Disruption of sphingolipid metabolism promotes tau seeding through endolysosomal membrane rigidification and rupture
- Chair of Neuroanatomy, Institute of Anatomy, Faculty of Medicine, Ludwig-Maximilians-University Munich (LMU Munich), Germany
- Center for Molecular Biology of Heidelberg University (ZMBH) and German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Germany
- Chemical Neurobiology Laboratory, Center for Genomic Medicine, Departments of Neurology and Psychiatry, Massachusetts General Hospital and Harvard Medical School, United States
- Institute Francois Jacob (MIRCen), CEA, and Laboratory of Neurodegenerative Diseases, CNRS, France
Figures
Figure 1 with 1 supplement
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-acylated 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 (Chitwood et al., 1995; Zhang et al., 2011; Zhu et al., 2013). 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 to 26 carbon atoms in length, with or without hydroxylation (Hänel et al., 2019; Scholz et al., 2021). It can also be derived from BCFAs, such as C15iso and C17iso (Chitwood et al., 1995; Zhang et al., 2011). 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 (Chitwood et al., 1995; Zhang et al., 2011). Each elongation cycle comprises four reactions (condensation, reduction, dehydration, and reduction), with the third reaction requiring very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase (encoded by hpo-8) (Zhang et al., 2011). 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) hydrolyze 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 phosphoethanolamine and (2E)-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.
-
Figure 1—source data 1
This Source Data contains raw data for Figure 1.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Sphk-1 and additional sphingolipid metabolic genes affect endolysosomal rupture.
(A) Genetic validation of the sphk-1 RNAi phenotype using a sphk-1 mutant. Quantification of sfGFP::LGALS3 foci per worm on day 5 (second day of adulthood) in wild-type (WT) and sphk-1 mutant animals. Each dot represents one animal; data are shown as mean ± SEM. n = 3 biological repeats with 15 animals per replicate. Statistical analysis was done using an unpaired Student’s t-test. (B, C) Co-KD of redundant genes involved in SL metabolism identifies additional regulators of endolysosomal rupture. Quantification of the percentage of animals on day 5 (second day of adulthood) expressing F3ΔK281::mCherry in touch receptor neurons with ≥3 hypodermal sfGFP::LGALS3 foci, upon indicated single and co-KDs. Percentages shown next to gene names indicate the relative amount of each RNAi bacterial clone in co-KD conditions. (B) Among the genes involved in SL biosynthesis, co-KDs of sptl-1 and -3 and hyl-1 and -2 significantly induce endolysosomal rupture compared with the empty vector control. (C) For genes related to SL degradation, co-KD of gba-2, -3, and -4 or gba-1,-2, -3, and -4 resulted in a significant increase in endolysosomal rupture. Data are shown as mean ± SEM. N = 45–60 animals from three biological replicates. Statistical analysis was done using one-way ANOVA with Dunnett’s post hoc test, **p < 0.01, ***p < 0.001.
-
Figure 1—figure supplement 1—source data 1
This Source Data contains raw data for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig1-figsupp1-data1-v1.xlsx
Figure 2 with 1 supplement
Knockdown of genes involved in sphingolipid metabolism decreases lysosomal membrane fluidity.
(A) Representative confocal single plane images from an 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%. 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 an 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%. 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.
-
Figure 2—source data 1
This Source Data contains raw data for Figure 2.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Disruption of SL metabolism decreases lysosomal membrane fluidity.
(A) Representative confocal single plane images from an FRAP experiment in animals on day 5 (second day of adulthood) expressing a mCherry-tagged Lysosomal Lysine/Arginine Transporter 1 (LAAT-1::mCherry) in the intestine grown either on empty vector or spl-1 RNAi plates. Dashed circles outline the bleach spots. Scale bar = 5 µm. (B) Combined FRAP curves of LAAT-1::mCherry in intestinal lysosomes of animals on day 5 (second day of adulthood) grown either on empty vector or spl-1 RNAi plates. Curves are normalized to the pre-bleach intensity as 100% and the first post-bleach intensity as 0%. Mean thalf (C) and maximal % recoverable fluorescence values (D) calculated from the LAAT-1::mCherry FRAP experiments. (E) FRAP curves of prenylated GFP enriched on the intestinal plasma membrane of animals on day 5 (second day of adulthood) 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%. Mean thalf (F) and maximal % recoverable fluorescence values (G) calculated from the GFP FRAP experiments. Data, including FRAP curves, represented as means ± SEM from 5–12 FRAP measurements on day 5 animals (second day of adulthood). Statistical analysis comparing RNAi conditions to the empty vector control was done using one-way ANOVA with Dunnett’s post hoc test. *p < 0.05.
