Pharmacological inhibition of MCL-1 disrupts mitochondrial cristae and depletes the human neural progenitor cell pool

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   Review Commons

   May 8, 2026

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   Reply to the reviewers

   __ __We thank all reviewers for the valuable feedback and critical insight on our study. We acknowledge the concern that the manuscript, in its initial form, appeared descriptive and did not provide the mechanistic insight inferred from the current data. In the revised manuscript, we will (i) more clearly delineate what mechanistic inferences can be drawn from the existing data, (ii) expand our discussion of the caspase-independent mechanisms, and (iii) incorporate additional experiments/analyses aimed at identifying downstream effectors that mediate the observed phenotypes. In this revision plan, we have included six new figures addressing some of the …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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   Reply to the reviewers

   __ __We thank all reviewers for the valuable feedback and critical insight on our study. We acknowledge the concern that the manuscript, in its initial form, appeared descriptive and did not provide the mechanistic insight inferred from the current data. In the revised manuscript, we will (i) more clearly delineate what mechanistic inferences can be drawn from the existing data, (ii) expand our discussion of the caspase-independent mechanisms, and (iii) incorporate additional experiments/analyses aimed at identifying downstream effectors that mediate the observed phenotypes. In this revision plan, we have included six new figures addressing some of the major issues raised by reviewers.

   1. Specifically, to address questions about mechanistic insight, we generated stable ACSL1:HaloTag expressing hESCs. Currently presented as __Figure 1A for reviewers____. __ACSL1 is a critical enzyme that catalyzes the first step of fatty acid oxidation at the outer mitochondrial membrane. Our previous analysis and work from the Opferman lab demonstrated that ACSL1 contains a BH3-like domain. Thus, we examined the effects of MCL-1 inhibition on the mitochondrial localization of this enzyme. Our findings pinpoint that MCL-1 inhibition is causing the displacement of ACSL1 from the mitochondria (Figures 1B-C for reviewers). Our interpretations of the effects of MCL-1 inhibition are 2-fold: 1) as we show in our data, MCL-1 inhibition causes disruption of the mitochondrial cristae, altering the microenvironment for fatty acid oxidation, and 2) as seen in cancer cells, the MCL-1 inhibitor may also displace ACSL1 from the mitochondria. In the new version of the manuscript, we will focus on these 2 mechanisms as mechanistic outcomes of MCL-1 inhibition.
   2. We have included data of cells treated with Perhexilin (CPT1/2 inhibitor), and Etomoxir (CPT1a inhibitor) (Figure 2 for reviewers). This experiment determines whether direct perturbation the FAO pathway mimics the effects of the MCL-1i.
   3. We have assayed the effects of MCL-1 inhibition on oxygen consumption rates in NPCs. Currently presented as Figure 3 for reviewers.
   4. We will perform MCL-1:MICOS proximity ligation assays and/or immunoprecipitation assays to determine whether MCL-1 inhibitors disrupt the association of MCL-1 with MICOS. Preliminary data suggesting an association (albeit, very weak) are shown in Figure 4 for reviewers. __Reviewer #1____ (Evidence, reproducibility and clarity (Required)): __

   Summary: This study claims that beyond its canonical anti-apoptotic function, MCL-1 has essential non-apoptotic roles in human neurodevelopment. Pharmacologic inhibition of MCL-1 in human neural stem cells disrupts mitochondrial inner membrane architecture by destabilizing cristae and the OPA1-MICOS complex, leading to swollen mitochondria with disorganized cristae. These structural defects impair fatty acid oxidation and lipid droplet homeostasis, linking cristae integrity to metabolic competence. Independently of apoptosis or proliferation, MCL-1 inhibition selectively depletes intermediate neural progenitors, indicating a direct role in lineage progression. Overall, the work positions MCL-1 as a key regulator of mitochondrial structure-metabolism coupling that instructs neural progenitor identity and human neurogenesis.

   *Overall: The study does a good job of using (in most assays) caspase inhibition (e.g., QVD treatment) to block apoptotic responses induced by MCL-1 inhibition. As a result, many of the phenotypes caused by inhibition are likely to be independent of caspase activation. As a result, this manuscript would be of interest to researchers that study the topics of the BCL-2 family and cell death signaling, mitochondrial bioenergetics and dynamics, neurodevelopment, and cellular metabolism. However, as currently presented the manuscript is only descriptive and lacks mechanistic insight. *

   We thank Reviewer 1 for the insightful evaluation of our work. We are encouraged that the reviewer finds the study relevant to investigators in the fields of BCL-2 family biology, mitochondrial dynamics and bioenergetics, neurodevelopment, and cellular metabolism. We also thank the reviewer for pointing out the need to increase the mechanistic insight of our findings. As mentioned above, in the revised manuscript, we are proposing to address this.

   *Major Concerns: *

   *1) The authors only use a single MCL-1 inhibitor and never use other non-targeting BH3-mimetics (such as venetoclax) as negative controls. This seems like a missed opportunity to demonstrate that the phenotypes observed are MCL-1 dependent. *

   This is an excellent point. We will include venetoclax (ABT-199) to examine their effect on intermediate progenitors (TBR2 +) and early born neurons (BIII tubulin +).

   *2) There is no mechanism proposed in this study other than reliance upon QVD as not affecting the phenotypes. As submitted, the manuscript only can speculate that these phenotypes are due to non-apoptotic roles of MCL-1 inhibition. The authors have missed an opportunity to explore MCL-1's non-apoptotic functions directly. *

   Mechanistically, we propose MCL-1 is acting in 2 ways: 1) as we show in our data, MCL-1 inhibition causes disruption of the mitochondrial cristae, altering the microenvironment for fatty acid oxidation, and 2) as seen in cancer cells, MCL-1 inhibitors may also displace ACSL1 from the mitochondria.

   In the past few weeks, since receiving the initial reviews, we have focused on testing the 2nd possibility, since the accumulation of lipids was also seen in cancer cells (see PMID: 38503284). We have successfully generated stable ACSL1:HaloTag expressing hESCs (Figure 1A for reviewers). Our findings included here, ACSL1 is displaced from the mitochondria by MCL-1 inhibition in NPCs (Figures 1B-C for reviewers).

