Oxidative stress‐induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2

Abstract Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson's disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4‐TRPML1‐ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca2+ fluxes tightly regulated this system. Increased vulnerability of JIP4 KO cells to acrolein indicated that lysosomal clustering and subsequent autophagy activation served as defence mechanisms against cytotoxicity of acrolein itself. Furthermore, the JIP4‐TRPML1‐ALG2 pathway was also activated by H2O2, indicating that this system acts as a broad mechanism of the oxidative stress response. Conversely, starvation‐induced lysosomal retrograde transport involved both the TMEM55B‐JIP4 and TRPML1‐ALG2 pathways in the absence of the JIP4 phosphorylation. Therefore, the phosphorylation status of JIP4 acts as a switch that controls the signalling pathways of lysosoma l distribution depending on the type of autophagy‐inducing signal.

5. The authors should check whether acrolein-induced lysosomal retrograde transport is inhibited by treatment with antioxidants such as N-acetyl-l-cysteine (NAC).
6. The interpretation of this study is that acrolein-mediated lysosomal retrograde transport results in mTORC1 inactivation, and this inactivation then contributes, at least in part, to autophagy induction. However, another possibility if that the observed mTORC1 inactivation is independent of lysosomal trafficking. The authors should measure mTORC1 activity in cells treated with acrolein + nocodazole (or ciliobrevin D) vs nocodazole alone. 7. Does TRPML1 depletion affect lysosomal clustering in response to starvation? Include these data in Figure 6f and add quantification and statistical analysis for all the conditions. 8. The authors do not show that JIP4 is a direct CaMK2G target or whether CaMK2G affects TRPML1-mediated calcium release in response to acrolein.
Minor comments: 1. Figure 1 seems a little bit disconnected from the rest of the study as the authors never assess whether acrolein actually has a positive (by enhancing autophagy) or negative (high toxicity) effect on the pathophysiology of PD.

