Inversely proportional myelin growth due to altered Pmp22 gene dosage identifies PI3K/Akt/mTOR signaling as a novel therapeutic target in HNPP

  1. Doris Krauter
  2. David Ewers
  3. Timon J Hartmann
  4. Stefan Volkmann
  5. Theresa Kungl
  6. Robert Fledrich
  7. Sandra Goebbels
  8. Klaus-Armin Nave
  9. Michael W Sereda

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Abstract

Duplication of the gene encoding the myelin protein PMP22 causes the hereditary neuropathy Charcot-Marie-Tooth disease 1A (CMT1A), characterized by hypomyelination of medium to large caliber peripheral axons. Conversely, haplo-insufficiency of PMP22 leads to focal myelin overgrowth in hereditary neuropathy with liability to pressure palsies (HNPP). However, the molecular mechanisms of myelin growth regulation by PMP22 remain obscure. Here, we found that the major inhibitor of the myelin growth signaling pathway PI3K/Akt/mTOR, phosphatase and tensin homolog (PTEN) is increased in abundance in CMT1A and decreased in HNPP rodent models. Indeed, treatment of DRG co-cultures from HNPP mice with PI3K/Akt/mTOR pathway inhibitors reduced focal hypermyelination and, importantly, treatment of HNPP mice with the mTOR inhibitor Rapamycin improved motor behavior, increased compound muscle amplitudes (CMAP) and reduced tomacula formation in the peripheral nerve. In Pmp22 tg CMT1A mice, we uncovered that the differentiation defect of Schwann cells is independent from PI3K/Akt/mTOR activity, rendering the pathway insufficient as a therapy target on its own. Thus, while CMT1A pathogenesis is governed by dys-differentiation uncoupled from PI3K/Akt/mTOR signaling, targeting the pathway provides novel proof-of-principle for a therapeutic approach to HNPP.

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    Reply to the reviewers

    Manuscript number: RC-2023-01910

    Corresponding author(s): Michael W. Sereda

    1. General Statements

    Reviewer #1:

    In this paper the authors report a direct correlation between PMP22 and PTEN expression levels in the nerve of CMT mutants. In CMT1A Pmp22tg rat nerves, PTEN levels are increased, whereas in Pmp22+/- mutants, a model of the HNPP neuropathy, PTEN levels decrease. Consistent with this, Pmp22tg nerves display lower Akt phosphorylation and, vice versa, Pmp22+/- nerves have higher Akt phosphorylation. The authors lowered PTEN in the transgenic and inhibited mTOR using Rapamycin in the Pmp22+/- to support the functional relevance of the PMP22-PTEN correlation. ... In conclusion, the correlation between PMP22 and PTEN is a potential interesting observation. However, in my opinion, experiments as shown don't support the conclusion that PMP22 controls PTEN expression level and activity, which is suggested at the basis of the pathogenesis of PMP22 dosage-related neuropathies.

    We thank Reviewer #1 for this detailed feedback. We appreciate the Reviewer’s assessment that our observation that PMP22 and PTEN* are correlated *in CMT1A and HNPP is of potential interest. In the revised manuscript we addressed this key point by adding additional quantifications (Figure 1a, d; Figure 5d) and novel Western Blot analyses (Figure 1a, d). Regarding the pathophysiological significance of the correlation, we point out that both the original as well as the partially revised manuscript contain multiple pieces of evidence demonstrating that altered PTEN activity is critical for both PMP22 gene-dosage related neuropathies:

