Translation of the hexanucleotide G4C2 expansion associated with C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) produces five different dipeptide repeat protein (DPR) species that can confer toxicity. There is yet much to learn about the contribution of a single DPR to disease pathogenesis. We show here that a short repeat length is sufficient for the DPR poly-GR to confer neurotoxicity in vitro, a phenomenon previously unobserved. This toxicity is also reported in vivo in our novel knock-in mouse model characterized by widespread central nervous system (CNS) expression of the short-length poly-GR. We observe sex-specific chronic ALS/FTD-like phenotypes in these mice, including mild motor neuron loss, but no TDP-43 mis-localization, as well as motor and cognitive impairments. We suggest that this model can serve as the foundation for phenotypic exacerbation through second-hit forms of stress.

A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.

Genetic mutations that cause amyotrophic lateral sclerosis (ALS), a progressively lethal motor neuron disease, are commonly found in ubiquitously expressed genes. In addition to direct defects within motor neurons, growing evidence suggests that dysfunction of non-neuronal cells is also an important driver of disease. Previously, we demonstrated that mutations in DNA/RNA binding protein fused in sarcoma (FUS) induce neurotoxic phenotypes in astrocytes in vitro, via activation of the NF-κB pathway and release of pro-inflammatory cytokine TNFα. Here, we developed an intraspinal cord injection model to test whether astrocyte-specific expression of ALS-causative FUSR521G variant (mtFUS) causes neuronal damage in vivo. We show that restricted expression of mtFUS in astrocytes is sufficient to induce death of spinal motor neurons leading to motor deficits through upregulation of TNFα. We further demonstrate that TNFα is a key toxic molecule as expression of mtFUS in TNFα knockout animals does not induce pathogenic changes. Accordingly, in mtFUS-transduced animals, administration of TNFα neutralizing antibodies prevents neurodegeneration and motor dysfunction. Together, these studies strengthen evidence that astrocytes contribute to disease in ALS and establish, for the first time, that FUS-ALS astrocytes induce pathogenic changes to motor neurons in vivo. Our work identifies TNFα as the critical driver of mtFUS-astrocytic toxicity and demonstrates therapeutic success of targeting TNFα to attenuate motor neuron dysfunction and death. Ultimately, through defining and subsequently targeting this toxic mechanism, we provide a viable FUS-ALS specific therapeutic strategy, which may also be applicable to sporadic ALS where FUS activity and cellular localization are frequently perturbed.

The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadruplex conformation of G-rich C9orf72 repeat RNA, we find that G-quadruplex motifs are abundant and conserved features of NEAT1. Furthermore, we determine that NONO binds NEAT1 G-quadruplexes with structural specificity and provide evidence that G-quadruplex motifs mediate NONO-NEAT1 association, with NONO binding sites on NEAT1 corresponding largely to G-quadruplex motifs, and treatment with a G-quadruplex-disrupting small molecule causing dissociation of native NONO-NEAT1 complexes. Together, these findings position G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1 and provide a framework for further investigation into the role of G-quadruplexes in paraspeckle formation and function.

The (G4C2)n nucleotide repeat expansion (NRE) mutation in C9orf72 is the most common genetic cause of ALS and FTD. The biological functions of C9orf72 are becoming understood, but it is unclear if this gene is regulated in a neural-specific manner. Neuronal activity is a crucial modifier of biological processes in health and neurodegenerative disease contexts. Here, we show that prolonged membrane depolarization in healthy human iPSC-cortical neurons leads to a significant downregulation of a transcript variant 3 (V3) of C9orf72, with a concomitant increase in variant 2 (V2), which leads to total C9orf72 RNA transcript levels remaining unchanged. However, the same response is not observed in cortical neurons derived from patients with the C9-NRE mutation. These findings reveal the impact of depolarization on C9orf72 transcripts, and how this response diverges in C9-NRE-carriers, which may have important implications in the underlying unique clinical associations of C9-NRE transcripts and disease pathogenesis.

A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.

The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention.

Huntington's disease (HD) is caused by an expanded CAG repeat in the Huntingtin (HTT) gene. The mechanism(s) by which mutant HTT (mHTT) causes disease is unclear. Nucleocytoplasmic transport, the trafficking of macromolecules between the nucleus and cytoplasm, is tightly regulated by nuclear pore complexes (NPCs) made up of nucleoporins (NUPs). Previous studies offered clues that mHTT may disrupt nucleocytoplasmic transport and a mutation of an NUP can cause HD-like pathology. Therefore, we evaluated the NPC and nucleocytoplasmic transport in multiple models of HD, including mouse and fly models, neurons transfected with mHTT, HD iPSC-derived neurons, and human HD brain regions. These studies revealed severe mislocalization and aggregation of NUPs and defective nucleocytoplasmic transport. HD repeat-associated non-ATG (RAN) translation proteins also disrupted nucleocytoplasmic transport. Additionally, overexpression of NUPs and treatment with drugs that prevent aberrant NUP biology also mitigated this transport defect and neurotoxicity, providing future novel therapy targets.

How huntingtin (HTT) triggers neurotoxicity in Huntington's disease (HD) remains unclear. We report that HTT forms a transcription-coupled DNA repair (TCR) complex with RNA polymerase II subunit A (POLR2A), ataxin-3, the DNA repair enzyme polynucleotide-kinase-3'-phosphatase (PNKP), and cyclic AMP-response element-binding (CREB) protein (CBP). This complex senses and facilitates DNA damage repair during transcriptional elongation, but its functional integrity is impaired by mutant HTT. Abrogated PNKP activity results in persistent DNA break accumulation, preferentially in actively transcribed genes, and aberrant activation of DNA damage-response ataxia telangiectasia-mutated (ATM) signaling in HD transgenic mouse and cell models. A concomitant decrease in Ataxin-3 activity facilitates CBP ubiquitination and degradation, adversely impacting transcription and DNA repair. Increasing PNKP activity in mutant cells improves genome integrity and cell survival. These findings suggest a potential molecular mechanism of how mutant HTT activates DNA damage-response pro-degenerative pathways and impairs transcription, triggering neurotoxicity and functional decline in HD.

The most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic hexanucleotide repeat expansion in the C9orf72 gene. In disease, RNA transcripts containing this expanded region undergo repeat-associated non-AUG translation to produce dipeptide repeat proteins (DPRs), which are detected in brain and spinal cord of patients and are neurotoxic both in vitro and in vivo paradigms. We reveal here a novel pathogenic mechanism for the most abundantly detected DPR in ALS/FTD autopsy tissues, poly-glycine-alanine (GA). Previously, we showed motor dysfunction in a GA mouse model without loss of motor neurons. Here, we demonstrate that mobile GA aggregates are present within neurites, evoke a reduction in synaptic vesicle-associated protein 2 (SV2), and alter Ca2+ influx and synaptic vesicle release. These phenotypes could be corrected by restoring SV2 levels. In GA mice, loss of SV2 was observed without reduction of motor neuron number. Notably, reduction in SV2 was seen in cortical and motor neurons derived from patient induced pluripotent stem cell lines, suggesting synaptic alterations also occur in patients.