Small-molecule Activation of TFEB Alleviates Niemann-Pick Disease Type C via Promoting Lysosomal Exocytosis and Biogenesis

Reviewer #1 (Public review):

Summary:

The authors are trying to determine if SFN treatment results in dephosphorylation of TFEB, subsequent activation of autophagy-related genes, exocytosis of lysosomes, and reduction in lysosomal cholesterol levels in models of NPC disease.

Strengths:

(1) Clear evidence that SFN results in translocation of TFEB to the nucleus.

(2) In vivo data demonstrating that SFN can rescue Purkinje neuron number and weight in NPC1-/- animals.

Weaknesses:

(1) Lack of molecular details regarding how SFN results in dephosphorylation of TFEB leading to activation of the aforementioned pathways. Currently, datasets represent correlations.

(2) Based on the manuscript narrative, discussion, and data it is unclear exactly how steady-state cholesterol would change in models of NPC disease following SFN treatment. Yes, there is good evidence that lysosomal flux to (and presumably across) the plasma membrane increases with SFN. However, lysosomal biogenesis genes also seem to be increasing. Given that NPC inhibition, NPC1 knockout, or NPC1 disease mutations are constitutively present and the cell models of NPC disease contain lysosomes (even with SFN) how could a simple increase in lysosomal flux decrease cholesterol levels? It would seem important to quantify the number of lysosomes per cell in each condition to begin to disentangle differences in steady state number of lysosomes, number of new lysosomes, and number of lysosomes being exocytosed.

(3) Lack of evidence supporting the authors' premise that "SFN could be a good therapeutic candidate for neuropathology in NPC disease".

Reviewer #2 (Public review):

Summary:

This study presents a valuable finding that the activation of TFEB by sulforaphane (SFN) could promote lysosomal exocytosis and biogenesis in NPC, suggesting a potential mechanism by SFN for the removal of cholesterol accumulation, which may contribute to the development of new therapeutic approaches for NPC treatment.

Strengths:

The cell-based assays are convincing, utilizing appropriate and validated methodologies to support the conclusion that SFN facilitates the removal of lysosomal cholesterol via TFEB activation.

Weaknesses:

(1) The in vivo experiments demonstrate the therapeutic potential of SFN for NPC. A clear dose-response analysis would further strengthen the proposed therapeutic mechanism of SFN. Additional data supporting the activation of TFEB by SFN for cholesterol clearance in vivo would strengthen the overall impact of the study

(2) In Figure 4, the authors demonstrate increased lysosomal exocytosis and biogenesis by SFN in NPC cells. Including a TFEB-KO/KD in this assay would provide additional validation of whether these effects are TFEB-dependent.

(3) For lysosomal pH measurement, the combination of pHrodo-dex and CF-dex enables ratiometric pH measurement. However, the pKa of pHrodo red-dex (according to Invitrogen) is ~6.8, while lysosomal pH is typically around 4.7. This discrepancy may account for the lack of observed lysosomal pH changes between WT and U18666A-treated cells. Notably, previous studies (PMID: 28742019) have reported an increase in lysosomal pH in U18666A-treated cells.

(4) The authors are also encouraged to perform colocalization studies between CF-dex and a lysosomal marker, as some researchers may be concerned that NPC1 deficiency could reduce or block the trafficking of dextran along endocytosis.

(5) In vivo data supporting the activation of TFEB by SFN for cholesterol clearance would significantly enhance the impact of the study. For example, measuring whole-animal or brain cholesterol levels would provide stronger evidence of SFN's therapeutic potential.

Reviewer #3 (Public review):

Summary:

The authors demonstrate that activation of TFEB facilitates cholesterol clearance in cell models of Niemann-Pick type C (NPC). This is done through a variety of approaches including activation of TFEB by sulforaphane (SFN), a naturally occurring small-molecule TFEB agonist. SFN induces TFEB nuclear translocation and promotes lysosomal exocytosis. In an NPC mouse model, SFN dephosphorylates/activates TFEB in the brain and rescues the loss of Purkinje cells.