    1. The inhibition of the PI3K/PTEN/AKT/mTOR axis upstream (LY294002) or downstream (Rapamycin) of decreased PTEN ameliorates myelin defects in an in vitro HNPP model (Figure 2b, c).
    2. Downstream of PTEN, Rapamycin treatment ameliorates myelin defects, motor behavior and electrophysiology in the HNPP mouse model in vivo (Figure 3c, d, e,____ g, i)
    • Targeting of increased PTEN directly by inhibiting its activity pharmacologically (VO-OHpic) in a CMT1A rat model or by depleting it genetically in a CMT1A model leads to ameliorated myelination in vitro (Figure 4b, c; Figure 5f, g).
    1. The genetic depletion of PTEN in a CMT1A mouse model increases myelination in vivo, albeit not in the long term (Figure 6a, b, c, d). We therefore feel that any additional evidence to show that "PMP22 controls PTEN activity" is not vital for supporting the major claims of the manuscript, i.e. that the observed correlation of PTEN levels with PMP22 gene dosage has relevance for the etiology of PMP22 dosage diseases and and that targeting the PI3K-PTEN-AKT-mTOR axis downstream of PTEN provides a potential pharmacological therapy of HNPP (while directly targeting PTEN ultimately fails to rescue CMT1A). However, we agree that the activity of PTEN on the molecular level is interesting, and such evidence would further strengthen our conclusions. Therefore, in the final revised version, we plan to add further Western Blots and explore possible downstream effects of altered PTEN levels.

    Reviewer #2:

    This study investigates the modulation, both genetically and pharmacologically, of the PI3K/Akt/mTOR signaling in preclinical animal models for the inherited peripheral neuropathies HNPP and CMT1A. These conditions result from a gene dosage abnormality of the peripheral myelin protein gene PMP22. The exact biological molecular mechanisms remain enigmatic despite it having been over 30 years since the major genetic lesions, the CMT1A duplication and HNPP deletion, were described. With respect to myelin biology one observes focally slowed nerve conduction at pressure palsies and local/segmental hypermyelination in HNPP whereas hypomyelination occurs in CMT1A. The study is nicely conducted, data illustrations very informative, and writing clear and concise. This paper will likely be of great interest to your readers. The authors provide convincing evidence that the HNPP pathobiology is ameliorated by PI3K/Akt/mTOR inhibitors. Interestingly they found radial myelin growth was most affected by this approach and suggest an interesting transdermal approach in injured nerves in the acute prevention of pressure palsies.

    We thank Reviewer #2 for this positive evaluation.

    Reviewer #3:

    *In this paper Sareda and co-workers demonstrate that the PTEN/mTOR pathway is indirectly involved in regulating myelin thickness and wrapping in models of altered PMP22 gene dosage both in vitro and in vivo. Inhibition of this pathway decreases myelin thickness in models of HNPP, while increasing myelin thickness in models of CMT1A. The evidence for these conclusions is complex but reasonably presented, and the conclusions mainly supported by the data. The abstract for this paper, however, presents a somewhat oversimplified conclusion that the PTEN pathway mainly modifies models of HNPP, where the paper clearly demonstrates that models of CMT1A are also affected by this same pathway. This should be clarified. *

    We thank Reviewer #3 for the feedback on the manuscript. We agree with the Reviewer that the same pathway (PI3K/Akt/mTOR) also affects CMT1A, but it is of importance for us to highlight that the disease mechanisms are -at least partly- different between HNPP and CMT1A. This is supported by our observation that PTEN reduction in CMT1A only transiently improves myelination in vivo (Figure 6) and the persistent alteration of differentiation markers despite PTEN reduction, which is not observed in HNPP (Figure 7).

    2. Description of the planned revisions

    Reviewer #1

    Regarding the activity of PTEN

    Figure 1

    • *Additional experiments are needed to support the conclusion of Figure 1 that, in the two mutants, Pten levels reversely correlate with PI3K-Akt-mTOR pathway activation, which represents the rationale of all further experiments. For example, it should be shown systematically in both mutants both Akt and ERK phosphorylation levels (Akt at both T308 and S473), and mTOR activity read outs. In the previously published paper (Fledrich et al.) only increased Akt phosphorylation in Pmp22+/- nerves was reported, whereas Pmp22tg analysis was focused on the interdependence between Akt and ERK without exploring mTOR activation, which is relevant here. *2) (Figure 4) A different model, the C61 mouse a Pmp22tg overexpressing PMP22 is used here (rather than the CMT1A rat). This should be explained in the results. Is also this model characterized by increased Pten levels in the nerve? And low Akt-mTOR activation for instance? 3) (Figure 5) How is Akt-mTOR signaling in the double mutant as compared to Pmp22tg? Is that increased at P18? * Response: We fully agree with the Reviewer that further exploration of PTEN downstream effects will add value to the manuscript. We already justified the usage of the C61 mouse model more clearly, added P-S6 staining of wildtype in addition to an improved representation in Figure 5e, and performed extra Western Blot analysis of PTEN expression (described in the next section “Incorporated *revisions”). Moreover, we will further evaluate the downstream signaling components of PTEN and will perform additional Western Blot analyses of peripheral nerves of HNPP mice, CMT1A rats as well as C61 and C61xPTENhKO mice.

