Abstract
A hexanucleotide repeat expansion in a noncoding region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reduction of select or total C9orf72 transcript and protein levels is observed in postmortem C9-ALS/FTD tissue, and loss of C9orf72 orthologues in zebrafish and C. elegans results in motor deficits. However, how the reduction in C9orf72 in ALS and FTD might contribute to the disease process remains poorly understood. It has been shown that C9orf72 interacts and forms a complex with SMCR8 and WDR41, acting as a guanine exchange factor for Rab GTPases. Given the known synaptosomal compartmentalization of C9orf72-interacting Rab GTPases, we hypothesized that C9orf72 localization to synaptosomes would be required for the regulation of Rab GTPases and receptor trafficking. This study combined synaptosomal and post-synaptic density preparations together with a knockout-confirmed monoclonal antibody for C9orf72 to assess the localization and role of C9orf72 in the synaptosomes of mouse forebrains. Here, we found C9orf72 to be localized to both the pre- and post-synaptic compartment, as confirmed by both post-synaptic immunoprecipitation and immunofluorescence labelling. In C9orf72 knockout (C9-KO) mice, we demonstrated that pre-synaptic Rab3a, Rab5, and Rab11 protein levels remained stable compared with wild-type littermates (C9-WT). Strikingly, post-synaptic preparations from C9-KO mouse forebrains demonstrated a complete loss of Smcr8 protein levels, together with a significant downregulation of Rab39b and a concomitant upregulation of GluR1 compared with C9-WT mice. We confirmed the localization of Rab39b downregulation and GluR1 upregulation to the dorsal hippocampus of C9-KO mice by immunofluorescence. These results indicate that C9orf72 is essential for the regulation of post-synaptic receptor levels, and implicates loss of C9orf72 in contributing to synaptic dysfunction and related excitotoxicity in ALS and FTD.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal adultonset neurodegenerative disease primarily affecting motor neurons of the motor cortex, brain stem, and spinal cord
Chromosome 9 open reading frame 72 (C9orf72) localizes to both the pre- and post-synapses in murine forebrains Given that different Rab proteins show unique localization to the pre- and post-synaptic compartment [22, 26], we reasoned that C9orf72 and its interacting partner syndrome Chromosomal Region candidate gene 8 (Smcr8) [11, 62, 64] would be present in post-synaptic densities
We could not detect any protein level changes between C9-WT and C9orf72 knockout (C9-KO) mice for glutamate receptor 2 (GluR2) and N-methyl-D-aspartic acid receptor 1 (NMDAR1) in PSDs. These results suggest that post-synaptic localization of C9orf72 together with its Rab GTPase Rab39b are important for the regulation of glutamatergic receptor levels in vivo
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal adultonset neurodegenerative disease primarily affecting motor neurons of the motor cortex, brain stem, and spinal cord. Hexanucleotide (G4C2) repeat expansions within the first intron of C9orf are the most common known genetic cause of both ALS and frontotemporal dementia (FTD). How the expanded G4C2 repeats in C9orf cause neurodegeneration in ALS and FTD remains largely uncertain. Three potential pathomechanisms have been proposed to result from the repeat expansions [21, 30, 52]: (1) RNAmediated toxicity through sequestration of RNA-binding proteins in nuclear repeat RNA foci; (2) accumulation of five dipeptide repeat (DPR) proteins, glycine-alanine (GA), glycine-arginine (GR), proline-alanine (PA), prolinearginine (PR), and glycine-proline (GP), by repeat-associated non-ATG (RAN) translation; and (3) loss of function through C9orf haploinsufficiency
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