Characterisation of the effects of disease-associated mutations in amyotrophic lateral sclerosis (ALS) using tissue expression and functional studies in cell culture
Author(s)
Bakhtiar, Intan Barul Akma
Type
Thesis
Abstract
Amyotrophic lateral sclerosis (ALS) also known as Lou Gehrig’s disease, is a neurodegenerative disease affecting lower and upper motor neurons without effective treatments. Sporadic ALS (SALS) accounts for the majority of ALS cases, whereas about 10% of ALS cases are familial cases (FALS). The objective of this project is to characterise pathogenic mechanisms in two genes; the most common FALS gene, C9ORF72 and a putative FALS gene identified from exome sequencing, ‘Kay’. In this study, the gene expression approach was applied through quantitative PCR, DNA, RNA and protein extractions and Western blotting in addition to functional characterisation of the genes using cell cultures, staurosporine treatment, ER stress treatment and apoptosis analysis. The C9ORF72 hexanucleotide repeat expansions (HRE) have been identified as being the most common mutation in ALS and frontotemporal dementia (FTD). Here, we reported that C9ORF72 isoform a and b mRNA levels are unaltered in the spinal cord from SALS subjects. However, we observed that in FTD C9-positive subjects, the mRNA levels of both C9ORF72 isoform a and b were downregulated in the temporal cortex whereas in the frontal cortex, C9ORF72 isoform b mRNA level was upregulated but not C9ORF72 isoform a. We were able to detect a single band at 54kDa corresponding to C9ORF72 long isoform (C9ORF72-L) in our lymphoblastoid cell lines (LCLs) that showed a trend of downregulation upon ER stress. We also found that Rab7 mRNA level to be significantly increased in C9ORF72 HRE carriers compared with healthy controls. Our study therefore further provides a strong evidence of autophagy involvement in C9ORF72-ALS...
Version
Open Access
Date Issued
2020-01-17
Date Awarded
01/12/2020
License URL
Advisor
Matthews, Paul
Sponsor
Universiti Teknologi MARA
Publisher Department
Department of Brain Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)