Sphingolipid imbalance aggravates tau pathology by endomembrane rigidification and rupture

Reviewer #1 (Public review):

Summary:

In this study, Tittelmeier et al. explored the role of sphingolipid metabolism in maintaining endolysosomal membrane integrity and its downstream effects on tau aggregation and toxicity, using both worms and human cell models. The authors showed that knockdown of sphingolipid metabolism genes reduced endolysosomal membrane fluidity, as revealed by FRAP and C-Laurdan imaging, leading to increased vesicle rupture. Furthermore, tau aggregates accumulated in endolysosomes and exacerbated membrane rigidity and damage, promoting seeded tau aggregation, likely by enabling tau seed escape into the cytosol. Importantly, unsaturated fatty acid supplementation restored membrane fluidity, suppressed tau propagation, and alleviated neurotoxicity in C. elegans. These findings provide insight into how lipid dysregulation contributes to tau pathology and highlight membrane fluidity restoration as a potential therapeutic avenue for Alzheimer's disease.

Strengths:

The study addresses the connection between sphingolipid metabolism, endolysosomal membrane integrity, and tau pathology, which is a relevant topic in the context of Alzheimer's disease and related tauopathies.

The use of both C. elegans and human cell models provides cross-species perspectives that help frame the findings in a broader biological context.

The combination of FRAP and C-Laurdan dye imaging offers a biophysical approach to investigate changes in membrane properties, which is a technically interesting aspect of the study.

The observation that unsaturated fatty acid supplementation can modulate membrane fluidity and influence tau-related phenotypes adds an element of potential therapeutic interest.

The study presents multiple experimental approaches to address the proposed mechanism, and efforts were made to examine both membrane behavior and tau aggregation dynamics.

Weaknesses:

In Figure 3, the authors used C-Laurdan imaging to assess membrane fluidity and showed that knockdown of SPHK2, the human ortholog of sphk-1, led to increased membrane rigidity. However, the authors did not co-stain with a lysosomal marker, making it unclear whether the observed effect is specific to lysosomal membranes or reflects general membrane changes. Co-staining with LysoTracker or applying segmentation masks to isolate lysosomal signals would significantly improve interpretation.

Line 173 states that Lipofectamine 2000 increases membrane fluidity based on GP index changes, but this is incorrect. A higher GP index indicates increased membrane order (i.e., reduced fluidity), so the statement should be revised. Additionally, Lipofectamine 2000 can itself alter membrane rigidity, posing a risk of false-positive interpretations. To confirm the role of SPHK2 in this phenotype, the authors should use a CRISPR/Cas9 knockout model instead of relying solely on siRNA transfection, which may be confounded by the delivery reagent. Without lysosomal co-staining and SPHK2 KO validation, the authors cannot conclusively claim that SPHK2 loss affects endolysosomal membrane integrity.

The section titled "Fibrillar tau increases membrane rigidity and exacerbates endolysosomal damage" (lines 177-215) requires substantial revision. The narrative jumps abruptly between worms and cell models, making it hard to follow the logic. The use of the F3ΔK281::mCherry strain is introduced without explanation or context. It is unclear whether this strain is relevant to lysosomal membrane rupture, as no reference or justification is provided. The authors should clarify whether this reporter is intended to detect lysosomal membrane permeabilization (LMP). If so, it would be more appropriate to use established LMP reporters, such as lysosome-targeted fluorescent sensors, galectin-based reporters, or dextran leakage assays. Based on the current data in Figure 3G, it is difficult to draw firm conclusions regarding membrane rupture levels.

To support the conclusion that sphingolipid metabolism gene knockdown alters membrane properties, the study would benefit from direct lipidomic analysis. Measuring changes in sphingolipid profiles in both C. elegans and cell models would provide biochemical evidence for the proposed disruption of lipid homeostasis. Given the availability of lipidomics platforms, this type of analysis should be feasible in both worms and human cells and would significantly strengthen the mechanistic claims regarding membrane fluidity and integrity.

The conclusions of the study rely heavily on imaging-based assays, including FRAP, C-Laurdan, and fluorescence microscopy. While these approaches provide valuable spatial and qualitative insights, they are inherently indirect and subject to interpretive limitations. To strengthen the mechanistic claims, the authors should incorporate additional biochemical or quantitative approaches. For example, lipidomics would allow direct measurement of membrane lipid composition changes, and western blotting or quantitative proteomics could assess levels of membrane-associated proteins involved in endolysosomal function or stress responses. Including such data would significantly improve the robustness and reproducibility of the study's conclusions.

The human cell experiments were performed exclusively in HEK293T cells, which are not physiologically relevant for modeling Alzheimer's disease or lysosomal function in neurons. Given that the study aims to draw conclusions related to tau aggregation and lysosomal membrane integrity, the use of a more disease-relevant cellular model is essential. There are several established AD-relevant cell models, including iPSC-derived neurons, neuroblastoma lines expressing tau, or microglial models, which would better reflect the cellular context of tauopathies. Validation of key findings in at least one of these systems would substantially enhance the biological relevance and translational impact of the study.

The authors reported that PUFA supplementation rescues neurotoxic phenotypes by increasing membrane fluidity. However, the data supporting this claim rely entirely on confocal imaging, shown in both the main and supplemental figures. To substantiate the mechanistic link between PUFA treatment and improved lysosomal membrane properties, the authors should include functional assays demonstrating that PUFAs are indeed incorporated into lysosomal membranes. Additionally, lipidomics analysis would be valuable to identify which lipid species are altered upon supplementation and correlate these changes with the observed phenotypic rescue. Furthermore, the conclusion that PUFAs rescue "neurotoxic phenotypes" is not appropriate based on data derived solely from HEK293T cells, which are not neuronal. To make claims about tau-related neurotoxicity, the authors should validate their findings in a more relevant neuronal model, such as SH-SY5Y neuroblastoma cells expressing tau or iPSC-derived neurons. This would better reflect the cellular environment of Alzheimer's disease and provide stronger support for the proposed therapeutic potential of PUFA supplementation.

