Activation of mTORC1 and c-Jun by Prohibitin1 loss in Schwann cells may link mitochondrial dysfunction to demyelination

  1. Gustavo Della-Flora Nunes
  2. Emma R Wilson
  3. Edward Hurley
  4. Bin He
  5. Bert W O'Malley
  6. Yannick Poitelon
  7. Lawrence Wrabetz
  8. M Laura Feltri

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    Evaluation Summary:

    This manuscript builds upon the recent observation that Schwann cell (SC)-specific loss of the mitochondrial protein Prohibitin-1 results in a rapid, progressive demyelinating peripheral neuropathy in mice associated with mitochondrial dysfunction. They establish pathways as downstream effectors of mitochondrial dysfunction in Schwann cells. The authors provide a comprehensive evaluation of these pathways following the loss of Prophibitin-1 and identify JUN and mTORC1 as potential mediators of myelin disruption.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

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Abstract

Schwann cell (SC) mitochondria are quickly emerging as an important regulator of myelin maintenance in the peripheral nervous system (PNS). However, the mechanisms underlying demyelination in the context of mitochondrial dysfunction in the PNS are incompletely understood. We recently showed that conditional ablation of the mitochondrial protein Prohibitin 1 (PHB1) in SCs causes a severe and fast progressing demyelinating peripheral neuropathy in mice, but the mechanism that causes failure of myelin maintenance remained unknown. Here, we report that mTORC1 and c-Jun are continuously activated in the absence of Phb1 , likely as part of the SC response to mitochondrial damage. Moreover, we demonstrate that these pathways are involved in the demyelination process, and that inhibition of mTORC1 using rapamycin partially rescues the demyelinating pathology. Therefore, we propose that mTORC1 and c-Jun may play a critical role as executioners of demyelination in the context of perturbations to SC mitochondria.

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  1. Version published to 10.7554/elife.66278 on eLife
  2. eLife

    Author Response:

    Reviewer #3 (Public Review):

    This manuscript was built on their recent observation that Schwann cell (SC)-specific loss of the mitochondrial protein Prohibitin-1 results in a rapid, progressive demyelinating peripheral neuropathy in mice associated with mitochondrial dysfunction. Although several mechanisms have been well-studied in SCs, the potential novelty here is establishing those pathways as downstream effectors of mitochondrial dysfunction in SCs. The authors provide a comprehensive evaluation of these pathways following the loss of SC Prophibitin-1 and identify JUN and mTORC1 as potential mediators of myelin disruption. This manuscript includes a substantial amount of data. However, some data are not directly related to the primary mechanistic conclusions. In addition, the manuscript relies heavily on descriptive, rather than mechanistic, data regarding the roles for JUN and mTORC1. Specific issues to be addressed are listed below:

    Thank you for the detailed comments and careful analysis of our manuscript!

    1. Figure 1: The authors suggest that increased JUN expression and mTORC1 activation are associated with the demyelinating in Phb1-SCKO mice with "peaking around P40 - P60" (Line 82). However, it appears the most profound effects on number of myelinated and demyelinated axons were observed at P90. Interestingly, immunoblots for JUN and mTORC1 targets suggest that increases in these signaling pathways are much greater at P20 and P40 when compared to P90. This may suggest that JUN and mTORC1 are important for early demyelination, but other mediators play a more prominent role in chronic changes. It would be nice to have data from a time point between P40 and P90 to further understand the time course of JUN and mTORC1 changes. If not, the authors should discuss these possibilities in further detail.

    Thank you for your suggestion. Following your comment, we included the P60 time point, which is now reported on Figure 1–figure supplement 2. We found that, at P60, the magnitude of the relative change (Phb1-SCKO vs Controls) is roughly the same as at P40. In our previous publication (Della-Flora Nunes et al., 2021), we conducted a careful analysis of the time course of demyelination and we found that the number of myelinated and demyelinated axons in nerves of Phb1-SCKO is roughly the same at P60 and P90 (Fig. 1e of the referred manuscript). Therefore, we believe that demyelination peaks between P40 and P60, and that mTORC1 and c-Jun activation is in line with the demyelination phenotype of Phb1-SCKO.

    1. Figure 4: Since these are teased nerve fibers, not adjacent sections, please describe the detailed methods for immunofluorescence detection of DAPI and protein targets (JUN, P0, MBP, p-S6).

