Necroptosis inhibition counteracts neurodegeneration, memory decline, and key hallmarks of aging, promoting brain rejuvenation

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1. Version published to 10.1111/acel.13814

   Mar 27, 2023
2. 

   Version published to 10.1101/2021.11.10.468052v3 on bioRxiv

   Nov 16, 2022
3. 

   Review Commons

   Jul 18, 2022

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

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   Reply to the reviewers

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

   This study is interesting on finding that necroptosis may regulate axonal degeneration in the dentate gyrus, which led to the loss of synaptic transmission and plasticity and impaired the performance of mice in water maze. Genetic ablation of MLKL, a key factor for necroptotic pathway, or pharmacological inhibition of necroptosis with GSK'872, the inhibitor to another necroptotic key factor RIPK3, prevented mice from axonal degeneration, synapse dysfunction, and memory loss. They also tested long term potentiation (LTP) and found LTP that was disrupted in aged mice, was rescued by MLKL …

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

   Learn more at Review Commons

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   Reply to the reviewers

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

   This study is interesting on finding that necroptosis may regulate axonal degeneration in the dentate gyrus, which led to the loss of synaptic transmission and plasticity and impaired the performance of mice in water maze. Genetic ablation of MLKL, a key factor for necroptotic pathway, or pharmacological inhibition of necroptosis with GSK'872, the inhibitor to another necroptotic key factor RIPK3, prevented mice from axonal degeneration, synapse dysfunction, and memory loss. They also tested long term potentiation (LTP) and found LTP that was disrupted in aged mice, was rescued by MLKL knockout or GSK'872 treatments. The authors further compared normally aged mice (more than 20 months old) with aged MLKL-knockout mice or aged mice with GSK'872 treatments for altered proteins by single-shot label-free mass spectrometry, and discovered that in 7000 detected proteins, 2516 proteins were increased while 2307 were decreased in the aging hippocampus. They carried out bioinformatic analysis and clustered these proteins for biofunctions of synaptic mechanisms, senescence, etc. With the bioinformatic analysis, they further examined cell senescence by SA-βgalactosidase (SA-βgal) staining and concluded that the cellular senescence was also rescued by necroptosis inhibition.

   Although it is an exciting idea that inhibiting necroptosis may be a potential approach to combating aging and rejuvenating the brain, I have many of concerns about the reliability and consistency of the data that did not show strong supports to the conclusion.

   Major comments:

   The authors used axonal degeneration as a major readout for brain aging. However, the conclusion of axonal degeneration was simply based on immunostaining. These staining results are not consistent in different parts, and their conclusion is hard to be supported by the representative images. It is not convincing that axonal integrity was altered as concluded by the authors as shown in the representative images of Fig. 1e, Fig. 2d, Fig. 3a, as well as those in the supplementary figures. Electron microscopy and other convincing means are necessary.

   We agree with the reviewer, we are performing new staining and incorporating new imaging techniques, including confocal microscopy to better define axons in different regions of the hippocampus. We propose to use Light Sheet Microscopy in clarified hippocampus in order to perform 3D analyses of the axons in the entire hippocampus (ongoing experiments). The Light Sheet microscope is currently available in our Center and we have settled the clarity protocol in brain tissue, particularly in the hippocampus (see pictures below of an hippocampi before and after the clarification protocol).

   As suggested by the reviewer, electron microscopy could be a very good addition, nevertheless this technique is not implemented in the laboratory at the moment. Nevertheless, we have initiated conversations with a possible collaborator in the UK to explore the possibility to perform 3D reconstructions at the EM level for a future publication.

   Similarly, they tested the involvement of necroptosis also simply by immunostaining of necroptotic key factors. These staining results were not consistent in different figures. Western blotting is better for the examination of protein level changes of MLKL, pMLKL, RIPK3, and pRIPK3.

   We will perform western blot for pMLKL and pRIPK3 in the different conditions, including different ages, aged Mlkl-KO and aged GSK-treated mice.