In Figures 6h and S7c
, also include the effect of increasing concentrations of acrolein on viability in cells depleted of either TMEM55B or TRPML1.
3. It is unclear why TMEM55B depletion prevents to some extent lysosomal clustering in response to acrolein. Could TMEM55B and TMEM55A show some redundancy under these experimental conditions? Does simultaneous depletion of both proteins prevent lysosomal transport to the cell center in acrolein-treated cells?
Referee #2: The study by Sasazawa and collaborators begins with the observation that the toxic metabolite acrolein (a product of spermidine metabolism) is elevated in the serum of Parkinson's disease patients. Studies in SH-SY5Y cells showed that acrolein induces perinuclear clustering of lysosomes as well as autophagy. Clustering occurred around the microtubule-organizing center, was abolished by inhibition of microtubule polymerization and dynein motors, and was not accompanied by lysosomal damage or lysosomal enzyme inhibition. Subsequent experiments showed that acrolein inhibits the activity of the autophagy inhibitor mTORC1, thereby increasing autophagic flux.
Testing of three candidate pathways for dynein-mediated lysosomal retrograde transport led to the identification of the TRPML1-ALG2 pathway as the one responsible for the observed acrolein-induced effects. Importantly, the authors show that also JIP4 plays an essential role in this pathway independently of its classic partner protein, TMEM55B. Biochemistry experiments and the screening of kinase inhibitor libraries identified CaMK2G as a kinase that (upon TRPML1-mediated calcium release) phosphorylates JIP4 at T217 to trigger lysosomal retrograde movement and autophagy.
Finally, JIP4 was found to be essential also in lysosomal retrograde transport triggered by H202 and nutrient starvation, even though additional experiments clarified that two different JIP4-mediated mechanisms are in play: Oxidative stress (acrolein, H202) triggers lysosomal retrograde transport via the phospho-JIP4-TRPML1-ALG2 pathway, whereas starvation triggers lysosomal retrograde transport via the TMEM55B-non-phosphorylated JIP4 (T217) pathway. Knockout of JIP4 in SH-SY5Y left the cells more vulnerable to acrolein exposure, indicating that JIP4 is part of a protective stress mechanism that acts through autophagy to counteract acrolein toxicity.
The study is very elegantly executed with a vast array of techniques, experiments mostly organized in a mechanistic sense, and appropriate controls. The presentation of the data is straightforward and the story is well built, referenced, and discussed. The data are novel and important and clarify the mechanisms by which JIP4 regulates lysosomal retrograde transport according to two different pathways, importantly establishing CaMK2G-mediated phosphorylation of JIP4 as the switch between the two pathways. This is a complete and compelling story and I don't have any experimental suggestion for this present manuscript (additional components of these pathways could be explored in subsequent manuscripts). My only observation is that the data in Appendix Figure 7d should be presented in the Results section rather than in the Discussion section.
Referee #3: Lysosomal retrograde transport or positioning is crucial in stress-induced autophagy activation. In the manuscript, Sasazawa et al show that JIP4 phosphorylation at T217 by CaMK2G is critical for lysosomal positioning/clustering under oxidative stress (e.g., acrolein, H2O2), and TRPL1 and ALG2 also engage with the process though mediating the interaction between the lysosome and the microtubule motor complex. The action of mechanism is vital for autophagy activation under the type of stress. On the other hand, starvation-induced lysosome positioning is independent of JIP4 phosphorylation, but depends on the TMEM55B-JIP4 pathway. The study is of high interest, as it, for the first time, provides direct evidence showing that JIP4 phosphorylation plays pivotal roles in lysosomal retrograde transport and autophagy activation under specific stress settings, while starvation-induced lysosomal retrograde transport is involved in the parallel TMEM55B-JIP4 pathway. The current study offers mechanistic insight into stress-induced lysosomal positioning and autophagy activation, suggesting a new approach to modulate autophagy by tackling JIP4 phosphorylation. Overall, the findings are novel, the manuscript is well written, experiments are properly designed and controlled. The conclusions are supported by the extensive data presented, and the study is a concrete piece of work.
The reviewer has a few specific comments as appended: 1. In Fig 3g, how was the percentage (cells with spikes) at 0 frequency defined? At frequency 1, no TRPML1 knockdown (hollow) graph bar can be seen. 2. In Fig 4, it is interesting that T217 at JIP4 was identified as the site of phosphorylation by CaMK2G. It should be useful to perform alignment analysis for the protein sequences around T217 of JIP4 from a variety of species, and see if the T217 site and its neighbouring sequences (potential CaMK2G phosphorylation consensus motif) are conserved across different species. 3. In Fig 4i, does the bar level indicate the phosphorylation level of each peptide as shown? Please clarify this. 4. Statistical analysis is indicated in the figures. Please detail statistical analysis in the figure legends or/and methods. 5. In the introduction, the content about autophagy is minimal. The background about autophagy process should be included. This will offer opportunities to introduce autophagosome maturation, which is involved in lysosomal transport and fusion. Such the content is relevant to the studies. 6. In Fig 1e and Fig S3, acrolein, Spd or Spm markedly reduces p70S6K phosphorylation marking mTOR activity. Does inhibition of mTOR activity contribute to JIP4 phosphorylation and lysosomal positioning, or does lysosomal positioning regulate mTOR activity, or both could be the case? It is worth discussing this in the discussion section.
Dear Dr. Ieva Gailite, Thank you for the review of our paper entitled "Phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2" (EMBOJ-2022-111476) and giving us the opportunity to revise the manuscript. We have carefully read the critiques and performed extensive revision experiments. Below we include point-by-point responses to the questions raised by the reviewers. We have also highlighted the revised sentences in the manuscript in red.
The numbers of pages and lines are indicated based on the MS Word revised manuscript. We believe this revision appropriately addresses the issues raised by Reviewers. Finally, all the authors would like to thank again the Editor and Reviewers for improving our manuscript by providing their invaluable comments and suggestions. As per Reviewer's comment, we had investigated our findings in light of the two excellent papers on RUFY3/4. Specifically, we investigated whether depletion of RUFY3 prevents acrolein-induced lysosomal clustering. We prepared 3 different sequences of siRNA (#3 is the same as in PMID: 35314681). We first checked their knockdown efficiency in SH-SY5Y cells by using RUFY3 antibody (Novus Biologicals, #NBP1-89614, same as in PMID: 35314681), and found that all of them could suppress RUFY3 protein expression as shown below. However, in our hands siRNA #3 showed high cytotoxicity in SH-SY5Y cells and thus we assessed lysosomal distribution by using siRNA #1 and #2. As shown in Fig. EV5b, acrolein induced-lysosomal clustering was not suppressed by RUFY3 depletion by either siRNA. We have added related sentences in the introduction (page 5, line 6 to line 8) and discussion sections (page 22, line 18 to page 23, line 6) and provided data in preferably by measuring changes in cumulative intensity distribution as described by Starling et al., 2016 (PMID: 27113757).

