Restoration of defective oxidative phosphorylation to a subset of neurons prevents mitochondrial encephalopathy

Oxidative Phosphorylation (OXPHOS) defects can cause severe encephalopathies and no effective treatment exists for these disorders. To assess the ability of gene replacement to prevent disease progression, we subjected two different CNS-deficient mouse models (Ndufs3/complex I or Cox10/complex IV conditional knockouts) to gene therapy. We used retro-orbitally injected AAV-PHP.eB to deliver the missing gene to the CNS of these mice. In both cases, we observed survival extension from 5–6 to more than 15 months, with no detectable disease phenotypes. Likewise, molecular and cellular phenotypes were mostly recovered in the treated mice. Surprisingly, these remarkable phenotypic improvements were achieved with only ~30% of neurons expressing the transgene from the AAV-PHP.eB vector in the conditions used. These findings suggest that neurons lacking OXPHOS are protected by the surrounding neuronal environment and that partial compensation for neuronal OXPHOS loss can have disproportionately positive effects.

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EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection.Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status.***** Reviewer's comments ***** Referee #1 (Comments on Novelty/Model System for Author): The chosen models for mitochondrial disease are mice conditional knockouts.Mice models of mito disease often do not display similar phenotypes to the human ones but in these cases the models do display encephalopathy and it is that presentation that is being rescued.
Referee #1 (Remarks for Author): The manuscript from Walker and colleagues reports the rescue of the neuron-specific deletion of NDUFS2 and COX10 by adeno-associated viral expression of the respective wild type genes in the CNS.The paper is well written, concise and clear.It is notable that this expression can rescue the encephalopathy associated with either deletion as well as rescue of OXPHOS and, partially, neuroinflammation.Perhaps the most important message from this impressive work is that rescue appears to only be required in a subset (~30%) of neurons.The authors suggest this data is consistent with the defective neurons being protected by the local environment and give several possibilities as to how this may occur.Overall, this is a thorough and impressive piece of work and I recommend publication.I have a couple of minor points that the authors may wish to address: 1.I Recommend that the authors briefly explain why retro-orbital injection is used.2. Fig 5E,F I guess the levels of CI in F is not related to the BN gels in E, or are they ?Irrespective, how were the steady state levels calculated?Why do the authors think the steady state level of CI is low in the COX10 KO when the activity is actually greater than wild type ? 3. 'IBA1 staining revealed similar levels of microglia (Fig. 6B).'I found this a confusing statement.Similar to what ?I think the authors meant that the microglia staining with IBA1 was also increased back to wild type levels in the rescued mice.Please clarify.

Referee #2 (Remarks for Author):
This is an interesting manuscript in which gene therapy is used to restore mitochondrial function to a subset of affected neurons.Mitochondrial function had been compromised via 2 mouse models, a complex I mutant subunit and a complex IV mutant assembly factor.Both models are well established.There is clear evidence for effective gene therapy and thus this paper if of considerable medical interest.
Despite its interest, the work reported is largely observational and would benefit from mechanistic enhancement.Some specific points are: 1.There are a number of sex-specific effects.For example, reintroduction of NDUFS3 in females (Fig. S1B) was not as successful as that observed in Fig. 3, yet there was a rescue of the phenotype.Also, mitochondrial mass (VDAC) is increased 30-40% (Fig. 3D) on reintroduction of NDUFS3 in males but trends lower in females (S1D).Can the authors clarify this? 2. Total protein was used to normalize the protein levels in immunoblots.The authors have selected one specific size for the normalization.Also, there are multiple bands shown in each figure.Could the authors provide details of which specific band was used to quantify and the rationale?Why was total protein better than the usual housekeeping markers? 3. Since the end product of OxPhos is ATP, it's surprising that mitochondrial ATP levels are not shown.Inclusion of ATP levels after gene reintroduction may help understand the individual complex levels and activities.
4. Some results are not consistent with previous observations.For example, in their Peralta et al. paper (JCI), COX I shows a 3fold increase at 4 mo in their Ndufs3 KO whereas in the current manuscript there is no increase (Fig. 3c).

5.
