Abstract
A pathogenic GGGCCC hexanucleotide expansion in the first intron/promoter region of the C9orf72 gene is the most common mutation associated with amyotrophic lateral sclerosis (ALS). The C9orf72 gene product forms a complex with SMCR8 (Smith-Magenis Syndrome Chromosome Region, Candidate 8) and WDR41 (WD Repeat domain 41) proteins. Recent studies have indicated roles for the complex in autophagy regulation, vesicle trafficking, and immune response in transgenic mice, however a direct connection with ALS etiology remains unclear. With the aim of increasing understanding of the multi-functional C9orf72-SMCR8-WDR41 complex, we determined by mass spectrometry analysis the proteins that directly associate with SMCR8. SMCR8 protein binds many components of the ubiquitin-proteasome system, and we demonstrate its poly-ubiquitination without obvious degradation. Evidence is also presented for localization of endogenous SMCR8 protein to cytoplasmic stress granules. However, in several cell lines we failed to reproduce previous observations that C9orf72 protein enters these granules. SMCR8 protein associates with many products of genes associated with various Mendelian neurological disorders in addition to ALS, implicating SMCR8-containing complexes in a range of neuropathologies. We reinforce previous observations that SMCR8 and C9orf72 protein levels are positively linked, and now show in vivo that SMCR8 protein levels are greatly reduced in brain tissues of C9orf72 gene expansion carrier individuals. While further study is required, these data suggest that SMCR8 protein level might prove a useful biomarker for the C9orf72 expansion in ALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that afflicts about 1 in 50,000 people each year and involves loss of upper and lower motor neurons [1]
We confirmed that C9orf72, SMCR8 and WDR41 proteins formed a stable complex in our overexpression experimental system using human embryonic kidney (HEK) 293T cells
We show that SMCR8 protein expression is reduced in the brains of C9orf72 repeat expansion ALS (C9ALS) patients compared with unaffected controls
Summary
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that afflicts about 1 in 50,000 people each year and involves loss of upper and lower motor neurons [1]. Three possible non-exclusive mechanisms have been proposed by which the C9orf repeat expansion may cause ALSFTD: 1) haploinsufficiency and loss of C9orf protein function, 2) repeat-associated non-AUG (RAN) translation of the hexanucleotide repeats generating dipeptide repeats that aggregate in toxic neuronal cytoplasmic and nuclear aggregates, and 3) toxic gain-of-function from repeat-containing RNA which forms nuclear foci that sequester hexanucleotode repeat-binding proteins (reviewed in [7–10]). Various studies have reported a reduction in C9orf mRNA and/or protein expression in brain and induced pluripotent stem cell (iPSC)-derived neuronal lines of some C9orf ALS (C9ALS) and FTD patients [4–6, 11–25]
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