The Troyer syndrome protein spartin mediates selective autophagy of lipid droplets

Lipid droplets (LDs) are crucial organelles for energy storage and lipid homeostasis. Autophagy of LDs is an important pathway for their catabolism, but the molecular mechanisms mediating LD degradation by selective autophagy (lipophagy) are unknown. Here we identify spartin as a receptor localizing to LDs and interacting with core autophagy machinery, and we show that spartin is required to deliver LDs to lysosomes for triglyceride mobilization. Mutations in SPART (encoding spartin) lead to Troyer syndrome, a form of complex hereditary spastic paraplegia1. Interfering with spartin function in cultured human neurons or murine brain neurons leads to LD and triglyceride accumulation. Our identification of spartin as a lipophagy receptor, thus, suggests that impaired LD turnover contributes to Troyer syndrome development.

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The reviewers were not yet convinced that spartin acts as a lipophagy receptor and their concerns were numerous and significant. We feel that the referees' concerns point to a premature dataset and concerns regarding the role of spartin in autophagy globally would need to be addressed thoroughly with experiments and data. Reconsideration of the study for this journal and re-engagement of the Rev#3 asked for a mutant that disrupts spartin's localization at LDs and its interaction with ATG8; would any of the figure 1 mutants (and/or in combination with Fig 2 mutants) fit for this line of analysis?
B. Please also check that spartin functions in lipophagy (Rev#3 #5) under other types of stress, and not in lipolysis (Rev#2 #6), with validation that is does specifically function to regulate lipophagy in neurons (Rev#2 #4), as opposed to LD biogenesis. We strongly encourage you to study the role of spartin in adipocytes (Rev#2 #1) and agree with Rev#2 that a function for spartin in bulk autophagy should be ruled out thoroughly (Rev#2 #8).
C. Given your access to the mice expressing spartin-DN, please answer Rev#2's question about their phenotypes (Rev#2 #2) to enrich the phenotypic analyses.
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We would be happy to consider a revised manuscript that would satisfactorily address these points, unless a similar paper is published elsewhere, or is accepted for publication in Nature Cell Biology in the meantime.
In contrast, although we agree with Rev#2 that exploring the role of spartin in other forms of selective autophagy (Rev#2 #9) would provide valuable insights, we do not think it is strictly needed to support the current conclusions. Similarly, testing human disease-linked Spartin mutations would be a significant addition to the paper (#3 Rev#2) but in our view this is not strictly required to support the role of spartin as a lipophagy receptor. Thus, addressing these comments experimentally will not be strictly necessary for reconsideration of the manuscript at this journal.
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Data & methodology: validity of approach, quality of data, quality of presentation The quality of the experimental work is a hallmark of this paper and the authors are to be congratulated on the robustness of their work. A particularly positive feature is that the authors have striven to show the endogenous relevance of their work at every opportunity, rather than relying on over-expression systems. The work is presented very nicely.

Appropriate use of statistics and treatment of uncertainties
For the most part that statistics are appropriate, with one general exception. The authors use paired ttest in many instances where more than two conditions are being compared on a histogram. This is not correct -ANOVA with pairwise comparisons (and post-hoc testing for multiple testing) should be used.

Conclusions: robustness, validity, reliability
Overall I think the conclusions of this paper are robust and reliable.
Suggested improvements: experiments, data for possible revision The paper is already very convincing, but I have a few suggestions for possible improvements. I regard all of these suggestions as addressing relatively minor points: 1. A surprising facet of this paper is that the lysosomal receptor consisted of autophagy proteins, rather than ESCRT proteins, which would be the natural candidate in view of their endolysosomal localisation and known interaction with spartin. Are the authors sure that the MIT domain plays no role in this process? It would be helpful to nail this point by including MIT-domain deleted (or better F24 mutated) forms of spartin in the rescue experiments for the lysosome-lipid droplet co-localisation assays used in figure 2 (or perhaps using the Keima-livedrop system with rescues of the spartin KO phenotype).
2. The experiments showing interaction between spartin and LC3 components are convincing, but would be made even more convincing if shown with the endogenous proteins.
3. Enhanced figure 3c suggests that some modest interaction between spartin and LC3 is maintained with the construct that expresses only the senescence domain. Can the authors comment/explain, as this doesn't quite fit with the model of only the UBR being involved. Figure 3b, I think it would be helpful to carry out a control experiment adding bafilomycin to the WT OA withdrawal condition -this would validate that the colour change is dependent on vacuolar ATPase activity (i.e. acidification of lysosomes). 5. While not strictly necessary (the human neuronal results are pretty convincing), the neuronal LD accumulation would be even more convincing if the authors could show LD accumulation in the brain of the published spartin knock out mouse. If this mouse is not easily available I would not hold up 6 publication of the important results of this paper. 6. A previous study (Eastman et al, JCB, 2009) suggested that spartin binds to the lipid droplet protein TIP47. Could the authors clarify whether TIP47 binding is involved in the mechanism of recruitment of spastin to LDs that they have described via amphipathic helices in the senescence domain, for example by depleting TIP47 and testing if spartin's recruitment to LDs is reduced? The Eastman study also highlighted the role of ubiquitin ligases, binding to spartin's PPXY motif, in regulating spartin's association with lipid droplets. Could the authors test whether the PPXY motif plays a role in regulating the lipophagy mechanism that they have described?