   *Other concerns exist that weaken the impact of the study. *

   1. * Figure 1 should include the fact that QVD inhibition (shown in Sup Fig 2) does not obviate the phenotype induced by pharmacological inhibition of MCL-1 on mitochondrial morphology. * We would like to clarify that QVD does prevent the phenotypes induced by MCL-1 inhibition on mitochondrial morphology. In Fig1B, we report an increase in volume and surface area at 24h and 48h along with a decrease in mitochondrial content at 48h when NPCs were treated with MCL-1i only. However, NPCs co-treated with QVD in Supp Fig 2B did not exhibit any significant morphological phenotypes on average or at min/max values. Reviewer 1 may be referring to Fig 1B’s corresponding min/max values presented in Supp Fig 2A where we reported an increase in __max __volume.

   Figure #

   Volume

   Surface Area

   Fig 1B (MCL-1i only, avg values)

   Increase (avg vol)

   increase (avg)

   Supp Fig 2B (MCL-1i+QVD)

   no change

   no change

   Supp Fig 2A (MCL-1i only, max/min values)

   increase (max vol)

   no change (max)

   For clarity, we will move Supplementary Fig 2A into Supplementary Fig 1.

   * Figure 2 would benefit from evidence that caspase inhibition does not repress the phenotype on mitochondrial cristae morphology (volume and area). Furthermore, the FIB-SEM data are very hard to appreciate as the size precludes visualization of individual mitochondria. *

   While we included the visualization of the segmented mitochondria and cristae (Figure 2C), as well as snapshots through the z-stack for segmented cristae only (Figure 2E) and segmented mitochondria separately (Supp Figure 3A) in the original manuscript, we are also now attaching the FIB-SEM 3D reconstruction videos (New Supplementary Videos 1-2 for reviewers) (1. Mito and cristae, 2. Cristae only, 3. Mito only) for ease of visualization purposes.

   * Figure 3 reports that MIC60 and OPA1 appear to be downregulated in response to MCL-1 inhibition, but these appear to be more significant only when QVD is added. Why would the phenotype be obscured in the non-QVD setting (Fig. 2B&C). How does MCL-1 inhibition lead to changes in MIC60/MICOS/OPA1? This seems quite preliminary at this point.*

   In Figures 3B and 3C, we report decreased protein levels of short-form OPA1 and MIC10 only, not MIC60. We argue that our data with QVD shows that the cell death function of MCL-1 (i.e., inhibiting cell death effectors from initiating the caspase cascade) is not the main trigger of the phenotypes we report (cristae dysregulation and fatty acid oxidation disruption), however, cells without a functional cristae and/or defects in FAO, may not be able to survive long-term. Thus, QVD treatment preserves these cells that may not survive the dismantling of such an essential structure. To confirm this, we have performed immunofluorescence of cleaved caspase 3 (Figure 5 for reviewers). These results show that indeed MCL-1 inhibition at the time points of our study doesn’t result in increased activation of Caspase-3. We reported similar results of MCL-1 inhibition in oligodendrocyte precursor cells (Gil and Hanna et al., Glia, 2025, PMID: 41420072)

   * The loss of MIC60 and OPA1 should repress electron transport chain function, are such impacts observed in the cultured cells? This could be shown by assessing oxygen consumption, etc. Such data would enhance the authors' conclusion that MCL-1 inhibition leads to defects in mitochondrial physiology**. *

   We completely agree with this comment by Reviewer 1. In our revision, we will include an assessment of mitochondrial oxygen consumption rate, using the Seahorse analyzer (mitochondrial stress test), of NPCs treated with MCL-1i. Preliminary data (n=3) are currently presented as Figure 3 for reviewers. Interestingly, these data show a more nuanced cellular response. Consistent with our conclusion that MCL-1 inhibition does not cause apoptotic cell death, MCL-1i did not affect mitochondrial respiration at baseline. The specific deficits appear in spare respiratory capacity and maximal respiration, meaning cells can sustain routine mitochondrial function but lose the ability to respond to increased energetic demand. This suggests MCL-1 loss creates a mitochondrial reserve deficiency rather than a generalized bioenergetic failure. The results with caspase inhibitors show a near-zero OCR across both 24h and 48h timepoints, and significant reductions in maximal respiration, spare respiratory capacity, and non-mitochondrial OCR. Remarkably, these conditions are not detrimental to newborn neurons, as shown in Figure 7. This is very interesting because it suggests that, under severe bioenergetic failure, neural stem cells (PAX6+) can differentiate into newborn neurons in a TBR2-independent manner. More relevant to this study, our results unequivocally demonstrate that TBR2-positive cells depend on the non-apoptotic function of MCL-1

   * In Figure 4, the differences between transcripts (qPCR data) and protein (immunoblot) data are often confusing and not well explained. Why do the authors propose that mRNA expression is decreasing whereas the protein expression is increasing? Example CPT1. Furthermore, it is unclear what these data mean functionally? Is this reflective of enhanced lipid oxidation or simply a response to inhibition of fatty acid oxidation? Clarification of the impact of these findings is necessary. *

   We agree with Reviewer 1 that the results could be hard to interpret. However, the effects of MCL-1 inhibitors on the transcription of fatty acid oxidation genes have been widely cited by the work of Opferman and Walensky (PMID: 36198266). We speculate that the effects on transcription are triggered by mitochondrial signaling. The mechanistic insight into this phenomenon would be an interesting next step.

   In the case of CPT1, we addressed this comment and found that the difference is due to differential expression of isoforms The RT-qPCR shown in Figure 4, is on CPT1c, whereas the western blot is on CPT1a. Unfortunately, after trying several products, we determined that there are no good antibodies for CPT1c. Thus, since we can’t compare gene and protein expression, we will include CPT1a RT-qPCR data to complement the western blot.

   * The increase in lipid droplet number induced by MCL-1 inhibition has been previously documented, but it is unclear whether this increase is related to an inability to oxidize lipid (defective fatty acid oxidation) that leads to increases in the cellular abundance or whether this indicates that MCL-1 inhibition leads to enhanced storage. Do other inhibitors of fatty acid oxidation lead to similar increases in lipid droplet size and abundance? Does QVD inhibition affect this phenotype? *

   This is a great point raised by Reviewer 1, and one we have also wondered about. We conducted an experiment using C16 BODIPY to address this point (Figure 6 for Reviewers). We observed no changes in C16 lipid droplet accumulation in count, volume, or surface area when cells were treated with MCL-1 inhibitor for 24 hours total with or without a starvation period in the last 6 hours of treatment. However, we observed significant pan-lipid droplet accumulation in the same conditions. This contrast suggests that FAO of exogenous LC-fatty acids is not reliant on MCL-1. This finding does not discount from the requirement of MCL-1 for other FAO processes especially given the major limitation of how much C16 BODIPY (fluorescent palmitate) can be administered to the cells (10µM) which was 10-fold less than what we exogenously supplied to the cells for the pan-BODIPY experiment (100µM, see Figure 5). It is entirely possible that this small dose was not enough to detect any lipid droplet accumulation.

