Age-dependent VAP(P58S) aggregation in a Drosophila model of Amyotrophic Lateral Sclerosis

Abstract

Protein aggregation is a common feature observed in several neurodegenerative disorders. Their presence can affect cellular homeostasis in several ways and could prove to be valuable clues to understand neural disease pathogenesis and progression. For my thesis I am investigating VAP (P58S) aggregation in the fatal, progressive neurodegenerative disorder, Amyotrophic Lateral Sclerosis (ALS). This disease is characterized by extensive loss of motor function , severely reduced life expectancy and protein aggregation caused by defects in regulation. VAPB is known to interact with several other cellular components like microtubules, ER and several proteins (Lev et al., 2008). The Vesicle Associated Membrane Protein (VAMP) Associated Protein B (VAPB) locus was the 8th locus identified out of 32 known ALS loci (Abel et al,2012,). A missense mutation in the VAPB gene which results in the substitution of the 56th conserved Pro to Ser causes protein misfolding and aggregation (Nishimura et al., 2004 ). For my thesis, I have established a system to study VAP (P58S) aggregation in a Drosophila model of ALS8 using immunostaining and confocal microscopy. Using this model, we have attempted to characterize aggregation of VAP in relation to the disease progression by using adult Drosophila that phenocopy the disease. Our study revealed interesting relationships between VAP wildtype protein and VAP (P58S), which result in differing aggregation behaviour. We also observed the relationship between VAP aggregation, ER stress and age. In order to understand the regulatory processes involved in VAP aggregate formation, we have also observed aggregation in the background of genetic and physical interactors of VAP previously identified in the lab. These include SOD1, Caspar and VCP/Ter94. To broaden the scope of available genetic tools to study more complex interactions, we have also successfully developed a genomic VAP (P58S) mutant using a CRISPR-Cas9 strategy.