    Figure S1

    • *Figure S1, page 4: what does it mean "in line with this finding we were unable to detect protein-protein...". May be the authors meant: since there is a direct correlation between Pmp22 and Pten expression levels in the mutants, the authors explored the possibility of an interaction between the two. Regarding the co-IPs, in panel a, the co-IP at the endogenous level, the immunoprecipitation efficiency of PMP22 is very low. May be a pull-down experiment using either exogenous purified PMP22 or PTEN and nerve lysates can help to rule out the possibility of an interaction. The experiments in b, c are performed in overexpression in a heterologous system (293 cells). * Response: We agree with the Reviewer that we might have missed a possible interaction between PMP22 and PTEN in the experiments performed so far. Indeed, pull-down experiments may prove helpful to rule out / reveal protein-protein interaction. Therefore, we will use purified PMP22 and perform pull-down experiments using nerve lysates of wildtype and CMT1A rats.

    Figure 5

    • *Pten Fl/+ Dhh-Cre cultures seem to have axonal fasciculation. * Response____: We thank the Reviewer for this observation. We will systematically inspected all recorded images for features of fasciculation. We will also assess whether fasciculation is a representative feature in cultures derived from any of the genotypes, and if so, whether the genotypes differ in this regard.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Changes in the text are highlighted in green in the revised manuscript

    Reviewer #1:

    Figure 1

    • *Panel a: the decrease of Pten expression should be quantified with at least n=3 taking into account the variability among different samples at the different time points indicated (the same applies in panel b, even if here the increase of Pten expression level in Pmp22tg nerves is more evident). * Response: We agree with the Reviewer that the timeline is not sufficient to demonstrate alteration in PTEN expression in PMP22 gene dosage diseases CMT1A and HNPP. Therefore, we performed new Western Blot experiments evaluating PTEN expression in (i) HNPP mice, (ii) CMT1A rat (iii) C61 mice and (iv) C61xPTENhKO mice with minimum n = 3 biological replicates and performed the respective quantification which is shown in Figure1 (i, ii) and Figure 5 (iii, iv). The results of the Western Blot analysis and quantification show an increase in PTEN abundance in CMT1A rat (Figure 1d) and C61 mice (Figure 5d) while a decrease is observed in HNPP mice (Figure 1a) and PTENhKOxC61 mice (Figure 5d) when compared to wildtype controls.

    • *Panel a and b: the statement that Pten is more expressed at P18 at the peak of myelination in wildtype nerves is not supported by the blots as shown. * Response: We agree that this observation is only partly supported by the Western Blot analysis, as seen in the HNPP mouse model, and deleted this part in the results section.

    • Figure S1, page 4: what does it mean "in line with this finding we were unable to detect protein-protein...". May be the authors meant: since there is a direct correlation between Pmp22 and Pten expression levels in the mutants, the authors explored the possibility of an interaction between the two. Response: We thank the Reviewer for pointing out the lack of clarity here. We changed the respective sentence accordingly:

    “Since there is a direct correlation between PMP22 and PTEN expression levels in the mutants, we explored the possibility of an interaction between the proteins. By immunoprecipitation experiments we were unable to detect protein-protein interaction between PMP22 and PTEN (Figure S1).” (Page 4)

    • *Page 4: "Taken together, Pmp22 dosage inversely correlates with the abundance of PTEN...": please revise this statement * Response: We thank the reviewer for spotting this mistake. We changed the sentence accordingly, which now reads:

    “Taken together, Pmp22 dosage directly correlates with the abundance of PTEN and presumably the activation level of the PI3K/Akt/mTOR pathway in myelinating Schwann cells (Figure 1i)." (Page 4, Line 23)

    Figure 2:

    • The aberrant myelin figures displayed are similar to myelin ovoids preceding degeneration rather than myelin outfoldings. It is also strange that these alterations are in the wildtype cultures treated with RAPA, that instead, in this system, has been reported to increase myelination as it improves protein homeostasis (autophagy, quality control, etc). Response: We thank the Reviewer for pointing this out. Indeed, in the way the images have been presented the aberrant myelin profiles can be mistaken for ovoids. However, a close inspection of the TUJ1 channel images revealed continuity of the axons below the aberrant myelin, thereby excluding ovoid formation. In the partially revised manuscript, we now also show the TUJ1 channel individually (Figure 2), so that it can be appreciated that the defects are confined to the myelin. Concerning the incidence of the myelin defects in RAPA treated wildtype cultures, our analysis can have missed a potential amelioration due to the rather high variability in the data.

    Figure 3

    *Panel c-e: aberrant fibers should be normalized on total number of fibers and on the area, particularly because RAPA is used. *

    Response: We agree with the Reviewer that number of tomacula and recurrent loops should be normalized to the total number of fibers on the area. We have quantified the total number of fibers in the whole sciatic nerve and normalized the tomacula and recurrent loops number accordingly. Results show a decrease in both tomacula and recurrent loops after Rapamycin treatment in the HNPP mice (Figure 3c, d, e, f).

    Figure 4

    The improvement in the number of myelin segments following PTEN inhibition in Pmp22tg co-cultures is very weak. The 500 nM has instead a consistent effect in reducing myelin segments in the wildtype and I think that these results overall don't support the conclusion that myelination is ameliorated by reducing PTEN activity in Pmp22tg co-cultures.

    Response: We thank the Reviewer for this important point. We like to emphasize that we treated whole cultures with the PTEN inhibitor and we cannot rule out a (probably) negative effect on axonal PTEN, resulting in only weak improvement of myelination in PMP22tg cultures and strong effects also on the wildtype co-cultures. Therefore, we decided against a treatment of CMT1A models in vivo and further explored the effects of PTEN reduction specifically in Schwann cells using the genetic model as described Figure 5. The Reviewer made clear to us that this is inappropriately explained in the results section and we therefore adapted this in the manuscript on page 6:

    “Similarly, the prolonged inhibition of PTEN with VO-OHpic (for 14 days) caused a dosage-dependent reduction in myelinated segments in wildtype co-cultures (Figure 4c, Figure S2). The mechanism is currently unexplained but cannot rule out a negative effect of PTEN inhibition on DRG neurons and myelination.”

    Figure 5:

    • *A different model, the C61 mouse a Pmp22tg overexpressing PMP22 is used here (rather than the CMT1A rat). This should be explained in the results. Is also this model characterized by increased Pten levels in the nerve? And low Akt-mTOR activation for instance? * Response: We agree with the Reviewer that it has not been clear in the text why we changed here to the C61 mouse model. We clarified this in the Results section which now reads on page 6:

    “To reduce Pten function in CMT1A models also in vivo, we applied a genetic approach (Figure 5a). As the genetic tools to specifically target Schwann cells were only available in the mouse and not the rat, we used the C61 mouse model of CMT1A. We reduced PTEN by about 50% selectively in CMT1A Schwann cells by crossbreeding Pmp22 transgenic mice with floxed *Pten *and Dhh-cre mice, yielding *PTENfl/+Dhhcre/+PMP22tg *experimental mutants (Figure 5b). Western blot analyses of sciatic nerve lysates confirmed the increase of PTEN in PMP22tg mice and the reduction of PTEN in the double mutants (Figure 5c, d).”

    Moreover, regarding the PTEN expression we added Western Blot analysis and quantification in Figure 5c, d showing increased PTEN expression in the C61 mouse model of CMT1A and decreased PTEN in the PTENhKOxC61 double mutants. Further analysis of the downstream signaling is planned (see “planned revision”).

    • *PTEN, Akt-mTOR expression/activation levels should be checked biochemically also in this model. And quantified (panel c). * Response: We added an explanation for the use of the C61 mouse model (see point Figure 5.1 above). Moreover, we quantified the Western Blot analysis and added it in Figure 5d. The expression of PTEN was included in the Western Blot analysis (Figure 5c) showing increased PTEN expression also in the C61 mouse model. Further biochemical analysis of the C61 mouse model is planned (see “planned revision”).