In the Keimsa experiments in
References: appropriate credit to previous work?
Yes, although the authors might make it clearer that endogenous spartin had already been localised to LDs in reference 14 (Edwards et al) Clarity and context: lucidity of abstract/summary, appropriateness of abstract, introduction and conclusions The paper is very nicely written and very clear. The abstract is appropriate.
Reviewer #2: Remarks to the Author: Lipophagy is one of selective autophagy and has an essential for lipid mobilization. However, the molecular mechanism regulating lipophagy is not well understood compared to other selective autophagy. Furthermore, the physiological relevance of lipophagy also remains largely elusive. In this study, Chung et al. found that the Troyer syndrome protein spartin as a novel regulator of lipophagy. They found that Spartin localizes on lipid droplets and interacts with autophagosomal proteins LC3A/C to serve as an autophagy receptor. Depletion of Spartin impaired the turnover of lipid droplet as revealed by Keima reporter assay and biochemical analysis of TG level. Moreover, motor neurons derived from spartin KO iPS cells and mouse brains injected with dominant negative spartin showed the accumulation of lipid droplets, suggesting that defect of lipophagy could contribute to Troyer syndrome development. Although the identification of a novel regulator of lipophagy contributes to understanding of lipophagy, the current study did not go beyond the functional analysis of spartin during the turnover of lipid droplets. Considering that spartin has been shown to be localized on lipid droplets (JCB, 2009), I personally doubt that it brings sufficient novelty and conceptual advance which are required for this journal and recommend a more specialized journal to submit. The lack of significance of findings in in vivo lipid metabolism also dampens the enthusiasm of this reviewer to support its publications. The followings are specific comments for authors. Major 1. Liver and brown adipose tissue play a central role in lipid metabolism. Is spartin essential for lipophagy in liver or brown adipose tissue? Previous studies showed that lipophagy occurs in liver or brown adipose tissue; however, the authors performed most of the experiments using SUM159 breast cancer cells. It is to be determined whether spartin is required for hepatic or adipose lipophagy. 8 recruitment to lipid droplets. They further demonstrated that show spartin interacts with ATG8 family protein LC3A, potentially through its ubiquitin-binding region (UBR). Knock out of spartin causes deficient lipid droplet turnover in cultured cells. They further show impairing spartin function by overexpression of a dominant negative spartin fragment leads to compromised lipophagy and TG accumulation in cultured human neurons and murine brain neurons. Overall, this study would be of interest for the readership as it would highlight a potential new lipophagy receptor. However, the conclusion of spartin as a lipophagy receptor is not fully supported by the data, more key evidences are needed to clarify the role of spartin in lipophagy.
Major points: 1. The authors investigated the function of spartin in lipophagy mainly by spartin knockout or by overexpressing a dominant ain negative fragment that with a large truncation of its N-terminal MIT domain and the UBR. It is hard to conclude that spartin acts as a cargo receptor based on the effects caused by either fully knockout or such a big truncation. Specific mutants without disrupting the global domain conformation that block its localization on lipid droplets and its interaction with ATG8 family proteins respectively, are need to determine the role of spartin in the turnover of lipid droplets.
2. Spartin was shown to interact with the Nedd4-family E3 ligase AIP4 and promote the ubiquitination of adipophilin on lipid droplets (Eastman SW, 2009;Hooper C, 2010). Given the authors analyzed the function of spartin in lipophagy mostly using spartin knock out cells, one possibility is that spartin is mainly involved in the ubiquitination of proteins on the lipid-droplet membrane, and the ubiquitinated proteins recruit the SAR family receptors such as p62, NBR1 that further mediate the engulfment of lipid droplet by autophagy. And actually, it was shown in macrophage lipid droplets are decorated with ubiquitin, and SARs including p62, OPTN and NBR could target to ubiquitinated lipid droplets (Karlsson AB, 2014;Robichaud S, 2021). The authors should test whether lipid droplets are ubiquitinated in the neuron system, and test the function of SAR receptors in spartin-mediated lipophagy. The authors need to clarify spartin functions directly as a receptor itself or indirectly by recruiting the SAR receptors.
3. One major evidence shown by the authors that supporting spartin acts as a lipophagy receptor is the interaction between spartin and ATG8 family members LC3A and LC3C. They claimed that the binding between spartin and LC3A is mediated through spartin UBR domain, but not a conventional LC3 interaction motif (LIR). The authors should show the data that the LIR is not required for its binding to LC3A in the manuscript. Have the authors checked whether the LIR is required for its binding with LC3C? Do the authors have particular reasons to pick LC3A? Would it be possible spartin binds to LC3C via the LIR and thus LC3C is the functional partner of spartin? If the consensus LIR motif is not required for its binding for neither LC3A nor LC3C, to prove a specific binding between spartin UBR and LC3A/C, a comprehensive mapping of the interaction or structural analysis that characterizing the interface between spartin UBR and LC3A/C is needed. A specific mutant of spartin disrupts its binding for LC3A/C is needed to be characterized and used to check the role of spartin in lipophagy. 9 5. The author developed an assay for lipophagy by treating cells with oleic acid first and then remove the oleic acid, it is important to determine whether spartin is generally required for lipophagy induced by other stresses, such as starvation.
Minor points: 1. Does spartin has an effect on lipid droplet biogenesis at basal condition? Although the authors describe that spartin knock out did not affect biogenesis by showing that there was no obvious difference of lipid droplet numbers between WT and KO cells after 30 min OA treatment in Extended Data Fig. 4, but lipid droplet numbers of WT and KO cells before OA treatment were not shown here. Indeed, the lipid droplet number was significantly increased in spartin KO cells compared to the WT cells in Extended Data Fig. 8. These data look counteract, given if spartin does not affect newly lipid droplet formation, after OA treatment, there should be more lipid droplets in KO cells because of the accumulated lipid droplets at basal condition. Otherwise, these data suggest that spartin knock out suppresses lipid droplet biogenesis. The authors need to double check and comment on this.
2. In Fig. 1a and b, the location of spartin under normal condition need to be shown as controls.
3. In Fig. 2a, the quantification data about ratio of spartin that colocalized with lysosomes under OA loading and OA withdraw conditions are needed. Does the colocalization increase upon lipophagy induction?
4. In Fig. 2d, f and g, it looks the binding affinity of GST-LC3A and spartin from cell lysate is much lower than that of GST-LC3A and purified spartin UBR, because 3 nM and 1 uM GST-LC3A protein were used for the pull-down experiments, respectively. Is this a typo? Or does it suggest that modifications of spartin in cells improve its binding with LC3A? 5. In Fig. 3b, images of WT and KO cells with or without OA loading need to be shown.