   It is very confusing which kind of neurons and which circuit is influenced by necroptosis. As emphasized in the description for Fig. 1b in Line 91, axonal degeneration was restricted to the hilus of dentate gyrus (revealed by Fluoro Jade C staining). However, synaptic transmission (Fig. 4a-f, Fig. 6a-e) and plasticity (Fig. 8c,d) were tested for CA3-CA1 projection, instead of DG-CA3 projection. Moreover, cellular senescence, as detected by SA-beta-gal in Fig. S11, was not in granule cells or hilar cells at the dentate gyrus.

   We agree with the reviewer. Considering his comments, we propose to extend our imaging analysis of axonal degeneration and necroptosis activation to the entire hippocampus, including CA1-CA3 subfields. We consider that recording CA1-CA3 circuit represents and overall response of the hippocampus, but also this subfield contains most of the axonal inputs of this brain region. We will now analyze by confocal microscopy Schaffer collaterals axons which correspond to those axons given off by CA3 pyramidal cells that project to CA1. We already showed by immunohistochemistry in Figure 2f that pMLKL levels are increased in Schaffer collaterals axons in aged mice, but we will perform 3D analysis of pMLKL in NF positive axons by immunofluorescence in this region.

   Axonal tracts for DG-CA3 projection were from granule cells at DG. However, pMLKL was found to be increased in hilar cells. In contrast, the authors concluded that pMLKL in granule cells at DG did not exhibit difference during aging (Line 115). The fact is pMLKL can be easily visualized in many cells including granule cells in adult mice that were not aged (Fig.2a, Fig. S1). Moreover, the signal of pMLKL in granule cells can be seen to be increased in aged mice, although they overlapped DAPI on it. These facts lead to a doubt that their immunostaining of pMLKL was not specific, or they did not analyze the signal accurately.

   As the reviewer remarked, we did not observe an increase in pMLKL levels in the granular cell layer of the DG (see the quantification below). Several reports have demonstrated that necroptosis is an axonal-self destruction program that is not necessarily involved in the death of the whole neuron, which suggests that pMLKL could be detected in aged axons without showing changes in the soma. We will include a paragraph in the discussion section to address this issue. By contrast, we did observe increased pMLKL in hilar cells, CA3 neurons and Schaffer collateral axons, as we demonstrated both by immunofluorescence and immunohistochemistry in Fig 2. In order to clarify the reviewer’s doubts regarding our images, we will include the same image presented in Fig 2, showing the pMLKL signal without DAPI. We will also include the pMLKL channel alone in main figures. Moreover, we believe that the new confocal analyses that we are currently performing will help us to better define necroptosis activation and axonal colocalization in the different subfields of the hippocampus.

   The pattern of non-pNF staining in Fig. 1c is not consistent with that in Fig. 3d, Fig. 5a.

   We will repeat these immunostainings and analyze the staining pattern of the non-pNF antibody to give a clear response to this comment, improving the extent of the analysis.

   Minor comments:

   For Fig. 3d,e and Fig. S4, GFAP staining is also suggested since astrocytes are the other glia that are easily reactive to inflammatory pathogenic conditions.

   This is an excellent suggestion. We are currently performing GFAP staining to establish astrocyte activation in the different conditions (aged wt, MLKL KO, and GSK intervention compared to vehicle in aged animals). This data will be included in a revised manuscript.

   Why was there no colocalization of pMLKL with NF in degenerating axonal tracts?

   We are performing confocal studies to study colocalization of these proteins. Indeed, colocalization was found but a better analysis is needed to demonstrate this, which will be included in a new version of the manuscript.

   Fig. 3a showed no hilar cells stained for pMLKL in aged mice, which is different from that shown in Fig. 2b.

   We have reviewed all the available images and there are some variabilities in aged mice that explain different patterns of pMLKL staining. This is not surprising considering the intrinsic heterogeneity of the aging process. Some mice show more axonal staining while other present clear staining in cell layer (soma) and axons. In a revised manuscript, we will include representative images of the different patterns observed.

   Images in Fig. S4 lack labels.

   Label on the figure indicates ‘Iba1’, but the color used does not allow to get a good view. Label will be changed to increase the contrast.

   Fig. S6, pRIPK3 staining pattern is different from that of pMLKL. They were not activated in the same cells?

   We observed pRIPK3 staining in the hilar cells of the hippocampus. We are currently performing double immunostaining against pMLKL and pRIPK3 to determine whether they colocalize within the same cell-type in the hippocampus.

