Reduced C9orf72 function leads to defective synaptic vesicle release and neuromuscular dysfunction in zebrafish

Abstract

A hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). Reduced levels of C9orf72 mRNA and protein have been found in ALS/FTD patients, but the role of this protein in disease pathogenesis is still poorly understood. Here, we report the generation and characterization of a stable C9orf72 loss-of-function (LOF) model in the zebrafish. We show that reduced C9orf72 function leads to motor defects, muscle atrophy, motor neuron loss and mortality in early larval and adult stages. Analysis of the structure and function of the neuromuscular junctions (NMJs) of the larvae, reveal a significant reduction in the number of presynaptic and postsynaptic structures and an impaired release of quantal synaptic vesicles at the NMJ. Strikingly, we demonstrate a downregulation of SV2a upon C9orf72-LOF and a reduced rate of synaptic vesicle cycling. Furthermore, we show a reduced number and size of Rab3a-postive synaptic puncta at NMJs. Altogether, these results reveal a key function for C9orf72 in the control of presynaptic vesicle trafficking and release at the zebrafish larval NMJ. Our study demonstrates a novel role for C9orf72 in ALS/FTD pathogenesis, where it regulates synaptic vesicle release and neuromuscular functions.

Zebrafish as a model organism for neurodegenerative disease

“…The first in vivo C9orf72 loss-of-function model was developed via a MO-induced knockdown leading to signs of motor neuron degeneration with axonopathy phenotypes and abnormal motor behavior ( Ciura et al, 2013 ). Recent studies mimicking C9orf72 loss of function involve the generation of novel transgenic lines which include the specific deletion of the functional DENN domain of C9orf72 ( Yeh et al, 2018 ) as well as a miRNA-based gene-silencing approach to achieve loss of over 50% C9orf72 function (C9-miR) ( Butti et al, 2020 ). The reduced C9orf72 expression resulted in aberrant axonal growth, increased neuronal apoptosis and muscle atrophy, as well as and locomotor deficits when compared to age-matched controls.…”

Recent Progress in Drug Repurposing Using Protein Variants and Amino Acids in Disease Phenotypes/Disorders

“…Most recently, due to the advances mentioned above, it led to the finding that a mutation in the C9orf72 gene (chromosome 9 open reading frame 72 genes) is the primary genetic cause of amyotrophic lateral sclerosis (ALS). These losses of function, induced by the mutation of the C9orf72 gene are thought to affect communication between motor neurons and muscles in people with ALS [31]. Further, this mutation is thought in part to be responsible for 40-50% of hereditary cases of ALS, and 5-10% of cases without family history.…”

“…Until now, it still remains unclear how this GGGGCC base repeat expansions cause neurodegeneration in ALS. Although, mechanistically, the C9orf72 protein function in a complex with the WDR41 and SMCR proteins (guanine exchange factors (GEF)) for Rab8 and Rab39 [31].…”

“…The average incidence rate of ALS worldwide is about one in 50,000 people per year and the average age of onset of the disease is about 60 years, with men at a slightly higher risk compared to women. FDA-approved treatments for ALS are only modestly effective and the disease still results in complete paralysis and death within the first 5 years after diagnosis [31,32].…”

Zebrafish, an In Vivo Platform to Screen Drugs and Proteins for Biomedical Use

“…The injection of human mutant FUS ( fused in sarcoma ) mRNA resulted in motor deficits and ventral root MN axonal abnormalities in embryos [ 97 ]. Reduced c9orf72 ( C9orf72-SMCR8 complex subunit ) function in zebrafish results in motor defects, such as muscle atrophy, MN loss and mortality in early larval and adult stages [ 98 ]. Vaccaro et al [ 99 ] demonstrated that the overexpression of mutant human TDP-43 in C. elegans and zebrafish models exhibited certain aspects of ALS symptoms, including MN degeneration, axonal deficits, and progressive paralysis.…”