A result like that in Fig. 4J showing about 70% overlap of COX1 and NeuN -does this mean that some neurons don't contain COX1 or is there a technical limitation?Also, what is the meaning of a result like ~30% COX1+NeuN in that figure or about 40% NDUFS3 expression in Fig. 3B?Is the knockout incomplete?And why is the level at 5 months so much higher than the ~20% level at only 4 months in the JCI paper?Referee #3 (Remarks for Author): The manuscript entitled "Restoration of Defective Oxidative Phosphorylation to a Subset of Neurons Prevents Mitochondrial Encephalopathy" describes a study in which gene replacement approaches are used to treat two distinct mouse models of mitochondrial encephalopathy.In general, the results are promising as each disease mouse model does appear to benefit from their respective treatment strategy.My major concerns are related to the presentation of male/female data -this may impact effect sizes as your cohort numbers are low.Also, in several instances throughout the manuscript a significant improvement is declared due to differences between affected and affected-treated mice despite there being no difference between WT heathy controls and affected mice to begin with.My specific comments are as follows: 1) Throughout the manuscript, at times male and female data are combined and at other times they are separated.Based upon your data, sex appears to be a significant biological variable.Male/female data should be presented separately (different bars) but displayed in the same graphs in the main figures of the manuscript.Exact numbers of males and females included per cohort or experiment need to be indicated in figure legends.
2) Present doses in vg/kg.3) Figure 2B -Asterisk markings are not clearly positioned -significance appears to be between treated and GFP-treated onlynot between GFP-treated and WT.Males and females should be separated for all data presented.Exact number included per cohort needs to be indicated.4) Figure 2E -As open field tests apparently show no significant differences between WT and affected mice at any age, this is not very interesting and could be supplemental data.5) Page 6 -1st paragraph -It is an overstatement to say that Ndufs-nKO mice show a trend in increase in mtDNA copy numbers based upon the data presented.Also, gene replacement does not appear to have any significant effect on this.6) The mouse cohort numbers used throughout this manuscript are very low.Significant differences are the only ones that should be called out -there are many instances where things are described as elevated or increased when the data show no significant differences.This needs to be corrected throughout the manuscript -almost every figure.The data that show no significant differences between groups can be moved to supplemental data.7) Cox10-nKO female treated/untreated data need to be provided.8) Figure 6 -female data need to be provided and presented alongside the male data.9) Figure 7 -female data need to be provided and presented alongside the male data.10) Figure S2 -males and females are not indicated and should be presented separately.11) Based upon Figure 7 data it seems there may be a loss of NueN+ cells over time in KO-GFP mouse brains.Unclear if this is true for both models or only one but loss of a cell type does sometimes result in altered AAV vg persistence in tissues.12) Figure 8 should be supplemental data and should include images from both models.13) Some supplemental figures and legends need editing.S1 title says female mice but the legend says male mice.S3 title only mentions one model not both.14) Some discussion on what other cell types may have been transduced by this vector is needed.15) The discussion over-states the results in many cases as the treatment effects in females are not as significant or were not presented.16) Some discussion needed for why are there differences between the treated and affected mice when there were no differences between WT and affected mice for many of the assessments presented.17) Some discussion needed regarding differences between cell type affected in the models used (only neurons) and those impacted in the human clinical presentation of these disorders.These are good mouse models but residual functional proteins in surrounding non-neurons or potential for overexpression from AAV transduction of other non-neuron cells may have an impact on the mouse phenotypes and can be discussed.
We would like to thank the reviewers for their constructive suggestions and corrections.Below, we address the specific points raised.
Referee #1 1) I recommend that the authors briefly explain why retro-orbital injection is used.Retro-orbital injection, an established delivery method, was used for intra-venous administration due to the size of the mice.Although tail vein administration allows for more volume to be injected, it can be difficult to perform in small mice, causing distress to the mice, and has a high rate of failure.Our lab, as well as several others, has successfully utilized retro-orbital injections for multiple mouse models and viral constructs.A review from 2001, describes the method and highlights its advantages (Yardeni et al, 2011).We added this explanation to the Methods (page 15, line 12).
2) Fig 5E,F I guess the levels of CI in F is not related to the BN gels in E, or are they ?Irrespective, how were the steady state levels calculated?Why do the authors think the steady state level of CI is low in the COX10 KO when the activity is actually greater than wild type ?The Complex I steady state levels were calculated via densiometric measurements of the CI+CIII supercomplex band and normalized to the CII band of the same blot (using a Chemidoc detection system, BioRad).Activity levels were calculated in a similar manner and normalized to the CII band of the blot run with the sample samples.After reviewing the Reviewers comments, we reanalyzed the activity gel and noticed a very uneven background.We re-analyzed the gel using better background correction and confirmed the visual inspection that KO+GFP mice had decreased CI activity, which is also in agreement with the CI SC steady state levels.Figure 5H was revised accordingly.