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Author Rebuttal to Initial comments
Point-by-point response to reviewers (NCB-LE48087):

Reviewer #1:
-Summary of the key results: This paper focuses on the function of spartin, a protein encoded by a gene mutated in hereditary spastic paraplegia. It confirms previous observations that spartin can localise to lipid droplets and endolysosomal organelles, but greatly extends these observations by i) defining a subset of lipid droplets that interact with spartin, ii) showing how spartin localises to lipid droplets (via amphipathic helices in the senescence domain), iii) showing that spartin is necessary for lipophagy of lipid droplets, iv) fleshing out the mechanism of this process (including employing a novel assay with Keima fusion proteins) to show that spartin links lipid droplets to the autophagy machinery via UBR-domain-mediated interactions with lc3 components, v) showing that this biology may be relevant to HSP by demonstrating that human neurons lacking spastin show lipid droplet accumulation, and that expression of dominant negative spartin in mouse cortex does the same.
-Originality and significance: This work is important and highly original. The existence of lipophagy has been known for some time, but the lipophagy receptors are not known -this work now provides strong evidence that spartin serves this purpose. In addition, the work may be clinically useful by suggesting mechanisms by which spartin disrupts neurons to cause spastic paraplegia.
-Data & methodology: The quality of the experimental work is a hallmark of this paper and the authors are to be congratulated on the robustness of their work. A particularly positive feature is that the authors have striven to show the endogenous relevance of their work at every opportunity, rather than relying on over-expression systems. The work is presented very nicely.
-Conclusions: Overall I think the conclusions of this paper are robust and reliable.
We thank this reviewer for his/her critical and helpful evaluation of our manuscript. In response to the reviewer's critique, our manuscript has undergone a major revision.
-Appropriate use of statistics and treatment of uncertainties: For the most part that statistics are appropriate, with one general exception. The authors use paired t-test in many instances where more than two conditions are being compared on a histogram. This is not correct -ANOVA with pairwise comparisons (and post-hoc testing for multiple testing) should be used.
We thank the reviewer for this comment and agree. We now provide ANOVA analyses and description for each histogram that has more than two conditions. Suggested improvements: The paper is already very convincing, but I have a few suggestions for possible improvements. I regard all of these suggestions as addressing relatively minor points: 1. A surprising facet of this paper is that the lysosomal receptor consisted of autophagy proteins, rather than ESCRT proteins, which would be the natural candidate in view of their endolysosomal localisation and known interaction with spartin. Are the authors sure that the MIT domain plays no role in this process? It would be helpful to nail this point by including MIT-domain deleted (or better F24 mutated) forms of spartin in the rescue experiments for the lysosome-lipid droplet co-localisation assays used in figure 2 (or perhaps using the Keimalivedrop system with rescues of the spartin KO phenotype).

We thank the reviewer for bringing up this important point. Following the suggestion from the reviewer, we tested the requirement of spartin's MIT domain lipophagy by analyzing co-localization between LAMP1 and a spartin mutant lacking the MIT domain. Our new results shown in Extended data Figs. 3c and 3d (new addition) reveal that the MIT domain is dispensable for spartin-mediated lipophagy.
2. The experiments showing interaction between spartin and LC3 components are convincing, but would be made even more convincing if shown with the endogenous proteins.
We agree with the reviewer and, although the interactions between LC3 and autophagy receptors are often somewhat weak, we, performed immunoprecipitation analysis between endogenous LC3 and endogenous spartin. In these experiments, now added in Extended data Fig. 2d (new addition), we observe co-precipitation specifically of spartin with LC3.
3. Extended figure 3c suggests that some modest interaction between spartin and LC3 is maintained with the construct that expresses only the senescence domain. Can the authors comment/explain, as this doesn't quite fit with the model of only the UBR being involved. (biochemistry?) This is an interesting question. The reviewer is correct that we detect some LC3 protein in this case, albeit much less than with the UBR domain present. We currently do not fully understand this result. It is possible that since the senescence domain still binds to LDs, a pulldown might immunoprecipitate some LDs that interact with LC3 via endogenous spartin.
To further test whether the senescence domain of spartin may function in recruiting LDs to lysosomes, we co-localized spartin and lysosomes in cells. As the reviewer can appreciate from the figure below, the senescence domain does not co-localize with lysosomes, consistent with the interpretation that it cannot function in recruiting LC3. Figure 3b, I think it would be helpful to carry out a control experiment adding bafilomycin to the WT OA withdrawal condition -this would validate that the color change is dependent on vacuolar ATPase activity (i.e. acidification of lysosomes). 5. While not strictly necessary (the human neuronal results are pretty convincing), the neuronal LD accumulation would be even more convincing if the authors could show LD accumulation in the brain of the published spartin knock out mouse. If this mouse is not easily available, I would not hold up publication of the important results of this paper.

In the Keima experiments in
We appreciate the reviewer's comment and we also wish to pursue this. With the generous support from Dr. Craig Blackstone who originally established the Spartin KO mouse line (Renvoisé et al, 2012), we are in the early stages of the mouse line recovery. Due to the time constraints and the requirement to age these mice for evaluation (LDs were found at age 3-6 months for Cravatt's lab DDHD2 knockout mice (Inloes et al, 2014), we are unable to include the mouse analyses in the current manuscript. (Eastman et al, JCB, 2009) suggested that spartin binds to the lipid droplet protein TIP47. Could the authors clarify whether TIP47 binding is involved in the mechanism of recruitment of spartin to LDs that they have described via amphipathic helices in the senescence domain, for example by depleting TIP47 and testing if spartin's recruitment to LDs is reduced? The Eastman study also highlighted the role of ubiquitin ligases, binding to spartin's PPXY motif, in regulating spartin's association with lipid droplets. Could the authors test whether the PPXY motif plays a role in regulating the lipophagy mechanism that they have described?

A previous study
Thank you for the interesting questions. In response, we performed additional experiments to clarify 1) the involvement of PLIN3/TIP47 in LD recruitment of Spartin and 2) the importance of the PPXY motif in lipophagy.
First, to test whether PLIN3/TIP47 is required for spartin localization, we knocked down PLIN3 in PLIN3/spartin double-knock-in cell lines. Spartin enrichment in LDs was not diminished by PLIN3 depletion, rather, there was a slight increase of spartin recruitment to LDs, suggesting that spartin and PLIN3 might compete to target to LD surface using their 3-11 amphipathic helices (Extended Data Fig.  1g, i, h; new addition). Fig. 3c and 3d (new addition), indicate that the PPAY motif is not required for spartinmediated lipophagy as assessed by lysosomal colocalization. Supporting this, we also found that spartin-mediated lipophagy is ubiquitination-independent (Extended Data Fig. 4b, c, d; new addition).

