Oligodendrocytes depend on MCL-1 to prevent spontaneous apoptosis and white matter degeneration

  1. Abigail H. Cleveland
  2. Alejandra Romero-Morales
  3. Laurent Alfonso Azcona
  4. Melisa Herrero
  5. Viktoriya D. Nikolova
  6. Sheryl Moy
  7. Orna Elroy-Stein
  8. Vivian Gama
  9. Timothy R. Gershon

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Abstract

Neurologic disorders often disproportionately affect specific brain regions, and different apoptotic mechanisms may contribute to white matter pathology in leukodystrophies or gray matter pathology in poliodystrophies. We previously showed that neural progenitors that generate cerebellar gray matter depend on the anti-apoptotic protein BCL-xL. Conditional deletion of Bcl-xL in these progenitors produces spontaneous apoptosis and cerebellar hypoplasia, while similar conditional deletion of Mcl-1 produces no phenotype. Here, we show that, in contrast, postnatal oligodendrocytes depend on MCL-1. We found that brain-wide Mcl-1 deletion caused apoptosis specifically in mature oligodendrocytes while sparing astrocytes and oligodendrocyte precursors, resulting in impaired myelination and progressive white matter degeneration. Disabling apoptosis through co-deletion of Bax or Bak rescued white matter degeneration, implicating the intrinsic apoptotic pathway in Mcl-1 -dependence. Bax and Bak co-deletions rescued different aspects of the Mcl-1 -deleted phenotype, demonstrating their discrete roles in white matter stability. MCL-1 protein abundance was reduced in eif2b5 -mutant mouse model of the leukodystrophy vanishing white matter disease (VWMD), suggesting the potential for MCL-1 deficiency to contribute to clinical neurologic disease. Our data show that oligodendrocytes require MCL-1 to suppress apoptosis, implicate MCL-1 deficiency in white matter pathology, and suggest apoptosis inhibition as a leukodystrophy therapy.

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    Reply to the reviewers

    We are grateful to the editors at Review Commons and to the reviewers for their thoughtful attention to our manuscript. Our work presents data showing that deletion of the apoptosis regulator Mcl-1 in CNS stem cells that give rise to neurons and glia resulted in specific degeneration of the white matter, beginning after postnatal day 7 (P7). Cellular analysis shows that oligodendrocytes were depleted while astrocytes persisted. Co-deletion of apoptosis effectors Bax or Bak rescued different aspects of the Mcl-1 deletion phenotype, confirming the role of apoptosis. Based on these observations, we conclude that oligodendrocytes require MCL-1 to prevent spontaneous apoptosis, and that MCL-1 depletion results in leukodystrophy, which resembles severe cases of the human disorder Vanishing White Matter Disease (VWMD). We further suggest that MCL-1 deficiency, caused by the eIF2B mutations of VWMD, may play a critical role in VWMD pathogenesis.

    The reviewers questioned the similarity of the Mcl-1 deletion phenotype to VWMD and were not convinced that MCL-1 deficiency is integral to VWMD. Based on reviewer feedback, we concede that a firm link to VWMD is not supported by the available data. We consider, however, that our findings that MCL-1 is required for oligodendrocyte survival and white matter stability remain highly significant. Accordingly, we propose to revise the work as suggested by Reviewer 1 to highlight the insight our data provide as to apoptosis regulation in glia and its importance for brain development, and to revise the title, as suggested by Reviewer 3, to remove the specific reference to VWMD.

    In the revision, we will make clear that the comparison to specific leukodystrophies is hypothetical and will require extensive follow-up experiments that are suggested by the findings of this work, as described in the reviews. Revising our work by removing the assertion that our data strongly implicate MCL-1 in VWMD pathogenesis will address the main reviewer concern, strengthen the logical flow, and highlight the potential for MCL-1 to be broadly relevant to white matter pathology. The significance of our findings that oligodendrocytes depend on MCL-1 protein to prevent their spontaneous apoptosis, and that MCL-1 deficiency produces white matter degeneration, will not be altered by these changes. Our data will continue to show that MCL-1 dependence is a physiologic vulnerability of oligodendrocytes that sets them apart from astrocytes and neurons and that this vulnerability is sufficient to cause white matter-specific brain degeneration when MCL-1 expression is blocked.

    The other issues raised by the reviewers are all tractable and can be addressed with new experiments that we can complete in a short time-frame, such as studies of retinal pathology and addition immunohistochemistry studies, or with changes to the text. We consider that with these revisions, the manuscript will be an important contribution to understanding glial biology and the pathogenesis of white matter-specific disorders. We describe in detail below our responses to reviewer feedback and planned changes to the manuscript.

    *Reviewer comments are in italics *and our responses are in plain text.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):*

    **Comments**

    1. MCL-1 promotes the survival of different cell lineages through its ability to inhibit the pro-apoptotic proteins BAK and BAX, the main effectors of cell death in mammalian cells. By depleting brain cells of MCL-1, apoptosis is promoted in these cells, as confirmed by histopathology of the mouse brain. This is, however, a nonspecific process. Depletion of MCL-1 in any tissue would promote apoptosis in cells of this tissue and general knock-out is known to cause embryonic lethality. So, it is no surprise that knock out of MCL-1 in brain cells leads to a brain disease.

    While we acknowledge many important points in this review, this first point is based on a premise that is inaccurate. Based on published data, we respectfully disagree with the statement that “Depletion of MCL-1 in any tissue would promote apoptosis in cells of this tissue”. Most cells do not require an anti-apoptotic protein to prevent spontaneous apoptosis; cells that depend on anti-apoptotic proteins are specifically referred to as “primed for apoptosis” (1-5). Our conditional deletion genotypes ablated Mcl-1 in neurons of the forebrain and cerebellum and in all subtypes of glial cells. The loss of oligodendrocytes in our Mcl-1-deleted mice shows that a specific subset of white matter cells in the postnatal brain require MCL-1. Together with the increase in apoptosis and the rescues by co-deletion of Bax or Bak, these data demonstrate that cells within the oligodendrocyte lineage are primed for apoptosis in a manner that is restricted by MCL-1. In contrast, we have shown in published data that we cite in this manuscript that conditional deletion of Mcl-1 cerebellar granule neurons, the largest neuronal population in the brain, does not cause apoptosis (6); these data provide direct evidence that large populations of cells in the brain do not depend on MCL-1. We therefore disagree with the characterization of the brain-specific Mcl-1 deletion phenotype as “non-specific”.