While the authors demonstrate that ALA supplementation mitigates neurotoxicity in C. elegans expressing aggregated tau (F3ΔK281::mCherry), the current data are not sufficient to conclude that ALA directly rescues tau aggregation toxicity via a lysosome-specific mechanism. It remains unclear how lipid composition is altered upon ALA treatment and whether these changes correlate with functional improvement of lysosomal pathways. The manuscript does not provide mechanistic insight into how ALA enhances lysosomal health or attenuates endolysosomal damage. Moreover, supplementation with PUFAs like ALA can activate a wide range of cellular processes beyond lysosomal function, including alterations in membrane fluidity, signaling cascades, and oxidative stress responses. The authors should clarify how they distinguish the lysosome-related effects from these alternative pathways. For example, did they observe specific lysosomal markers or structural improvements in lysosomes upon ALA treatment? Additional data or controls would be necessary to support a lysosome-specific protective mechanism and to exclude the involvement of other PUFA-responsive pathways in the observed phenotypes.

Reviewer #2 (Public review):

Tittelmeier et al. investigated the role of sphingolipid (SL) metabolism in the maintenance of endolysosomal vesicle integrity. They find that both impaired SL biosynthesis and degradation in C. elegans, decrease the fluidity of endolysosomal membranes and promote their rupture, while it has little effect on plasma membrane fluidity. Endolysosomal membrane fluidity is also negatively affected in human cells upon knockdown (KD) of a gene (SPHK2) involved in the SL degradation pathway. Aggregated forms of tau in both models (C. elegans and human cells) can also cause rigidification of the endolysosomal membrane, with SL homeostasis disruption having an additive effect, exacerbating endolysosomal rupture. Notably, KD of SPHK2 also increased the formation of tau foci, suggesting that compromised endolysosomal integrity may promote tau aggregation. These data provide a clearer understanding of how genetic manipulation of SL metabolism affects endolysosomal membranes and their rigidification in the context of tau aggregation. Supplementation of polyunsaturated fatty acids (PUFAs), which has a beneficial effect on Alzheimer's patients, improved membrane fluidity and reduced tau propagation in human cells and tau-associated neurotoxicity in C. elegans, suggesting a possible mechanism of action.

Overall, the conclusions of this paper are supported by the data, with a few aspects requiring further clarification and elaboration.

(1) A reference to Figure S2E-G, which shows that KD of SL biosynthesis genes do not affect the plasma membrane, is missing from the main text.

(2) In Figure 3C, lipofectamine alone shows that it increases membrane rigidity (increased GP values), not membrane fluidity.

(3) In Figure 3F, the EV cntl condition expressing F3:mCh tau should have increased LGALS3 foci compared to the mCh EV cntl according to Ref (20) and its Figure 2G (at least for Day 5 animals), which would be indicative of the tau spreading in hypodermal tissue. What C. elegans age was examined in Figure 3F? Can the authors provide evidence of the transmission of the F3:mCh tau from the touch receptor neurons to the hypodermis in the EV [similar to Figure 2C & D from Ref (20)] and compare it to the KDs? Otherwise, it seems that KD of SL genes impacts not only endolysosomal rupture but significantly affects tau accumulation/spreading as well (e.g., shown later in HEK cells, where SPHK2 KD increases the formation of tau-Venus foci).

(4) Sphingolipids are essential membrane components and signaling molecules. Does KD of SL genes in C. elegans and the subsequent endolysosomal rupture cause any major, intermediate, or minor defects/phenotypes (in non-aggregation prone models, w/t..)?

Reviewer #3 (Public review):

Summary:

The authors set off with an analysis of the lysosomal integrity upon knockdown of genes of the sphingolipid metabolic pathway that they identified in a previous (yet unpublished) work of an RNA screen using a new C. elegans Tau model. They then used cell culture and C. elegans experiments to study the link between lysosomal rupture and Tau propagation.

Strengths:

The authors use two complementary model systems and use probes to assess membrane rigidity that allow a quick assessment of the membrane dynamics and offer the opportunity to treat the cells with lipids, RNAi. Tau seeds, etc.

Weaknesses:

The main weakness is that this work builds on not-yet-peer-reviewed manuscript that established a new C. elegans Tau model and RNAi screen that aimed to identify genes involved in the propagation of Tau.

This reviewer misses essential information of the C. elegans Tau strain (not included in the method section): e.g., promoter used for the expression, information on the used Tau variant, expression pattern, and aggregation, etc.

Throughout the study, I missed data on:

(1) Effect of the knockdown on Tau expression, localisation (with lysosomal membrane?), aggregation, and proteotoxicity. The effect of the RNAi-mediated knockdown could also simply lead to a reduced expression of Tau that, in turn, leads to suppressed propagation.

(2) A quantification of RNAi knockdown is needed to judge the efficiency of the RNAi, in particular for the combinatorial RNAi experiments involving 2 and even 4 genes in parallel. Ideally, these analyses should be validated with mutants for these genes.

Further:

(3) Figure 4 H, I: Would Tau also aggregate in the absence of externally added Tau?

(4) How specific is the effect for Tau? It would help if the authors could assess other amyloid proteins.

(5) The connection between sphingolipids and AD is not new. See He et al, 2010, Neurobiol. Aging + numerous publications and also not between Tau seeding and lysosomal rupture: Rose et al., PNAS 2024 (that has been cited by the authors).