-
Figure 2—figure supplement 1—source data 1
This Source Data contains raw data for Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 1 supplement
KD of SPHK2 and aggregated tau increase 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 after transfection 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 of 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.
-
Figure 3—source data 1
This Source Data contains raw data for Figure 3.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
KD of SPHK2 and exposure to fibrillar tau decrease membrane fluidity in human cells.
(A) Upper panels: pseudo-colored images of SH-SY5Y cells untreated or transfected with Lipofectamine showing the C-Laurdan GP Index. Lower panels: LysoTracker staining. Scale bar = 10 µm. (B) Quantification of GP values in SH-SY5Y cells that were untreated or treated with Lipofectamine alone. GP values were measured either across the whole cell (left) or specifically within LysoTracker-positive (LTR) regions (right). No significant differences were observed (see also Figure 3C). Statistical analysis was performed 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) Western blot analysis of total cell lysates from SH-SY5Y cells after 48 or 72 hr of no treatment, Lipofectamine treatment alone, or transfection with control or SPHK2 siRNA using Lipofectamine. SPHK2 and GAPDH protein levels were assessed using anti-SPHK2 and anti-GAPDH antibodies, respectively (see methods section). Quantification of SPHK2 protein levels relative to GAPDH after 48 hr (D) or 72 hr (E) of indicated controls or SPHK2 siRNA treatment. Each dot represents an independent experiment, with lines indicating mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. (F) Upper panels: pseudo-colored images of HEK293T cells exposed to PBS control or 1N4R tau fibrils showing the C-Laurdan GP Index. Lower panels: LysoTracker staining. Scale bar = 10 µm. (G) Quantification of GP values in HEK293T cells exposed to PBS control or 1N4R tau fibrils. Increased GP values indicate increased membrane rigidity following tau fibril treatment. Statistical analysis was performed 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. (H) Pseudo-colored images of HEK293T cells exposed to PBS control or monomeric 1N4R tau displaying the C-Laurdan GP Index. Scale bar = 10 µm. (I) Quantification of GP values in HEK293T cells exposed to PBS control or monomeric 1N4R tau. Statistical analysis was performed using Student’s t-test. n = 3 independent experiments, with 10 images analyzed per experiment. (J) Max. intensity projection of confocal z-stacks of HEK293T cells expressing sfGFP-LGALS3 following control or SPHK2 siRNA treatment, with or without 1N4R tau fibril seeding. White box indicates the zoomed in section depicted in Figure 3F. Scale bar = 20 µm. (K) Max. intensity projection of confocal z-stacks of P301S mutant tau-Venus biosensor cell line following control or SPHK2 siRNA treatment, with or without 1N4R tau fibril seeding. White box indicates the zoomed in section depicted in Figure 3H. Scale bar = 20 µm. n.s.: not significant, *p < 0.05, **p < 0.01.
-
Figure 3—figure supplement 1—source data 1
This Source Data contains raw data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig3-figsupp1-data1-v1.xlsx
-
Figure 3—figure supplement 1—source data 2
This Source Data contains Original membranes corresponding to Figure 3, panel C.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig3-figsupp1-data2-v1.zip
-
Figure 3—figure supplement 1—source data 3
This Source Data contains Original membranes corresponding to Figure 3, panel C.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig3-figsupp1-data3-v1.pdf
Figure 4
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 (gray) 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 Sidak’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, ****p < 0.0001.
-
Figure 4—source data 1
This Source Data contains raw data for Figure 4.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig4-data1-v1.xlsx
Figure 5 with 2 supplements
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.