   We have now also included experiments using etomoxir and perhexiline to assess their effects on TBR2/PAX6 (Figure 2 for reviewers). The results indicate that inhibiting the FAO pathway does not fully mimic the effects of MCL-1i on TBR2. However, we show that MCL-1i displaces ACSL1 from the mitochondria, a step that is upstream of CPT1/2. We suggest a model in which the coordinated non-apoptotic function of MCL-1 at the outer mitochondrial membrane promotes ACSL1 activity and, in the inner mitochondrial membrane, regulates mitochondrial cristae morphology. While our data point to this model, we are limited by the tools to investigate it further, but it will be a great direction for future experiments.

   * For Figure 6, while these data may be very meaningful, as presented they are very hard to appreciate. Insets that show the neuronal populations would help to convey the point that the differentiation is impacted. Also, are there other methods that could confirm these observations (qPCR to show changes in differentiation). *

   We agree with Reviewer 1. In the new version of the manuscript, we will include panels that zoom into the cell populations we quantified. The current panels will go to a new Supplemental figure. We will also add the TUBB3 to the qPCR panel in the new version.

   * Figure 7 is also very hard to appreciate. What is the reader to see? Can these be quantified? It seems that QVD may be rescuing in this figure, does this suggest that MCL-1 inhibition might be inducing death. All of this needs to be quantified. *

   We will provide quantification of BIII tubulin branching, and it will be included next to the images provided.

   * BCL-XL has also been implicated in affecting mitochondrial electron transport chain function (See PMID: 19255249, 21926988, 21987637). Can BCL-XL inhibitors affect any of the phenotypes associated here? *

   We will include experiments to test the effect of BCL-2 and BCL-XL inhibitors on TBR2 cells to address this comment.

   * Please be carefully avoid using the term "MCL-1 loss", when talking about pharmacological inhibition. Only genetic ablation (e.g. knockout, silencing, etc.) should be termed loss. *

   We have now removed the reference to MCL-1 loss in line 199.

   __*Reviewer #1 (Significance (Required)):

   The study advances in human cells the impacts of MCL-1 inhibition. They replicate many impacts previously observed in mouse systems and refine analyses to impacts on MICOS complex, lipid droplet storage, and neuronal differentiation. While these findings are important and would be well received by a wide audience, the study fails to provide almost any mechanistic insight into how these phenotypes are being induced. The only common theme is that blocking caspase activation in many assays fails to block the phenotype.

   *__

   __Reviewer #2____* (Evidence, reproducibility and clarity (Required)): ____

   Summary: This manuscript by Hanna et al. investigates non-apoptotic roles of MCL-1 in human neural stem cells and connects MCL-1 inhibition to mitochondrial cristae formation and beta-oxidation. Connecting these roles to brain development, the authors also show a reduction in the number of progenitor cells upon MCL-1 inhibition, independently of caspase activity. Throughout their work, the authors make use of an impressive array of imaging techniques. While the methods used offer sufficient evidence to connect MCL-1 inhibition to cristae architecture, the mechanistic underpinnings of this effect remain unexplored. *__

   We thank Reviewer 2 for the thoughtful and positive assessment of our manuscript. We appreciate the reviewer’s recognition that our study reveals non-apoptotic roles of MCL-1 in human neural stem cells. We are also grateful for the acknowledgment of the imaging approaches employed, which allowed us to connect MCL-1 function to cristae architecture with multiple complementary techniques. We acknowledge the reviewer’s point that the mechanistic basis by which MCL-1 influences cristae structure remains insufficiently defined. In the revised manuscript, we will clarify the limitations of the current data, expand our discussion of potential mechanisms, and incorporate additional analyses to identify downstream effectors that mediate these structural and metabolic changes.

   *Major comments: *

   *- In Fig. 1B, the very same representative images are shown for both conditions (DMSO and S63845) at 48 hours. *

   We deeply appreciate Reviewer 2 for catching this unintentional duplication that occurred during figure preparation. We have now corrected this issue.

   *- For Western Blot analysis, it looks like the authors only quantified the band density of their proteins of interest without considering varying levels of control protein (Actin) levels. Normalizing the protein levels to actin would account for any differences in loaded protein amounts (although a Ponceau staining might be preferable still to exclude this). This is especially relevant for Fig. 4E, where actin levels visibly differ between the conditions. *

   All WB quantifications were normalized to Actin (this detail is now added to the y-axis of all band density graphs and figure legends). In addition, we will transform the data to a logarithmic scale to “normalize” for gel-to-gel variability.

   *- The authors offer evidence that MCL-1 inhibition impedes proteolytic cleavage of OPA1-L into the OPA-1-S isoforms, yet do not explore the mechanism behind this. Since OPA1 is cleaved by both OMA1 and YME1L, determination of the levels of these proteases could help shed some light on the mechanism leading to cristae reorganization. *

   We will follow up on Reviewer 2's comment with a WB analysis of OMA1 and YMEL in cells treated with an MCL-1 inhibitor.

   *- Generally speaking, while the authors show all those effects (cristae defects, FAO dysfunction) upon MCL-1 inhibition, it would be interesting to see whether any of those effects can be rescued by blocking FA import e.g. through carnitine palmitoyl- transferase 1a (CPT1a) inhibition with etomoxir to understand if they are downstream of altered Fa supply. This could affect cristae morphology through altered Cardiolipin biogenesis. *

   This is an excellent point, which was also raised by reviewer 1. We have now included experiments using etomoxir and perhexiline to assess their effects on TBR2/PAX6 (Figure 2 for Reviewers). As mentioned above, the results indicate that inhibiting the FAO pathway does not fully mimic the effects of MCL-1i on TBR2. However, we show that MCL-1i displaces ACSL1 from the mitochondria, a step that is upstream of CPT1 and 2. We suggest a model in which the coordinated non-apoptotic function of MCL-1 at the outer mitochondrial membrane promotes ACSL1 activity and, in the inner mitochondrial membrane, regulates mitochondrial cristae morphology. While our data point to this model, we are limited by the tools to investigate it further, but it will be a great direction for future experiments. The suggestion of Reviewer 2 that the effects on FAO could impact cardiolipin biogenesis is a very exciting possibility. However, difficult to test with the tools available.