Strengths:

NPC is a severe disease and there is little in the way of treatment. The manuscript points towards some treatment options. However, the title, the title "Small-molecule activation of TFEB Alleviates Niemann-Pick Disease..." is far too strong and should be changed.

Weaknesses:

(1) The manuscript is extremely hard to read due to the writing; it needs careful editing for grammar and English.

(2) There are a number of important technical issues that need to be addressed.

(3) The TFEB influence on filipin staining in Figure 1A is somewhat subtle. In the mCherry alone panels there is a transfected cell with no filipin staining and the mCherry-TFEBS211A cells still show some filipin staining.

(4) Figure 1C is impressive for the upregulation of filipin with U18666A treatment. However, SFN is used at 15 microM. This must be hitting multiple pathways. Vauzour et al (PMID: 20166144) use SFN at 10 nM to 1microM. Other manuscripts use it in the low microM range. The authors should repeat at least some key experiments using SFN at a range of concentrations from perhaps 100 nM to 5 microM. The use of 15 microM throughout is an overall concern.

Author Response:

Thank you for your interest in our paper. We would also like to thank the anonymous reviewers for their critical and constructive comments. Although the reviewers found our work interesting, they raised several important concerns about our study. To address these concerns, mostly we will perform new experiments as following.

1. Examine whether antioxidant-NAC can block SFN-induced TFEB-nuclear translocation in NPC cells;

2. Examine whether calcineurin inhibitor (FK506+CsA) or Ca 2+ inhibitor (Bapta-AM) can block SFN-induced TFEB-nuclear translocation in NPC cells.

3. Investigate whether cholesterol was cleared by activation of TFEB by SFN in vivo tissues.

4. Investigate whether SFN-evoked the lysosomal exocytosis is TFEB-dependent by using TFEB-KO cells.

5. Examine the effect of NPC1 deficiency on dextran trafficking by studying the localization of CF- dex and Lamp1.

6. Perform cytotoxicity experiments to examine whether SFN used in this study is cytotoxic in various cell lines

In addition, according to the reviewers’ suggestions, we will make clarifications and corrections wherever appropriate in the manuscript. Below please find our point-by-point responses and plans to the reviewers’ comments.

Reviewer #1 (Public review):

Summary:

The authors are trying to determine if SFN treatment results in dephosphorylation of TFEB, subsequent activation of autophagy-related genes, exocytosis of lysosomes, and reduction in lysosomal cholesterol levels in models of NPC disease.

Strengths:

(1) Clear evidence that SFN results in translocation of TFEB to the nucleus.

(2) In vivo data demonstrating that SFN can rescue Purkinje neuron number and weight in NPC1-/- animals.

Thank you for the support!

Weaknesses:

(1) Lack of molecular details regarding how SFN results in dephosphorylation of TFEB leading to activation of the aforementioned pathways. Currently, datasets represent correlations.

Thank you for this constructive comment. The reviewer is right that in this manuscript the molecular mechanism of SFN-activated TFEB has not been discussed in details. Because previously we have shown that SFN induces TFEB nuclear translocation via a Ca 2+ - dependent but MTOR (mechanistic target of rapamycin kinase)-independent mechanism through a moderate increase in reactive oxygen species (ROS). And calcineurin-mediated TFEB dephosphorylation underlies SFN-induced TFEB activation. These data have been published in 2021 autophagy (Li, Shao et al. 2021) . Therefore, in this study we did not mention this part. We will add the molecular mechanism of TFEB activation by SFN in the discussion part. And to further confirm this mechanism in NPC cells, we will also perform experiments including: 1) examine whether antioxidant-NAC can block SFN-induced TFEB-nuclear translocation in NPC cells; 2) examine whether calcineurin inhibitor (FK506+CsA) can block SFN-induced TFEB-nuclear translocation in NPC cells.