    Thank you for your comment! We have now included an expanded description of the immunofluorescence method used in our analysis.

    1. For Figure 2 and Figure 3, the authors state "mTORC1 and JUN may be activated in different stages of the SC response to mitochondrial damage, with mTORC1 preceding JUN temporally" (Lines 293 - 294). However, the data presented here are somewhat confusing for this conclusion. The immunoblots provided in Figure 1 suggest a similar time course for both JUN and mTORC1 activation after Phb1 loss in SCs at both P20 and P40. However, in Figure 3, teased nerve from Phb1-SCKO at P40 shows reduced JUN but not p-56 expression. The authors may consider repeating the PhAM-DAPI-JUN/p-S6 studies at the P20 and P90 time points to clarify this issue.

    Thank you for your suggestion. We repeated the co-staining experiments at P90 and the new data is reported on Figure 3 – figure supplement 1. These data go in line with our previous results at P40, suggesting an association of mitochondrial damage with c-Jun expression, but not with p-S6. Unfortunately, mitochondrial loss is not visible at P20, so a similar analysis cannot be carried out at this time point. However, due to this comment and suggestions from Reviewer #2, we have removed inferences to which of these pathways is activated first since our experiments do not allows us to reach a firm conclusion. Our hypothesis would be that mTORC1 is activated earlier in the presence of subtler mitochondrial dysfunction, while high c-Jun expression happens later, following mitochondrial loss and preceding demyelination.

    1. Figure 7: Significant recovery of the demyelinating phenotype and nerve conduction velocity were noted after blockade of the mTORC1 pathway using rapamycin in Phb1-SCKO mice. However, this did not result in recovery of CMAP or overall functional improvement using the Rotarod assay. Given that demyelination was reversed, does this suggest that an important trophic function of SC mitochondria for associated axons is disrupted in Phb1-SCKO mice? Alternatively, could rapamycin delivery at P20 be too late to rescue degenerating axons, leading to incomplete functional recovery? The authors should discuss these possibilities in further detail.

    Thank you very much for this comment! In Phb1-SCKO mice, clear axonal degeneration seems to happen fast; so, it is challenging to detect these events. According to our data on Figure 7, Phb1-SCKO mice treated with rapamycin from P20 to P40 showed a trend towards amelioration of axonal degeneration in tibial nerves at P40 as assessed in semithin sections. To explore this data in more detail, but also to substantiate the data on rescue of demyelination by rapamycin, we now performed an analysis of the same tissue in electron microscopy. Our new results reported in Figure 7 – figure supplement 2 suggest that, although rapamycin is efficient at reducing the demyelination in Phb1-SCKO, it did not significantly alter the axonal degeneration as quantified from the electron micrographs. Therefore, we believe that the main beneficial effect of rapamycin on nerve conduction velocity is mediated by its capacity to prevent the demyelination on Phb1-SCKO mice. However, we cannot entirely rule out the possibility that maintenance of myelin sheaths by rapamycin can also have a small indirect effect on axon survival (which could be what we picked up in our previous quantification from semithin images). We adjusted the results and discussion sections to convey that the effect of rapamycin on axonal integrity is small or even non-present in our paradigm. We also believe that earlier inhibition of mTORC1 (before P20) could be beneficial to Phb1-SCKO mice. However, mTORC1 is known to be essential for SC proliferation during development, and, therefore, an earlier treatment could also affect myelin formation. This is one of the main reasons guiding our choice for the starting point of rapamycin application.

  3. eLife

    Evaluation Summary:

    This manuscript builds upon the recent observation that Schwann cell (SC)-specific loss of the mitochondrial protein Prohibitin-1 results in a rapid, progressive demyelinating peripheral neuropathy in mice associated with mitochondrial dysfunction. They establish pathways as downstream effectors of mitochondrial dysfunction in Schwann cells. The authors provide a comprehensive evaluation of these pathways following the loss of Prophibitin-1 and identify JUN and mTORC1 as potential mediators of myelin disruption.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    This manuscript is an interesting extension and major follow-up on a previous paper by some of the same authors, currently published as a preprint (Reference as listed by the authors in the current manuscript: DELLA-FLORA NUNES, G., WILSON, E. R., MARZIALI, L. N., HURLEY, E., SILVESTRI, N., HE, B., O'MALLEY, B. W., BEIROWSKI, B., POITELON, Y., WRABETZ, L. & FELTRI, M. L. 2020. Prohibitin 1 is essential to preserve mitochondria and myelin integrity in Schwann cells. Available at Research Square, Preprint (Version 1)).