3) IBA1 staining revealed similar levels of microglia (Fig. 6B).'I found this a confusing statement.Similar to what ?I think the authors meant that the microglia staining with IBA1 was also increased back to wild type levels in the rescued mice.Please clarify.Our wording was unclear regarding the IBA1 staining.We have corrected the text to state that the pattern was similar to the GFAP pattern (i.e.increased in the KO and normalized in the treated).Page 8, line 12-13.

Referee #2
1.There are a number of sex-specific effects.For example, reintroduction of NDUFS3 in females (Fig. S1B) was not as successful as that observed in Fig. 3, yet there was a rescue of the phenotype.Also, mitochondrial mass (VDAC) is increased 30-40% (Fig. 3D) on reintroduction of NDUFS3 in males but trends lower in females (S1D).Can the authors clarify this?Although Ndufs3-nKO+Ndufs3 females did not have as great of a molecular recovery of NDUFS3 (by western blots), we did see an approximately 30% increase in Complex I supercomplex assembly and activity.Besides the fact that western blots are semi quantitative assays, this observation further supports our later point that a relatively small percentage of neurons transduced can have a disproportional beneficial phenotypic effect.It is unclear why 7th Jun 2024 1st Authors' Response to Reviewers VDAC trends lower in female Ndufs3-nKO+eGFP.Mouse data can be variable, and the small differences between males and females for Ndufs3 and VDAC1 may not be sex-related.
2. Total protein was used to normalize the protein levels in immunoblots.The authors have selected one specific size for the normalization.Also, there are multiple bands shown in each figure .Could the authors provide details of which specific band was used to quantify and the rationale?Why was total protein better than the usual housekeeping markers?Regarding total protein, the entire lane was quantified and used for normalization; however, to have a more proportional figure panel, we cropped the image to show the 25-35kDa range, where our two proteins of interest lie.There are different views on what the best loading controls are for western blots, but some housekeeping proteins, such as β-actin, GAPDH, and α-tubulin have been shown to not be good loading controls for neuropathological samples, as they can change in response to different stresses (Goasdoue et al, 2016).Other studies showed that beta-actin is not a reliable marker (Dittmer & Dittmer, 2006).Total protein normalization is arguably more accurate as it includes a large number of endogenous proteins (Aldridge et al, 2008).
3. Since the end product of OxPhos is ATP, it's surprising that mitochondrial ATP levels are not shown.Inclusion of ATP levels after gene reintroduction may help understand the individual complex levels and activities.Although we agree with the Reviewer that data on ATP levels would be interesting, ATP assays are more accurate when using fresh or freshly frozen samples.Moreover, ATP levels generated from glial cells, which contain a large amount of glycolytic astrocytes(Takahashi, 2021) and oligodendrocytes (Funfschilling et al, 2012), could mask differences in homogenates.
4. Some results are not consistent with previous observations.For example, in their Peralta et al. paper (JCI), COX I shows a 3-fold increase at 4 mo in their Ndufs3 KO whereas in the current manuscript there is no increase (Fig. 3c).We have noted some small differences in COX1 levels between this group of Ndufs3-nKO mice when compared to our previous paper; however, in Peralta et al. we normalized protein levels to -actin, whereas we now normalized western bands to total protein loading.Additionally, we now analyzed mice at a slightly later time point, which may affect these changes.5.A result like that in Fig. 4J showing about 70% overlap of COX1 and NeuNdoes this mean that some neurons don't contain COX1 or is there a technical limitation?Also, what is the meaning of a result like ~30% COX1+NeuN in that figure or about 40% NDUFS3 expression in Fig. 3B?Is the knockout incomplete?And why is the level at 5 months so much higher than the ~20% level at only 4 months in the JCI paper?This is likely a technical issue.Sometimes the confocal plane in WT neurons shows part of the neuron with nuclear NeuN staining but very little (thin) cytoplasm.Regarding the nKO samples, the knockout occurs only in CamKII-positive neurons, leaving a population of CamKIInegative neurons unaffected and Cox+.As discussed above, our subunit quantification results sligthly varied from Peralta et al. due to differences in normalization methods.Moreover, western blots are prone to variability if perfomed in different conditions/different days.Figure 3B shows the quantification of proteins in cortex homogenates, which contain neuronal and nonneuronal cells.The fact that we analyzed samples at 5 months could have influenced the levels of Ndufs3 or COX1, as there are more astrocytes at 5 months than at 4 months.
