Ceramides And Stress Signalling Intersect With Autophagic Defects In Neurodegenerative Drosophila blue cheese (bchs) Mutants

Sphingolipid metabolites are involved in the regulation of autophagy, a degradative recycling process that is required to prevent neuronal degeneration. Drosophila blue cheese mutants neurodegenerate due to perturbations in autophagic flux, and consequent accumulation of ubiquitinated aggregates. Here, we demonstrate that blue cheese mutant brains exhibit an elevation in total ceramide levels; surprisingly, however, degeneration is ameliorated when the pool of available ceramides is further increased, and exacerbated when ceramide levels are decreased by altering sphingolipid catabolism or blocking de novo synthesis. Exogenous ceramide is seen to accumulate in autophagosomes, which are fewer in number and show less efficient clearance in blue cheese mutant neurons. Sphingolipid metabolism is also shifted away from salvage toward de novo pathways, while pro-growth Akt and MAP pathways are down-regulated, and ER stress is increased. All these defects are reversed under genetic rescue conditions that increase ceramide generation from salvage pathways. This constellation of effects suggests a possible mechanism whereby the observed deficit in a potentially ceramide-releasing autophagic pathway impedes survival signaling and exacerbates neuronal death.


S1, related to Figure 2
Survey of changes in phospholipid species across genotypes. Grey shaded cells indicate no change; red indicates a significant decrease whereas green indicates a significant increase at p<0.05 as determined by ANOVA followed by post-hoc Tukey test. Numbers indicate percent change when compared to relevant genetic control. were identified based on LC-Ms 3 generated signature product. Peak heights were used to compute absolute quantities in picomoles/brain unit. For a better representation of the MS results, related genotypes were normalized to a suitable genetic control (fixed at 100% indicated by the horizontal line). Bar graphs represent percent mean ± SEM (with respect to genetic controls) in ceramide levels. The genetic controls include eve-Gal4 driving UASmCD8GFP (eve>GFP) alone or in combination with C155-Gal4 (C155; eve>GFP).

S4, related to Figure 2
Comparison of levels of major ceramide species in bchs mutants and interactors. Cer32:1, 34:1 are changed in bchs mutants and the modifier combinations. In contrast, Cer 36:1 levels are only changed in combinations that display the highest increases in ceramide levels.
Bar graphs represent mean ± SEM of percent change (with respect to relative genetic controls) for total Cer32:1 (A), Cer34:1(B) and Cer36:1 (C) levels (quantified as picomoles/brain) in manipulations of CDase (green), nSMase (lavender/pink) and lace/Spt2 (blue). For a better representation of the mass spectrometric results related genotypes were normalized to a suitable genetic control (fixed at 100% indicated by the horizontal line). The genetic controls include eve-Gal4 driving UASmCD8GFP (eve>GFP; grey hatched bars) alone or in combination with C155-Gal4 (C155; eve>GFP; white hatched bars). Bar colors represent different genetic perturbations, hatched bars indicate combination of multiple genetic perturbations. Numbers represent mean percent change relative to suitable genetic control whose lipid levels are fixed at 100% (indicated by horizontal line). *p<0.05, **p<0.005 and ***p<0.0005 between 2 genotypes indicated by black bar as determined by ANOVA followed by post-hoc Tukey analyses.

S5, related to Figure 2
Trends for diene-ceramides (34:2 and 36:2). Cer 34:2 (A) and Cer 36:2 (B) levels remain unaltered in most genotypes with the exception of slab combinations. Bar graphs represent mean ± SEM of percent change (with respect to relative genetic controls) for total Cer34:2 (A) and Cer36:2 (B) levels (quantified as picomoles/brain) in manipulations of CDase, nSMase and lace/Spt2. For a better representation of the mass spectrometric results related genotypes were normalized to a suitable genetic control (fixed at 100% indicated by the horizontal line).
The genetic controls include eve-Gal4 driving UASmCD8GFP ( UAS-nCDase(slab)/+; lace = lace k05305 /+). No significant differences are observed from wild type, given in the graphs in A.

S9, related to Experimental Procedures
Genotypes used in the lipidomics study.

S11: Adult head LCB and LCB-P analysis
Samples were prepared and analysed as described in Narayanaswamy et al, 2014, with some modifications. 50 heads from adult flies (4 days old) for each genotype were collected in triplicate, flash frozen in liquid nitrogen, and stored at -80 C until lipid extraction.
After addition of LCB and LCB-P (d18:1 LCB-P 13 C 2 D 2 ) standards (4 ng/ml) in 200 μl of butanol:methanol (1:1) the samples were sonicated at room temperature for 30 min. After centrifugation at 14000 g for 10 min the supernatant was split into 2 equivalent aliquots. One aliquot was directly analysed (2 μl injection) by LC-MSMS