Second, concerning whether the PPAY motif is required for spartin-mediated lipophagy, we examined co-localization between LAMP1 and a spartin mutant lacking the PPAY motif. Our results, shown in Extended Data
7. References: Yes, although the authors might make it clearer that endogenous spartin had already been localised to LDs in reference 14 (Edwards et al) Thanks. We have made this clearer.

Reviewer #2:
Remarks to the Author: Lipophagy is one of selective autophagy and has an essential for lipid mobilization. However, the molecular mechanism regulating lipophagy is not well understood compared to other selective autophagy. Furthermore, the physiological relevance of lipophagy also remains largely elusive. In this study, Chung et al. found that the Troyer syndrome protein spartin as a novel regulator of lipophagy. They found that Spartin localizes on lipid droplets and interacts with autophagosomal proteins LC3A/C to serve as an autophagy receptor. Depletion of Spartin impaired the turnover of lipid droplet as revealed by Keima reporter assay and biochemical analysis of TG level. Moreover, motor neurons derived from spartin KO iPS cells and mouse brains injected with dominant negative spartin showed the accumulation of lipid droplets, suggesting that defect of lipophagy could contribute to Troyer syndrome development. Although the identification of a novel regulator of lipophagy contributes to understanding of lipophagy, the current study did not go beyond the functional analysis of spartin during the turnover of lipid droplets. Considering that spartin has been shown to be localized on lipid droplets (JCB, 2009), I personally doubt that it brings sufficient novelty and conceptual advance which are required for this journal and recommend a more specialized journal to submit. The lack of significance of findings in in vivo lipid metabolism also dampens the enthusiasm of this reviewer to support its publications. The followings are specific comments for authors.
We thank this reviewer for her/his critical and helpful evaluation of our manuscript. In response to the reviewer's critique, our manuscript has undergone a major revision.
We respectfully disagree that our paper does not bring sufficient novelty and conceptual advance for this journal. Previous studies (Eastman et al, 2009;Hooper et al, 2010) showed spartin can co-localize with lipid droplets but they did not demonstrate a functional requirement in lipophagy or show mechanistically how this occurs. Here we identify Spartin as a selective autophagy receptor for lipid droplets (i.e., the first clearly identified lipophagy receptor), we show how spartin mechanistically works in this process, and we demonstrate its functional requirement for TG turnover in cultured cells and neuronal lipid metabolism. Given the very limited mechanistic insights into LD autophagy currently available, the unclear function of spartin, and the emerging role of LDs in the brain (with pertinent links to neurodegenerative hereditary spastic paraplegias), we believe that this study constitutes an important contribution to lipid droplet cell biology and its application to medical questions.
Major points: 1. Liver and brown adipose tissue play a central role in lipid metabolism. Is spartin essential for lipophagy in liver or brown adipose tissue? Previous studies showed that lipophagy occurs in liver or brown adipose tissue; however, the authors performed most of the experiments using SUM159 breast cancer cells. It is to be determined whether spartin is required for hepatic or adipose lipophagy.
We thank the reviewer for these interesting questions. We use SUM159 as a cell culture model enabling rapid progress on mechanistic questions, but of course agree that it will be important to determine the physiological relevance of spartin-mediated lipophagy. Unfortunately, little is yet known about the mechanisms of LD autophagy in any tissue, including liver or brown adipose tissue. Since Spartin is prominently expressed in neurons and in since mutations in this protein lead to a neurological disease, spastic paraplegia, we elected to analyze its function there first. It will be interesting of course to determine whether spartin's role in lipophagy is neuron-specific or also present in other cell types and tissues. Yet, because this would require a series of long experiments in mice, finding the answer to this fascinating question is beyond the scope of this first report of spartin function in lipophagy.