Referee #3 1) Throughout the manuscript, at times male and female data are combined and at other times they are separated.Based upon your data, sex appears to be a significant biological variable.Male/female data should be presented separately (different bars) but displayed in the same graphs in the main figures of the manuscript.Exact numbers of males and females included per cohort or experiment need to be indicated in figure legends.In toto, our data suggests that sex is not a significant biological variable in these analyses.However, as requested, we have separated out the data (Fig. 2).We have updated the figure legend to specify males/females per cohort.
3) Figure 2B -Asterisk markings are not clearly positionedsignificance appears to be between treated and GFP-treated onlynot between GFP-treated and WT.Males and females should be separated for all data presented.Exact number included per cohort needs to be indicated.We have corrected and clarified the weight graphs to better show the statistical differences.4) Figure 2E -As open field tests apparently show no significant differences between WT and affected mice at any age, this is not very interesting and could be supplemental data.We agree with the Reviewer and have moved this data to supplemental figures.5) Page 6 -1st paragraph -It is an overstatement to say that Ndufs-nKO mice show a trend in increase in mtDNA copy numbers based upon the data presented.Also, gene replacement does not appear to have any significant effect on this.We agree with the Reviewer and removed these data.
6) The mouse cohort numbers used throughout this manuscript are very low.Significant differences are the only ones that should be called outthere are many instances where things are described as elevated or increased when the data show no significant differences.This needs to be corrected throughout the manuscriptalmost every figure.The data that show no significant differences between groups can be moved to supplemental data.We agree with the Reviewer and have modified those instances accordingly.The numbers were not high because of a combination of factors, such as obtaining enough mice (at the same time) with the required multiple alleles for both treatement and control groups as well as the need to inject high titers of rAAV.Nonetheless, based on the robust effect we reached significance in the majority of the analyses.An unfortunate exception was females Cox10-nKO, as described below.7) Cox10-nKO female treated/untreated data need to be provided.….(also in 7 and S2).Due to issues with our mouse colony (unrelated to the study), we lost some animals in the course of the experiment and were unable to obtain enough Cox10-nKO females needed to complete the 6-month-old full cohort.For this reason, we did not provide molecular data for Cox10-nKO females.Nevertheless, we believe that the data we have with Ndufs3 (males and females) and Cox10 (males) are strong enough to support our conclusions.8) Figure 6 female data need to be provided and presented alongside the male data.We agree with the Reviewer and have prepared a figure containing these data (Ndufs3-nKO), for an expanded view figure (Fig. EV2).11) Based upon Figure 7 data it seems there may be a loss of NeuN+ cells over time in KO-GFP mouse brains.Unclear if this is true for both models or only one but loss of a cell type does sometimes result in altered AAV vg persistence in tissues.Neuronal loss was not observed in the Ndufs3-nKO mice at 5 months.We did observe neuronal loss in the Cox10 nKO males at 6 months.Neuronal loss could certainly play a role in the AAV persistence in brain.However, because we quantified GFP+ cells among NeuN+ cells, neuronal loss was accounted for.
Figure 8 should be supplemental data and should include images from both models.We believe Figure 8 backs up one of the most important conclusions of this work, as it shows that a relatively small percentage of neurons expressing the rAAV transgene is sufficient to prevent the phenotype.We have moved several parts of the other figures to extended view, but we would like to keep Figure 8 as a main figure.Representative images were included to illustrate the quantification, and the full data sets are provided for both models.13) Some supplemental figures and legends need editing.S1 title says female mice but the legend says male mice.S3 title only mentions one model not both.We have corrected the Supplemental figures and legend errors identified.
14) Some discussion on what other cell types may have been transduced by this vector is needed.AAV-PHP.eBdoes transduce other cell types, however the synapsin promoter is neuronal specific (Kugler et al, 2003).In clinical settings, where multiple tissues are affected, broad transduction could be beneficial and a ubiquitous promoter could be used.In our work, the genes of interest, when fully knocked out, are embryonic lethal.By utilizing a cell specific knockout, we were able to address whether a neuron that is experiencing a severe OXPHOS complex deficiency can be rescued via gene replacement.We used the synapsin promoter to assure robust expression in neurons, the affected cells.As discussed, there may be neuroprotection either via mitochondrial transfer, the neural milia, stem cells, or other mechanisms.We hope to explore the role of mitochondrial transfer in the future, however this is beyond the scope of the current work.We did add this clarification to the Discussion (page 12, lines 27-32).
