrating other proteins to interfere rRNA synthesis, ribosome biogenesis, translation, and nucleocytoplasmic transport. The C9ORF72 functions through binding to its partners-Smith-Magenis chromosome regions 8 (SMCR8) and WD repeat-containing protein (WDR41). Loss of C9ORF72 function results in impairment of autophagy, deregulation of autoimmunity, increased stress, and disruption of nucleocytoplasmic transport. Further insight into the mechanism in C9ORF72 HRE pathogenesis will facilitate identifying novel and effective therapeutic targets for ALS.

The hookup model of the HOPS complex in autophagosome-lysosome fusion.

Macroautophagy/autophagy is a highly conserved process that involves the degradation of proteins, damaged organelles, and other cytoplasmic macromolecules. Autophagosome-lysosome fusion is critical for successful substrate degradation and is mediated by SNARE proteins. The fusion process requires additional vesicle docking and tethering-regulating factors. Our recent work has uncovered a functional model of autophagosome-lysosome fusion. We demonstrated that the six-subunit homotypic fusion and vacuole protein sorting (HOPS) complex can be assembled by two subcomplexes, the VPS39-VPS11 subcomplex (HOPS-2) and the VPS41-VPS16-VPS18-VPS33A subcomplex (HOPS-4). VPS39 binds with RAB2 on the autophagosome and VPS41 binds with RAB39A on the lysosome, which then promotes membrane tethering and autophagic SNARE-mediated membrane fusion. Moreover, we have revealed that ALS- and FTD-related C9orf72 is a guanine exchange factor (GEF) for RAB39A. In this punctum, we discuss how the C9orf72-RAB39A-HOPS axis function regulates autophagosome-lysosome fusion.

Artificial microRNA suppresses C9ORF72 variants and decreases toxic dipeptide repeat proteins in vivo.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons, causing progressive muscle weakness and respiratory failure. The presence of an expanded hexanucleotide repeat in chromosome 9 open reading frame 72 (C9ORF72) is the most frequent mutation causing familial ALS and frontotemporal dementia (FTD). To determine if suppressing expression of C9ORF72 gene products can reduce toxicity, we designed a set of artificial microRNAs (amiRNA) targeting the human C9ORF72 gene. Here we report that an AAV9-mediated amiRNA significantly suppresses expression of the C9ORF72 mRNA, protein, and toxic dipeptide repeat proteins generated by the expanded repeat in the brain and spinal cord of C9ORF72 transgenic mice.