    • The white matter disease is interpreted as similar to VWM; VWM is specifically investigated and MCL-1 is found to be decreased in VWM brain tissue. The decrease is most likely nonspecific. Decrease in MCL-1 is most likely part of a general mechanism of degeneration of brain tissue or white matter. That is a different but also important conclusion. It is essential that other progressive leukodystrophies and acquired brain diseases with tissue degeneration, such as encephalitis, are investigated as well to see whether MCL-1 is also decreased in these disorders. If so, the MCL-1 decrease in white matter disease and other brain degenerative disease should be described as a final common pathway rather than specifically applicable to VWM.*

    We agree that MCL-1 is likely to be a final common point in multiple disease processes that affect white matter. As described in our response to point 3 below, we are persuaded by the reviewers that the proposed similarity of the Mcl-1 deletion phenotype and VWMD is not sufficiently supported by the available evidence. We will revise the text to make clear that we consider that impaired MCL-1 is “likely part of a general mechanism of degeneration of… white matter”.

    • Adding to point 2 is the fact that the pathology of the brain-specific MCL-1 knock-out mouse does not resemble the pathology of VWM at all. The central features of VWM are abnormal astrocyte morphology with astrocytes having a few stunted processes, lack of reactive astrogliosis, lack of microgliosis, increase in number of oligodendrocytes and presence of foamy oligodendrocytes. The increase in oligodendrocytes in VWM may be such that the high cellularity leads to diffusion restriction on MRI. Bergmann glia are typically ectopic, but not reduced in number. By contrast, the brain-specific MCL-1 knock-out mouse is characterized by decreased numbers of oligodendrocytes, increased numbers of microglia, reactive astrogliosis, decreased numbers of Bergmann glia and ectopic granule cells. No morphological abnormalities of oligodendrocytes and astrocytes are observed. So, histopathologically the only shared feature is preferential involvement of the brain white matter.*

    We are persuaded by the reviewers that our assertion of a high degree of similarity between the Mcl-1 deletion phenotype and VWMD was not adequately supported by our available data. In the revision, we will state that a role for MCL-1 deficiency in VWMD pathogenesis is hypothetical, and that additional studies beyond the scope of this project will be needed to test this hypothesis. However, we reassert that the white matter specificity of the Mcl-1-deletion phenotype is important.

    The reviewer accurately characterizes the pathology of the Mcl-1 deletion phenotype and notes “the preferential involvement of the white matter”. We consider that the preferential involvement of white matter, and of oligodendrocytes within the white matter are highly significant. We will revise the work to focus on the Mcl-1 deletion phenotype, the white matter specificity, and the potential relevance to diverse white matter-specific disease.

    While we concede that more data would be needed to firmly connect MCL-1 to VWMD, we do not agree that the Mcl-1 phenotype “does not resemble the pathology of VWM at all”. There is a diversity of published observations of pathology in VWMD and not all published reports support the descriptions in the reviewer comment. This diversity of findings is highly relevant to our work. For example, while autopsy studies of humans with end stage VWMD show lack of microgliosis (7), studies of mice with a mutation known to cause VWMD in humans, that clearly recapitulate VWMD, show robust microgliosis earlier in the disease process (8). These different observations raise the possibility that microgliosis occurs during the period of active neurodegeneration or at least that in murine brain, the VWMD process activates a microglial reaction. Either interpretation would support a likeness between Mcl-1-deleted mice and VWMD mouse models. Another study of cerebellar pathology in twin human fetuses with characteristic VWMD mutations showed complete absence of Bergmann glia (9). We propose in the revision to address the reviewer’s concerns by presenting the diversity of perspectives on microglial reaction and Bergmann glial changes in VWMD, including all of the citations above.

    • The clarity of the work would benefit from a different approach to introduce the study. It would help the reader to know that (1) gray matter cell specific Mcl-1 deletion in mice did not cause apoptosis and (2) apoptosis may have different effector proteins. This important information is now in the discussion. The switch to another cell type in the brain (hGFAP+ cells) would be logical and the significance of the work may improve. When approaching the topic from the field of leukodystrophies one would not necessarily think of deleting the Mcl-1 gene, especially as this gene is not associated with any known leukodystrophy and tends to associate with preneoplastic and neoplastic disease.*

    We appreciate these suggestions, which we agree will enhance the logical flow and the significance, in line with our response to point 3. We will revise the Introduction as suggested.

    • The authors claim that the ISR is activated in VWM, which means that eIF2α phosphorylation levels are increased, general protein synthesis is decreased and a transcription pathway is regulated by ATF4 and other factors. However, this is not what is seen in VWM. Increased eIF2α phosphorylation and reduced general protein synthesis are not observed in VWM; strikingly, the level of eIF2α phosphorylation is reduced, general protein synthesis appears at a normal rate, and only the ATF4-regulated transcriptome is continuously expressed in VWM astrocytes. *

    This point is not well-settled, as published studies show that the ISR is activated in VWMD despite decreased eIF2α phosphorylation (10, 11). Published scRNA-seq studies of mice with VWMD mutations moreover, show that the ISR transcriptome is activated in oligodendrocytes, as well as neurons, endothelial cells and microglia (8). We will address this concern in the revision by citing these published reports that show both decreased eIF2α phosphorylation and lines of evidence that support ISR activation.