    • *In panel d overactivation of mTOR (PS6 staining) in Schwann cells is not evident. * Response: We agree with the Reviewer that the way the image was displayed is not sufficient to show P-S6 activation in the double mutants. We have now split the image (Figure 5e) to better visualize the P-S6 staining alone compared to the co-staining with P0 (marker for compact myelin) and DAPI (nuclei). Further, we added staining of wildtype nerve. We hope this way the differences in P-S6 activation can be easier appreciated.

    Figure 6:

    *G-ratio analysis: which are the mean values (numbers) with SEM in the three groups analyzed wildtype, Pmp22tg and Pmp22tg; Pten fl/+; Dhh-Cre? *

    Response: We thank the Reviewer for pointing this out. We added the quantification of the mean g-ratios in Figure 6d, f.

    Figure 7:

    • *If more fibers are committed to myelinate in the double mutant as compared to the single Pmp22tg at P18 ,particularly, it is unclear why there is no difference in differentiation marker expression in Figure 7 (Oct6 and Hmgcr). * Response: We thank the reviewer for this comment. We do not necessarily expect to see a strong difference in the expression of differentiation markers given the mild increase in myelination in the double mutants. Similarly, we do not observe alterations in the expression of differentiation markers in HNPP mice, while these fibers produce more myelin. Therefore, we concluded that alterations in PTEN-PI3K/Akt/mTOR signaling do not influence differentiation in the mouse models while in the PMP22 overexpressing situation of CMT1A other mechanisms alter differentiation of the Schwann cells. We also note that experiments were performed at postnatal day 18 and we cannot rule out possible alterations in differentiation marker expression at earlier time points in development in the double mutants.

    • In conclusion, the correlation between PMP22 and PTEN is a potential interesting observation. However, in my opinion, experiments as shown don't support the conclusion that PMP22 controls PTEN expression level and activity, which is suggested at the basis of the pathogenesis of PMP22 dosage-related neuropathies. Response: Please also see section 1. In order to avoid any overstatement that "*PMP22 controls PTEN expression level and activity", *in our revised version we have clarified this point and changed the wording in the main text:

    "The mechanisms that link the abundance of PMP22 to that of PTEN are still unclear and we here neither show direct nor indirect control of PTEN expression by PMP22." (Page 8)

    Reviewer #2:

    1. Regarding in the Introduction: "...the molecular mechanisms causative for the abnormal myelination remain largely unknown and still no therapy is available." Suggest consider modifying to perhaps: '...no small molecule or pharmacological therapeutic intervention exist.' To say "no therapy" exist is 'myopic' and untrue.

    *Suggest adding question mark to end of sentence or changing ‘asked’ to “investigated” for following thought: “Here, we asked whether PI3K/Akt/mTOR signaling provides therefore a therapeutic target to treat the consequences of altered Pmp22 gene-dosage.” *

    Rather than attempt to establish PRIORITY perhaps ‘softening’ the INTRODUCTION concluding statement “Our results thus identify a potential pharmacological target for this inherited neuropathy.

    *[This makes thePI3K/Akt/mTOR pathway a promising target for a preventive treatment of affected nerves also in human patients.] **Does this belong in RESULTS? Or rather DISCUSSION? *

    Response: We thank the Reviewer for the suggestions. We changed the sentences accordingly in the manuscript (1.: Page 3, Line 23; 2.: Page 3, Line 26; highlighted in green). Regarding point 3, we are convinced that identifying pharmacological targets for peripheral neuropathies should be given priority. Indeed, the aspect concerning point 4 is already highlighted in the discussion therefore we removed the sentence from the result section.

    Reviewer #3:

    *The abstract for this paper, however, presents a somewhat oversimplified conclusion that the PTEN pathway mainly modifies models of HNPP, where the paper clearly demonstrates that models of CMT1A are also affected by this same pathway. This should be clarified. *

    We agree with the Reviewer that the same pathway (PI3K/Akt/mTOR) also affects CMT1A, but it is of importance for us to highlight that the disease mechanisms are -at least partly- different between HNPP and CMT1A. This is supported by our observation that PTEN reduction in CMT1A only transiently improves myelination in vivo (Figure 6) and the persistent alteration of differentiation markers despite PTEN reduction, which is not observed in HNPP (Figure 7). For clarification we have altered the wording in the abstract which now reads: "In contrast, we found that CMT1A pathogenesis was only transiently ameliorated by altered PI3K/Akt/mTOR signaling, which drives radial but not longitudinal growth of peripheral myelin sheaths".

