Functional analysis of amyotrophic lateral sclerosis mutations using Drosophila cell culture models

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterised by the degeneration of motor neurons, resulting in paralysis and eventually death. This thesis examined the molecular mechanisms of ALS by analysing three key ALS-associated genes: FUS, SOD1, and TDP43, along with their respective three mutations (FUS: H517Q, R521C, P525L; SOD1: A4V, G85R, E100G; TDP43: G298S, Q331K, M337V) using Drosophila S2R+ cell models. This project combines molecular biology, high-throughput genetic screening, and transcriptomics analysis to identify potential therapeutic targets of ALS. Transient Drosophila S2R+ cell models expressing wild-type or mutant forms of the ALS-associated genes FUS, SOD1, TDP43 were generated to evaluate protein expression, subcellular protein localisation, and cytotoxicity through immunostaining, western blot, and RT-qPCR. Pooled variable dose analysis (VDA) was employed to identify genetic interactions that rescue cellular viability. This study focused on kinases and highlighted the effectiveness of pooled VDA in identifying synthetic rescue effects, especially in cells expressing TDP43 wild-type and mutants. Key therapeutic targets, such as aurB and bon have been identified, providing potential therapeutic targets for ALS. Bulk RNA sequencing was employed to investigate transcriptomic changes in Drosophila S2R+ cells expressing TDP43 wild-type and mutant. Differential gene expression and pathway analysis revealed dysregulation in RNA metabolism, mitochondrial function, and immune-related pathways, providing insights into the molecular mechanisms involved in ALS pathogenesis. Gene ontology analysis revealed that stress granule dynamics and ion channel regulation are significantly overexpressed as critical processes disrupted in TDP43 mutant expressing Drosophila S2R+ cells. This thesis enhances the understanding of genetic interactions and molecular pathways associated with ALS through the combination of genetic screening and transcriptomic profiling. The findings contribute to the identification of potential therapeutic targets and highlights the use of Drosophila S2R+ cells as a model system in ALS research.<p></p>

Author dc:creator

• Jeong Yeol Lee (21038513)

• ALS
• amyotrophic lateral sclerosis
• Drosophila
• high-throughput screening
• HTS
• kinome-wide screen
• VDA
• variable dose analysis
• TDP43

Statement dc:rights

• CC BY-NC-ND
• Open Access after 2027-12-01

Identifier

10779/exe.32566923.v1

OAI identifier oai:identifier

oai:figshare.com:article/32566923