Did mice expressing the DN mutant spartin in neurons show any defect in lipid metabolism and neuronal functions?
Yes, we detected neuronal LD accumulations and accumulation of triglyceride lipids in brain samples from mice expressing the DN mutant spartin. This is shown in Figures 5f, 5g, 5h, 5i. We agree it would be quite interesting to study a variety of neurological functions with spartin deficiency. However, due to the requirements of larger numbers of animals and the complexity of stereotaxic injections in the mouse motor cortex and uneven DN mutant overexpression, we are unfortunately not able to test neuronal functions adequately in this model. We plan to do such experiments in future experiments with knock-out animals when they become available in the next year.
3. It is critical to examine actual human SPART mutations impair lipophagy. human SPART mutations affects the localization of spartin on lipid droplets? Author should also check if SPART mutation constructs can rescue the LD turnover in iPS derived spartin KO human motor neuron.
We agree that this is an important and interesting question. Almost all pathogenic mutations in spartin are nonsense mutations that are predicted to result spartin lacking the senescence (lipid dropletbinding) domain. Thus, if pathogenic mutants are expressed, they would show similar behavior of Spartin (1-380) that we included in Figure 1f. To further clarify this point, we now also performed the localization analysis of the patient mutant (Spartin-1110delA). As expected, Spartin-1110delA does not localize to LDs (Extended Data Fig. 1k; new addition) 4. The in vivo experiment using the DN mutant is confusing. Although mouse brain is not incubated with fatty acids, the DN mutant spartin induced LD formation in neurons. Both LD formation and lipophagy constitutively occur in neurons? It is also conceivable that the mutant spartin promotes LD formation with unknown mechanisms. The authors need to clarify if the DN mutant inhibits lipophagy per se in neurons.
We appreciate the reviewer's concern and share the view that currently, LD biology in neurons is not well understood. Our data, including new experiments measuring the cellular TG biosynthesis rate in cells expressing the DN mutant spartin (Extended Data Fig. 9c) show that impairment of spartin function interferes with TG clearance, not TG synthesis for LD formation. Thus, we believe the most parsimonious explanation of our results for dominant-negative spartin is that there is some level of lipophagy in wildtype neurons, and when this is interfered with, TG-rich LDs accumulate. We note that although neurons do not normally exhibit many LDs at baseline, this appears to be due to a high turnover rate and resultant low pool size. There are now two studies, one by the Cravatt lab and ours that show that if TG clearance is interfered with, TGs and LDs accumulate. This suggests high flux through a small pool of TG.
5. Because lipophagy is one of cellular "degradation" systems, it is to be determined if spartin is required for LD degradation. The authors performed the radioisotope labeling assays to see TG degradation. To further examine this, the authors should show the detailed kinetics of LD degradation in spartin WT and KO cells using live imaging.
We appreciate the reviewer's suggestion. In response, we now included more detailed analyses of LD degradation rates in WT and spartin KO cells, showing delayed LD turnover in spartin deficiency (Figure 4g and 4h; new addition).
6. Does deletion of spartin impair lipolysis? The authors need to dissect the role of spartin in lipolysis and lipophagy.
We thank the reviewer for this important comment. To test a spartin function in lipolysis, we examined the LD degradation rate in the presence and absence of a lipase ATGL. The result previously shown in Figure 4j (Figure 3g in original submission) demonstrated that spartin does not affect lipolysismediated clearance of TG.
7. During lipophagy, ubiquitin or other ATG proteins are recruited onto LDs? The authors claim that the spartin-LC3 interaction mediates lipophagy. But, in other selective autophagy, the ubiquitinated substrates recruit autophagy receptors and ATG proteins. For example, an autophagy receptor NDP52 recruits the ULK1 complex during mitophagy.
We thank the reviewer for the comment and have performed additional experiments to investigate involvement of ubiquitination or other ATG proteins in spartin-mediated lipophagy. To test whether ubiquitination of spartin or other ubiquitinated LD proteins mediate lipophagy, we blocked the ubiquitination pathway using a small molecular inhibitor of the ubiquitin activating enzymes TAK-243 (Hyer et al, 2018). We verified successful inhibition of ubiquitination reactions (Extended Data Fig.   4b; new addition), and found that LD delivery (labeled by LiveDrop) to lysosomes was not largely affected by E1 inhibition (Extended Data Fig. 4c, d; new addition). We therefore think that ubiquitination does not play a major role in spartin-mediated autophagy. We have added this to the discussion.
Furthermore, to address an involvement of known selective autophagy receptors (SARs) in spartinmediated lipophagy, we knocked down three key SARs (NBR1, OPTN, and SQSTM1) and examined lipophagy flux using our Keima-LiveDrop reporter system. As now shown in Extended Data Fig. 4e, f (new addition), the lipophagy pathway was not affected by knockdown of these SARs.
Taken together, along with our data showing direct protein interactions with LDs and the autophagy machinery, these results suggest that spartin directly mediates lipophagy reaction as a lipophagy receptor protein.
8. Observation of conversion from LC-I to LC3-II is not sufficient to exclude the possible involvement of spartin in starvation induced bulk autophagy (Extended data. Fig.2). Authors also need to examine the actual autophagy flux with/without BafilomycinA1.
We thank the reviewer for this comment, agree and have performed additional experiments using a Keima-LC3B autophagy reporter system. New results show that spartin appears not to be involved in bulk autophagy (Extended Data Fig. 5b; new addition).
9. Is spartin only involved in lipophagy? It is worth checking the role of spartin in other selective autophagy such as mitophagy and aggrephagy. This is an intriguing idea, and we thank the reviewer for suggesting this interesting possibility. Spartin appears not to be involved in bulk autophagy, and we were not yet able to systematically test a function for spartin in other selective autophagy pathways but will examine this in future studies. Fig.1f, Fig.2e, Fig. 3b is missing.

The quantification data in
We thank the reviewer for the comment, apologize for the oversight and have added the quantification data of Fig. 1f (added on the bottom of panel), Fig. 2e (Figure 3h), and Fig. 3b (Figure 4c).

Minor
1. Some data lack controls. The following controls are to be included: 30min OA in Fig. 1c; Before the OA withdrawal in Fig. 2a; WT cells in Fig. 2h; Before the OA withdrawal in Fig. 3c.
We appreciate this comment. All requested controls were added in the new figures: 30 min OA in Fig.  1c (original Extended Data Fig. 1d); Before the OA withdrawal in Fig. 2a (Extended Data Fig. 2c and Fig.  2b); WT cells in Fig. 2h (Figure 2f top panel); Before the OA withdrawal in Fig. 3c (Figure 4d). Fig.4f, the label of bottom low is missing (probably Spartin-FL?).

In
Thank you for catching this! We corrected the label in new Figure 5f (original Figure 4f).

Reviewer #3:
Remarks to the Author: In this manuscript, Chung et al describe a role for the Troyer syndrome protein spartin as a lipophagy receptor that mediates the turnover of lipid droplets. Spartin is demonstrated by previous studies to target to lipid droplets, the authors show the C-terminal amphipathic helices were required for its recruitment to lipid droplets. They further demonstrated that show spartin interacts with ATG8 family protein LC3A, potentially through its ubiquitin-binding region (UBR). Knock out of spartin causes deficient lipid droplet turnover in cultured cells. They further show impairing spartin function by overexpression of a dominant negative spartin fragment leads to compromised lipophagy and TG accumulation in cultured human neurons and murine brain neurons. Overall, this study would be of interest for the readership as it would highlight a potential new lipophagy receptor. However, the conclusion of spartin as a lipophagy receptor is not fully supported by the data, more key evidences are needed to clarify the role of spartin in lipophagy.
We thank this reviewer for his/her critical and helpful evaluation of our manuscript. In response to the reviewer's critique, our manuscript has undergone a major revision.
Major points: 1. The authors investigated the function of spartin in lipophagy mainly by spartin knockout or by overexpressing a dominant-negative fragment that with a large truncation of its N-terminal MIT domain and the UBR. It is hard to conclude that spartin acts as a cargo receptor based on the effects caused by either fully knockout or such a big truncation. Specific mutants without disrupting the global domain conformation that block its localization on lipid droplets and its interaction with ATG8 family proteins respectively, are needed to determine the role of spartin in the turnover of lipid droplets.
We thank the reviewer for this comment and agree. We added a substantial amount of new data that have strengthened our conclusions. Specifically, we added data to show that a mutation that disrupts Spartin binding to LC3 impairs lipophagy in our model systems.
For this, we determined the motif in spartin responsible for LD targeting. Initially, we narrowed down the spartin region required for LD binding (original Figure 1f). Once we identified the repeats of 3-11 amphipathic helices in the senescence domain, we identified point mutations of hydrophobic residues changed to alanine in 3-11 helices in the context of full-length spartin (current Figure 1h and 1i). The results indicate the importance of the amphipathic helices in mediating LD binding. (Figure 3g and 3h). In addition, a recombinant UBR-ΔLIR impaired the interaction of LC3A and spartin ( Figure 3F).