   *- In line 262 the authors discuss that mitochondria lose metabolic function upon MCL-1 inhibition. This claim would require additional experiments. While the authors look at lipid droplet accumulation and FAO enzymes, there are many more aspects to mitochondrial metabolic function that should be investigated. While measuring the oxygen consumption rate via Seahorse might require additional resources (optional), measurements of ATP production, ROS generation or determination of the mitochondrial membrane potential should be feasible. *

   We fully agree with Reviewer 2's comment, which was also raised by Reviewer 1. In our revision, we will include an assessment of the mitochondrial oxygen consumption rate of NPCs treated with MCL-1i, measured using the Seahorse analyzer (mitochondrial stress test). These data are presented as Figure 3 for reviewers. Interestingly, these data show a more nuanced cellular response. While MCL-1i does not globally collapse mitochondrial respiration at baseline, the specific deficits appear in spare respiratory capacity and maximal respiration, meaning cells can sustain routine mitochondrial function but lose the ability to respond to increased energetic demand. This suggests MCL-1 loss creates a mitochondrial reserve deficiency rather than a generalized bioenergetic failure. The results with caspase inhibitors show a near-zero OCR across both 24h and 48h timepoints, and significant reductions in maximal respiration, spare respiratory capacity, and non-mitochondrial OCR. These conditions are detrimental for TBR2-positive NPCs (Figure 6) , but not for newborn neurons (Figure 7).

   *- While the authors "propose a model in which MCL-1 associates with MICOS", they do not offer direct scientific to support this hypothesis. Co-immunoprecipitation experiments or e.g. proximity ligation assays would better support the proposed model. *

   We agree with this statement. Preliminary, we have performed proximity ligation assays and immunoprecipitation analyses to test for this interaction (see below and ____Figure 4 for reviewers), and the results indicate an interaction, albeit very weak. In the revised version of the manuscript, we will attempt to repeat these experiments with MCL-1i.

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   *- While Fig. 7 shows representative images, quantification e.g. for the truncation of neuronal processes is missing. *

   We will provide quantification of BIII tubulin branching, which will be included alongside the images provided.

   *- In lines 219f. the authors state that they "observed a significant downregulation of PAX6 and EOMES at 24 hours that was not rescued by QVD co-treatment". While there is still a trend towards a downregulation, there is no statistical significance anymore. In fact, PAX6 levels almost mirror those of SOX2 which is not described as "downregulated" by the authors. In order to be more consistent, I would suggest rephrasing this part, or at least reword it to be less absolute. *

   In the new version, we will clarify that while QVD rescued TBR2 and PAX6 transcript levels at 24h, it did not rescue them at 48h. We will also mention the downregulation of SOX2 at 48h that persists with co-treatment.

   *- Brinkmann et al. (2025) also investigated cristae structure upon MCL-1 deletion in vivo and found no effect when MCL-1 was replaced with other Bcl-2 family members. It would be interesting to combine MCL-1 inhibition with overexpression of MCL-1 versus BCL-XL to reconsolidate some of the discrepant findings. *

   While this is a great suggestion for future studies, there are some complications. Specifically, it is likely that the inhibitor may also target the overexpressed MCL-1 and thus, a mutant form is needed.

   To address this, we generated a Flag-tagged MCL-1 construct with a mutated BH3 domain, previously described by Kotschy et al. Nature 2016. We validated the construct in HeLa cells, but unfortunately the mutant protein appears to be significantly less stable than the WT construct, complicating analysis of this experiment.

   *Minor comments: *

   *- In Supp. Fig. 1C the MCL-1 protein is shown both to run above 37kDa (upper panel) and below 37 kDa (lower panel). Could the authors please comment on why this is the case? *

   The observed variation is caused by drift in the gel during electrophoresis. In Fig 1C, the protein ladder is on the edge of the gel, whereas in Fig 1E, the protein ladder is in the middle of the gel, and the last sample is on the edge and also exhibits edge drift.

   *- In line 64 of the introduction the authors mention clinical trials yet do not give a citation for these trials making it hard to judge whether the content of these trials is actually related to the brain. *

   This information is anecdotal, based on an Amgen press release.

   *- MCL-1 as well as ACSL-1 are sometimes written without the hyphen both in the text and figures. *

   We will carefully check the manuscript before submission.

   *- Lines 92-94 and 106-108 essentially highlight the same existing knowledge gap. Maybe the content of these two paragraphs could be combined in order to avoid repetition. *

   We thank Reviewer 2 for this suggestion. We will do this in the new version of the manuscript.

   *- In Fig. 1A, the authors provide a schematic for their experimental design. While the figure legend is very thorough, some of this information (like the days of collection) could also be included in the figure itself. The same is true for schematics in the following figures. *

   We agree with this and will incorporate the suggestion in the new version.

   *- Fig. 2A includes a typo (analyze) but would maybe also be more suitable for the supplement figures or could even be combined with Fig. 1A as not much new content is added. *

   We already incorporated these changes in the new version of the manuscript.

   *- Regarding statistical analysis, could the authors please comment on why they did not consider one-sample t-tests suitable for the cases where control values were set at 1 (e.g. Fig. 4B, C for the relative expression). *

   This is a valid suggestion. We will rerun RT-qPCR data using a one-sample t-test.

   *- In lines 247f. the authors state that "inhibition of MCL-1 leads to [...] and disassembly of the MICOS complex as well as OPA1". This sounds like OPA1 is still cleaved upon MCL-1, which is not at all what the authors showed and further discuss. Rewording of the sentence would help in avoiding any misunderstandings. *

   We agree with this comment and have now reworded the paragraph: “Inhibition of MCL-1 leads to structural collapse of the cristae likely due to the possible disassembly of the MICOS complex, as suggested by decreased MIC10 levels, and interruption of OPA1 cleavage, as suggested by decreased short-form OPA1, two scaffolds required for cristae maintenance.”

