SUMMARY Expanded GGGGCC nucleotide repeats within the C9ORF72 gene are the most common genetic mutation associated with both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Sense and antisense transcripts of these expansions are translated to form five dipeptide repeat proteins (DRPs). We employed primary cortical and motor neuron cultures, live-cell imaging, and transgenic fly models and found that the arginine-rich dipeptides, in particular Proline-Arginine (PR), are potently neurotoxic. Factors that anticipated their neurotoxicity included aggregation in nucleoli, decreased number of processing bodies, and stress granules formation, implying global translational dysregulation as path accountable for toxicity. Nuclear PR aggregates were also found in human-induced motor neurons and postmortem spinal cord tissues from C9ORF72 ALS and ALS/FTD patients. Intronic G4C2 transcripts, but not loss of C9ORF72 protein, are also toxic to motor and cortical neurons. Interestingly, G4C2 transcript-mediated neurotoxicity synergizes with that of PR aggregates, suggesting convergence of mechanisms.

Summary An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its pathogenic mechanism remains unclear. Here we use human induced motor neurons (iMNs) to show that repeat-expanded C9ORF72 is haploinsufficient in ALS. We show that C9ORF72 interacts with endosomes and is required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduces C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms leads to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescues patient neuron survival and ameliorates neurodegenerative processes in both gain- and loss-of function C9ORF72 mouse models. Thus, modulating vesicle trafficking can rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN, and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS/FTD.

SUMMARY Aberrant hexanucleotide repeat expansions in C9orf72 are the most common genetic change underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). RNA transcripts containing these expansions undergo repeat associated non-ATG (RAN) translation to form five dipeptide repeat proteins (DPRs). DPRs are found as aggregates throughout the CNS of C9orf72-ALS/FTD patients and some cause degeneration when expressed in vitro in neuronal cultures and in vivo in animal models. The spread of characteristic disease-related proteins drives the progression of pathology in many neurodegenerative diseases. While DPR toxic mechanisms continue to be investigated, the potential for DPRs to spread has yet to be determined. Utilizing different experimental cell culture platforms, including spinal motor neurons derived from induced pluripotent stem cells from C9orf72-ALS patients, we found evidence for cell-to-cell spreading of DPRs via exosome-dependent and independent pathways, which may potentially be relevant to disease.

Nucleotide repeat expansions (NREs) are prevalent mutations in a multitude of neurodegenerative diseases. Repeat‐associated non‐AUG (RAN) translation of these repeat regions produces mono or dipeptides that contribute to the pathogenesis of these diseases. However, the mechanisms and drivers of RAN translation are not well understood. Here we analyzed whether different cellular stressors promote RAN translation of dipeptide repeats (DPRs) associated with the G4C2 hexanucleotide expansions in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We found that activating glutamate receptors or optogenetically increasing neuronal activity by repetitive trains of depolarization induced DPR formation in primary cortical neurons and patient derived spinal motor neurons. Increases in the integrated stress response (ISR) were concomitant with increased RAN translation of DPRs, both in neurons and different cell lines. Targeting phosphorylated‐PERK and the phosphorylated‐eif2α complex reduces DPR levels revealing a potential therapeutic strategy to attenuate DPR‐dependent disease pathogenesis in NRE‐linked diseases.