    3. Description of analyses that authors prefer not to carry out

    Reviewer #1:

    Figure 1:

    *Figure 1, Panel e: may be with this experiment the authors aim to suggest that Pten and Pmp22 are unlikely to interact directly or indirectly since Pten is cytosolic and Pmp22 myelin-membrane enriched. However, this myelin purification shows that Pmp22 as P0 expression levels are also abundant in the cytosol, may be also because P18 has been chosen as time point. What about a different type of membrane-cytosol fractionation experiment and/or another time point? *

    Response: We want to clarify that in this experiment not myelin and cytosol fractions were separated but myelin and whole sciatic nerve lysate (which is the input before isolation of the myelin fraction, called “lysate”). Therefore, the analysis aimed at showing an enrichment of PMP22 and P0 in the myelin fraction while PTEN and TUJ (as a control) are not, which makes it more unlikely for PTEN and PMP22 to interact directly. This experiment, together with the immunohistochemical analysis in Figure 1h should highlight the location of PMP22 and PTEN in the Schwann cell. Together with the newly suggested experiments of the Reviewer for Figure S1 (see planned Revision point 1) we do not see the need for extra membrane-cytosol fractionations and/ or another timepoint as the more detailed as the improved experiment on protein-protein interaction using nerve lysate (not only cell culture) is the experiment of choice to clarify whether we have a direct interaction or not.

    Regarding in vitro Schwann cell- DRG co-culture experiments:

    (Figure 2, Figure 4 and Figure 5e)

    1. *(Figure 2) For this experiment, pulse treatment may be beneficial rather than in continuous. Is Akt-mTOR phosphorylation-signaling increased also in Pmp22+/- co-cultures as in mutant nerves? Is the treatment reducing the overactivation? *
    2. *(Figure 4) Similarly to Figure 2, is PTEN level increased in Pmp22tg cultures along with Akt-mTOR downregulation? *
    3. *(Figure 5) Panel e: co-cultures are established using ex vivo Dhh-Cre recombination. The downregulation of Pten in the cultures should be documented. * Response: We agree with Reviewer #1 that a deeper analysis of the co-culture system regarding the downstream signaling of PTEN would increase the value of the experiments. Unfortunately, the experiments were designed in a very small scale with the intention of only evaluating myelin alterations on a histological level and we did have enough tissue to collect cells for deeper protein expression analysis. Moreover, we tried to use the co-culture system as a proof-of-principle experiment in parallel to our in vivo studies which we value more important due to the still quite artificial co-culture setup. We hope that the Reviewer can understand our approach with the focus we set on the in vivo work.

    Figure 3:

    1. *The RAPA treatment seems to increase Pten level in the mutant even above wildtype levels (panel b), which can result in decreased myelin thickness due to downregulation of Akt-mTOR. A different method to normalize expression levels should be used. * Response: Comparing the mean, relative expression levels resulting from our quantification as plotted in the graph (panel b) revealed no increase above wildtype level after Rapamycin treatment in the HNPP mouse. Further, we decided for whole protein staining as the superior approach to loading control because we have observed alterations in the expression of other frequently used “housekeepers” such as GAPDH, Actin and Vinculin in the CMT1A rodent models.

    *Panel c-e: Can these data also be reproduced in quadriceps nerves as tomacula are more prominent in these Pmp22+/- nerves showing less variability due to the prevalence of large caliber axons? *

    Response: Unfortunately, quadriceps nerves were not collected for histology in the experiment and therefore we cannot redo the quantification. Nevertheless, we agree that the quadriceps nerves have less variability than the sciatic nerve and will definitely include the tissue in our future experiments.