15) The discussion over-states the results in many cases as the treatment effects in females are not as significant or were not presented.We have clarified that the data are based on both sexes for Ndufs3 (Paragraph starting at line 18, page 5) and males for Cox10 (page 6, line 20).16) Some discussion needed for why are there differences between the treated and affected mice when there were no differences between WT and affected mice for many of the assessments presented.
Between treated and not treated mice, we found clear and significant differences for the key players in the pathogenesis, including: levels of the knocked out protein (NDUFS3 or COX1 [surrogate of COX10]), levels of the affected OXPHOS complexes, neuropathology and behavioral phenotypes.As most work with mice, there is some variability.Still, we believe this work provides solid evidence that the onset of the neuropathy could be prevented by using the AAV-PHP.eBvectors in the neuronal KO models.As an extreme example, we had a group of treated Ndufs3 nKO mice sacrificed at 15 months, and they showed essentially no phenotype.We have never had a Ndufs3-nKO living beyond 7 months (usually they have overt phenotypes by 5 months and all dead by 6 months with massive neurodegeneration).Thank you for the submission of your revised manuscript to EMBO Molecular Medicine.I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) Please address all the points and implement all suggestions raised by the referee #3.Acceptance of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript.For this reason, and to save you from any frustrations in the end, I would strongly advise against returning an incomplete revision.

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Currently, our records indicate that the ORCID for your account is 0000-0002-8077-7092.This revised manuscript is improved but there remain a few points that should be addressed.Figure 2 (and several other figures) -In general, it would make more sense to have each figure's story flow from left to right and then down as opposed to top to bottom and then back up to the top.Also, for figure 2, your reader is more likely to compare top of B to top of D (to see when weight phenotype -or corrected one -correlates to a functional deficit or correction) as your two mouse models are not directly compared in the analyses.In other words, try to keep Ndufs3 data together and COX10 data together in panels or label each graph separately with panel letters.Figure 2B and Page 4 Line 15 -Why do the female COX10 KO AAV-COX10 treated mice weigh so much more than their WT controls?This divergence appears to unique to females and thus indicates that sex is a biological variable and should be mentioned and discussed.The statement that the treated groups "continued to gain weight, comparable to the WT mice" is not accurate as your data indicates the treatment has a peculiar effect on female COX10 mice. Figure 2C -Survival curves should be presented with male and female data separated.This is especially important for the COX10 cohort.Figure 2D -statistics should be run between males and females in these graphs -then you can say more definitively whether there is or isn't a M/F difference in this assessment.Figure 3 and onwards -When cropped images are used, the full blot images are typically provided as supplemental or appendix data -perhaps I missed this, but I do not see them.Page 6, Line 18 -I understand the need to use COX1 as a surrogate for COX10, however, the wording of this section should be changed because COX10 levels are not directly measured.There may be COX10 expression present in neurons that was below your detection level based upon the COX1 surrogate -or not enough expressed to correct the COX1 localization phenotype.I understand your general point, but it isn't accurate to title this subsection "COX10 levels are increased" when that is not actually assessed.Page 7, Line 9 -Was this actually COX10 staining or COX1?This needs to be accurately described.
Reviewers 1 and 2 were satisfied with the previous responses and did not have further concerns.
Reviewer 3 suggestions are addressed below.1) Figure 2 (and several other figures) -In general, it would make more sense to have each figure's story flow from left to right and then down as opposed to top to bottom and then back up to the top.Also, for figure 2, your reader is more likely to compare top of B to top of D (to see when weight phenotypeor corrected onecorrelates to a functional deficit or correction) as your two mouse models are not directly compared in the analyses.In other words, try to keep Ndufs3 data together and COX10 data together in panels or label each graph separately with panel letters.We changed Figures 2 and 8 as requested.We believe the other figures have a good flow.
2) Figure 2B and Page 4 Line 15 -Why do the female COX10 KO AAV-COX10 treated mice weigh so much more than their WT controls?This divergence appears to unique to females and thus indicates that sex is a biological variable and should be mentioned and discussed.The statement that the treated groups "continued to gain weight, comparable to the WT mice" is not accurate as your data indicates the treatment has a peculiar effect on female COX10 mice.Most of the Cox10 nKO females were indeed heavier than the WT females at early age.We do not have an explanation for this (other than mouse variability) as it is not a consistent finding with this line.What the data showed clearly is that both males and females with a Cox10 neuronal KO start to lose weight at 4 months.This was also observed in our previous report (Diaz et al., 2012) for the Cox10 nKO.A similar pattern was observed for the Ndufs3 nKO (Fig. 2C).In contrast, AAV-PHP.eB-Cox10injected mice did not show weight loss (Fig. 2E).We changed the text to read: "On the other hand, after administration of the respective conditionally deleted genes, both OXPHOS-nKO models showed normal posture and no weight loss".