    *Fritsh et al. show that MCL-1 protein synthesis is reduced by increased eIF2α phosphorylation due to reduced translation rates at the Mcl-1 mRNA and not due to differences in Mcl-1 mRNA levels. *

    We agree with this interpretation of Fritsh et al, which is fully compatible with our proposed mechanism. We suggest that ISR activation in VWMD decreases translation of Mcl-1 mRNA, leading to reduced MCL-1 protein expression. MCL-1 protein is rapidly degraded and may therefore be a more sensitive detector of impaired translation than other readouts. We currently cite published work documenting altered translation in VWMD in the manuscript and in the revision will add the reference Moon et al, which is directly on point (11).

    *One would a priori not expect to find altered MCL-1 synthesis rates in the mildly affected VWM mouse model Eif2B5R132H/R132H. *

    The model does not show reduced global translation under normal conditions, but rather hypo-activity of eIF2B affects the translation of specific mRNAs (12). We will make this point clear in the revision.

    *Actually, ISR deregulation has not been reported in the Eif2B5R132H/R132H VWM mouse model. The authors need to rephrase this part of their study taking this information into account, when explaining their experiments and interpreting their results. *

    Consistent with the data that the ISR is activated in VWMD, mice show ATF4 up-regulation and other evidence of ISR activation (13) and impaired responses to physiologic stress (14, 15). In the revision, we will add these citations. To address the reviewer concerns, we will state in the revision that ISR activation is one of many potential mechanisms of reduced MCL-1 expression.

    The authors now imply that their study adds mechanistic insight into the VWM field and that is not the case.

    As we describe in response to point 3, we will acknowledge in the revision that the assertion that MCL-1 deficiency causes VWMD is hypothetical.

    In addition, Figure 7C shows differences in actin signal rather than MCL-1 signal, suggesting that transfer of the actin protein from the gel to the blot was not optimal for the middle lanes. MCL-1 protein may thus not be reduced in these samples from Eif2B5R132H/R132H VWM mice.

    We stand by our Western blot data that show that MCL-1 levels are lower in the Eif2B5R132H/R132H VWM mouse model, coincident with the onset of symptoms. The Western blot shown is a representative image that includes 3 biological replicates for each condition and of a total of 12 mice. The quantification demonstrates the reproducibility of the finding.

    • Can the authors show in which cell type was apoptosis found (lines 315-316)? Their study uses the hGFAP - Cre mouse model to generate conditional Mcl-1 knock-out mice. The original paper by Zhuo et al. describing the hGFAP - promoter mouse model suggests that Mcl-1 expression is also affected in neurons and ependymal cells. The authors can investigate this further to assess which cell types (1) are sensitive to apoptosis by Mcl-1 deletion and (2) depend on Bax and Bak.*

    Apoptosis may occur at different times in different cell populations, and asynchronous apoptosis can be difficult to detect at any point in time, which can complicate the suggested studies. Despite significant effort, we have not been able to co-localize any markers with dying cells in our model.

    To address the question of neuronal involvement, the revised manuscript will refer to prior published studies (16-18) which show that Mcl-1 deletion affects forebrain neural progenitors. In this context, we will discuss that our Mcl-1 deletion studies show that significant neural progenitor populations survive prenatal *Mcl-1 *deletion and generate appropriate cortical and hippocampal architecture in Mcl-1-deleted mice at P7, prior to the onset of white matter degeneration.

    To identify involved glial cells, we quantified the cells that were depleted or persisted in the Mcl-1 deleted brain. These studies identified oligodendrocytes and Bergmann glial as cell types depleted during P7-P15, when postnatal degeneration occurs in Mcl-1 deleted mice. In contrast, astrocytes persisted, indicating that astrocytes are not MCL-1-dependent. In the review, we will add new data quantifiying the immature, PDGFRA-expression subset of oligodendrocytes, which will increase the specification of which cells are depleted by Mcl-1 deletion.

    We share the reviewer’s interest in the question of which subsets of Mcl-1 dependent cells are rescued by co-deletion of Bax or Bak. As known markers may not be sufficient to distinguish these subsets, we consider that scRNA-seq studies are an ideal approach to identify these subsets and their specific gene expression patterns. However, these studies are outside the scope of the present work, which establishes that specific white matter cells depend on Mcl-1.

    • Heterozygous deletion of Bak greatly reduces the number of Bak-expressing cells (Fig. 3C, line, 331-333). Authors need to explain this remarkable finding. *

    As we state in the text, the reduced Bak expression in the heterozygous Bak +/- mice is consistent with a gene dosage effect, which has been observed for other genes.

    *Please provide raw IHC data. *

    Our IHC data is “raw” in the sense of unaltered. We are happy to include a supplementary figure with additional low power and high-power images of BAK staining.

    *Co-staining with neuronal, astrocytic or oligodendrocytic markers would be insightful. *

    To address this point, we have successfully performed double labeling with antibodies to BAK and with antibodies to the oligodendrocyte marker SOX10 and the astrocyte marker GFAP. We will add these images to the revision. These images show that BAK+ cells include oligodendrocytes and astrocytes. The position and morphology of the BAK+ cells show that they are not neurons.

    *In addition, what does the Western blot signal for the BAX protein represent in Bax homozygous knock out mice (Fig. 3C)? *

    We will add text stating that the small residual BAX protein detected in the conditional Bax-deleted mice can be attributed to BAX expression in cells outside the *Gfap *lineage, including endothelial cells, vascular fibroblasts, and microglia.

    Can the percentage of BAX+ cells in Mcl-1/BaxdKO corpus callosum be determined, similarly as was done for BAK? Co-staining with neuronal, astrocytic or oligodendrocytic markers would be insightful here as well. The legend of Fig. 3D does not state what staining is shown (H&E?).

    We were not able to label BAX protein in individual cells using immunohistochemistry. In contrast, BAK immunohistochemistry worked well, allowing us to analyze the cellular distribution of BAK protein. We will revise the legend in 3D to state the staining is H&E.