   *- In lines 210f. the authors state that "quantitative imaging increased the average and maximum volume of lipid droplets". While there is definitely a trend towards an increase for the maximum volume, the increase is in fact not statistically significant. This should be reflected in the wording. *

   We have reworded this to “Quantitative imaging revealed a significant increase in average lipid droplet volume and a trending increase in maximum volume of lipid droplets.”

   *- In Fig. 6 the overlap between TBR2 and PAX6 is hard to judge when printed out. Including a zoom-in may make it easier to judge. *

   We agree with Reviewer 2. In the new version of the manuscript, we will include panels that zoom into the cell populations we quantified. The current panels will go to a new Supplemental figure. We will also add the TUBB3 to the qPCR panel in the new version.

   *- In Fig. 7 the color-coding is listed in the figure legend but is missing from the figure itself. If the authors could include this, as they did for the other figures, it would further improve this figure. *

   We agree. We have specified the channel color in the new figure.

   *- Line 238 should reference Fig. 7A, as Fig 7B does not exist. *

   Thanks for catching this. It is already corrected

   *- In the figure legends the authors state that biological replicates were used. Were technical replicates also performed? *

   Yes, technical replicates were performed for RT-qPCR.

   Reviewer #2 (Significance (Required)):____* Significance*

   *The authors make use of a wide array of imaging techniques to further elucidate non-apoptotic roles of MCL-1. The study has the potential to offer new insights into mitochondrial biology on the level of basic research rather than translational. While the methods used offer sufficient evidence to connect MCL-1 inhibition to cristae architecture, the mechanistic underpinnings of this effect remain unexplored. Nevertheless, the study offers additional knowledge on the role of MCL-1 in human neural stem cells, whereas previous research mostly focused on cardiomyocytes or cancer cells. *

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

   *Summary: ____ In this study, Gama et al. describe a non-canonical role for the anti-apoptotic protein Myeloid Cell Leukemia-1 (MCL-1) in mitochondrial cristae organization and suggest a role of MCL-1 in regulating metabolism and neuronal differentiation. Using fluorescence microscopy imaging and electron microscopy, the authors show changes to mitochondrial morphology upon treatment with MCL-1 inhibitor S63845. MCL-1 inhibition results in altered protein and transcript levels of some key proteins involved in mitochondrial cristae organization and fatty acid metabolism. While some of the findings are interesting and indeed point towards a non-canonical role of MCL-1, several key conclusions of the authors are not sufficiently supported by the data shown in the manuscript. *

   We thank Reviewer 3 for the careful evaluation of our manuscript. We appreciate the reviewer’s recognition that our study identifies a potential non-canonical role for MCL-1 in mitochondrial cristae organization, metabolism, and neuronal differentiation. As with Reviews 1 and 2, we are encouraged that the reviewer finds these observations interesting and suggestive of previously unappreciated functions for MCL-1. We agree that stronger evidence is required to firmly link MCL-1 inhibition to specific changes in MICOS organization and metabolic regulation. In the revised manuscript, we will (i) more clearly distinguish between observations and mechanistic inferences, (ii) temper conclusions where appropriate, and (iii) incorporate additional analyses and controls to better substantiate the proposed model.

   *Major comments: *

   1. __* The authors try to disentangle the apoptotic and non-apoptotic role of MCL-1 through addition of a caspase inhibitor. However, I am not convinced that phenotypes found under the addition of caspase inhibitor are necessarily caused by non-canonical functions independent of apoptosis. It could also be that the observed changes happen upstream of caspase activation. In addition, many of the described finding, such as CPT1 expression changes, only happen in the presence of the caspase inhibitor. If one follows the logic of the authors, changes associated by non-canonical MCL-1 functions should happen under MCL-1 inhibition and caspase inhibition, but not with MCL-1 inhibition only*____. __ The reviewer is right that we expected non-canonical functions to happen under MCL-1 inhibition and caspase inhibition. Our data with QVD shows that the cell death function of MCL-1 (i.e., inhibiting cell death effectors from initiating the caspase cascade) is not the main trigger of the phenotypes we report (cristae dysregulation and fatty acid oxidation disruption), however, cells without a functional cristae and/or defects in FAO, may not be able to survive long-term. Thus, QVD treatment preserves these cells that may not survive the dismantling of such an essential structure. To confirm this, we performed immunofluorescence of cleaved caspase 3 (Figure 5 for reviewers). These results show that, indeed, MCL-1 inhibition at the time points of our study doesn’t result in increased Caspase-3 activation. We reported similar results of MCL-1 inhibition in oligodendrocyte precursor cells (Gil and Hanna et al., Glia, 2025, PMID: 41420072).

   * The authors show no data on the viability of the cells in response to the MCL-1 inhibitor. To exclude secondary effects of the inhibitor, at least some of the results should be validated with an MCL-1 knock down. *

   We will include this experiment in our revised manuscript. To check the effects of MCL-1 knockdown on TBR2 positive cells, we tested 5 different ASOs for MCL-1. Knockdown efficiency with ASOs was very low (on average * In Figure 1, the authors show immunofluorescence data of mitochondria and nucleus staining and conclude that MCL-1 inhibition alters mitochondrial morphology. Based on the images shown in Fig. 1, I do not think that individual mitochondria can be segmentd to measure their volume and length. In addition, some metrics such as mitochondrial content are not explained in the text or methods. *

   We can achieve mitochondrial segmentation with a SoRa Spinning Disk Confocal Microscope, which has a lateral (XY) resolution of approximately 120 nm to 150 nm and an axial (Z) resolution of approximately 300 nm–320 nm. All images are first denoised prior to sharpening using the Richardson-Lucy deconvolution algorithm. Additionally, the FIB-SEM data are consistent with the IF data (both show increase in mitochondrial volume and surface area).

   We agree with Reviewer 3 that we need to explain some metrics in the revised version. We will specify the meaning of mitochondrial content (count of all mitochondria in FOV, not normalized to Hoechst).

   * In Fig. 2 B-D, the authors show TEM and FIB-SEM imaging to demonstrate alterations in the cristae architecture upon treatment with MCL-1 inhibitor. However, based on the images shown, it looks that cristae area and density is reduced under S63845 treatment in TEM images, while the FIB-SEM data come to the opposite conclusion. In addition, the quantification of cristae volume quantified as cristae volume in percentage is unclear to me. *

   We apologize for the confusion. No conclusions about the cristae area and density were made using the TEM data, because TEM data represent a single snapshot section of a mitochondrion without a discernible orientation. Cristae from TEM were described as “aberrant” and preliminarily revealed changes in cristae and were followed up with FIB-SEM, 3D reconstruction of intact mitochondria, and quantification of volume.