We now also map a LIR motif within UBR domain. Deletion of the LIR motif (residue 193-200) in fulllength spartin abolished co-localization of spartin and LC3a
2. Spartin was shown to interact with the Nedd4-family E3 ligase AIP4 and promote the ubiquitination of adipophilin on lipid droplets (Eastman SW, 2009;Hooper C, 2010). Given the authors analyzed the function of spartin in lipophagy mostly using spartin knock out cells, one possibility is that spartin is mainly involved in the ubiquitination of proteins on the lipid-droplet membrane, and the ubiquitinated proteins recruit the SAR family receptors such as p62, NBR1 that further mediate the engulfment of lipid droplet by autophagy. And actually, it was shown in macrophage lipid droplets are decorated with ubiquitin, and SARs including p62, OPTN and NBR could target to ubiquitinated lipid droplets (Karlsson AB, 2014; Robichaud S, 2021). The authors should test whether lipid droplets are ubiquitinated in the neuron system, and test the function of SAR receptors in spartin-mediated lipophagy. The authors need to clarify spartin functions directly as a receptor itself or indirectly by recruiting the SAR receptors. Fig. 4b; Fig. 4c, d; new addition).

new addition), did not impair LD delivery (labeled by LiveDrop) to lysosomes (Extended Data
To address an involvement of selective autophagy receptors (SARs) in spartin-mediated lipophagy, we knocked down three key SARs (NBR1, OPTN, and SQSTM1) and examined lipophagy flux using Keima-LiveDrop reporter system. As shown in Extended Data Fig. 4e, f (new addition), lipophagy was not affected by knockdown of these SARs.
We appreciate the suggestion to examine ubiquitination of LDs in the nervous system. However, we no longer have the fractions to analyze this. Additionally, since our new data suggest ubiquitination is not necessary for spartin-mediated lipophagy, we feel the results will not change the major conclusion and don't warrant the additional months of experiments.
3. One major evidence shown by the authors that supporting spartin acts as a lipophagy receptor is the interaction between spartin and ATG8 family members LC3A and LC3C. They claimed that the binding between spartin and LC3A is mediated through spartin UBR domain, but not a conventional LC3 interaction motif (LIR). The authors should show the data that the LIR is not required for its binding to LC3A in the manuscript. Have the authors checked whether the LIR is required for its binding with LC3C? Do the authors have particular reasons to pick LC3A? Would it be possible spartin binds to LC3C via the LIR and thus LC3C is the functional partner of spartin?
If the consensus LIR motif is not required for its binding for neither LC3A nor LC3C, to prove a specific binding between spartin UBR and LC3A/C, a comprehensive mapping of the interaction or structural analysis that characterizing the interface between spartin UBR and LC3A/C is needed. A specific mutant of spartin disrupts its binding for LC3A/C is needed to be characterized and used to check the role of spartin in lipophagy. Figure 3b). These data have been added to the manuscript.

4.
To further verify spartin serves as a lipophagy receptor, it is essential to determine the lysosomal turnover of spartin protein, as the cargo receptors are engulfed by the autophagosome and delivered to lysosomes. Although the authors designed a Keima-Spartin reporter, it is important to show the protein degradation by western blot, and show that the spartin degradation is mediated by the autophagy-lysosome pathway. Besides, time course images of Keima-Spartin before OA loading, OA loading and OA withdraw need to be shown to prove the turnover of spartin.
We thank the reviewer for bringing up this important point, which we addressed experimentally. First, we tested degradation of spartin by autophagy using western blot analyses (Data Extended Fig. 4a). We used Baf A1 treatment and ATG7 KO as controls to validate selective degradation of spartin and find that spartin is indeed degraded by a lysosome-dependent process. Second, we monitored Keima-Spartin during a time course and representative images of 'before OA loading', 'OA loading', and 'OA withdrawal' are now included in new Figure 4d. Fig. 6 (new addition), spartin mediates lipophagy in various metabolic stresses, but lipid deficiency actives spartin-mediated lipophagy most. This suggests a possibility of distinct cellular mechanism activating lipophagy apart from bulk autophagy.

We agree with the reviewer and, in response, performed Keima-Spartin flux measurement to address which cellular stresses requires spartin-mediated lipophagy activity. As shown in Extended Data
Minor points: 1. Does spartin has an effect on lipid droplet biogenesis at basal condition? Although the authors describe that spartin knock out did not affect biogenesis by showing that there was no obvious difference of lipid droplet numbers between WT and KO cells after 30 min OA treatment in Extended Data Fig. 4, but lipid droplet numbers of WT and KO cells before OA treatment were not shown here. Indeed, the lipid droplet number was significantly increased in spartin KO cells compared to the WT cells in Extended Data Fig. 8. These data look counteract, given if spartin does not affect newly lipid droplet formation, after OA treatment, there should be more lipid droplets in KO cells because of the accumulated lipid droplets at basal condition. Otherwise, these data suggest that spartin knock out suppresses lipid droplet biogenesis. The authors need to double check and comment on this. Fig. 5b since we are only counting the number of LDs. Fig. 1a and b, the location of spartin under normal condition need to be shown as controls. Fig. 1d (new addition). Fig. 2a, the quantification data about ratio of spartin that colocalized with lysosomes under OA loading and OA withdraw conditions are needed. Does the colocalization increase upon lipophagy induction? Fig. 2c and Fig. 2b (new addition). We observed that association between spartin and lysosomes increased upon lipophagy induction. Fig. 2d, f and g, it looks the binding affinity of GST-LC3A and spartin from cell lysate is much lower than that of GST-LC3A and purified spartin UBR, because 3 nM and 1 uM GST-LC3A protein were used for the pulldown experiments, respectively. Is this a typo? Or does it suggest that modifications of spartin in cells improve its binding with LC3A?