    • What explains the strong GFAP expression in processes of Mcl-1 KO astrocytes? Are these cells refractory to apoptosis or to hGFAP-driven Cre expression and recombination? Do they lack BAK or BAX or other apopotic-regulating protein? Or do specific factors compensate for the loss of MCL-1?*

    As we discuss in our response to point 1 above, not all cells require MCL-1 to prevent spontaneous apoptosis. The persistence of GFAP+ astrocytes in Mcl-1-deleted mice shows that astrocytes do not require MCL-1 to maintain their survival. These data do not mean that these astrocytes are refractory to apoptosis, but rather they are not primed for apoptosis in a way that is critically restricted by MCL-1. We will add a discussion of these implications to the revision.

    • Which developing symptoms do the authors refer to in line 468? Please specify and introduce appropriate references.*

    We will add a description of symptoms to the revision.

    • The definition of leukodystrophies given in the paper is outdated. Leukodystrophies are not invariably progressive and fatal disorders. For more recent definition of leukodystrophies see Vanderver et al., Case definition and classification of leukodystrophies and leukoencephalopathies, Mol Genet Metab 2015, and van der Knaap et al., Leukodystrophies a proposed classification system based on pathology, Acta Neuropathol 2017.*

    We appreciate this advice. We will revise the Introduction accordingly and cite the recommended work.

    • It is not correct that there is no specific targeted therapy clinically implemented to arrest progression of the disease in any leukodystrophy. Perhaps hematopoietic stem cell transplantation is not specific targeted, although curative if applied in time in adrenoleukodystrophy and metachromatic leukodystrophy, but certainly genetically engineered autologous hematopoietic stem cells would qualify the definition. In any case, the suggestion that no leukodystrophy is treatable is not correct.*

    We appreciate this correction. We will revise the text to provide a more detailed description of treatment options while underscoring the need for mechanistic insight.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, the authors characterize the phenotype associated with brain-specific deletion of the mcl-1 gene in mice as a model for vanishing white matter-like disease in humans. Unfortunately, the gfap gene is expressed in many cell types during development which are outside of the intended cell type for this study, so functional data presented from the mutant mice is open to interpretation. The authors have not ruled out other interpretations of their results. The authors need to address major shortcomings in their data interpretation by addressing the following issues.

    We appreciate that concerns related to vision and hearing in the Mcl-1 deleted mice, and address these concerns as described below.

    On line 57, the authors indicate that seizures are common in leukodystrophy. This is controversial. Patients may have attacks that look like seizures, but without EEG recordings there is no way to distinguish these events from myoclonus. The authors should note this ambiguity.

    We will note this ambiguity in the revision.

    On line 58, the authors indicate the absence of treatments for leukodystrophies. The authors should review the following articles: PMID: 7582569, 15452666 and 27882623, and moderate the text.*

    We will cite these papers and moderate the text as recommended

    *The methods section is lacking in details in several areas. For example beginning line 136, there is virtually no indication of the MRI details without going to secondary literature. The authors should provide a brief description including magnet strength, type of imaging and the general sequence, software used to collect and analyze the images. *

    We will include these details in the revision.

    Were the brains actually harvested fresh, where mechanical stresses easily deform brain structure, prior to immersion fixation for 48h? This could be troubling despite the method being previously published.

    Brains were harvested fresh and drop fixed. We have extensive experience over more than ten years in handling brain tissue from neonatal mice and subsequently analyzing MRI images and sections. These methods have allowed us to make quantitative volumetric comparisons of the 3-dimensional architecture of the developing brain using MRI in prior studies, that detected genotypic differences in brain growth without confounding fixation artefact (19). We can confirm that no mechanical stress of handling can reproduce the white matter specific changes that we see in the Mcl-1-deleted brain. We did not detect any abnormalities in control brains subjected to the same handling techniques.

    Beginning on line126, the authors could at least indicate the fixative details and whether the mice were perfused or tissue was immersion fixed. Compare this lack of detail with the description of lysis buffer beginning on line 158.*

    We will add fixation details to the revision.

    Behavioral testing at young ages is rather problematic regarding data interpretation. For example, open field testing (Fig. 2B) at postnatal day 7, which relies on visual cues, is rather dubious when mice do not open their eyes until 12-13 days after birth. How would the pups know if they were in the middle of an open field and exhibit thigmotaxis, even if they were capable of the behavior at such a young age? Thus, the P7 data likely cannot be interpreted in terms of the knockouts being normal.

    We fully agree with the reviewer on the challenges with behavioral analysis of such young mice. The rationale for the open field test was that, at P7, mouse pups are gaining greater control of hind limb function, which can be observed as a transition from pivoting in one place to forward locomotion. Thus, we measured the number of pivots and distance traveled in the open field as indicators for maturation of motor function. Center time was presented to show that, at P7, both WT and knockout mice stayed in the middle (i.e., the groups were at the same stage of limited mobility). We consider that these measures, together with geotaxis and latency to righting (Table 1), provide a developmentally-appropriate neurologic assessment for an age when behaviors are very limited. We will make clear in the revision that these specific tests must be considered together in order to be informative.

    By P14, when the mutants exhibit a phenotype, they are already significantly underweight, which can lead to non-specific phenotypes such as retinal dysfunction or degeneration. Did the authors look for pathological changes in the retina?

    Further, GFAP is expressed in retina of many vertebrate species (PMID 1283834) which would inactivate mcl1 in that tissue and possibly lead to blindness. Indeed, the table at the following link provides a list of tissues in which the gfap-cre transgene is expressed during development. The authors need to address this major issue. http://www.informatics.jax.org/allele/MGI:2179048?recomRibbon=open

    We appreciate this suggestion and we will look for pathology in the retina and optic nerve. Such pathology, if we find it, is likely to be specific, as the optic nerve is myelinated and we have already noted extensive myelination abnormalities in the Mcl-1-deleted mice. If we find retinal or optic nerve abnormalities, we will note the potential for these abnormalities to impact on open field testing.

    For the startle response, which relies on normal hearing, did the authors check to determine if the mutants are deaf? This is very difficult at such a young age, especially prior to tight junction assembly in the lateral wall at around P14. Again, GFAP is expressed in the cochlea at an early age (see PMID 20817025) and may have caused degenerative pathology in this tissue. The authors need to address this major issue.

