Knockdown of sphingolipid metabolism genes promotes endolysosomal vesicle rupture.

(A) General overview of sphingolipid metabolism with a particular focus on the genes identified in the genome-wide screen. Sphingolipids constitute a group of amphipathic lipids featuring a polar head group and a sphingoid base backbone that is N-acetylated with a (very) long-chain fatty acid ((V)LCFA) side chain. In contrast to mammals, where the sphingoid base is conventionally derived from palmitic acid and serine, C. elegans sphingolipids usually contain a characteristic C17iso branched chain sphingoid base75–77. Its synthesis involves the branched chain FA (BCFA) elongation pathway to yield C15iso-CoA, which then condenses with L-serine to form 3-ketosphinganine. This reaction is catalyzed by serine palmitoyltransferase (encoded by sptl-1, -2, and -3). The serine incorporator (SERINC) protein family (encoded by R11H6.2) is believed to assist in the incorporation of L-serine into specific membranes. 3-ketodihydrosphingosine reductase (KDSR, in C. elegans predicted to be encoded by Y37E11AM.3) then catalyzes the reduction of 3-keto sphinganine to sphinganine. The (V)LCFA side chain is primarily comprised of a straight saturated FA chain, ranging from 20-26 carbon atoms in length, with or without hydroxylation30, 78. It can also be derived from BCFAs, such as C15iso and C17iso75, 76. However, most of the side chain FA moieties originate from palmitoyl-CoA via the canonical de novo FA biosynthesis pathway, involving the sequential addition of C2 moieties from malonyl-CoA through the LCFA elongation cycle75, 76. Each elongation cycle comprises four reactions (condensation, reduction, dehydration, reduction), with the third reaction requiring very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase (encoded by hpo-8)76. Finally, ceramide synthases (encoded by hyl-1 and hyl-2) catalyze the addition of various acyl side chains to the sphingoid base to yield dihydroceramide, which is then desaturated to ceramide. The latter reaction is catalyzed by dihydroceramide desaturases, which require electrons from NAD(P)H provided by cytochrome b5 reductases (encoded by hpo-19 and T05H4.4). All complex sphingolipids, such as sphingomyelin and glycosphingolipids (including cerebrosides and gangliosides) originate from ceramide. Degradation of complex sphingolipids takes place in the lysosome. Essential for this process are saposins or sphingolipid activator proteins (PSAPs, encoded by spp-10), which serve as crucial bridges between the lipid substrate and hydrophilic hydrolases. Glucocerebrosidases (encoded by gba-1, gba-2, gba-3, and gba-4) hydrolyse glucosylceramide into ceramide and glucose. Sphingosine may be either recycled and metabolized back into ceramide or phosphorylated by sphingosine kinase (encoded by sphk-1) to generate sphingosine-1-phosphate (S1P). S1P lyase (encoded by spl-1) irreversibly cleaves S1P into phospho-ethanolamine and hexadecenal. The C. elegans genes identified in the primary screen, along with their human orthologs, are framed with color. Genes identified in subsequent co-RNAi experiments and their human orthologs are framed in gray. (B) Schematic of lysosomal rupture detected by the galectin puncta assay. (C) Widefield fluorescence images of day 5 (second day of adulthood) animals expressing F3ΔK281::mCherry in touch receptor neurons and hypodermal sfGFP::LGALS3 and RNAi mediated KD of indicated sphingolipid metabolism genes. Numerous foci are visible indicating lysosomal rupture. Zoomed in image is indicated in overview image by a white box. Scale bar 100 µm. (D) Mean percentage of day 5 (second day of adulthood) animals positive for lysosomal rupture (defined as three or more sfGFP::LGALS3 foci in the hypodermis). Data shown as means of three technical replicate plates with 17-30 animals per plate ± SEM. Statistical analysis comparing RNAi conditions to the empty vector control (EV ctrl) was done using one-way ANOVA with Dunnett’s post-hoc test. *** = p < 0.001.

Knockdown of genes involved in sphingolipid metabolism decreases lysosomal membrane fluidity.

