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

Familial Amyotrophic Lateral Sclerosis (F-ALS) is an incurable, late onset motor neuron disease, linked strongly to various causative genetic loci. ALS8 codes for a missense mutation, P56S, in VAMP-associated Protein B (VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase our understanding of the disease and lead to potential therapeutics.\n\nHere, we develop a quantitative high-throughput, Drosophila S2R+ cell-based kinetic assay coupled with fluorescent microscopy to score for genes involved in the modulation of aggregates of fly ortholog, VAP(P58S), tagged with GFP. As proof of principle, we conducted a targeted RNAi screen against 900 genes, consisting of VAP genetic interactors, other ALS loci, as also genes involved in proteostasis. The screen identified 150 hits that modify aggregation, including the ALS loci SOD1, TDP43 and also genes belonging to the TOR pathway.\n\nTo validate these modifiers, we developed a system to measure the extent of VAP(P58S) aggregation in the Drosophila third instar larval brain using the UAS-GAL4 system, followed by quantitative imaging of cellular inclusions. We find that reduction of SOD1 activity or decreased TOR signalling reduces aggregation. Interestingly, we find that increase in cellular reactive oxygen species (ROS) levels, assessed by measuring oxidation of cellular lipids and proteins, in response to SOD1 knockdown or by inhibition of TOR signalling appears to be the trigger for clearing of aggregates. The mechanism of aggregate clearance is, primarily, the proteasomal machinery, and not autophagy. Increase in VAP, but not VAP(P58S) levels, appears to elevate ROS, which may in turn regulate VAP transcription in a feedback loop.\n\nWe have thus uncovered an interesting interplay between SOD1, ROS and TOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead us to a better understanding of initiation and progression of ALS.