   Fig. S7, pMLKL staining pattern is different from that in Fig. 2a,f?

   As we have detailed in point 8, this could be explained by the variability of the pMLKL staining in aged mice. In a revised manuscript we will review these images and include a supplementary image with the different staining patters found.

   Resolution for signaling pathway annotations in Fig. 8, Fig. S12, Fig. S13, and Fig. S14, is too low.

   We will increase resolution for this data. In addition, we will include the original images in our final version to avoid loss of quality during file conversion to PDF.

   The titles for Table S1 and S2 should be on the top of the tables.

   This has been corrected.

   Reviewer #1 (Significance (Required)):

   The finding that systemic administration of GSK'872 improved synaptic plasticity and mouse performances in water maze is exciting, indicating a potential medicine for brain rejuvenation.

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

   The authors (in the research group of Filipe Court in Chile) previously studied the contributions of the necroptosis pathway to the degeneration of axons following nerve damage. In this paper, the authors ask whether necroptosis pathway contributes to axon loss, inflammation and cognitive decline in naturally aging mice. The study presents some promising observations that suggest necroptosis is activated in the brain of aged mice and that inhibiting necroptosis through genetics or pharmacology can rescue some cognitive defects in aged mice. The potential implications are exciting, however the scope of what is presented thus far is preliminary. I'll list below several issues with both the experimental design and the presentation of data that strongly diminish from the potential conclusions and significance of this work.

   Major comments:

   1. The n is limited to 3 mice per condition in most of the experiments in this study, and it is not mentioned what sex the animals were. If data was pooled from both sexes than the n is not large enough to take into account potential sex differences. The absence of discussion of sex in the methods weakens my trust in the experimental design.

   WT mice of different ages are all male, purchased from Jackson Laboratory and shipped to Chile (USA). In a revised version, we will specify the sex of the mice in the methods section of the manuscript. This also applies for the group of mice used to pharmacologically inhibit necroptosis with GSK’872, which were also purchased from Jackson Labs. Regarding Mlkl-KO and their WT littermates we used both sexes. We will include a table detailing all the animals used and their age and sex in a revised manuscript. Moreover, as the reviewer suggested, we are currently increasing the n for morphological analysis from 3 to 5, which will be included in the new version of this work. For the behavioral experiments, we have used large group of animals. All details about this and sex of the KO animals will be included in a table with the raw data files.

   2)The studies are not thorough. For instance, there is only one age presented for the mlkl-KO mice. Do these mice still age-dependent changes in axon degeneration or inflammation markers?

   We have the data for other ages in the Mlkl-KO animals, which will be included in the revised manuscript.

   The strain background of the mlkl-KO mice is not mentioned and it is not clear what steps (if any) have been taken to control for strain background and rearing conditions. For instance, WT mice of different ages were purchased from Jackson labs while mlkl-KO mice were apparently bred in house.

   We will include this information in the revised manuscript. Mlkl knockout mice (Mlkl-KO) were kindly provided by Dr Douglas Green (St. Jude Children’s Research Hospital, Memphis, TN, USA). As we have described in the manuscript, the details regarding Mlkl-KO mice are cited in reference 71, which details the generation of Mlkl deficient mice and background. Age-matched control mice correspond to WT mice obtained by Mlkl heterozygous breeding in our animal facility. In addition, we systemically check genotype of mice (PCR-based genotyping protocol is now included in the method section of our manuscript).

   Reference 71. Murphy, J. M. et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39, 443–453 (2013).

   1. For the inhibitor studies, use of littermates for the vehicle control would have been feasible, but it is not mentioned if this was done.

   As we detailed in point 1, we have used Jackson mice for the inhibitor studies. Mice were selected randomly for both vehicle and GSK’872 groups. We are currently increasing the number of mice (10 more animals per condition) for behavioral and morphological analyses in order to control for eventual variations.

   1. The Morris water tank test is a stressful condition for aged mice. Differences in performance could be confounded by differences in swimming ability and potentially stress response. Its a pity that this is the only behavioral test shown, since there are many others (eg Y-maze or novel object recognition) that would be appropriate for the questions posed.