    The current manuscript uses mainly a mouse model lacking Prohibitin 1 specifically in Schwann cells to elucidate the mechanistic connections between mitochondrial dysfunction and demyelination (much of the basic analyses of these mice is described in the preprint mentioned above). Employing an elaborate combination of mouse genetics, pharmacological interventions and cell culture experiments, the authors provide evidence that the mTORC1 and JUN pathways are involved. Furthermore, individual contributions of these pathways have been examined.

    The major strengths of the manuscript lay in the thorough genetic approaches in vivo and the extensive data analysis. Furthermore, the presented data are sound and the approaches suitable.

  5. eLife

    Reviewer #2 (Public Review):

    Here the authors genetically perturb mitochondria in Schwann cells (SCs) by conditional knockout of Phb1 in mice. The work aims to address mechanisms by which mitochondrial dysfunction in SCs affects myelin maintenance. Prohibitin 1 (and prohibitin 2) are ubiquitously expressed proteins, found in the cytosol, nucleus and mitochondria, that play roles in oxidative phosphorylation, mitochondrial biogenesis, the unfolded protein response and mitochondrial dynamics, amongst others. The authors find that conditional knockout of Phb1 leads to progressive failure of myelin maintenance (demyelination) and secondary axon degeneration; demonstrated convincingly using electron microscopy of sciatic nerve. To probe the pathological mechanisms, the authors examine candidate signalling pathways using western blotting of whole peripheral nerve lysates. Quantification of their high quality blots show increased steady state levels of JUN and increased levels of total and phosphorylated TORC1 targets, S6 and 4E-BP1. The last could be ameliorated slightly by pharmacologically blocking the intracellular stress response (ISR), in vivo, suggesting the ISR partially contributes to activation of 4E-BP1. In contrast, levels of AKT/p-AKT and mTOR/p-mTOR in whole nerve lysates are unchanged. Importantly, others have shown that very highly elevated levels of JUN lead to a hypomyelination pathology in vivo.

    To confirm that primary mitochondrial dysfunction directly activate these pathways, the authors demonstrate, using one of each of three compounds, that pharmacologically interfering with mitochondrial function in primary Schwann cells grown in high glucose medium, can activate mTORC1 and pathways involved in cell stress; 7 days' intervention being more potent than 24 h. Notably, levels of JUN were significantly decreased after 7 days in the presence of two of these factors, which the authors suggest might reflect already elevated levels of JUN in vitro.

    Comment: Possibly, replacing glucose with pyruvate would have been appropriate here, to prevent the cells relying on glycolysis for ATP synthesis, and might have resulted in response more compatible with the in vivo observations.

    As western blotting does not provide spatial resolution, the authors turned to histological evaluation to show that mitochondrial damage, assessed using a fluorescent reporter, was significantly associated with JUN expression in the SC nucleus in teased fibres from the conditional mutant. However, they found no similar correlation for mTORC1 activation. This, despite that p-S6 was detected by immunostaining in the conditional mutant fibres.

    Comment: This histological evaluation raises the possibility that JUN and S6 are each activated in different fibres, or at different time points in the same fibres, despite that western blotting shows both are simultaneously elevated. In this respect, the majority of JUN positive fibres contained myelin ovoids, whereas the association was more tenuous for p-S6 staining.

    Next, the authors demonstrate convincingly by western blotting that steady state levels of several targets of JUN are up- or down-regulated in nerve lysates from conditional knockout mice, including myelin genes and factors involved in autophagy. They then genetically knockout or deplete JUN in SCs in conditional Phb1 mice, leading to partially reduced levels of p-S6 and p-4E-BP1, but not total S6 or 4E-BP1. The authors interpret this to suggest the possibility that mTORC1 is activated downstream of JUN activation.

    Comment: However, in the histological analyses described above, p-S6 staining did not correlate with mitochondrial disruption, which would be expected to be the case if (i) JUN activation is secondary to mitochondrial disruption, as suggested (Fig 3A-C) and (ii) mTORC1 is activated downstream of JUN.

