Description

Caenorhabditis elegans uv1 cells undergo a dramatic cell death in a pnc-1 NAD+ salvage deficient mutant (Huang and Hanna-Rose, 2006). uv1 cell death is a result of accumulation of the PNC-1 substrate nicotinamide (NAM), which overactivates the OCR-4 OSM-9 transient receptor potential cation V (TRPV) channel, causing excitotoxicity (Upadhyay, et al., 2016, Vrablik, et al., 2009). Reduced uv1 cell survival is almost completely restored in pnc-1 mutant animals by sustained activation of the Epidermal Growth Factor (EGF) receptor LET-23, either by overexpression of the ligand LIN-3 or by a gain of function mutation in the receptor (Huang and Hanna-Rose, 2006). We presume that the constitutively activated receptor acts cell autonomously in the uv1 cells to promote survival, but have no direct evidence in support of that presumption, besides a prior report showing that a let-23(sa62) gain of function allele was able to induce uv1 specification in the absence of LIN-3 (Chang, et al., 1999). To find genes required for uv1 survival in a let-23(sa62gf); pnc-1(pk9605) background, we performed an RNAi screen and found that phosphatidylcholine synthesis via PMT-1 or PCYT-1 is required for LET-23 to promote uv1 cell survival in pnc-1 background (Crook, et al., 2016). Moreover, treatment with exogenous phosphatidylcholine alone is partially sufficient to promote cell survival in the pnc-1 mutant (Crook, et al., 2016). The requirement of phosphatidylcholine synthesis indicates that membrane phospholipid composition, and by extension disruption of lipid homeostasis, may play a role in preventing TRPV-induced excitotoxic death.

Lipid synthesis homeostasis in general and phosphatidylcholine synthesis in particular are regulated by the srebp-1 homolog sbp-1 in C. elegans (Walker, et al., 2011). SBP-1 is activated by low phosphatidylcholine levels resulting from pmt-1 or pcyt-1 RNAi (Walker, et al., 2011). We hypothesized that uv1 survival in a let-23gf: pnc-1 background decreases when SBP-1 is activated through reduced phosphatidylcholine synthesis via pmt-1 or pcyt-1 RNAi. We predicted that inactivation of SBP-1 when pmt-1 or pcyt-1 were knocked down by RNAi would restore uv1 survival to that seen in a let-23gf: pnc-1 background.

We tested the role of SBP-1 by reducing its activity in a let-23(sa62gf); pnc-1(pk9605) background with reduced phosphatidylcholine synthesis. We took two complementary approaches. First we carried out double RNAi experiments (Ahringer, 2006) where both sbp-1 and pmt-1 or pcyt-1 were knocked down in a let-23(ga62gf); pnc-1(pk9605) background using an extrachromosomal ida-1p::gfp marker. Each RNAi was carried out twice with an empty vector (L4440) as control. Double RNAi experiments were carried out by mixing equal volumes of overnight LB-Amp RNAi cultures before spotting NGM-Carb-Tet plates. We found that double RNAi of sbp-1 with either pmt-1 or pcyt-1 resulted in a significant increase in uv1 survival compared with pmt-1 or pcyt-1 RNAi alone (Fig. 1A). However, uv1 survival was not restored to the level seen in a let-23(sa62gf); pnc-1(pk9605) background, which may have been due to incomplete knockdown in the double RNAi experiments. To address this possibility, we created a let-23(ga62gf); sbp-1(ep79); pnc-1(pk9605) strain with an integrated ida-1p::gfp marker. We then knocked down pmt-1 or pcyt-1 in this background and compared its effect on uv1 survival to the same gene knockdowns in a let-23(ga62gf); pnc-1(pk9605) strain, using an integrated ida-1p::gfp marker to count living uv1 cells. Each RNAi was carried out three times. We found that the pmt-1 or pcyt-1 knockdown induced reduction of uv1 survival in a let-23(ga62gf); pnc-1(pk9605) background did not occur when these genes were knocked down in a let-23(ga62gf); sbp-1(ep79); pnc-1(pk9605) background (Fig. 1B). There was no effect of either sbp-1 RNAi (Fig. 1A) or the sbp-1(ep79) mutant allele (Fig. 1B) alone on uv1 cell survival in a pnc-1 mutant background. Thus, both of our approaches support the hypothesis that sbp-1 is required for the reduction in uv1 survival seen when phosphatidylcholine synthesis is reduced in a let-23(ga62gf); pnc-1(pk9605) background.

let-23(sa62gf) mediated uv1 survival requires phosphatidylcholine synthesis and reducing phosphatidylcholine synthesis reduces cell survival. Our work shows that the reduction in uv1 survival when pcyt-1 or pmt-1 are knocked down is dependent on SBP-1. We propose the following model based on these results and our previous work on TRPV-induced excitotoxic death (Upadhyay, et al., 2016). An elevated level of NAM in a pnc-1 mutant activates the OCR-4/OSM-9 TRPV channel and results in uv1 cell death. Constitutive activation of LET-23 promotes phosphatidylcholine synthesis via PMT-1 and PCYT-1. Elevated phosphatidylcholine levels result in a cell membrane in which the OCR-4/OSM-9 TRPV channel is nonfunctional, which in turn prevents its activation by elevated NAM levels and results in uv1 survival. Reduction of phosphatidylcholine levels via pmt-1 or pcyt-1 RNAi activates SBP-1, which restores lipid homeostasis and cell membrane phospholipid composition, resulting in a functional OCR-4/OSM-9 TRPV channel and cell death. Support for our model also comes from the observation that exogenous phosphatidylcholine restores OLQ survival in a pnc-1 background, even though constitutive activation of LET-23 did not (Crook, et al., 2016). The precise mechanism by which elevated phosphatidylcholine levels disrupt OCR-4/OSM-9 TRPV channel function is not yet known, but our work supports an interaction between phosphatidylcholine levels, ion channel function and SBP-1-mediated lipid homeostasis.