   We evaluated swimming ability of mice and we did not observe differences between WT and KO mice of same age (see figure below), as we discussed in the manuscript. However, we agree with the reviewer that the use of other behavioral test to evaluate memory in aged mice without a stressful condition will improve the quality of our work and will help to support our data. Therefore, we will perform Y-maze and NOR in aged MLKL WT and KO mice as well as in aged animals treated with GSK-872.

   Minor comments:

   1. It is striking that some very relevant citations are absent. For instance, PMID: 34515928 (october 2021) noted a compelling increase in necroptosis markers in the aging CNS, and effects on neuroinflammation in aging mice. These conclusions have some overlap with conclusions in this study. The other study did not address contributions of necroptosis to cognitive or synaptic defects, so the current study still has some novelty, and is supported by other work that should be cited.

   This article was included in the introduction and discussion section of the original manuscript. As there are two version of the manuscript uploaded in bioRxiv file, is possible that the reviewer is referring to the first version (November 11). In fact, the second version (April 18) was uploaded to specifically include this reference.

   https://www.biorxiv.org/content/10.1101/2021.11.10.468052v2.full

   1. The methods used for analysis need to be described with more detail and rigor. For instance, how many sections are analyzed and where and how? How is normalization done and how is it determined that analogous regions are compared across animals and conditions? Likewise, the method for scoring axonal fragmentation needs to be described, and clarified where in the brain this is analyzed.

   We thank the reviewer for this suggestion. We will include a detailed description of the analysis performed, including the details referred by the reviewer and the new analyses.

   1. There are numerous typos, including impactful ones (such as legend of figure 1 where 'old' mice are 12-25 months).

   We will check and correct for typos in a revised version of the manuscript.

   1. Figure 8A is not legible. Perhaps the findings can be highlighted in a merged form on a single pathway cartoon.

   We will change the image as reviewer suggests. Moreover, we will include original images with better quality as raw data.

   Reviewer #2 (Significance (Required)):

   The investigation of the role of necroptosis in the CNS during aging is of high impact and has translational relevance, since necroptosis is a viable pathway for pharmaceutical targeting.

   The idea that it contributes to axonal degeneration and/or synaptic changes in the aging brain is novel and under-explored.

   Read the original source
4. 

   Review Commons

   Jul 18, 2022

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

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   The authors (in the research group of Filipe Court in Chile) previously studied the contributions of the necroptosis pathway to the degeneration of axons following nerve damage. In this paper, the authors ask whether necroptosis pathway contributes to axon loss, inflammation and cognitive decline in naturally aging mice. The study presents some promising observations that suggest necroptosis is activated in the brain of aged mice and that inhibiting necroptosis through genetics or pharmacology can rescue some cognitive defects in aged mice. The potential implications are exciting, however the scope of what is presented thus far is …

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

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   The authors (in the research group of Filipe Court in Chile) previously studied the contributions of the necroptosis pathway to the degeneration of axons following nerve damage. In this paper, the authors ask whether necroptosis pathway contributes to axon loss, inflammation and cognitive decline in naturally aging mice. The study presents some promising observations that suggest necroptosis is activated in the brain of aged mice and that inhibiting necroptosis through genetics or pharmacology can rescue some cognitive defects in aged mice. The potential implications are exciting, however the scope of what is presented thus far is preliminary. I'll list below several issues with both the experimental design and the presentation of data that strongly diminish from the potential conclusions and significance of this work.

   Major comments:

   1. The n is limited to 3 mice per condition in most of the experiments in this study, and it is not mentioned what sex the animals were. If data was pooled from both sexes than the n is not large enough to take into account potential sex differences. The absence of discussion of sex in the methods weakens my trust in the experimental design.

   2. The studies are not thorough. For instance, there is only one age presented for the mlkl-KO mice. Do these mice still age-dependent changes in axon degeneration or inflammation markers? The strain background of the mlkl-KO mice is not mentioned and it is not clear what steps (if any) have been taken to control for strain background and rearing conditions. For instance, WT mice of different ages were purchased from Jackson labs while mlkl-KO mice were apparently bred in house.

   3. For the inhibitor studies, use of littermates for the vehicle control would have been feasible, but it is not mentioned if this was done.