    The reviewer brings up the potential issue of deafness as a confounding factor for acoustic startle testing. Our results showed that startle responses in the mutant mice were increased at P14, which clearly indicates the mice were able to hear the acoustic stimuli. Further, at P14 and P21, both WT and knockout mice had orderly patterns of prepulse inhibition, providing confirmation of good hearing ability at each timepoint. We will make these points clear in the revision.

    *Reviewer #2 (Significance (Required)):

    Unknown.*

    The reviewer has not raised specific issues with the significance. We consider the significance of our work to be the finding that oligodendrocyte-lineage glial cells depend on MCL-1 and thus are primed for apoptosis, such that disrupting MCL-1 expression results in catastrophic degeneration of the cerebral white matter. Addressing the reviewer’s concerns described in the section on Evidence, reproducibility and clarity will support this significance.*

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Cleveland et al. tried to argue that brain-specific depletion of apoptosis regulator MCL-1 reproduces Vanishing White Matter Disease (VWMD) in mice. The authors show that brain-specific MCL-1 deficiency leads to brain atrophy, increased brain cell apoptosis, decreased oligodendrocytes, decreased MBP immunoreactivity, and activation of astrocytes and microglia. It is known that VWMD is a hypomyelinating disorder caused by mutation of eIF2B subunits, which displays severe myelin loss but minimal oligodendrocyte apoptosis or loss in the CNS white matter. In fact, a number of studies show increased oligodendrocyte numbers in the CNS white matter.*

    Published reports show decreased normal oligodendrocytes and increased immature oligodendrocyte populations (20).

    *The characteristic oligodendrocyte pathology is foamy oligodendrocytes (Wong et al., 2000), rather than apoptosis. *

    Foamy oligodendrocyte pathology and increased oligodendrocyte apoptosis are not mutually exclusive. The above referenced paper, Wong et al, in addition to foamy oligodendrocytes, also describes a “decrease in numbers of cells with oligodendroglial phenotype, both normal and abnormal” (21); this decrease is compatible with increased apoptosis. Moreover, published reports specifically describe apoptotic oligodendrocytes in human brains with VWMD (22). To address this point, we propose to include both of these citations in the revision to reference foamy oligodendrocyte pathology in VWMD and to state that this pathologic finding does exclude a role for apoptosis in VWMD pathogenesis.

    *Since the CNS pathology of brain-specific MCL-1 deficient mice is drive by brain cell apoptosis, the relevance of this mouse model to VWMD is very limited. *

    Whether apoptosis plays a mechanistic role in VWMD is less clear than this comment suggests, as described in multiple publications (22, 23).

    *The title of this manuscript is misleading, and should be changed. *

    We accept that our statement that Mcl-1-deletion recapitulates VWMD is premature and not adequately supported by the available data. We will revise the title, introduction and discussion accordingly, to focus on the white matter specificity of the Mcl-1-deletion phenotype.

    Moreover, there are a number of major concerns.*

    1. Figure 1 clearly shows severe atrophy of neocortex in Mcl-1 cKO mice; however, the white matter appears largely normal in the cerebellum and brain stem. Mcl-1 cKO mice also display ventricular dilation and possible atrophy of corpus callosum. The authors should discuss severe atrophy of neocortex in Mcl-1 cKO mice and the possibility that ventricular dilation and corpus callosum atrophy result from severe atrophy of neocortex?*

    The cortical atrophy that the reviewer notes begins after P7 and is minimal at P14 when white matter loss is already pronounced. At P21, when there is clear cortical thinning, the white matter loss is extreme. Based on the time course, we consider that the white matter loss is the primary pathology, and the cortical thinning is secondary. Importantly, glial cells populate the cortex as well as the white matter and our cellular data show that oligodendrocytes are reduced in the cortex as well as in the white matter structures. Based on these lines of evidence, we consider that the primary cell type affected is the oligodendroglial population of the glia. We will add a discussion along these lines to the revision.

    We agree that the brain stem is preserved. Our data show that the hGFAP-Cre promoter is least efficient in the brain stem and midbrain regions (Sup Fig.1). We will note this differential efficiency in the revision.

    • The motor and sensory tests in Figure 2 are potential interesting, but their relevance to myelin abnormalities is limited. The authors should perform the behaviors tests that are highly relevant to myelin abnormalities.*

    The tests presented show progressive neurologic impairment, correlating with the onset of neuropathology. In the revision we will note that ataxia and tremor are common features of leukodystrophies and the Mcl-1-deleted mice show both ataxia and tremor.

    • It is well expected that there are increased apoptotic cells in the brain of Mcl-1 cKO mice. The authors should perform double labeling to demonstrate which cell types undergo apoptosis: neurons, oligodendrocytes, or other cell types? On the other hand, Figure 3A shows that there are substantial apoptotic cells in the cerebral cortex, which is consistent with severe cerebral cortex atrophy in Mcl-1 cKO mice, suggesting neuron apoptosis in the cerebral cortex. Neuron apoptosis would further rule out the relevance of Mcl-1 cKO mice to VWMD.*

    These studies would be of interest, but we have not been able to co-label apoptotic cells in the Mcl-1-deleted mice with any marker. In the advanced state of apoptosis when dying cells are detectable by TUNEL staining, the relevant marker proteins have been degraded beyond recognition by IHC. In contrast, the apoptotic marker cleaved caspase-3, which is positive earlier in the apoptotic process and might allow marker co-labeling, was not detectably elevated in the Mcl-1-deleted mice. We attribute the lack of cleaved caspase-3+ cells to the asynchronous nature of the increased cell death, and to the short duration in which dying cells are cleaved caspase-3+. While double label studies of dying cells have been problematic, our studies quantifying each cell type provide information to address the reviewer’s question. Our cell counts show clearly that oligodendrocytes are the primary cell type reduced in number in the Mcl-1 deleted mice.

