A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5–10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions.We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci – TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, whose knockdown reversed the large bouton phenotype associated with VAP(P58S) expression in neurons. A similar reversal was seen by over-expressing Tuberous Sclerosis Complex (Tsc1,2) that negatively regulates TOR signaling as also by reduction of S6K activity. In comparison, the small bouton phenotype associated with VAP(wt) expression was reversed with Tsc1 knock down as well as S6K-CA expression. Tor therefore interacts with both VAP(wt) and VAP(P58S), but in a contrasting manner. Reversal of VAP(P58S) bouton phenotypes in larvae fed with the TOR inhibitor Rapamycin suggests upregulation of TOR signaling in response to VAP(P58S) expression.The VAPB network and further mechanistic understanding of interactions with key pathways, such as the TOR cassette, will pave the way for a better understanding of the mechanisms of onset and progression of motor neuron disease.

SOD1 activity thresholds and TOR signalling modulate VAP(P58S) aggregation via ROS-induced proteasomal degradation in a Drosophila model of Amyotrophic Lateral Sclerosis

“…201 202 Drosophila SOD1 is a modifier of VAP(P58S) aggregation 203 SOD1, first known ALS locus (ROSEN et al 1993), has been implicated in both 204 sporadic as well as familial cases and was our first choice for validation of the S2R+ 205 based screen, in the animal. We previously identified SOD1 as a genetic interactor of 206 VAP in a fly-based reverse genetics screen (DEIVASIGAMANI et al 2014). Here, we 207 individually knocked down SOD1 using three independent RNAi lines in the CI55-GAL4/+; 208 UAS-VAP(P58S)/+ background and observed a significant decrease in aggregation 209 density in the ventral nerve cord (Fig.…”

“…Thirdly, this screen 361 highlighted specific chaperones that could be involved in the misfolding and formation of 362 VAP(P58S) aggregates providing insight into the initiation of the disease condition. Most 363 importantly, through our previous study (DEIVASIGAMANI et al 2014), and our cell-based 364 screen followed by subsequent experimentation, we have established mTOR signalling 365 as a strong modulator of VAP(P58S) aggregation. mTOR signalling responds and 366…”

“…The next category 151 included 273 genes from a VAP Drosophila GRN comprising of 406 genes (DEIVASIGAMANI 152 et al 2014). As mTOR was identified as a major interactor of VAP in our previous study 153 (DEIVASIGAMANI et al 2014), we chose 22 genes of the extended mTOR pathway. To 154 explore the functional aspects of VAP(P58S), we also screened genes involved in lipid 155 biosynthesis (92 genes) and FFAT motif interactors of VAP (34 genes).…”

“…ALS loci,165 notably SOD1 and TBPH, were found as interesting modulators perturbing 166 VAP(P58S):GFP aggregation. Targets belonging to the VAP genetic network, as defined 167 by (DEIVASIGAMANI et al 2014), were also enriched. As identified earlier (DEIVASIGAMANI et 168 al.…”

“…In order to validate targets from the screen in vivo, we used the UAS-GAL4 system 185 to specifically overexpress wild-type VAP or VAP(P58S) in the brain using a pan-neuronal 186 driver, C155 (elav) (RATNAPARKHI et al 2008;DEIVASIGAMANI et al 2014). Based on anti-187 VAP immunostaining, unlike wild-type VAP (Suppl.…”