Theses Doctoral

Novel functions of C9ORF72, a gene involved in ALS/FTD

Fomin, Vitalay

The discovery that the (GGGCC)n>30 repeat expansion in the non-coding region of C9orf72 (C9) is the most prevalent mutation in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) has led to a massive effort to discover the mechanism by which the expansion causes ALS. One effect of the repeat expansion is the reduction in C9 mRNA and protein levels. Therefore, we chose to concentrate on studying the function of C9 and how its reduction contributes to ALS progression. First, we show that C9 short (C9S) and long (C9L) isoforms have different cellular localization, and that C9 knockdown in several cell lines, results in significant changes in gene expression. Specifically, we show that differentially expressed genes were enriched in immune system activation pathway. Additionally, we observed gene expression changes in important glutamate-glutamine cycling genes and show that C9 knockdown results in accumulation of intracellular glutamate. We also show that C9S isoform may regulate gene expression as it interacts with chromatin, and can be ChIPed on the promoter of endothelin-1 (EDN1).
C9 knockdown also leads to significant morphological changes that include increased cell sizes and nucleus, massive vacuolization, and results in reduced cell viability. Investigation into the vacuoles revealed that they originate from hyperactivation of macropinocytosis. The hyperactivation of macropinocytosis results in a caspase-independent cell death known as methuosis. We show that vacuolization is a p53-dependent process, and we present evidence that p53 mediates vacuolization via the repression of the mevalonate pathway. Furthermore, we found that inhibition of isoprenylation, a process depending on mevalonate pathway products (geranylgeranyl pyrophosphate and farnesyl pyrophosphate), participates in the induction of vacuoles. Importantly, we also reveal that C9 knockdown leads to mitochondrial dysfunction, increased ROS, increased DNA-damage and p53 activation, all of which are seen in C9 ALS patient samples or in C9 patient derived motor neurons (C9 iMNs). Our results reveal several previously unknown pathways which are affected by C9 knockdown , which have potential therapeutic implications, that include endothelin signaling and macropinocytosis, both of which can be blocked with FDA approved drugs.


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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Prives, Carol L.
Manley, James L.
Ph.D., Columbia University
Published Here
September 27, 2019