-
Figure 5—source data 1
This Source Data contains raw data for Figure 5.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Saturated fatty acids decrease membrane fluidity and exacerbate seeded tau aggregation.
(A) Left: saturated fatty acid membrane scheme. Right: upper panels: pseudo-colored images of SH-SY5Y cells pre-loaded with BSA or 50 µM PA conjugated to BSA (BSA–PA) displaying the C-Laurdan GP Index. Lower panels: LysoTracker staining. Scale bar = 10 µm. (B) Quantification of GP values in SH-SY5Y cells upon PA treatment. GP values were measured either across the whole cell (left) or specifically within 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) Quantification of GP values in HEK293T cells after PA treatment. GP values were measured either across the whole cell (left) or specifically within LysoTracker-positive (LTR) regions (right). Statistical analysis was performed 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 panels: Pseudo-colored images of SH-SY5Y cells pre-loaded with BSA or 50 µM PA conjugated to BSA (BSA–PA) and subsequently exposed to 1N4R tau fibrils displaying the C-Laurdan GP Index. Lower panels: LysoTracker staining. Scale bar = 10 µm. (E) Quantification of GP values in SH-SY5Y cells pre-loaded with PA and subsequently exposed to 1N4R tau fibrils. GP values were measured either across the whole cell (left) or specifically within 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) Quantification of GP values in HEK293T cells preloaded with PA and subsequently exposed to 1N4R tau fibrils. GP values were measured either across the whole cell (left) or specifically within LysoTracker-positive (LTR) regions (right). Statistical analysis was performed using a two-way mixed-model ANOVA, followed by pairwise comparisons of estimated marginal means with Sidak correction for multiple comparisons. n = 2 independent experiments, with 10 images analyzed per experiment. (G) Max. intensity projection of confocal z-stacks of HEK293T cells expressing sfGFP-LGALS3 pre-loaded with BSA or BSA–PA and subsequently exposed to PBS control or 1N4R tau fibrils. White box indicates the zoomed in section depicted in lower panel. Scale bar = 10 µm. (H) Quantification of sfGFP-LGALS3 foci in HEK293T cells pre-loaded with BSA or BSA–PA and subsequently exposed to PBS control or 1N4R tau fibrils. Statistical analysis comparing BSA + tau to other conditions was done using one-way ANOVA with Dunnett’s post hoc test. n = 3 independent experiments, with 10 images analyzed per experiment. (I) Max. intensity projection of confocal z-stacks of tau biosensor cells expressing Venus-tagged full-length P301S tau pre-loaded with BSA or BSA–PA with or without exposure to 1N4R tau fibrils. White box indicates the zoomed in section depicted in lower panel. Scale bar = 10 µm. (J) Quantification of P301S tau-Venus 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 analyzed per experiment. *p < 0.05, **p < 0.01, ***p < 0.001.
-
Figure 5—figure supplement 1—source data 1
This Source Data contains raw data for Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 2
PUFA supplementation restores lysosomal membrane integrity and reduces seeded tau aggregation.
(A) Upper panels: Pseudo-colored images of SH-SY5Y cells pre-loaded with BSA control or 150 µM ALA conjugated to BSA (BSA–ALA) displaying the GP Index. Lower panels: LysoTracker staining. Scale bar = 10 µm. (B) Quantification of GP values in SH-SY5Y cells pre-loaded with BSA or BSA–ALA. GP values were measured either across the whole cell or specifically within LysoTracker-positive (LTR) regions. Statistical analysis was conducted using a two-way mixed-model ANOVA with each experiment/image as random effects, with pairwise comparisons of estimated marginal means with Sidak correction for multiple comparisons. n = 3 independent experiments, 10 images each. Quantification of GP values in HEK293T cells pre-loaded with BSA or BSA–ALA and subsequently exposed to PBS control (C) or 1N4R tau fibrils (D). GP values were measured either across the whole cell or specifically within LysoTracker-positive (LTR) regions. 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. (E) Max 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. White box indicates the zoomed in section depicted in Figure 5C. Scale bar = 20 µm. (F) Max intensity projection of whole confocal z-stacks of P301S tau-Venus biosensor cell line pre-loaded with BSA or BSA–ALA with or without exposure to 1N4R tau fibrils. The white boxes delineate the zoomed images presented in Figure 5E. Scale bar = 20 µm. **p < 0.01, ***p < 0.001.