   In the new version of the manuscript, we will specify that the cristae volume is normalized to the volume of its respective mitochondria (i.e., how much of the mitochondrial volume is attributed to cristae).

   * The change in CPT1/2 protein levels (Fig. 4) is interesting but does not directly proof that fatty acid oxidation is altered, as concluded by the authors. For this, the authors would need to directly measure fatty acid oxidation for example using Seahorse or metabolic tracing experiments. Also, to prove that the MCL-1 inhibition affects neural differentiation through fatty acid oxidation, a rescue experiment should be performed through CPT1 overexpression. *

   We agreed that this is an important point. We have optimized the fatty acid oxidation test using Seahorse and will make sure to include it in the revised version of the manuscript.

   * In Figure 6, the authors show decreased intermediate progenitor cells after MCL-1 inhibition by immunofluorescence staining. I am not convinced that this can be concluded from the data shown, since the concentration of intermediate progenitor cells is very close to the noise levels. Since the MCL-1 treated cells look much less sparse, I don't think the percentages can be compared (total counts are between 2-20). Although this data might give some indication that differentiation could be impaired, the measured effect could be very well due to lower viability of the cells. The authors need to control for this or come up with a different method for measuring differentiation. *

   The number of TBR2 is low, but we disagree with the reviewer’s assessment of noise levels. We focused on cells expressing only TBR2 and rigorously examined this population of cells. The percentages are compared to account for the lower density of the MCL-1i-treated cultures, as the IPC counts are normalized to the Hoechst total cell count within the FOV. Moreover, the immunofluorescence images are complemented with RT-qPCR, which shows significant downregulation of *EOMES *(gene encoding TBR2).

   * Figure 7 is missing quantification *

   We will include this quantification in the revised version of the manuscript.

   *Reviewer #3 (Significance (Required)): *

   General assessment____*: The manuscript reports an interesting finding, which suggest a non-canonical role of MCL-1 in mitochondrial remodeling, regulation of fatty acid oxidation and neuronal fate. While this finding would be highly interesting and relevant, the presented data do not sufficiently support this conclusion. Further experiments would have to be performed to proof causality. ____ Advance: Should the authors manage to proof their hypothesis by additional experiments, this would indeed advance the field on mitochondrial remodeling and its effect on neuronal differentiation by *

   identifying a novel molecular player. ____ Audience: mitochondrial biology, cell biology, developmental neuroscience Own expertise: mitochondrial biology, cell biology, advanced imaging techniques

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   In this study, Gama et al. describe a non-canonical role for the anti-apoptotic protein Myeloid Cell Leukemia-1 (MCL1) in mitochondrial cristae organization and suggest a role of MCL1 in regulating metabolism and neuronal differentiation. Using fluorescence microscopy imaging and electron microscopy, the authors show changes to mitochondrial morphology upon treatment with MCL1 inhibitor S63845. MCL1 inhibition results in altered protein and transcript levels of some key proteins involved in mitochondrial cristae organization and fatty acid metabolism. While some of the findings are interesting and indeed point towards a …

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   Referee #3

   Evidence, reproducibility and clarity

   Summary:

   In this study, Gama et al. describe a non-canonical role for the anti-apoptotic protein Myeloid Cell Leukemia-1 (MCL1) in mitochondrial cristae organization and suggest a role of MCL1 in regulating metabolism and neuronal differentiation. Using fluorescence microscopy imaging and electron microscopy, the authors show changes to mitochondrial morphology upon treatment with MCL1 inhibitor S63845. MCL1 inhibition results in altered protein and transcript levels of some key proteins involved in mitochondrial cristae organization and fatty acid metabolism. While some of the findings are interesting and indeed point towards a non-canonical role of MCL1, several key conclusions of the authors are not sufficiently supported by the data shown in the manuscript.

   Major comments:

   1. The authors try to disentangle the apoptotic and non-apoptotic role of MCL1 through addition of a caspase inhibitor. However, I am not convinced that phenotypes found under the addition of caspase inhibitor are necessarily caused by non-canonical functions independent of apoptosis. It could also be that the observed changes happen upstream of caspase activation. In addition, many of the described finding, such as CPT1 expression changes, only happen in the presence of the caspase inhibitor. If one follows the logic of the authors, changes associated by non-canonical MCL1 functions should happen under MCL1 inhibition and caspase inhibition, but not with MCL1 inhibition only.
   2. The authors show no data on the viability of the cells in response to the MCL1 inhibitor. To exclude secondary effects of the inhibitor, at least some of the results should be validated with an MCL1 knock down.
   3. In Figure 1, the authors show immunofluorescence data of mitochondria and nucleus staining and conclude that MCL1 inhibition alters mitochondrial morphology. Based on the images shown in Fig. 1, I do not think that individual mitochondria can be segmentd to measure their volume and length. In addition, some metrics such as mitochondrial content are not explained in the text or methods.
   4. In Fig. 2 B-D, the authors show TEM and FIB-SEM imaging to demonstrate alterations in the cristae architecture upon treatment with MCL1 inhibitor. However, based on the images shown, it looks that cristae area and density is reduced under S63845 treatment in TEM images, while the FIB-SEM data come to the opposite conclusion. In addition, the quantification of cristae volume quantified as cristae volume in percentage is unclear to me.
   5. The change in CPT1/2 protein levels (Fig. 4) is interesting but does not directly proof that fatty acid oxidation is altered, as concluded by the authors. For this, the authors would need to directly measure fatty acid oxidation for example using Seahorse or metabolic tracing experiments. Also, to prove that the MCL1 inhibition affects neural differentiation through fatty acid oxidation, a rescue experiment should be performed through CPT1 overexpression.
   6. In Figure 6, the authors show decreased intermediate progenitor cells after MCL1 inhibition by immunofluorescence staining. I am not convinced that this can be concluded from the data shown, since the concentration of intermediate progenitor cells is very close to the noise levels. Since the MCL1 treated cells look much less sparse, I don't think the percentages can be compared (total counts are between 2-20). Although this data might give some indication that differentiation could be impaired, the measured effect could be very well due to lower viability of the cells. The authors need to control for this or come up with a different method for measuring differentiation.
   7. Figure 7 is missing quantification

   Significance

   General assessment: The manuscript reports an interesting finding, which suggest a non-canonical role of MCL1 in mitochondrial remodeling, regulation of fatty acid oxidation and neuronal fate. While this finding would be highly interesting and relevant, the presented data do not sufficiently support this conclusion. Further experiments would have to be performed to proof causality.