    • Figure1, 4 the authors use H&E staining to demonstrate white matter loss. H&E staining is good to show general CNS morphology; however, it is impossible to use H&E staining to quantify the integrity of the white matter. The authors should perform specific staining to quantify white matter loss in the mouse models.*

    Our MBP stains later in the paper are used to quantify white matter loss.

    • Figure 5, MBP IHC is good to show general myelin staining, but is not a reliable assay to quantify myelin integrity in the CNS. The authors should perform electron microscopy analysis to quantify myelin integrity in the CNS in the mouse models.*

    Our studies of MBP staining show that the myelinated area in cross sections is significantly reduced in the Mcl-1-deleted mice. Electron microscopy studies cannot show whether the myelinated area is reduced and studies of myelin integrity are not needed to prove that reduced oligodendrocytes correlate with reduced myelination.

    • Figure 6, SOX10 is a marker of oligodendrocytes and OPCs. The authors should quantify the number of oligodendrocytes (using oligodendrocyte markers, such as CC1) and the number of OPCs (using OPC markers, such as NG2). Does deletion of BAK or BAX reduce oligodendrocyte apoptosis in the CNS of Mcl-1 cKO mice?*

    We agree that this is an important question, and we are working to quantify OPCs in the Mcl-1-deleted mice by counting cells labelled with the OPC marker PDGFRA. We will add these data to the revision and discuss their significance when we know what they show.

    • The authors show that the level of MCL-1 is comparable in brain lysates of wildtype and eIF2B5 R132H/R132H mice at the age of 7 months, and moderately decreased in eIF2B5 R132H/R132H mice at the age of 10 months. VWMDis a developmental disorder. Similarly, brain-specific MCL-1 deficiency causes developmental abnormalities in the CNS. The normal level of MCL-1 in 7-month-old eIF2B5 R132H/R132H mice strongly suggests that MCL-1 is not a major player involved in the pathogenesis of VWMD. Does brain-specific MCL-1 deficiency starting at the age of 10 months (using CreERT mice) cause CNS abnormalities in adult mice?*

    *We agree that Mcl-1 deletion in our model disrupts postnatal brain development. Our studies show that in early life, oligodendrocytes depend on MCL-1 to prevent spontaneous apoptosis. It is an interesting, but separate question whether Mcl-1 deletion induced in the adult would also cause a similar phenotype. The suggested studies would take over a year to conduct, and while they are of interest, they are not required to prove our main point, which is that developmental leukodystrophies may result from the dependence of oligodendrocytes on MCL-1. In the revision, we will state that our comparison on the Mcl-1-deletion phenotype to VWMD is hypothetical, and that additional studies are needed to test this hypothesis.

    • Does MCL-1 deletion exacerbate the pathology in eIF2B5 R132H/R132H mice? Moreover, does MCL-1 overexpression rescue the pathology in eIF2B5 R132H/R132H mice? These two experiments are necessary to demonstrate the involvement of MCL-1 in VWMDpathogenesis.*

    *We agree that these are interesting and important studies; however, these studies will require years to complete and extensive resources. These studies are not needed to show that Mcl-1 deletion produces early onset white matter degeneration, which is our main point. As in our response to point 7 above, we will state in the revision that our comparison on the Mcl-1-deletion phenotype to VWMD is hypothetical, and list these experiments as follow up studies that are needed to test this hypothesis.

    *Reviewer #3 (Significance (Required)):

    The study will not significantly advance the understanding of VWMD pathogenesis.*

    We recognize that our assertion of a direct relevance to VWMD was premature, and that additional studies, beyond the scope to this project, are needed to determine if MCL-1 deficiency contributes to VWMD pathology. We agree that the available data do not yet inform VWMD pathogenesis, but these data may become relevant to VWMD as follow-up studies are conducted. The data remain highly relevant to the broad group of leukodystrophies as they demonstrate a physiologic vulnerability of oligodendrocytes that sets them apart from astrocytes and neurons, and thus may play a role in disorders in which oligodendrocyte pathology is central.

    Neuroscientists may be interested in the reported findings.

    We appreciate the reviewer noting the significance for neuroscience.

    My field of expertise: oligodendrocyte, myelin, neurodegeneration, ER stress

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    9. A. Trimouille, F. Marguet, F. Sauvestre, E. Lasseaux, F. Pelluard, M. L. Martin-Negrier, C. Plaisant, C. Rooryck, D. Lacombe, B. Arveiler, O. Boespflug-Tanguy, S. Naudion, A. Laquerriere, Foetal onset of EIF2B related disorder in two siblings: cerebellar hypoplasia with absent Bergmann glia and severe hypomyelination. Acta Neuropathol Commun 8, 48 (2020).
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  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Cleveland et al. tried to argue that brain-specific depletion of apoptosis regulator MCL-1 reproduces Vanishing White Matter Disease (VWMD) in mice. The authors show that brain-specific MCL-1 deficiency leads to brain atrophy, increased brain cell apoptosis, decreased oligodendrocytes, decreased MBP immunoreactivity, and activation of astrocytes and microglia. It is known that VWMD is a hypomyelinating disorder caused by mutation of eIF2B subunits, which displays severe myelin loss but minimal oligodendrocyte apoptosis or loss in the CNS white matter. In fact, a number of studies show increased oligodendrocyte numbers in the CNS white matter. The characteristic oligodendrocyte pathology is foamy oligodendrocytes (Wong et al., 2000), rather than apoptosis. Since the CNS pathology of brain-specific MCL-1 deficient mice is drive by brain cell apoptosis, the relevance of this mouse model to VWMD is very limited. The title of this manuscript is misleading, and should be changed. Moreover, there are a number of major concerns.