-
Figure 5—figure supplement 2—source data 1
This Source Data contains raw data for Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig5-figsupp2-data1-v1.xlsx
Figure 6 with 1 supplement
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. Data were analyzed using a repeated-measures model of rank-transformed neurotoxicity scores with Type III Wald χ2 tests, followed by Bonferroni-adjusted pairwise comparisons of estimated marginal means. 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, ***p < 0.001.
-
Figure 6—source data 1
This Source Data contains raw data for Figure 6.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
The impact of ALA on anterior touch response.
Anterior 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. Two-way ANOVA showed significant effects of treatment (p = 0.0236) and age (p = 0.0391), but no treatment-by-age interaction (p = 0.4485). Bonferroni-adjusted comparisons between control and ALA at the individual ages were not significant. n = 3 independent experiments, with 10 animals analyzed per replicate. n.s.: not significant.
-
Figure 6—figure supplement 1—source data 1
This Source Data contains raw data for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/106865/elife-106865-fig6-figsupp1-data1-v1.xlsx
Figure 7
Proposed model linking membrane fluidity to seeded tau aggregation.
Sphingolipid dysregulation or supplementation with saturated fatty acids reduces lysosomal membrane fluidity, promoting membrane rupture, tau seed escape, and seeded tau aggregation. In contrast, supplementation with polyunsaturated fatty acids increases membrane fluidity and protects lysosomal integrity.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (Caenorhabditis elegans) | See Supplementary file 1 for details. | See Supplementary file 1 for details. | See Supplementary file 1 for details. | See Supplementary file 1 for details. |
| Strain (Escherichia coli OP50 and HT115 RNAi clones) | See Supplementary file 2 for details. | See Supplementary file 2 for details. | See Supplementary file 2 for details. | See Supplementary file 2 for details. |
| Cell line (Homo sapiens) | SH-SY5Y | ATCC | ATCC:CRL-2266 RRID:CVCL_0019 | Human neuroblastoma cell line. |
| Cell line (Homo sapiens) | HEK293T | ATCC | ATCC:CRL-3216 RRID:CVCL_0063 | Human embryonic kidney-derived cell line. |
| Cell line (Homo sapiens) | HEK293T sfGFP-LGALS3 | Sandhof et al., 2020; DOI:10.1080/15548627.2019.1643657 | Stable HEK293T reporter cell line expressing sfGFP-LGALS3. | |
| Cell line (Homo sapiens) | HEK293T 0N4R tauP301S-Venus (tau-Venus biosensor) | Dr. William A. McEwan, Cambridge University; published (McEwan et al., 2017; Nachman et al., 2020) | HEK293T biosensor line expressing Venus-tagged full-length P301S mutant 0N4R tau. | |
| Recombinant DNA reagent | rgef-1p::sfGFP::LGALS3::tbb2-3′UTR expression plasmid | This paper | pCFJ150-based vector backbone with rgef-1 promoter, sfGFP::LGALS3, and tbb-2 3′UTR. | |
| Recombinant DNA reagent | pPD49.26-sfGFP::LGALS3 | Dr. Bin Liu and Dr. Marja Jäättelä, University of Copenhagen | Expression plasmid coding for sfGFP::LGALS3, shared by Bin Liu and Marja Jäättelä. | |
| Recombinant DNA reagent | pLenti PGK Puro DEST (w529-2) | Addgene; Campeau et al., 2009; DOI:10.1371/journal.pone.0006529 | Addgene:19068 RRID:Addgene_19068 | Gift from Dr. Eric Campeau and Dr. Paul Kaufman; ordered from Addgene. |
| Sequence-based reagent | ON-TARGETplus Human SPHK2 siRNA SMARTpool | Horizon Discovery (Dharmacon) | Horizon Discovery:M-004831-00-0005 | Used at 20 nM final concentration for SPHK2 knockdown in human cells. |
