Sphingolipid metabolism dysregulation by KD is linked to of endolysosomal vesicle rupture.

(A) General overview of sphingolipid metabolism with a particular focus on the genes identified in the screen we performed. 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 base 68–70. Its synthesis involves the branched chain FA (BCFA) elongation pathway to yield C15iso-CoA, which then condensates 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, ranching from 20-26 carbon atoms in length, with or without hydroxylation 30,71. It can be also derived from BCFAs, such as C15iso and C17iso 68,69. 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 cycle 68,69. 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) 69. 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 animals expressing hypodermal sfGFP::LGALS3. Numerous foci are visible upon RNAi-mediated KD of sphingolipid metabolisms genes. Zoomed in image is indicated in overview image by a white box.

(D) Mean percentage of 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 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.

(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.

(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 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 reduces membrane fluidity leading to lysosomal rupture.

(A) Scheme of the fluorescence properties of C-Laurdan. The dye is excited using illumination at 405 nm. The dye fluoresces with a peak emission wavelength around 450 nm (red) when residing in the ordered phase and ∼500 nm in the disordered phase (blue). Two-channel acquisition is conducted in the wavelength bands indicated by shaded boxes.

(B) Pseudo-color images showing the GP index of C-Laurdan dye at each pixel position in HEK293T cells upon KD of SPHK2.

(C) Increase in GP Index of HEK293T cells upon KD of SPHK2. Statistical analysis was done using one-way ANOVA with Bonferroni’s post-hoc test.

(D) Max. intensity projection of confocal z-stacks of HEK293T cells expressing sfGFP-LGALS3 upon KD of SPHK2 and seeding with 1N4R tau fibrils.

(E) Average number of foci per number of cells. n= 3 independent experiments, 10 images each. Statistical analysis done using oKruskal-Wallis with a Dunn’s post-hoc test.

(F) Quantification of the percentage of animals with ≥ 3 hypodermal sfGFP::LGALS3 foci, representing endolysosomal damage, expressing either the F3ΔK281::mCherry (red) or only the mCherry tag (grey) in touch receptor neurons under knockdown of the indicated genes.

(G) Dilution of the indicated RNAi cultures with the empty vector control culture was done to reduce the strength of the knockdown. Data represented as mean ± SEM. Besides knockdown of sphk-1 in C n = 3 – 7 with F 40 – 50 or G 20 – 30 animals per replicate. 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 RNAi conditions to the empty vector control done using one-way ANOVA with Dunnett’s post-hoc test.

(H) Worms expressing F3ΔK281::mCherry or mCherry in touch receptor neurons stained with C-Laurdan. 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 Index.

(I) Increase in rigidity of membranes in animals expressing F3ΔK281::mCherry compared to mCherry. Statistical analysis was done using a t-test. n=4, total of at least 28 worms.

(J) Pseudocolored HEK293T cells exposed to tau 1N4R fibrils visualizing the C-Laurdan dye GP Index.

(K) Increase rigidity of membranes of cells seeded with tau fibrils as indicated by the GP index.

(L) GP index of cells seeded with monomeric tau. Statistical analysis for C-Laurdan in cells was done using a t-test. n= 3 independent experiments, 10 images each. Scale bar 10 µm. n.s.: not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Saturated fatty acids decrease membrane fluidity and exacerbate seeded tau aggregation.

(A) Left: saturated fatty acid membrane scheme. Right: pseudocolored HEK293T cells pre-loaded with 50 µM PA conjugated to BSA (BSA-PA) displaying the C-Laurdan dye GP Index.

(B) GP Index indicates increased rigidity of membranes with BSA-PA. Statistical analysis was done using the Mann-Whitney-test. n= 3 independent experiments, 10 images each.

(C) Pseudocolored HEK293T cells pre-loaded with BSA-PA and seeded with 1N4R tau fibrils.

(D) GP Index indicates an additive impact of tau fibrils on membrane rigidity when added to HEK293T cells pre-loaded with BSA-PA. Statistical analysis was done using t-test. n= 3 independent experiments, 10 images each.

(E) Quantification of hypodermal sfGFP-LGALS3 foci in animals grown on plates supplemented with PA reveal an increase in lysosomal rupture. Statistical analysis was done using Mann-Whitney test. n= 3 independent experiments, 15 worms per replicate.

(F) Max. intensity projection of confocal z-stacks of HEK293T cells expressing sfGFP-LGALS3 pre-loaded with BSA-PA and seeded with 1N4R tau fibrils.

(G) Quantification of sfGFP-LGALS3 foci in HEK293T cells following indicated treatments.

(H) Max. intensity projection of confocal z-stacks of Venus-tagged full-length P301S tau biosensor cells pre-loaded with BSA-PA and seeded with 1N4R tau fibrils.

(I) Quantification of Venus-P301S tau foci following indicated treatments. Statistical analysis comparing BSA + tau to other conditions was done using one-way ANOVA with Dunnett’s post-hoc test. n= 3 independent experiments, 10 images each. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Scale bar 10 µm.

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

(A) Left: unsaturated fatty acid membrane scheme. Right: C-Laurdan GP Index of HEK293T cells pre-loaded with 150 µM ALA conjugated to BSA (BSA-ALA) indicating an increase in membrane fluidity. Statistical analysis was done with a t-test. n= 3 independent experiments, 10 images each.

(B) Pseudocolored HEK293T cells pre-loaded with BSA-ALA and seeded with 1N4R tau fibrils.

(C) GP Index indicates that BSA-ALA reduces tau-induced rigidification of the membrane. Statistical analysis was done using the Mann-Whitney U-test. n= 3 independent experiments, 10 images each.

(D) Max. intensity projection of confocal z-stacks of HEK293T cells expressing sfGFP-LGALS3 pre-loaded with BSA-ALA and seeded with 1N4R tau fibrils. Scale bar 10 µm.

(E) Quantification of sfGFP-LGALS3 foci following indicated treatments. n= 3 independent experiments, 10 images each.

(F) Max. intensity projection of confocal z-stacks of tau-Venus biosensor cell line pre-loaded with BSA-ALA and seeded with 1N4R tau fibrils. Scale bar 10 µm.

(G) 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, 10 images each.

(H) Posterior response to touch during aging of animals expressing F3ΔK281::mCherry grown on plates supplemented with ALA or only the ethanol solvent. Statistical analysis was done using two-way ANOVA with Bonferroni’s multiple comparison test.

(I) Max. intensity projection of confocal z-stacks of day 6 old animals expressing F3ΔK281::mCherry.

(J) Neurotoxicity score of PLM neurons of animals grown on control or ALA plates. Statistical analysis was done using 2-way ANOVA with a Holm-Sidak’s multiple comparison test. n= 3 independent experiments, 15 worms per replicate. Scale bar 20 µm. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.