    1. Figure 1 clearly shows severe atrophy of neocortex in Mcl-1 cKO mice; however, the white matter appears largely normal in the cerebellum and brain stem. Mcl-1 cKO mice also display ventricular dilation and possible atrophy of corpus callosum. The authors should discuss severe atrophy of neocortex in Mcl-1 cKO mice and the possibility that ventricular dilation and corpus callosum atrophy result from severe atrophy of neocortex?
    2. The motor and sensory tests in Figure 2 are potential interesting, but their relevance to myelin abnormalities is limited. The authors should perform the behaviors tests that are highly relevant to myelin abnormalities.
    3. It is well expected that there are increased apoptotic cells in the brain of Mcl-1 cKO mice. The authors should perform double labeling to demonstrate which cell types undergo apoptosis: neurons, oligodendrocytes, or other cell types? On the other hand, Figure 3A shows that there are substantial apoptotic cells in the cerebral cortex, which is consistent with severe cerebral cortex atrophy in Mcl-1 cKO mice, suggesting neuron apoptosis in the cerebral cortex. Neuron apoptosis would further rule out the relevance of Mcl-1 cKO mice to VWMD.
    4. Figure1, 4 the authors use H&E staining to demonstrate white matter loss. H&E staining is good to show general CNS morphology; however, it is impossible to use H&E staining to quantify the integrity of the white matter. The authors should perform specific staining to quantify white matter loss in the mouse models.
    5. Figure 5, MBP IHC is good to show general myelin staining, but is not a reliable assay to quantify myelin integrity in the CNS. The authors should perform electron microscopy analysis to quantify myelin integrity in the CNS in the mouse models.
    6. Figure 6, SOX10 is a marker of oligodendrocytes and OPCs. The authors should quantify the number of oligodendrocytes (using oligodendrocyte markers, such as CC1) and the number of OPCs (using OPC markers, such as NG2). Does deletion of BAK or BAX reduce oligodendrocyte apoptosis in the CNS of Mcl-1 cKO mice?
    7. The authors show that the level of MCL-1 is comparable in brain lysates of wildtype and eIF2B5 R132H/R132H mice at the age of 7 months, and moderately decreased in eIF2B5 R132H/R132H mice at the age of 10 months. VWMD is a developmental disorder. Similarly, brain-specific MCL-1 deficiency causes developmental abnormalities in the CNS. The normal level of MCL-1 in 7-month-old eIF2B5 R132H/R132H mice strongly suggests that MCL-1 is not a major player involved in the pathogenesis of VWMD. Does brain-specific MCL-1 deficiency starting at the age of 10 months (using CreERT mice) cause CNS abnormalities in adult mice?
    8. Does MCL-1 deletion exacerbate the pathology in eIF2B5 R132H/R132H mice? Moreover, does MCL-1 overexpression rescue the pathology in eIF2B5 R132H/R132H mice? These two experiments are necessary to demonstrate the involvement of MCL-1 in VWMD pathogenesis.

    Significance

    The study will not significantly advance the understanding of VWMD pathogenesis.

    Neuroscientists may be interested in the reported findings.

    My field of expertise: oligodendrocyte, myelin, neurodegeneration, ER stress

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, the authors characterize the phenotype associated with brain-specific deletion of the mcl-1 gene in mice as a model for vanishing white matter-like disease in humans. Unfortunately, the gfap gene is expressed in many cell types during development which are outside of the intended cell type for this study, so functional data presented from the mutant mice is open to interpretation. The authors have not ruled out other interpretations of their results. The authors need to address major shortcomings in their data interpretation by addressing the following issues.

    On line 57, the authors indicate that seizures are common in leukodystrophy. This is controversial. Patients may have attacks that look like seizures, but without EEG recordings there is no way to distinguish these events from myoclonus. The authors should note this ambiguity.

    On line 58, the authors indicate the absence of treatments for leukodystrophies. The authors should review the following articles: PMID: 7582569, 15452666 and 27882623, and moderate the text.

    The methods section is lacking in details in several areas. For example beginning line 136, there is virtually no indication of the MRI details without going to secondary literature. The authors should provide a brief description including magnet strength, type of imaging and the general sequence, software used to collect and analyze the images. Were the brains actually harvested fresh, where mechanical stresses easily deform brain structure, prior to immersion fixation for 48h? This could be troubling despite the method being previously published.

    Beginning on line126, the authors could at least indicate the fixative details and whether the mice were perfused or tissue was immersion fixed. Compare this lack of detail with the description of lysis buffer beginning on line 158.

    Behavioral testing at young ages is rather problematic regarding data interpretation. For example, open field testing (Fig. 2B) at postnatal day 7, which relies on visual cues, is rather dubious when mice do not open their eyes until 12-13 days after birth. How would the pups know if they were in the middle of an open field and exhibit thigmotaxis, even if they were capable of the behavior at such a young age? Thus, the P7 data likely cannot be interpreted in terms of the knockouts being normal. By P14, when the mutants exhibit a phenotype, they are already significantly underweight, which can lead to non specific phenotypes such as retinal dysfunction or degeneration. Did the authors look for pathological changes in the retina?

    Further, GFAP is expressed in retina of many vertebrate species (PMID 1283834) which would inactivate mcl1 in that tissue and possibly lead to blindness. Indeed, the table at the following link provides a list of tissues in which the gfap-cre transgene is expressed during development. The authors need to address this major issue. http://www.informatics.jax.org/allele/MGI:2179048?recomRibbon=open

    For the startle response, which relies on normal hearing, did the authors check to determine if the mutants are deaf? This is very difficult at such a young age, especially prior to tight junction assembly in the lateral wall at around P14. Again, GFAP is expressed in the cochlea at an early age (see PMID 20817025) and may have caused degenerative pathology in this tissue. The authors need to address this major issue.

    Significance

    Unknown.