| Sequence-based reagent | ON-TARGETplus non-targeting control siRNA pool | Horizon Discovery (Dharmacon) | Used as control for siRNA-mediated knockdown experiments. | |
| Peptide, recombinant protein | Full-length human 1N4R tau monomer | Prepared as described in Tardivel et al., 2016; DOI:10.1186/s40478-016-0386-4 | Purified recombinant human Tau-412 (1N4R) used for monomeric tau controls and fibril assembly. | |
| Peptide, recombinant protein | Full-length human 1N4R tau fibrils | This paper; prepared from purified recombinant 1N4R tau | Assembled at 40 μM with 10 μM heparin for 5 days at 37°C; fragmented by sonication; used at 400 nM for seeding and membrane-fluidity experiments. | |
| Antibody | Rabbit polyclonal anti-SPHK2 | Proteintech (Thermo Fisher/Life technology) | 17096-1-AP RRID:AB_10598479 | Immunoblotting primary antibody; 1:5000. |
| Antibody | Mouse monoclonal anti-GAPDH (clone GAPDH-71.1) | Sigma-Aldrich/Merck | Sigma-Aldrich:G8795; clone:GAPDH-71.1 RRID:AB_1078991 | Immunoblotting loading-control primary antibody; 1:5000. |
| Antibody | Goat anti-mouse IgG (H+L), HRP conjugate | Bio-Rad | Bio-Rad:1706516 RRID:AB_2921252 | Secondary antibody for ECL-based immunoblot detection. |
| Antibody | Goat anti-rabbit IgG (H+L), HRP conjugate | Bio-Rad | Bio-Rad:1706515 RRID:AB_11125142 | Secondary antibody for ECL-based immunoblot detection. |
| Commercial assay or kit | Mycoplasma testing | GATC Biotech | Cell lines were regularly tested for Mycoplasma contamination. | |
| Commercial assay or kit | Protein assay dye reagent concentrate | Bio-Rad | Bio-Rad:5000006 | Used to determine protein concentration before SDS-PAGE/immunoblotting. |
| Chemical compound, drug | C-Laurdan | Tocris Bioscience/Bio-Techne | Tocris:7273; CAS:959839-06-6 | Membrane-order/fluidity dye; used at 10 mM for live worms and 15 μM for cells. |
| Chemical compound, drug | LysoTracker Red DND-99 | Invitrogen/Thermo Fisher Scientific | Invitrogen:L7528 | Used at 50 nM with C-Laurdan to identify LysoTracker-positive regions in cells. |
| Chemical compound, drug | α-Linolenic acid (ALA) | Sigma-Aldrich/Merck | Sigma-Aldrich:L2376; CAS:463-40-1 | Omega-3 polyunsaturated fatty acid; conjugated to fatty-acid-free BSA for cells and supplemented in C. elegans NGM at 0.3 mM. |
| Chemical compound, drug | Palmitic acid (PA) | Sigma-Aldrich/Merck | Sigma-Aldrich:P0500; CAS:57-10-3 | Saturated fatty acid; conjugated to fatty-acid-free BSA for cells or used to enrich OP50 bacterial food source for C. elegans experiments. |
| Chemical compound, drug | Fatty-acid-free BSA | Sigma-Aldrich/Merck | Sigma-Aldrich:A8806; CAS:9048-46-8 | Used to conjugate ALA and PA for cell treatments. |
| Chemical compound, drug | NP-40 substitute | Sigma-Aldrich/Merck | Sigma-Aldrich:74385; CAS:9016-45-9 | Used at 0.001% (vol/vol) to improve fatty acid distribution in C. elegans NGM plates. |
| Chemical compound, drug | Lipofectamine 2000 Transfection Reagent | Thermo Fisher Scientific | Thermo Fisher Scientific:11668019 | Transfection reagent used for siRNA delivery into human cells; medium exchanged after 6 hr. |
| Chemical compound, drug | ATTO 550 NHS ester | ATTO-TEC GmbH (Leica Microsystems) | Used to label 1N4R tau fibrils. | |
| Chemical compound, drug | Levamisole hydrochloride | AppliChem | AppliChem:A4341; CAS:16595-80-5 | Used at 2% (wt/vol) in mounting mix for live C. elegans imaging. |
| Chemical compound, drug | Nanosphere Size Standard, 100 nm | Thermo Scientific | Thermo Scientific: 3100A; Fisher Scientific: 10671531 | Used at 50% (vol/vol) in mounting mix for live C. elegans imaging. |