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    Referee #3

    Evidence, reproducibility and clarity

    In this paper Sareda and co-workers demonstrate that the PTEN/mTOR pathway is indirectly involved in regulating myelin thickness and wrapping in models of altered PMP22 gene dosage both in vitro and in vivo. Inhibition of this pathway decreases myelin thickness in models of HNPP, while increasing myelin thickness in models of CMT1A. The evidence for these conclusions is complex but reasonably presented, and the conclusions mainly supported by the data. The abstract for this paper, however, presents a somewhat oversimplified conclusion that the PTEN pathway mainly modifies models of HNPP, where the paper clearly demonstrates that models of CMT1A are also affected by this same pathway. This should be clarified.

    Significance

    These data are significant, since they would provide new targets for treating inherited neuropathy associated with altered PLP22 gene dosage.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    This study investigates the modulation, both genetically and pharmacologically, of the PI3K/Akt/mTOR signaling in preclinical animal models for the inherited peripheral neuropathies HNPP and CMT1A. These conditions result from a gene dosage abnormality of the peripheral myelin protein gene PMP22. The exact biological molecular mechanisms remain enigmatic despite it having been over 30 years since the major genetic lesions, the CMT1A duplication and HNPP deletion, were described. With respect to myelin biology one observes focally slowed nerve conduction at pressure palsies and local/segmental hypermyelination in HNPP whereas hypomyelination occurs in CMT1A.

    The study is nicely conducted, data illustrations very informative, and writing clear and concise. This paper will likely be of great interest to your readers. A few things the authors may want to consider:

    1. Regarding in the Introduction: "...the molecular mechanisms causative for the abnormal myelination remain largely unknown and still no therapy is available." Suggest consider modifying to perhaps: '...no small molecule or pharmacological therapeutic intervention exist.' To say "no therapy" exist is 'myopic' and untrue.
    2. Suggest adding question mark to end of sentence or changing 'asked' to "investigated" for following thought: "Here, we asked whether PI3K/Akt/mTOR signaling provides therefore a therapeutic target to treat the consequences of altered Pmp22 gene-dosage."
    3. Rather than attempt to establish PRIORITY perhaps 'softening' the INTRODUCTION concluding statement "Our results thus identify a potential pharmacological target for this inherited neuropathy.
    4. [This makes thePI3K/Akt/mTOR pathway a promising target for a preventive treatment of affected nerves also in human patients.] Does this belong in RESULTS? Or rather DISCUSSION?

    Significance

    The authors provide convincing evidence that the HNPP pathobiology is ameliorated by PI3K/Akt/mTOR inhibitors. Interestingly they found radial myelin growth was most affected by this approach and suggest an interesting transdermal approach in injured nerves in the acute prevention of pressure palsies.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    In this paper the authors report a direct correlation between PMP22 and PTEN expression levels in the nerve of CMT mutants. In CMT1A Pmp22tg rat nerves, PTEN levels are increased, whereas in Pmp22+/- mutants, a model of the HNPP neuropathy, PTEN levels decrease. Consistent with this, Pmp22tg nerves display lower Akt phosphorylation and, viceversa, Pmp22+/- nerves have higher Akt phosphorylation. The authors lowered PTEN in the transgenic and inhibited mTOR using Rapamycin in the Pmp22+/- to support the functional relevance of the PMP22-PTEN correlation.

    I have major concerns on the data as shown, which, in my opinion, don't support the main conclusion of this paper. In more detail:

    Figure 1 Panel a: the decrease of Pten expression should be quantified with at least n=3 taking into account the variability among different samples at the different time points indicated (the same applies in panel b, even if here the increase of Pten expression level in Pmp22tg nerves is more evident) Panel a and b: the statement that Pten is more expressed at P18 at the peak of myelination in wildtype nerves is not supported by the blots as shown

    Figure S1, page 4: what does it mean "in line with this finding we were unable to detect protein-protein...". May be the authors meant: since there is a direct correlation between Pmp22 and Pten expression levels in the mutants, the authors explored the possibility of an interaction between the two. Regarding the co-IPs, in panel a, the co-IP at the endogenous level, the immunoprecipitation efficiency of PMP22 is very low. May be a pull-down experiment using either exogenous purified PMP22 or PTEN and nerve lysates can help to rule out the possibility of an interaction. The experiments in b, c are performed in overexpression in a heterologous system (293 cells).