   Advance: Should the authors manage to proof their hypothesis by additional experiments, this would indeed advance the field on mitochondrial remodeling and its effect on neuronal differentiation by identifying a novel molecular player.

   Audience: mitochondrial biology, cell biology, developmental neuroscience

   Own expertise: mitochondrial biology, cell biology, advanced imaging techniques

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   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   This manuscript by Hanna et al. investigates non-apoptotic roles of MCL-1 in human neural stem cells and connects MCL-1 inhibition to mitochondrial cristae formation and beta-oxidation. Connecting these roles to brain development, the authors also show a reduction in the number of progenitor cells upon MCL-1 inhibition, independently of caspase activity. Throughout their work, the authors make use of an impressive array of imaging techniques.While the methods used offer sufficient evidence to connect MCL-1 inhibition to cristae architecture, the mechanistic underpinnings of this effect remain unexplored.

   Major comments:

   • In …

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   Referee #2

   Evidence, reproducibility and clarity

   Summary:

   This manuscript by Hanna et al. investigates non-apoptotic roles of MCL-1 in human neural stem cells and connects MCL-1 inhibition to mitochondrial cristae formation and beta-oxidation. Connecting these roles to brain development, the authors also show a reduction in the number of progenitor cells upon MCL-1 inhibition, independently of caspase activity. Throughout their work, the authors make use of an impressive array of imaging techniques.While the methods used offer sufficient evidence to connect MCL-1 inhibition to cristae architecture, the mechanistic underpinnings of this effect remain unexplored.

   Major comments:

   • In Fig. 1B, the very same representative images are shown for both conditions (DMSO and S63845) at 48 hours.
   • For Western Blot analysis, it looks like the authors only quantified the band density of their proteins of interest without considering varying levels of control protein (Actin) levels. Normalizing the protein levels to actin would account for any differences in loaded protein amounts (although a Ponceau staining might be preferable still to exclude this). This is especially relevant for Fig. 4E, where actin levels visibly differ between the conditions.
   • The authors offer evidence that MCL-1 inhibition impedes proteolytic cleavage of OPA1-L into the OPA-1-S isoforms, yet do not explore the mechanism behind this. Since OPA1 is cleaved by both OMA1 and YME1L, determination of the levels of these proteases could help shed some light on the mechanism leading to cristae reorganization.
   • Generally speaking, while the authors show all those effects (cristae defects, FAO dysfunction) upon MCL-1 inhibition, it would be interesting to see whether any of those effects can be rescued by blocking FA import e.g. through carnitine palmitoyl- transferase 1a (CPT1a) inhibition with etomoxir to understand if they are downstream of altered Fa supply. This could affect cristae morphology through altered Cardiolipin biogenesis.
   • In line 262 the authors discuss that mitochondria lose metabolic function upon MCL-1 inhibition. This claim would require additional experiments. While the authors look at lipid droplet accumulation and FAO enzymes, there are many more aspects to mitochondrial metabolic function that should be investigated. While measuring the oxygen consumption rate via Seahorse might require additional resources (optional), measurements of ATP production, ROS generation or determination of the mitochondrial membrane potential should be feasible.
   • While the authors "propose a model in which MCL-1 associates with MICOS", they do not offer direct scientific to support this hypothesis. Co-immunoprecipitation experiments or e.g. proximity ligation assays would better support the proposed model.
   • While Fig. 7 shows representative images, quantification e.g. for the truncation of neuronal processes is missing.
   • In lines 219f. the authors state that they "observed a significant downregulation of PAX6 and EOMES at 24 hours that was not rescued by QVD co-treatment". While there is still a trend towards a downregulation, there is no statistical significance anymore. In fact, PAX6 levels almost mirror those of SOX2 which is not described as "downregulated" by the authors. In order to be more consistent, I would suggest rephrasing this part, or at least reword it to be less absolute.
   • Brinkmann et al. (2025) also investigated cristae structure upon MCL-1 deletion in vivo and found no effect when MCL-1 was replaced with other Bcl-2 family members. It would be interesting to combine MCL-1 inhibition with overexpression of MCL-1 versus BCL-XL to reconsolidate some of the discrepant findings.

   Minor comments:

   • In Supp. Fig. 1C the MCL-1 protein is shown both to run above 37kDa (upper panel) and below 37 kDa (lower panel). Could the authors please comment on why this is the case?
   • In line 64 of the introduction the authors mention clinical trials yet do not give a citation for these trials making it hard to judge whether the content of these trials is actually related to the brain.
   • MCL-1 as well as ACSL-1 are sometimes written without the hyphen both in the text and figures.
   • Lines 92-94 and 106-108 essentially highlight the same existing knowledge gap. Maybe the content of these two paragraphs could be combined in order to avoid repetition.
   • In Fig. 1A, the authors provide a schematic for their experimental design. While the figure legend is very thorough, some of this information (like the days of collection) could also be included in the figure itself. The same is true for schematics in the following figures.
   • Fig. 2A includes a typo (analyze) but would maybe also be more suitable for the supplement figures or could even be combined with Fig. 1A as not much new content is added.
   • Regarding statistical analysis, could the authors please comment on why they did not consider one-sample t-tests suitable for the cases where control values were set at 1 (e.g. Fig. 4B, C for the relative expression).
   • In lines 247f. the authors state that "inhibition of MCL-1 leads to [...] and disassembly of the MICOS complex as well as OPA1". This sounds like OPA1 is still cleaved upon MCL-1, which is not at all what the authors showed and further discuss. Rewording of the sentence would help in avoiding any misunderstandings.
   • In lines 210f. the authors state that "quantitative imaging increased the average and maximum volume of lipid droplets". While there is definitely a trend towards an increase for the maximum volume, the increase is in fact not statistically significant. This should be reflected in the wording.
   • In Fig. 6 the overlap between TBR2 and PAX6 is hard to judge when printed out. Including a zoom-in may make it easier to judge.
   • In Fig. 7 the color-coding is listed in the figure legend but is missing from the figure itself. If the authors could include this, as they did for the other figures, it would further improve this figure.
   • Line 238 should reference Fig. 7A, as Fig 7B does not exist.
   • In the figure legends the authors state that biological replicates were used. Were technical replicates also performed?