| Chemical compound, drug | NheI restriction enzyme | New England Biolabs | NEB:R0131 | Used for restriction digestion during cloning. |
| Chemical compound, drug | BglII restriction enzyme | New England Biolabs | NEB:R0144S | Used for restriction digestion during cloning. |
| Chemical compound, drug | Deoxynucleotide (dNTP) Solution Set | New England Biolabs | NEB:N0446S | Used to prepare the Gibson assembly reaction mix. |
| Chemical compound, drug | β-Nicotinamide adenine dinucleotide (NAD+) | New England Biolabs | NEB:B9007S | Used in Gibson assembly reaction mix. |
| Chemical compound, drug | T5 Exonuclease | New England Biolabs | NEB:M0663S | Used in Gibson assembly reaction mix. |
| Chemical compound, drug | Phusion High-Fidelity DNA Polymerase | Thermo Fisher Scientific | Thermo Fisher Scientific:F530 | Used in Gibson assembly reaction mix. |
| Chemical compound, drug | Taq DNA Ligase | New England Biolabs | NEB:M0208L | Used in Gibson assembly reaction mix. |
| Software, algorithm | FIJI/ImageJ | Schindelin et al., 2012; https://imagej.net/software/fiji/ | RRID:SCR_002285; RRID:SCR_003070 | Used for image processing, thresholding, particle analysis, and foci quantification. |
| Software, algorithm | Automated ImageJ macro for C-Laurdan GP analysis | Published guidelines cited as (Owen et al., 2011); this paper | Used to calculate GP values and extract LysoTracker-positive regions. | |
| Software, algorithm | GraphPad Prism | GraphPad Software | Version 6h; version 10.1.1 RRID:SCR_002798 | Used for statistical analyses and graphing. |
| Software, algorithm | R | R Foundation for Statistical Computing | Version 4.3.1 RRID:SCR_001905 | Used for statistical analyses and graphing. |
| Software, algorithm | R package nlme | CRAN | Version 3.1-166 | Used for statistical analyses and graphing. |
| Software, algorithm | R package lme4 | CRAN | Version 1.1-35.5 | Used for statistical analyses and graphing. |
| Software, algorithm | R package emmeans | CRAN | Version 1.10.6 | Used for statistical analyses and graphing. |
| Software, algorithm | R package dplyr | CRAN | Version 1.1.4 | Used for statistical analyses and graphing. |
| Software, algorithm | Leica acquisition software | Leica Microsystems | Used for C-Laurdan image acquisition on DMI6000 and Leica SP8X WLL microscopes. | |
| Other | Olympus IXplore SpinSR confocal microscope | Olympus/Evident | Used for confocal imaging of galectin puncta and tau-Venus foci. | |
| Other | Zeiss LSM 780 confocal microscope | Carl Zeiss | Used for FRAP measurements of C. elegans membranes. | |
| Other | Leica M205 FA widefield binocular microscope | Leica Microsystems | Used for scoring hypodermal sfGFP::LGALS3 foci in C. elegans. | |
| Other | Leica SP8X WLL microscope | Leica Microsystems | Used for C-Laurdan imaging. | |
| Other | Hielscher Vial Tweeter/ultrasonic processor UIS250v | Hielscher Ultrasonics | UIS250v | Used to fragment 1N4R tau fibrils for seeding experiments. |
Additional files
-
Supplementary file 1
C. elegans strains used in this study.
- https://cdn.elifesciences.org/articles/106865/elife-106865-supp1-v1.xlsx
-
Supplementary file 2
Main RNAi target genes of the bacterial clones used in this study.
- https://cdn.elifesciences.org/articles/106865/elife-106865-supp2-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/106865/elife-106865-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Disruption of sphingolipid metabolism promotes tau seeding through endolysosomal membrane rigidification and rupture
eLife 14:RP106865.
https://doi.org/10.7554/eLife.106865.3