    Appropriately, the authors show that JUN deletion per se does not affect p-S6 and p-4E-BP1 levels, suggesting that JUN depletion in the conditional Phb1 mutant works through lowering the JUN elevation. Notwithstanding that depletion of JUN led to a decrease in the numbers of demyelinated axons, it did not rescue the numbers of myelinated axons or the neuropathy phenotype, possibly because of unrelated primary effects.

    Finally and most importantly, the authors find that pharmacological inhibition of mTORC1 in vivo restores myelin maintenance in conditional Phb1 knockout mice, and improves conduction velocity, whilst also reducing levels of JUN (albeit not significantly) and ISR pathway components.

    The authors convincingly demonstrate that physical mitochondrial dysfunction correlates with upregulation of JUN in the SC nucleus. However, the evidence that activation of JUN is causally related to demyelination is tenuous and is somewhat difficult to prove with the available models. In contrast, the evidence that the mTORC1 pathway is causally involved in demyelination is strong. The suggestion that mTORC1 is activated downstream of JUN is not well supported by the data, based on the histological observations.

    The work has wider relevance for mitochondrial dysfunction in other disorders.

  6. eLife

    Reviewer #3 (Public review):

    This manuscript was built on their recent observation that Schwann cell (SC)-specific loss of the mitochondrial protein Prohibitin-1 results in a rapid, progressive demyelinating peripheral neuropathy in mice associated with mitochondrial dysfunction. Although several mechanisms have been well-studied in SCs, the potential novelty here is establishing those pathways as downstream effectors of mitochondrial dysfunction in SCs. The authors provide a comprehensive evaluation of these pathways following the loss of SC Prophibitin-1 and identify JUN and mTORC1 as potential mediators of myelin disruption. This manuscript includes a substantial amount of data. However, some data are not directly related to the primary mechanistic conclusions. In addition, the manuscript relies heavily on descriptive, rather than mechanistic, data regarding the roles for JUN and mTORC1. Specific issues to be addressed are listed below:

    1. Figure 1: The authors suggest that increased JUN expression and mTORC1 activation are associated with the demyelinating in Phb1-SCKO mice with "peaking around P40 - P60" (Line 82). However, it appears the most profound effects on number of myelinated and demyelinated axons were observed at P90. Interestingly, immunoblots for JUN and mTORC1 targets suggest that increases in these signaling pathways are much greater at P20 and P40 when compared to P90. This may suggest that JUN and mTORC1 are important for early demyelination, but other mediators play a more prominent role in chronic changes. It would be nice to have data from a time point between P40 and P90 to further understand the time course of JUN and mTORC1 changes. If not, the authors should discuss these possibilities in further detail.

    2. Figure 4: Since these are teased nerve fibers, not adjacent sections, please describe the detailed methods for immunofluorescence detection of DAPI and protein targets (JUN, P0, MBP, p-S6).

    3. For Figure 2 and Figure 3, the authors state "mTORC1 and JUN may be activated in different stages of the SC response to mitochondrial damage, with mTORC1 preceding JUN temporally" (Lines 293 - 294). However, the data presented here are somewhat confusing for this conclusion. The immunoblots provided in Figure 1 suggest a similar time course for both JUN and mTORC1 activation after Phb1 loss in SCs at both P20 and P40. However, in Figure 3, teased nerve from Phb1-SCKO at P40 shows reduced JUN but not p-56 expression. The authors may consider repeating the PhAM-DAPI-JUN/p-S6 studies at the P20 and P90 time points to clarify this issue.

    4. Figure 7: Significant recovery of the demyelinating phenotype and nerve conduction velocity were noted after blockade of the mTORC1 pathway using rapamycin in Phb1-SCKO mice. However, this did not result in recovery of CMAP or overall functional improvement using the Rotarod assay. Given that demyelination was reversed, does this suggest that an important trophic function of SC mitochondria for associated axons is disrupted in Phb1-SCKO mice? Alternatively, could rapamycin delivery at P20 be too late to rescue degenerating axons, leading to incomplete functional recovery? The authors should discuss these possibilities in further detail.

  7. Version published to 10.1101/2020.11.25.398032v1 on bioRxiv