(RESPONSE)
According to the Reviewer's comment, we have attempted to quantify lysosomal distribution as described by Starling et al., 2016 (PMID: 27113757). Unfortunately, we were unable to apply the suggested protocol to SH-SY5Y cells, which have relatively large nuclei to the whole cytoplasm. Therefore, we have established a novel method to evaluate lysosomal clustering by calculating the ratio of lysosomes existing in the vicinity of MTOC relative to the total. We have re-stained cells with lysosomal marker LAMP2 and MTOC marker γ-tubulin and analysed the lysosomal clustering using FIJI software. Based on the additional experiments, we reconfirmed the effects of acrolein on the lysosomal distribution changes and added these data in the Results section ( Fig. 2a, 3d, 6d and 6f) and methods in Materials and Methods section (page 28, line 12 to page 29, line 3). Next, to examine the localization of TMEM55B and TRPML1 by electron microscopy, we have purchased all available antibodies as shown below table and checked their reactivity. Unfortunately, only one TMEM55B antibody purchased form Proteintech could detect target protein by immunocytochemistry, but others could not. Therefore, we abandoned the electron microscopy analysis using TRPML1 and TMEM55B antibodies and thus we evaluated whether TMEM55B localized in the lysosomes by immunostaining using super-resolution microscopy. As shown below, endogenous TMEM55B exhibited a patchy and clustered distribution in all LAMP2-positive lysosomes consistent with a previous report (J Cell Sci 135: jcs258566, 2022). Although endogenous TRPML1 could not be observed, we could obtain the data showing that TMEM55B distributed on the lysosomes homogenously, implying no association of TMEM55B distribution with the selection of the two pathways. As we described in discussion section, we propose that phosphorylation of JIP4 may alter the affinity of its binding partners (page 21, line 16 to page 22, line 2).

A caveat of this study is that it fails to show a phosphorylation-dependent interaction
between JIP4 and the TRPML1-ALG2 complex by immunoprecipitation, even when using recombinant proteins. Furthermore, the co-localization between JIP4 and ALG2 shown in Figure 5d is not very convincing and there is not a clear clustering of We have tried to detect JIP4 and the TRPML1-ALG2 complex by immunoprecipitation. GFP or GFP-ALG2 transfected TRPML1-mCherry stably expressing cells were treated with acrolein for 2h, lysed and immunoprecipitated with anti-RFP magnetic beads, and then co-immunoprecipitated JIP4 and GFP-ALG2 were detected. As shown below, co-immunoprecipitated GFP-ALG2 was detected only in conditions of acrolein treatment, indicating that ALG2 interacts with TRPML1 in response to acrolein.
However, JIP4 was detected as several smeared bands even though its amount in the IP fraction appeared to be upregulated in response to acrolein treatment. TRPML1 is a

GFP-ALG2 interacts with TRPML1-mCherry in response to acrolein treatment.
Moreover, we performed Lyso-IP, a method for the rapid isolation of lysosomes

The authors should check whether acrolein-induced lysosomal retrograde transport is
inhibited by treatment with antioxidants such as N-acetyl-l-cysteine (NAC).
We have examined effect of nocodazole on acrolein-induced mTORC1 inactivation.
As shown in Fig. EV2b, nocodazole did not affect the acrolein-suppressed phosphorylation levels of p70S6K and S6, indicating that acrolein induces lysosomal clustering and inactivates mTORC1 independently. These data suggested that acrolein promotes autophagy in the dual effects: the mTORC1 inhibition contributes to upregulation of autophagosome synthesis, and lysosomal clustering enhances the autophagosome-lysosomal fusion. We added related sentences in the Results section (page 9, line 6 to line 10 and page 10, line 3 to 7) and the data in Fig. EV2b.
7. Does TRPML1 depletion affect lysosomal clustering in response to starvation?
Include these data in Figure 6f and add quantification and statistical analysis for all the conditions.
We In this study, we showed that starvation did not induce lysosomal clustering in JIP4 KO cells, suggesting that JIP4 is an essential factor for starvation-induced lysosomal clustering, whilst the phosphorylated JIP4 was not involved under starvation conditions.
These observations imply that starvation-induced lysosomal clustering is controlled by TRPML1-ALG2 and TMEM55B-JIP4 complexes, both of which involve non-phosphorylated JIP4. On the other hand, we identified in this study that oxidative stress-induced lysosomal clustering is mainly controlled by TRPML1-ALG2 pathway that was regulated by phosphorylated JIP4. Perhaps the adaptor molecule connecting JIP4 and TRPML1-ALG2 may be different between oxidative stress and starvation stress. One possible adaptor candidate in starvation condition may be RUFY3 because RUFY3 KD did not affect acrolein-induced lysosomal clustering in our experiments but suppressed starvation-induced lysosomal clustering as reported by others (Nat Commun, 13:1506, 2022Nat Commun, 13:1540, 2022. Although the precise molecular mechanisms of lysosomal retrograde transport in response to starvation remains to be elucidated further, at least our study has identified the indispensable role of phosphorylated JIP4 (T217) in the oxidative stress conditions. Minor comments: 1. Figure 1 seems a little bit disconnected from the rest of the study as the authors never assess whether acrolein actually has a positive (by enhancing autophagy) or negative (high toxicity) effect on the pathophysiology of PD.
We believe that the marked increase of the serum acrolein levels during any stage of PD provides a strong rationale for mechanistic studies of the acrolein effect on cells. We completely agree with the Reviewer that equivocal interpretation, protective or toxic to the human, is indeed still required. However, based on the cell-based experiments of the study, we can conclude that acrolein might have partial beneficial effect via autophagy enhancement and we would like to highlight this potential role in the disease. Therefore, we would prefer to keep the PD data which suggests that our studies could have relevance to human pathology.