(A) Representative confocal single plane images from a FRAP experiment in day 5 (second day of adulthood) animals expressing a mCherry tagged Lysosomal Lysine/Arginine Transporter 1 (LAAT-1::mCherry) in the hypodermis grown either on empty vector control (EV ctrl), spl-1 or sphk-1 RNAi plates. Dashed circles outline the bleach spots. Scale bar = 10 µm. (B) Combined FRAP curves of LAAT-1::mCherry in hypodermal lysosomal membranes. Curves are normalized to the pre-bleach intensity as 100% and the first post-bleach intensity as 0%. (C, D) Increase (C) in the mean time until half of the maximal signal is recovered (thalf,) and decrease (D) in the maximal % recoverable fluorescence values upon KD of sphk-1 and spl-1 indicate a reduction in lysosomal membrane fluidity. (E) FRAP curves of LAAT-1::mCherry in hypodermal lysosomal membranes of animals grown either on empty vector or the indicated RNAi plates. Curves are normalized to the pre-bleach intensity set as 100% and the first post-bleach intensity as 0%. (F) Mean thalf upon KD of sphingolipid metabolism genes. (G) Maximal % recoverable fluorescence values upon KD of sphingolipid metabolism genes. (H) Representative confocal single plane images from a FRAP experiment in animals expressing prenylated GFP for lipid membrane anchorage in the intestine. Scale bar = 5 µm. (I) Combined FRAP curves of prenylated GFP enriched on the intestinal plasma membrane of animals grown either on empty vector, sphk-1 or spl-1 RNAi plates. Curves are normalized to the pre-bleach intensity as 100% and the first post-bleach intensity as 0%. (J, K) Mean thalf (J) and maximal % recoverable fluorescence values (K). Data represented as means ± SEM of 5 - 12 FRAP measurements per condition in animals on day 5 (second day of adulthood) collected in five biological replicates. Statistical analysis comparing RNAi conditions to the empty vector control was done using one-way ANOVA with Dunnett’s post-hoc test. n.s.: not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

KD of SPHK2 and aggregated tau increases membrane rigidity leading to lysosomal rupture.

(A) Scheme of the fluorescence properties of C-Laurdan. The dye is excited at 405 nm and exhibits peak emission at 450 nm (red) in ordered membrane phases and ∼500 nm in the disordered phase (blue). Two-channel acquisition is conducted in the wavelength bands indicated by shaded boxes. (B) Upper panels: Pseudo-colored images of SH-SY5Y cells transfected with control or SPHK2 siRNA showing the C-Laurdan GP Index at each pixel position. Lower panels show LysoTracker staining. Scale bar = 10 µm. (C) Quantification of GP values in SH-SY5Y cells upon transfected with control or SPHK2 siRNA. GP values were measured across the whole cell (left) or restricted to LysoTracker-positive (LTR) regions (right). Statistical analysis was conducted using a two-way mixed-model ANOVA, followed by pairwise comparisons of estimated marginal means with Sidak correction for multiple comparisons. n = 3 independent experiments, with 10 images analyzed per experiment. (D) Upper panel: Pseudo-colored images of SH-SY5Y cells exposed to PBS control or 1N4R tau fibrils showing the C-Laurdan GP Index at each pixel position. Lower panels show LysoTracker staining. Scale bar = 10 µm. (E) Quantification of GP values in SH-SY5Y cells exposed to PBS control or 1N4R tau fibrils. GP values were measured across the whole cell (left) or restricted to LysoTracker-positive (LTR) regions (right). Statistical analysis was conducted using a two-way mixed-model ANOVA followed by pairwise comparisons of estimated marginal means with Sidak correction for multiple comparisons. n = 3 independent experiments, with 10 images analyzed per experiment. (F) Maximum intensity projection of confocal z-stacks of HEK293T cells expressing sfGFP-LGALS3 upon treatment with control or SPHK2 siRNA, exposed to PBS control or 1N4R tau fibrils. Scale bar = 10 µm. (G) Quantification of sfGFP-LGALS3 foci per cell in HEK293T cells upon treatment with control or SPHK2 siRNA, exposed to PBS control or 1N4R tau fibrils. Data represent the mean number of foci per cell. Statistical analysis was done using Kruskal-Wallis with a Dunn’s post-hoc test. N = 3 independent experiments, with 10 images analyzed per experiment. (H) Maximum intensity projection of confocal z-stacks of a P301S tau-Venus biosensor cell line upon treatment with control or SPHK2 siRNA with and without exposure to 1N4R tau fibrils. Scale bar = 10 µm. (I) Quantification of tau-Venus foci upon treatment with control or SPHK2 siRNA with and without exposure to 1N4R tau fibrils. Data were analyzed by a two-way mixed-model ANOVA, followed by pairwise comparisons were performed using estimated marginal means with Sidak correction. n = 3 independent experiments, with 10 images analyzed per experiment. n.s.: not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Aggregated tau promotes endolysosomal rupture.