(2) Based on the manuscript narrative, discussion, and data it is unclear exactly how steady-state cholesterol would change in models of NPC disease following SFN treatment. Yes, there is good evidence that lysosomal flux to (and presumably across) the plasma membrane increases with SFN. However, lysosomal biogenesis genes also seem to be increasing. Given that NPC inhibition, NPC1 knockout, or NPC1 disease mutations are constitutively present and the cell models of NPC disease contain lysosomes (even with SFN) how could a simple increase in lysosomal flux decrease cholesterol levels? It would seem important to quantify the number of lysosomes per cell in each condition to begin to disentangle differences in steady state number of lysosomes, number of new lysosomes, and number of lysosomes being exocytosed.

Thank you for the suggestion. It is important to define the three states 1) original number of lysosomes, 2) number of new lysosomes, and 3) number of lysosomes being exocytosis. However, we have checked literature, so far it seems that there is no good method that could clearly differentiate the three states of lysosomes.

(3) Lack of evidence supporting the authors' premise that "SFN could be a good therapeutic candidate for neuropathology in NPC disease".

Suggestion was taken! We will investigate whether cholesterol was reduced by activation of TFEB by SFN in vivo to strength the point that SFN could be a potential therapeutic compound for NPC treatment. And to avoid confusion, we have removed this sentence.

Reviewer #2 (Public review):

Summary:

This study presents a valuable finding that the activation of TFEB by sulforaphane (SFN) could promote lysosomal exocytosis and biogenesis in NPC, suggesting a potential mechanism by SFN for the removal of cholesterol accumulation, which may contribute to the development of new therapeutic approaches for NPC treatment.

Strengths:

The cell-based assays are convincing, utilizing appropriate and validated methodologies to support the conclusion that SFN facilitates the removal of lysosomal cholesterol via TFEB activation.

Weaknesses:

(1) The in vivo experiments demonstrate the therapeutic potential of SFN for NPC. A clear dose-response analysis would further strengthen the proposed therapeutic mechanism of SFN. Additional data supporting the activation of TFEB by SFN for cholesterol clearance in vivo would strengthen the overall impact of the study

We understand the reviewer’s point. We examined two doses of SFN-30 and 50mg/kg. As shown in Fig.6, SFN (50mg/kg), but not 30mg/kg prevents a degree of Purkinje cell loss in the lobule IV/V of cerebellum, suggesting a dose-correlated preventive effect of SFN. In vivo experiments with higher concentrations of SFN and optimized dosage form of SFN were planned in the future study, but will not be included in this study.

We will investigate whether cholesterol was cleared by activation of TFEB by SFN in vivo.

(2) In Figure 4, the authors demonstrate increased lysosomal exocytosis and biogenesis by SFN in NPC cells. Including a TFEB-KO/KD in this assay would provide additional validation of whether these effects are TFEB-dependent.

Thank you for this valuable suggestion. We will investigate whether SFN-evoked the lysosomal exocytosis is TFEB-dependent by using TFEB-KO cells.

(3) For lysosomal pH measurement, the combination of pHrodo-dex and CF-dex enables ratiometric pH measurement. However, the pKa of pHrodo red-dex (according to Invitrogen) is ~6.8, while lysosomal pH is typically around 4.7. This discrepancy may account for the lack of observed lysosomal pH changes between WT and U18666A-treated cells. Notably, previous studies (PMID: 28742019) have reported an increase in lysosomal pH in U18666A-treated cells.

We understand the reviewer’s point. But we used pHrodo™ Green-Dextran (P35368, Invitrogen), but not pHrodo red-dex to measure the lysosomal luminal acidity. According to the product information from Invitrogen, pHrodo Green-dex conjugates are non-fluorescent at neural pH, but fluorescence bright green at acidic pH ranges 4-9, such as those in endosomes and lysosomes. Therefore, pHrodo Green-dex can be used to monitor the acidity of lysosome (Hu, Li et al. 2022) . We also used LysoTracker Red DND-99 (Thermo Scientific, L7528) to measure lysosomal pH (Fig. 4G, H), which is consistent with results of pHrodo Green/CF measurement. Overall, in our hands, we have not detected pH change of lysosomes in U18666A-treated NPC1 cell models.