    Panel e: may be with this experiment the authors aim to suggest that Pten and Pmp22 are unlikely to interact directly or indirectly since Pten is cytosolic and Pmp22 myelin-membrane enriched. However, this myelin purification shows that Pmp22 as P0 expression levels are also abundant in the cytosol, may be also because P18 has been chosen as time point. What about a different type of membrane-cytosol fractionation experiment and/or another time point?

    Page 4: "Taken together, Pmp22 dosage inversely correlates with the abundance of PTEN...": please revise this statement

    Additional experiments are needed to support the conclusion of Figure 1 that, in the two mutants, Pten levels reversely correlate with PI3K-Akt-mTOR pathway activation, which represents the rationale of all further experiments. For example, it should be shown systematically in both mutants both Akt and ERK phosphorylation levels (Akt at both T308 and S473), and mTOR activity read outs. In the previously published paper (Fledrich et al.) only increased Akt phosphorylation in Pmp22+/- nerves was reported, whereas Pmp22tg analysis was focused on the interdependence between Akt and ERK without exploring mTOR activation, which is relevant here.

    Figure 2 The aberrant myelin figures displayed are similar to myelin ovoids preceding degeneration rather than myelin outfoldings. It is also strange that these alterations are in the wildtype cultures treated with RAPA, that instead, in this system, has been reported to increase myelination as it improves protein homeostasis (autophagy, quality control, etc). Also for this experiment, pulse treatment may be beneficial rather than in continuous. Is Akt-mTOR phosphorylation-signaling increased also in Pmp22+/- co-cultures as in mutant nerves? Is the treatment reducing the overactivation?

    Figure 3 The RAPA treatment seems to increase Pten level in the mutant even above wildtype levels (panel b), which can result in decreased myelin thickness due to downregulation of Akt-mTOR. A different method to normalize expression levels should be used. Panel c-e: aberrant fibers should be normalized on total number of fibers and on the area, particularly because RAPA is used. Can these data also be reproduced in quadriceps nerves as tomacula are more prominent in these Pmp22+/- nerves showing less variability due to the prevalence of large caliber axons?

    Figure 4 A different model, the C61 mouse a Pmp22tg overexpressing PMP22 is used here (rather than the CMT1A rat). This should be explained in the results. Is also this model characterized by increased Pten levels in the nerve? And low Akt-mTOR activation for instance?

    The improvement in the number of myelin segments following PTEN inhibition in Pmp22tg co-cultures is very weak.. The 500 nM has instead a consistent effect in reducing myelin segments in the wildtype and I think that these results overall don't support the conclusion that myelination is ameliorated by reducing PTEN activity in Pmp22tg co-cultures. Similarly to Figure 2, is PTEN level increased in Pmp22tg cultures along with Akt-mTOR downregulation?

    Figure 5 As for Figure 4, the use of the mouse transgenic instead of the CMT1A rat should be specified and PTEN, Akt-mTOR expression/activation levels should be checked biochemically also in this model. And quantified (panel c). In panel d overactivation of mTOR (PS6 staining) in Schwann cells is not evident. Panel e: co-cultures are established using ex vivo Dhh-Cre recombination. The downregulation of Pten in the cultures should be documented. Pten Fl/+ Dhh-Cre cultures seem to have axonal fasciculation.

    Figure 6 G-ratio analysis: which are the mean values (numbers) with SEM in the three groups analyzed wildtype, Pmp22tg and Pmp22tg; Pten fl/+; Dhh-Cre? How is Akt-mTOR signaling in the double mutant as compared to Pmp22tg? Is that increased at P18? If more fibers are committed to myelinate in the double mutant as compared to the single Pmp22tg at P18 ,particularly, it is unclear why there is no difference in differentiation marker expression in Figure 7 (Oct6 and Hmgcr).

    Significance

    In conclusion, the correlation between PMP22 and PTEN is a potential interesting observation. However, in my opinion, experiments as shown don't support the conclusion that PMP22 controls PTEN expression level and activity, which is suggested at the basis of the pathogenesis of PMP22 dosage-related neuropathies.

  5. Version published to 10.1101/2021.11.08.467756v1 on bioRxiv