(A, B) Quantification of the percentage of animals with ≥ 3 hypodermal sfGFP::LGALS3 foci, upon expression of either F3ΔK281::mCherry (red) or mCherry control (grey) in touch receptor neurons and KD of the indicated genes on day 5 (second day of adulthood) (A). Quantification of endolysosomal rupture under sub-saturating RNAi conditions. RNAi cultures were diluted with the empty vector (EV) control bacteria to reduce the KD strength (B). Data represented as mean ± SEM. n = 3 - 7 independent experiments with 40 - 50 (A) or 20 - 30 (B) animals analyzed per experiment. Since even a dilution down to 5% of the sphk-1 RNAi resulted in 100% of animals being scored as positive in two independent replicates this condition was not repeated further. Statistical analysis comparing mCherry to F3ΔK281::mCherry under individual RNAi conditions was done using two-way ANOVA with Sidaks’s post-hoc test. ** = p < 0.01, *** = p < 0.001. (C) Representative images of hypodermal F3ΔK281::mCherry signal in EV control or sphk-1 RNAi treated animals on day 5 (second day of adulthood). Scale bar = 10 µm. (D) Quantification of hypodermal F3ΔK281::mCherry fluorescence intensity (integrated density, IntDen) in EV control or sphk-1 RNAi treated animals on day 5 (second day of adulthood), indicating sphk-1 KD does not alter tau transmission levels. Statistical analysis was done using Student’s t-test. n = 3 independent experiments with 35 animals analyzed in total. (E) C-Laurdan staining of animals expressing F3ΔK281::mCherry or mCherry in touch receptor neurons on day 4 (first day of adulthood). The mCherry signal was used to select a region of interest (ROI) around the soma of the posterior touch receptor neurons (PLM) to determine the GP value. Scale bar = 10 µm. (F) Quantification of GP values in animals expressing F3ΔK281::mCherry compared to mCherry on day 4 (first day of adulthood). Statistical analysis was done using a Student’s t-test. n = 4 independent experiments, with at least 28 animals analyzed in total. (G) Quantification of the percentage of animals expressing mCherry or F3ΔK281::mCherry in touch receptor neurons with ≥1 sfGFP::LGALS3 puncta in touch receptor neurons on day 5 (second day of adulthood). Each dot represents an independent experiment, and lines indicate the mean ± SEM. n = 3 independent experiments, with 10-14 animals per experiment. Statistical analysis was done using a Student’s t-test. n.s.: not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

PUFA supplementation restores lysosomal membrane integrity and reduces seeded tau aggregation.

(A) Left: unsaturated fatty acid membrane scheme. Right: Pseudo-colored images of SH-SY5Y cells pre-loaded with BSA control or 150 µM ALA conjugated to BSA (BSA-ALA) and treated with 1N4R tau fibrils showing the C-Laurdan GP Index at each pixel position. Lower panels show LysoTracker staining. Scale bar = 10 µm. (B) Quantification of GP values in SH-SY5Y cells pre-loaded with BSA control or 150 µM ALA conjugated to BSA (BSA-ALA) and exposed to 1N4R tau fibrils. GP values were measured across the whole cell (left) or restricted to LysoTracker-positive (LTR) regions (right). Statistical analysis was conducted using a two-way mixed-model ANOVA, followed by pairwise comparisons of estimated marginal means with Sidak correction for multiple comparisons. n= 3 independent experiments, with 10 images analyzed per experiment. (C) Maximum intensity projection of confocal z-stacks of HEK293T cells expressing sfGFP-LGALS3 pre-loaded with BSA or BSA-ALA with or without exposure to 1N4R tau fibrils. Scale bar = 10 µm. (D) Quantification of sfGFP-LGALS3 foci following indicated treatments. Statistical analysis comparing BSA + tau to other conditions was done using Kruskal-Wallis with Dunn’s post-hoc test. n = 3 independent experiments, with 10 images analyzed per experiment. *** = p < 0.001. (E) Maximum intensity projection of confocal z-stacks of tau-Venus biosensor cell line pre-loaded with BSA or BSA-ALA with or without exposure to 1N4R tau fibrils. Scale bar = 10 µm. (F) Quantification of tau-Venus foci following indicated treatments. Statistical analysis comparing BSA + tau to other conditions was done using Kruskal-Wallis with Dunn’s post-hoc test. n = 3 independent experiments, with 10 images analyzed per experiment. *** = p < 0.001.

ALA improves neuronal function and reduces toxicity during aging.

(A) Posterior touch response of animals expressing F3ΔK281::mCherry at indicated ages when grown on plates supplemented with ALA or ethanol solvent only control. Statistical analysis was done using two-way ANOVA with Bonferroni’s multiple comparison test. n = 3 independent experiments, with 10 animals analyzed per experiment. (B) Maximum intensity projection of confocal z-stacks of day 6 old animals expressing F3ΔK281::mCherry grown on EtOH solvent control or ALA plates. Scale bar = 20 µm. (C) Neurotoxicity score of PLM neurons of animals expressing F3ΔK281::mCherry grown on EtOH solvent control or ALA plates at indicated ages. Statistical analysis was done using two-way ANOVA with a Holm-Sidak’s multiple comparison test. n = 3 independent experiments, with 15 animals analyzed per experiment. (D) Quantification of the percentage of animals with ≥1 sfGFP::LGALS3 puncta in touch receptor neurons, upon co-expression of either mCherry control or F3ΔK281::mCherry and growth on EtOH solvent control or ALA-supplemented plates on day 5 (second day of adulthood). Statistical analysis was done using two-way ANOVA with Bonferroni’s multiple comparison test. Each dot represents an independent experiment, lines indicate the mean ± SEM. n= 3 independent experiments, with 10 animals analyzed per experiment. n.s.: not significant, * = p < 0.05, ** = p < 0.01.