In
Thank you for the comment, but we think that binding affinity in two conditions are difficult to compare since we cannot estimate the concentration of spartin in our 'cell lysate' experiments. It is also possible that modifications of spartin in cells affects binding, as suggested. To address this will require more, future investigation.

Decision Letter, first revision:
Dear Tobi, Thank you for submitting your revised manuscript "The Troyer syndrome protein spartin mediates selective autophagy of lipid droplets" to the journal. It has now been seen by the original reviewers, whose comments are pasted below. In light of their advice, we regret that we cannot offer to publish the study in Nature Cell Biology.
As you will see, the reviewers appreciated the significant revisions to bolster the core conclusions linking spartin to lipophagy. While the reviewers continued to find this work interesting, and although Reviewer #1 remains especially positive (and raises points that could likely be addressed in a straightforward manner), Reviewers #2 and #3 have overlapping, persisting concerns that the key claim of showing that spartin acts as a lipophagy receptor has not been sufficiently demonstrated. We have discussed the points from these reviewers in depth editorially and find them significant. Importantly, we are not sure that these points would be straightforward to address in a final minor revision. As we make every effort to limit all papers to one round of major experimental revision, we must regrettably return the manuscript to you.
We are very sorry that we could not be more positive on this occasion, but we thank you for the opportunity to consider this work.

With kind regards, Melina
Melina Casadio, PhD Senior Editor, Nature Cell Biology ORCID ID: https://orcid.org/0000-0003-2389-2243 Reviewers' comments: Reviewer #1 (Remarks to the Author): Thank you to the authors for addressing my comments so diligently. All of my previous remarks have been dealt with in a satisfactory way. In my initial review I said: "This work is important and highly original. The existence of lipophagy has been known for some time, but the lipophagy receptors are not known -this work now provides strong evidence that spartin serves this purpose. In addition, the work may be clinically useful by suggesting mechanisms by which spartin disrupts neurons to cause spastic paraplegia" ....and my enthusiasm for this work has been enhanced by the revisions that have been made. I find the story convincing, novel and a significant advance for the field.
I have two very minor remaining comments and one more significant one: 1. Between lines 90 and 95 the text on the Troyer syndrome-related frameshift mutation are somewhat misleading. Such mutations are almost certainly subject to nonsense mediated decay (Craig Blackstone published a paper validating this) and so it is likely that no protein is produced. I think this should be made clear to avoid misunderstanding. 2.In extended data figure 2d, tubulin is spelled incorrectly throughout. 3. The more significant comment relates to the unit of repeat used for "n" values in the presentation and stats for the microscopy experiments (and many apologies for not picking this up on my first review and so introducing this at a late stage). The "n" used is "number of fields". While this is an improvement on the "n=number of cells" that is sometimes (and almost always incorrectly) used , it does raise the question of whether these can be considered true biological repeats. While I find the results convincing as they are demonstrated through multiple orthogonal approaches, it would be comforting to know for the key experiments that the data arose from more than one (preferably at least 3) experiments, with the "n" used being number of biological repeats. This is perhaps a philosophical question for the journal editor to decide on their preferred approach.
Reviewer #2 (Remarks to the Author): The authors responded to part of my concerns I raised for the original manuscript in a satisfactory manner. However, there remains some critical points that should be resolved for accepting publication of the manuscript in the NCB.
Regarding comment #1, in most main figures (from Fig. 1 to Fig. 4), the authors used SUM159 as a cell culture model to show the mechanism by which Spartin acts as a lipophagy receptor. While many previous studies demonstrated that lipophagy occurs in liver or brown adipocytes (R Singh et al., Nature 2009;N Martinez-Lopez et al., Cell Metab 2016;MB Schott et al., JCB 2019), there is no report showing that lipophagy can occur in neurons. Therefore, to show the physiological relevance of these findings, the authors should determine if Spartin is required for lipophagy in liver or brown adipocytes. Or, the authors must show that lipophagy occurs also in neurons. As noted in comment #4, there is no evidence showing that Spartin-DN inhibits lipophagy, even in SUM159 cells. Because of the lack of this evidence, it is still undetermined if the LD accumulation by the DN mutant is due to loss of lipophagy, i.e., we still don't know if lipophagy occurs in neurons or not.

Reviewer #3 (Remarks to the Author):
The authors have addressed or responded to most of my comments raised in the previous round of review.
However, the authors did not pick up from my previous review was that 'A specific mutant of spartin disrupts its binding for LC3 is needed to be used to check the role of spartin in lipophagy'. It was nice that the authors characterized a LIR mutant disrupted spartin binding to LC3, but they did not provide evidences that this mutant would impair lipophagy. It would be essential to check whether LIR mutant could rescue LD turnover in spartin KO cells. Such evidence is needed to support spartin acts as a lipophagy receptor.
Minor point: Extended data Fig.2c was not showing the quantification data about ratio of spartin that colocalized with lysosomes as indicated by the authors. **Although we cannot publish your paper, it may be appropriate for another journal in the Nature Portfolio. If you wish to explore the journals and transfer your manuscript please use our manuscript transfer portal. You will not have to re-supply manuscript metadata and files, but please note that this link can only be used once and remains active until used. For more information, please see our manuscript transfer FAQ page.
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Reviewer #1:
Thank you to the authors for addressing my comments so diligently. All of my previous remarks have been dealt with in a satisfactory way. In my initial review I said: "This work is important and highly original. The existence of lipophagy has been known for some time, but the lipophagy receptors are not known -this work now provides strong evidence that spartin serves this purpose. In addition, the work may be clinically useful by suggesting mechanisms by which spartin disrupts neurons to cause spastic paraplegia" ....and my enthusiasm for this work has been enhanced by the revisions that have been made. I find the story convincing, novel and a significant advance for the field.
We thank this reviewer for his/her critical and positive evaluation of our manuscript.
I have two very minor remaining comments and one more significant one: 1. Between lines 90 and 95 the text on the Troyer syndrome-related frameshift mutation are somewhat misleading. Such mutations are almost certainly subject to nonsense mediated decay (Craig Blackstone published a paper validating this) and so it is likely that no protein is produced. I think this should be made clear to avoid misunderstanding.
We thank the reviewer for this comment and agree. We now added further explanation about 1110delA mutant and a new reference to the manuscript.