   Significance

   The authors make use of a wide array of imaging techniques to further elucidate non-apoptotic roles of MCL-1. The study has the potential to offer new insights into mitochondrial biology on the level of basic research rather than translational. While the methods used offer sufficient evidence to connect MCL-1 inhibition to cristae architecture, the mechanistic underpinnings of this effect remain unexplored. Nevertheless, the study offers additional knowledge on the role of MCL-1 in human neural stem cells, whereas previous research mostly focused on cardiomyocytes or cancer cells.

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   Referee #1

   Evidence, reproducibility and clarity

   Summary: This study claims that beyond its canonical anti-apoptotic function, MCL-1 has essential non-apoptotic roles in human neurodevelopment. Pharmacologic inhibition of MCL-1 in human neural stem cells disrupts mitochondrial inner membrane architecture by destabilizing cristae and the OPA1-MICOS complex, leading to swollen mitochondria with disorganized cristae. These structural defects impair fatty acid oxidation and lipid droplet homeostasis, linking cristae integrity to metabolic competence. Independently of apoptosis or proliferation, MCL-1 inhibition selectively depletes intermediate neural progenitors, indicating a direct …

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   Referee #1

   Evidence, reproducibility and clarity

   Summary: This study claims that beyond its canonical anti-apoptotic function, MCL-1 has essential non-apoptotic roles in human neurodevelopment. Pharmacologic inhibition of MCL-1 in human neural stem cells disrupts mitochondrial inner membrane architecture by destabilizing cristae and the OPA1-MICOS complex, leading to swollen mitochondria with disorganized cristae. These structural defects impair fatty acid oxidation and lipid droplet homeostasis, linking cristae integrity to metabolic competence. Independently of apoptosis or proliferation, MCL-1 inhibition selectively depletes intermediate neural progenitors, indicating a direct role in lineage progression. Overall, the work positions MCL-1 as a key regulator of mitochondrial structure-metabolism coupling that instructs neural progenitor identity and human neurogenesis.

   Overall: The study does a good job of using (in most assays) caspase inhibition (e.g., QVD treatment) to block apoptotic responses induced by MCL-1 inhibition. As a result, many of the phenotypes caused by inhibition are likely to be independent of caspase activation. As a result, this manuscript would be of interest to researchers that study the topics of the BCL-2 family and cell death signaling, mitochondrial bioenergetics and dynamics, neurodevelopment, and cellular metabolism. However, as currently presented the manuscript is only descriptive and lacks mechanistic insight.

   Major Concerns:

   1. The authors only use a single MCL-1 inhibitor and never use other non-targeting BH3-mimetics (such as venetoclax) as negative controls. This seems like a missed opportunity to demonstrate that the phenotypes observed are MCL-1 dependent.

   2. There is no mechanism proposed in this study other than reliance upon QVD as not affecting the phenotypes. As submitted, the manuscript only can speculate that these phenotypes are due to non-apoptotic roles of MCL-1 inhibition. The authors have missed an opportunity to explore MCL-1's non-apoptotic functions directly.

   Other concerns exist that weaken the impact of the study.

   1. Figure 1 should include the fact that QVD inhibition (shown in Sup Fig 2) does not obviate the phenotype induced by pharmacological inhibition of MCL-1 on mitochondrial morphology.
   2. Figure 2 would benefit from evidence that caspase inhibition does not repress the phenotype on mitochondrial cristae morphology (volume and area). Furthermore, the FIB-SEM data are very hard to appreciate as the size precludes visualization of individual mitochondria.
   3. Figure 3 reports that MIC60 and OPA1 appear to be downregulated in response to MCL-1 inhibition, but these appear to be more significant only when QVD is added. Why would the phenotype be obscured in the non-QVD setting (Fig. 2B&C). How does MCL-1 inhibition lead to changes in MIC60/MICOS/OPA1? This seems quite preliminary at this point.
   4. The loss of MIC60 and OPA1 should repress electron transport chain function, are such impacts observed in the cultured cells? This could be shown by assessing oxygen consumption, etc. Such data would enhance the authors' conclusion that MCL-1 inhibition leads to defects in mitochondrial physiology.
   5. In Figure 4, the differences between transcripts (qPCR data) and protein (immunoblot) data are often confusing and not well explained. Why do the authors propose that mRNA expression is decreasing whereas the protein expression is increasing? Example CPT1. Furthermore, it is unclear what these data mean functionally? Is this reflective of enhanced lipid oxidation or simply a response to inhibition of fatty acid oxidation? Clarification of the impact of these findings is necessary.
   6. The increase in lipid droplet number induced by MCL-1 inhibition has been previously documented, but it is unclear whether this increase is related to an inability to oxidize lipid (defective fatty acid oxidation) that leads to increases in the cellular abundance or whether this indicates that MCL-1 inhibition leads to enhanced storage. Do other inhibitors of fatty acid oxidation lead to similar increases in lipid droplet size and abundance? Does QVD inhibition affect this phenotype?
   7. For Figure 6, while these data may be very meaningful, as presented they are very hard to appreciate. Insets that show the neuronal populations would help to convey the point that the differentiation is impacted. Also, are there other methods that could confirm these observations (qPCR to show changes in differentiation).
   8. Figure 7 is also very hard to appreciate. What is the reader to see? Can these be quantified? It seems that QVD may be rescuing in this figure, does this suggest that MCL-1 inhibition might be inducing death. All of this needs to be quantified.
   9. BCL-XL has also been implicated in affecting mitochondrial electron transport chain function (See PMID: 19255249, 21926988, 21987637). Can BCL-XL inhibitors affect any of the phenotypes associated here?
   10. Please be carefully avoid using the term "MCL-1 loss", when talking about pharmacological inhibition. Only genetic ablation (e.g. knockout, silencing, etc.) should be termed loss.

   Significance

   The study advances in human cells the impacts of MCL-1 inhibition. They replicate many impacts previously observed in mouse systems and refine analyses to impacts on MICOS complex, lipid droplet storage, and neuronal differentiation. While these findings are important and would be well received by a wide audience, the study fails to provide almost any mechanistic insight into how these phenotypes are being induced. The only common theme is that blocking caspase activation in many assays fails to block the phenotype.

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5. 

   Version published to 10.64898/2025.12.12.694056 on bioRxiv

   Dec 14, 2025