(4) The authors are also encouraged to perform colocalization studies between CF-dex and a lysosomal marker, as some researchers may be concerned that NPC1 deficiency could reduce or block the trafficking of dextran along endocytosis.

Suggestion was taken! We will examine the effect of NPC1 deficiency on dextran trafficking by studying the localization of CF-dex and Lamp1.

(5) In vivo data supporting the activation of TFEB by SFN for cholesterol clearance would significantly enhance the impact of the study. For example, measuring whole-animal or brain cholesterol levels would provide stronger evidence of SFN's therapeutic potential.

We really appreciate the reviewer’s suggestions. We will investigate whether cholesterol was cleared by activation of TFEB by SFN in vivo.

Reviewer #3 (Public review):

Summary:

The authors demonstrate that activation of TFEB facilitates cholesterol clearance in cell models of Niemann-Pick type C (NPC). This is done through a variety of approaches including activation of TFEB by sulforaphane (SFN), a naturally occurring small-molecule TFEB agonist. SFN induces TFEB nuclear translocation and promotes lysosomal exocytosis. In an NPC mouse model, SFN dephosphorylates/activates TFEB in the brain and rescues the loss of Purkinje cells.

Strengths:

NPC is a severe disease and there is little in the way of treatment. The manuscript points towards some treatment options. However, the title, the title "Small-molecule activation of TFEB Alleviates Niemann-Pick Disease..." is far too strong and should be changed.

Weaknesses:

(1) The manuscript is extremely hard to read due to the writing; it needs careful editing for grammar and English.

We will thoroughly check grammar to improve the manuscript.

(2) There are a number of important technical issues that need to be addressed.

We will address the technical issues mentioned in the following.

(3) The TFEB influence on filipin staining in Figure 1A is somewhat subtle. In the mCherry alone panels there is a transfected cell with no filipin staining and the mCherry-TFEBS211A cells still show some filipin staining.

We understand the reviewer’s point. We will investigate whether cholesterol is cleared by activation of TFEB by SFN in vivo.

(4) Figure 1C is impressive for the upregulation of filipin with U18666A treatment. However, SFN is used at 15 microM. This must be hitting multiple pathways. Vauzour et al (PMID: 20166144) use SFN at 10 nM to 1microM. Other manuscripts use it in the low microM range. The authors should repeat at least some key experiments using SFN at a range of concentrations from perhaps 100 nM to 5 microM. The use of 15 microM throughout is an overall concern.

We understand the reviewer’s point. See RESPONSE #1, previously we have shown that SFN (10–15 μM, 2–9 h) induces robust TFEB nuclear translocation in a dose- and time-dependent manner in HeLa GFP-TFEB stable cells as well as in other human cell lines without cytotoxicity (Li, Shao et al. 2021) . According to previous results, in this study, we chose SFN (15 μM) to examine its effect on cholesterol clearance. We will add the information in the discussion part. In this study, we will perform dose-response TFEB nuclear translocation in NPC model cells as well as cytotoxicity experiments to examine whether the concentrations of SFN used in various cell lines are toxic.

References:

Hu, M. Q., P. Li, C. Wang, X. H. Feng, Q. Geng, W. Chen, M. Marthi, W. L. Zhang, C. L. Gao, W. Reid, J. Swanson, W. L. Du, R. Hume and H. X. Xu (2022). "Parkinson's disease-risk protein TMEM175 is a proton-activated proton channel in lysosomes.” Cell 185(13): 2292-+.

Li, D., R. Shao, N. Wang, N. Zhou, K. Du, J. Shi, Y. Wang, Z. Zhao, X. Ye, X. Zhang and H. Xu (2021). “Sulforaphane Activates a lysosome-dependent transcriptional program to mitigate oxidative stress.” Autophagy 17(4): 872-887.