In extended data figure 2d, tubulin is spelled incorrectly throughout.
Thank you for catching up the misspelling. We have corrected the error.
3. The more significant comment relates to the unit of repeat used for "n" values in the presentation and stats for the microscopy experiments (and many apologies for not picking this up on my first review and so introducing this at a late stage). The "n" used is "number of fields". While this is an improvement on the "n=number of cells" that is sometimes (and almost always incorrectly) used, it does raise the question of whether these can be considered true biological repeats. While I find the results convincing as they are demonstrated through multiple orthogonal approaches, it would be comforting to know for the key experiments that the data arose from more than one (preferably at least 3) experiments, with the "n" used being number of biological repeats. This is perhaps a philosophical question for the journal editor to decide on their preferred approach.
We thank the reviewer for bringing up this important point. The key microscopy experiments showed in the manuscript were repeated at least three times to ensure reproducibility. To clarify, we now state the number of biological replicates used for the quantification of each experiment in the figure legend.

Decision Letter, second revision:
Dear Tobi, Thank you for submitting your appeal and revised manuscript, "The Troyer syndrome protein spartin mediates selective autophagy of lipid droplets", and thank you very much for your patience with the re-review process. I sincerely apologize for the very long delay.
We have editorially assessed your responses to Rev#1's points and found them very helpful to resolve this referee's final points.
Unfortunately, Rev#3 was not available to re-review the study. As we were interested in expert input to evaluate the LIR-mutant data, and given that this falls within the expertise of our general selective autophagy expert Rev#2, we asked Rev#2 (who agreed to re-review) to evaluate those data.
As you will see from their comments (attached below), they found the new data provided in response to Reviewer #3 fully satisfactory and convincing. Rev#2 also shared their view on a final experiment needed for publication, which we believe should be addressed before we can consider publication in Nature Cell Biology.
We discussed the reviewer's request in great detail editorially. Reviewer #2 feels that it's important to establish that the spartin-DN mutant blocks lipophagy using the lipophagy reporter in SUM159 cellswhich would elegantly tie in the in vivo data using the mutant and the in vitro studies establishing that spartin controls lipophagy in this cell model. This comment relates to Rev#2's longstanding question as to whether there is enough evidence supporting the concept that lipophagy occurs in neurons, and whether the effects of spartin in vivo in Figure 5 are therefore dependent on lipophagy. This is not an issue that is new to this round of review.
As mentioned, we have been discussing the need for this experiment editorially, and we strongly recommend carrying out the experiment suggested by Rev#2 in SUM159 cells. In the event that you are unable to provide these data, please do let me know and we'll be happy to discuss, as always.
As typically, in revision, please also pay close attention to our guidelines on statistical and methodological reporting (listed below), as failure to do so may delay the reconsideration of the revised manuscript. In particular, please provide: -a Supplementary Figure including unprocessed images of all gels/blots in the form of a multi-page pdf file. Please ensure that blots/gels are labeled and the sections presented in the figures are clearly indicated.
-a Supplementary Table including all numerical source data in Excel format, with data for different figures provided as different sheets within a single Excel file. The file should include source data giving rise to graphical representations and statistical descriptions in the paper and for all instances where the figures present representative experiments of multiple independent repeats, the source data of all repeats should be provided.
We therefore invite you to take these points into account when revising the manuscript. In addition, when preparing the revision please: -ensure that it conforms to our format instructions and publication policies (see below and www.nature.com/nature/authors/).
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I apologize once again for the long delay in communicating our decision to you. We hope that you will find our referees' comments and editorial guidance helpful. Please do not hesitate to contact me if there is anything you would like to discuss.
Best wishes,

Melina
Melina Casadio, PhD Senior Editor, Nature Cell Biology ORCID ID: https://orcid.org/0000-0003-2389-2243 Reviewers' Comments: Reviewer #2: Remarks to the Author: This reviewer is convinced with most of the data shown in the revised manuscript. But, there still remains one point to needs be evaluated. The authors should show lipophagy occurs in neurons. Because a lipophagy receptor is previously undetermined, the two publications (PMCID 21803288; PMCID 33713908) lack the actual evidence showing that lipophagy (LD-specific autophagy) can occur in neurons. Therefore, the authors should determine if Spartin-DN causes LD accumulation via inhibiting lipophagy in neurons. To do this, the authors can express Spartin-DN in SUM159 cells to easily see whether the mutant inhibits lipophagy in a Keima-LiveDrop system. In the manuscript, the authors just showed Spartin-DN caused LD accumulation, which does not directly indicate that Spartin-DN inhibits lipophagy. Once the paper is published, researchers must use Spartin-DN as a lipophagy inhibitory factor. That's why the authors need to examine if the mutant truly inhibits lipophagy.
COMMENTS ABOUT THE RESPONSES TO REV#3'S POINTS: I have read the response to reviewer #3 and agree that the authors have adequately addressed the concerns raised by reviewer #3 in this round. The authors showed that lipophagic activity was lost when spartin lacks its LD localization or its binding to LC3 family proteins. I think reviewer #3 would be convinced. I am convinced that spartin acts as a lipophagy receptor. The only thing the authors need to do is to test whether the dominant negative mutant suppresses lipophagy in the Keima LiveDrop system.

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Decision Letter, Third Revision:
18th April 2023 Dear Tobi, Thank you very much for submitting your revised manuscript "The Troyer syndrome protein spartin mediates selective autophagy of lipid droplets" (NCB-LE48087C). We have editorially evaluated the final revision and greatly appreciated your efforts to provide evidence that the DN spartin mutant, in cells, is associated with reduced levels of Keima-LiveDrop and accumulation of LDs. Analyses of the mutant's association with acidic compartments (using Keima-Spartin constructs) and its interference with association of LAMP1-labled endolysosomes and lipidTOX-stained LDs are also important new pieces of data. Therefore, we'll be happy in principle to publish the study in Nature Cell Biology, pending minor revisions to comply with our editorial and formatting guidelines.
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