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    Referee #1

    Evidence, reproducibility and clarity

    Comments

    1. MCL-1 promotes the survival of different cell lineages through its ability to inhibit the pro-apoptotic proteins BAK and BAX, the main effectors of cell death in mammalian cells. By depleting brain cells of MCL-1, apoptosis is promoted in these cells, as confirmed by histopathology of the mouse brain. This is, however, a nonspecific process. Depletion of MCL-1 in any tissue would promote apoptosis in cells of this tissue and general knock-out is known to cause embryonic lethality. So, it is no surprise that knock out of MCL-1 in brain cells leads to a brain disease.
    2. The white matter disease is interpreted as similar to VWM; VWM is specifically investigated and MCL-1 is found to be decreased in VWM brain tissue. The decrease is most likely nonspecific. Decrease in MCL-1 is most likely part of a general mechanism of degeneration of brain tissue or white matter. That is a different but also important conclusion. It is essential that other progressive leukodystrophies and acquired brain diseases with tissue degeneration, such as encephalitis, are investigated as well to see whether MCL-1 is also decreased in these disorders. If so, the MCL-1 decrease in white matter disease and other brain degenerative disease should be described as a final common pathway rather than specifically applicable to VWM.
    3. Adding to point 2 is the fact that the pathology of the brain-specific MCL-1 knock-out mouse does not resemble the pathology of VWM at all. The central features of VWM are abnormal astrocyte morphology with astrocytes having a few stunted processes, lack of reactive astrogliosis, lack of microgliosis, increase in number of oligodendrocytes and presence of foamy oligodendrocytes. The increase in oligodendrocytes in VWM may be such that the high cellularity leads to diffusion restriction on MRI. Bergmann glia are typically ectopic, but not reduced in number. By contrast, the brain-specific MCL-1 knock-out mouse is characterized by decreased numbers of oligodendrocytes, increased numbers of microglia, reactive astrogliosis, decreased numbers of Bergmann glia and ectopic granule cells. No morphological abnormalities of oligodendrocytes and astrocytes are observed. So, histopathologically the only shared feature is preferential involvement of the brain white matter.
    4. The clarity of the work would benefit from a different approach to introduce the study. It would help the reader to know that (1) gray matter cell specific Mcl-1 deletion in mice did not cause apoptosis and (2) apoptosis may have different effector proteins. This important information is now in the discussion. The switch to another cell type in the brain (hGFAP+ cells) would be logical and the significance of the work may improve. When approaching the topic from the field of leukodystrophies one would not necessarily think of deleting the Mcl-1 gene, especially as this gene is not associated with any known leukodystrophy and tends to associate with preneoplastic and neoplastic disease.
    5. The authors claim that the ISR is activated in VWM, which means that eIF2α phosphorylation levels are increased, general protein synthesis is decreased and a transcription pathway is regulated by ATF4 and other factors. However, this is not what is seen in VWM. Increased eIF2α phosphorylation and reduced general protein synthesis are not observed in VWM; strikingly, the level of eIF2α phosphorylation is reduced, general protein synthesis appears at a normal rate, and only the ATF4-regulated transcriptome is continuously expressed in VWM astrocytes. Fritsh et al. show that MCL-1 protein synthesis is reduced by increased eIF2α phosphorylation due to reduced translation rates at the Mcl-1 mRNA and not due to differences in Mcl-1 mRNA levels. One would a priori not expect to find altered MCL-1 synthesis rates in the mildly affected VWM mouse model Eif2B5R132H/R132H. Actually, ISR deregulation has not been reported in the Eif2B5R132H/R132H VWM mouse model. The authors need to rephrase this part of their study taking this information into account, when explaining their experiments and interpreting their results. The authors now imply that their study adds mechanistic insight into the VWM field and that is not the case. In addition, Figure 7C shows differences in actin signal rather than MCL-1 signal, suggesting that transfer of the actin protein from the gel to the blot was not optimal for the middle lanes. MCL-1 protein may thus not be reduced in these samples from Eif2B5R132H/R132H VWM mice.
    6. Can the authors show in which cell type was apoptosis found (lines 315-316)? Their study uses the hGFAP - Cre mouse model to generate conditional Mcl-1 knock-out mice. The original paper by Zhuo et al. describing the hGFAP - promoter mouse model suggests that Mcl-1 expression is also affected in neurons and ependymal cells. The authors can investigate this further to assess which cell types (1) are sensitive to apoptosis by Mcl-1 deletion and (2) depend on Bax and Bak.
    7. Heterozygous deletion of Bak greatly reduces the number of Bak-expressing cells (Fig. 3C, line, 331-333). Authors need to explain this remarkable finding. Please provide raw IHC data. Co-staining with neuronal, astrocytic or oligodendrocytic markers would be insightful. In addition, what does the Western blot signal for the BAX protein represent in Bax homozygous knock out mice (Fig. 3C)? Can the percentage of BAX+ cells in Mcl-1/BaxdKO corpus callosum be determined, similarly as was done for BAK? Co-staining with neuronal, astrocytic or oligodendrocytic markers would be insightful here as well. The legend of Fig. 3D does not state what staining is shown (H&E?).
    8. What explains the strong GFAP expression in processes of Mcl-1 KO astrocytes? Are these cells refractory to apoptosis or to hGFAP-driven Cre expression and recombination? Do they lack BAK or BAX or other apopotic-regulating protein? Or do specific factors compensate for the loss of MCL-1?
    9. Which developing symptoms do the authors refer to in line 468? Please specify and introduce appropriate references.
    10. The definition of leukodystrophies given in the paper is outdated. Leukodystrophies are not invariably progressive and fatal disorders. For more recent definition of leukodystrophies see Vanderver et al., Case definition and classification of leukodystrophies and leukoencephalopathies, Mol Genet Metab 2015, and van der Knaap et al., Leukodystrophies a proposed classification system based on pathology, Acta Neuropathol 2017.
    11. It is not correct that there is no specific targeted therapy clinically implemented to arrest progression of the disease in any leukodystrophy. Perhaps hematopoietic stem cell transplantation is not specific targeted, although curative if applied in time in adrenoleukodystrophy and metachromatic leukodystrophy, but certainly genetically engineered autologous hematopoietic stem cells would qualify the definition. In any case, the suggestion that no leukodystrophy is treatable is not correct.

    Significance

    see above

  5. Version published to 10.1101/2020.12.02.408138 on bioRxiv