3) Figure 2C -Survival curves should be presented with male and female data separated.This is especially important for the COX10 cohort.We separated by sex as requested (new Fig. 2B).
4) Figure 2D statistics should be run between males and females in these graphsthen you can say more definitively whether there is or isn't a M/F difference in this assessment.
Statistics were run between male and females for each group.Only COX10-nKO+GFP animals showed a significant difference in RotaRod performance and only at 4 months of age (exact P values now provided in supplementary table S3).Therefore, most data points showed that males and females behaved similarly.
5) Figure 3 and onwards -When cropped images are used, the full blot images are typically provided as supplemental or appendix dataperhaps I missed this, but I do not see them.
Our apologies.The source data was compiled but not attached to the submission.These are now provided as "Source Data." 6) Page 6, Line 18 -I understand the need to use COX1 as a surrogate for COX10, however, the wording of this section should be changed because COX10 levels are not directly measured.There may be COX10 expression present in neurons that was below your detection level based upon the 14th Jul 2024 2nd Authors' Response to Reviewers COX1 surrogateor not enough expressed to correct the COX1 localization phenotype.I understand your general point, but it isn't accurate to title this subsection "COX10 levels are increased" when that is not actually assessed.We agree with the Reviewer and have corrected the title to read "COX1 levels" 7) Page 7, Line 9 -Was this actually COX10 staining or COX1?This needs to be accurately described.
We agree with the Reviewer and have corrected the manuscript to state "COX1 staining" 16th Jul 2024 2nd Revision -Editorial Decision 16th Jul 2024 Dear Prof. Moraes, We are pleased to inform you that your manuscript is accepted for publication and is now being sent to our publisher to be included in the next available issue of EMBO Molecular Medicine.Your manuscript will be processed for publication by EMBO Press.It will be copy edited and you will receive page proofs prior to publication.Please note that you will be contacted by Springer Nature Author Services to complete licensing and payment information.
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Aldridge GM, Podrebarac DM, GreenoughWT, Weiler IJ (2008)  The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting.J Neurosci Methods 172: 250-254 Dittmer A, Dittmer J (2006) Beta-actin is not a reliable loading control in Western blot analysis.Electrophoresis 27: 2844-2845 Funfschilling U, Supplie LM, Mahad D, Boretius S, Saab AS, Edgar J, Brinkmann BG, Kassmann CM, Tzvetanova ID, Mobius W et al (2012) Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity.Nature 485: 517-521 Goasdoue K, Awabdy D, Bjorkman ST, Miller S (2016) Standard loading controls are not reliable for Western blot quantification across brain development or in pathological conditions.Electrophoresis 37: 630-634 Kugler S, Kilic E, Bahr M (2003) Human synapsin 1 gene promoter confers highly neuron-specific longterm transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area.Gene Ther 10: 337-347 Takahashi S (2021) Neuroprotective Function of High Glycolytic Activity in Astrocytes: Common Roles in Stroke and Neurodegenerative Diseases.Int J Mol Sci 22 Yardeni T, Eckhaus M, Morris HD, Huizing M, Hoogstraten-Miller S (2011) Retro-orbital injections in mice.Lab Anim (NY) 40: 155-160 -6C and Figure4A,E-7E).Please indicate this clearly in the figure legends and make sure that the blots displayed in different figures are indeed form the same experiment.3)In the main manuscript file, please do the following: -Please address all comments suggested by our data editors listed below: o Figurelegends:1.Please note that the exact p values are not provided in the legends of figures 2b, d; 3b-c, e-h; 4b-d, f, j; 5b, d, f; 6d; 7b, f-g; 8b; EV 1b-c, f-j, l. 2. Please note that in figures 2b, d; 4b-d, f, j; 5b, d, f; 7b, f-g; EV 2b; EV 3c, e; EV 5o; there is a mismatch between the annotated p values in the figure legend and the annotated p values in the figure file that should be corrected.3. Please note that for the figures EV 4d-e; p-values and statistical tests are indicated in the legends.However, comparison for the same, "****/***/**/*" has not been represented in the figures.Please rectify this in the figures or legends as applicable.
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