Abstract
Hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis, but the pathogenic mechanism of this mutation remains unresolved. Haploinsufficiency has been proposed as one potential mechanism. However, insights if and how reduced C9orf72 proteins levels might contribute to disease pathogenesis are still limited because C9orf72 expression, localization and functions in the central nervous system (CNS) are uncertain, in part due to the poor specificity of currently available C9orf72 antibodies.Here, we generated and characterized novel knock-out validated monoclonal rat and mouse antibodies against C9orf72. We found that C9orf72 is a low abundant, cytoplasmic, highly soluble protein with the long 481 amino acid isoform being the predominant, if not exclusively, expressed protein isoform in mouse tissues and human brain. As consequence of the C9orf72 repeat expansion, C9orf72 protein levels in the cerebellum were reduced to 80% in our series of C9orf72 mutation carriers (n = 17) compared to controls (n = 26). However, no associations between cerebellar protein levels and clinical phenotypes were seen. Finally, by utilizing complementary immunohistochemical and biochemical approaches including analysis of human iPSC derived motor neurons, we identified C9orf72, in addition to its association to lysosomes, to be localized to the presynapses and able to interact with all members of the RAB3 protein family, suggestive of a role for C9orf72 in regulating synaptic vesicle functions by potentially acting as guanine nucleotide exchange factor for RAB3 proteins.In conclusion, our findings provide further evidence for haploinsufficiency as potential mechanism in C9orf72 pathogenesis by demonstrating reduced protein levels in C9orf72 mutation carriers and important novel insights into the physiological role of C9orf72 in the CNS. Moreover, the described novel monoclonal C9orf72 antibodies will be useful tools to further dissect the cellular and molecular functions of C9orf72.
Highlights
In 2011, abnormal expansion of a GGGGCC hexanucleotide repeat in a predicted non-coding region of the chromosome 9 open reading frame 72 (C9orf72) gene was identified as the most common genetic cause of familial and sporadic forms of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) including families in which both conditions co-occur [13, 40]
The specificity of identified monoclonal antibodies (mAbs) was first demonstrated by immunoblot analysis of protein lysates from HEK293 cells transiently expressing either untagged or myc-DDK-tagged human C9orf72 short (C9-S) and long (C9-L) isoforms as well as murine C9orf72 isoform 1 and isoform 2 (Fig. 1b; Additional file 1: Figure S1a)
In line with the respective epitopes recognized by the different mAbs, rat clone 12E7 detected C9-S, C9-L and murine C9orf72 isoform 1 (mC9–1); mouse clone 1C1 detected C9-S, C9-L, mC9–1 and mC9–2; rat clones 5F6 and 12G10 labeled human C9-S and C9-L but not murine C9orf72; and rat clones 2H7 and 15C5 labeled C9-L, mC9–1 and mC9–2 but not C9-S
Summary
In 2011, abnormal expansion of a GGGGCC hexanucleotide repeat in a predicted non-coding region of the chromosome 9 open reading frame 72 (C9orf72) gene was identified as the most common genetic cause of familial and sporadic forms of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) including families in which both conditions co-occur [13, 40]. Neuronal inclusions composed of these DPR proteins are highly specific neuropathological hallmark features of C9orf mutation carriers [2, 15, 33, 35, 60] and have shown neurotoxic effects in various model systems upon overexpression under artificial AUG start codon [8, 29, 31, 37]. No consistent correlation of RNA foci and DPR protein pathology with the regional pattern of neurodegeneration and/or presence of TDP-43 pathology, the neuropathological hallmark feature of ALS and FTD including cases with C9orf mutations [36], has emerged despite extensive quantitative analysis [11, 12, 26, 27].
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