In Figures 6h and S7c, also include the effect of increasing concentrations of acrolein on viability in cells depleted of either TMEM55B or TRPML1.
We have examined the cytotoxicity of acrolein in TMEM55B-or TRPML1-knockdown cells. Both TMEM55B-and TRPML1-KD resulted in increased vulnerability to acrolein treatment compared to control KD as well as JIP4 KO cells.
Depletion of TMEM55B is known to induce lysosomal stress (Genes Cells 23:418-434. 2018), which may prevent accurate assessment of acrolein-induced cytotoxicity. For this reason, we would like to include only the results using TRPML1 siRNA to avoid confusion in the Results section. We added these data in Fig.7c (page 19, line 2).

Knockout of JIP4 in SH-SY5Y left the cells more vulnerable to acrolein exposure,
indicating that JIP4 is part of a protective stress mechanism that acts through autophagy to counteract acrolein toxicity.
The study is very elegantly executed with a vast array of techniques, experiments mostly organized in a mechanistic sense, and appropriate controls. The presentation of the data is straightforward and the story is well built, referenced, and discussed.
The data are novel and important and clarify the mechanisms by which JIP4 regulates lysosomal retrograde transport according to two different pathways, importantly establishing CaMK2G-mediated phosphorylation of JIP4 as the switch between the two pathways. This is a complete and compelling story and I don't have any experimental suggestion for this present manuscript (additional components of these pathways could be explored in subsequent manuscripts). My only observation is that the data in Appendix Figure 7d should be presented in the Results section rather than in the Discussion section.
>> >> Thank you so much for your kind comments on our study. According to your comment we moved Fig. 7d to Results section at Appendix Fig. S4a. >> >> Thank you so much for your astute comments on our study. Based on your comments and suggestions, we revised texts and figures.
The reviewer has a few specific comments as appended: 1. In Fig 3g, how was the percentage (cells with spikes) at 0 frequency defined? At frequency 1, no TRPML1 knockdown (hollow) graph bar can be seen.

(RESPONSE)
We plotted the fluorescence ratio (F340/F380) values for each of more than 100 cells, and spikes with amplitudes greater than 0.05 were counted for each cell."0 frequency" means no spikes in the cell for 40 min. For example, the value in "0 frequency" in Fig. 3g represents the percentage of cells without spikes relative to the total cells. We replaced the  5. In the introduction, the content about autophagy is minimal. The background about autophagy process should be included. This will offer opportunities to introduce autophagosome maturation, which is involved in lysosomal transport and fusion. Such the content is relevant to the studies.

(RESPONSE)
We have added the background about autophagy process in the Introduction section as per Reviewer's suggestion (page 4, line 9 to line 16).
6. In Fig 1e and Fig S3, acrolein, Spd or Spm markedly reduces p70S6K phosphorylation marking mTOR activity. Does inhibition of mTOR activity contribute to JIP4 phosphorylation and lysosomal positioning, or does lysosomal positioning regulate mTOR activity, or both could be the case? It is worth discussing this in the discussion section.

(RESPONSE)
Starvation rapidly inactivates mTORC1 as shown below (left). On the other hand, starvation did not affect JIP4 phosphorylation as shown in Fig 6e. These data indicate that, inhibition of mTORC1 activity did not contribute to JIP4 phosphorylation. Moreover, Torin-1, a mTOR specific inhibitor, did not show neither JIP4 phosphorylation (below right) nor lysosomal distribution change (Fig. EV2c). On the other hand, nocodazole, which inhibits lysosomal movement, did not affect the acrolein-suppressed phosphorylation level of p70S6K and S6 (Fig. EV2b), indicating that acrolein induces lysosomal clustering and inactivates mTORC1, independently. We propose that these two effects of acrolein promote autophagy more effectively, because mTORC1 inactivation contributes to autophagosome upregulation, and lysosomal clustering contributes to autophagosome-lysosomal fusion. We added related sentences in the Results section (page 9, line 6 to line 10 and page 10, line 3 to 7) and in Fig.   EV2b,c.

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