   4. The Morris water tank test is a stressful condition for aged mice. Differences in performance could be confounded by differences in swimming ability and potentially stress response. Its a pity that this is the only behavioral test shown, since there are many others (eg Y-maze or novel object recognition) that would be appropriate for the questions posed.

   Minor comments:

   1. It is striking that some very relevant citations are absent. For instance, PMID: 34515928 (october 2021) noted a compelling increase in necroptosis markers in the aging CNS, and effects on neuroinflammation in aging mice. These conclusions have some overlap with conclusions in this study. The other study did not address contributions of necroptosis to cognitive or synaptic defects, so the current study still has some novelty, and is supported by other work that should be cited.

   2. The methods used for analysis need to be described with more detail and rigor. For instance, how many sections are analyzed and where and how? How is normalization done and how is it determined that analogous regions are compared across animals and conditions? Likewise, the method for scoring axonal fragmentation needs to be described, and clarified where in the brain this is analyzed.

   3. There are numerous typos, including impactful ones (such as legend of figure 1 where 'old' mice are 12-25 months).

   4. Figure 8A is not legible. Perhaps the findings can be highlighted in a merged form on a single pathway cartoon.

   Significance

   The investigation of the role of necroptosis in the CNS during aging is of high impact and has translational relevance, since necroptosis is a viable pathway for pharmaceutical targeting.

   The idea that it contributes to axonal degeneration and/or synaptic changes in the aging brain is novel and under-explored.

   Read the original source
5. 

   Review Commons

   Jul 18, 2022

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

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   This study is interesting on finding that necroptosis may regulate axonal degeneration in the dentate gyrus, which led to the loss of synaptic transmission and plasticity, and impaired the performance of mice in water maze. Genetic ablation of MLKL, a key factor for necroptotic pathway, or pharmacological inhibition of necroptosis with GSK'872, the inhibitor to another necroptotic key factor RIPK3, prevented mice from axonal degeneration, synapse dysfunction, and memory loss. They also tested long term potentiation (LTP) and found LTP that was disrupted in aged mice, was rescued by MLKL knockout or GSK'872 treatments. The authors …

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

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   This study is interesting on finding that necroptosis may regulate axonal degeneration in the dentate gyrus, which led to the loss of synaptic transmission and plasticity, and impaired the performance of mice in water maze. Genetic ablation of MLKL, a key factor for necroptotic pathway, or pharmacological inhibition of necroptosis with GSK'872, the inhibitor to another necroptotic key factor RIPK3, prevented mice from axonal degeneration, synapse dysfunction, and memory loss. They also tested long term potentiation (LTP) and found LTP that was disrupted in aged mice, was rescued by MLKL knockout or GSK'872 treatments. The authors further compared normally aged mice (more than 20 months old) with aged MLKL-knockout mice or aged mice with GSK'872 treatments for altered proteins by single-shot label-free mass spectrometry, and discovered that in 7000 detected proteins, 2516 proteins were increased while 2307 were decreased in the aging hippocampus. They carried out bioinformatic analysis and clustered these proteins for biofunctions of synaptic mechanisms, senescence, etc.. With the bioinformatic analysis, they further examined cell senescence by SA-βgalactosidase (SA-βgal) staining, and concluded that the cellular senescence was also rescued by necroptosis inhibition.

   Although it is an exciting idea that inhibiting necroptosis may be a potential approach to combating aging and rejuvenating the brain, I have many of concerns about the reliability and consistency of the data that did not show strong supports to the conclusion.

   Major comments:

   1. The authors used axonal degeneration as a major readout for brain aging. However, the conclusion of axonal degeneration was simply based on immunostaining. These staining results are not consistent in different parts, and their conclusion is hard to be supported by the representative images. It is not convincing that axonal integrity was altered as concluded by the authors as shown in the representative images of Fig. 1e, Fig. 2d, Fig. 3a, as well as those in the supplementary figures. Electromicroscopy and other convincing means are necessary.
   2. Similarly, they tested the involvement of necroptosis also simply by immunostaining of necroptotic key factors. These staining results were not consistent in different figures. Western blotting is better for the examination of protein level changes of MLKL, pMLKL, RIPK3, and pRIPK3.
   3. It is very confusing which kind of neurons and which circuit is influenced by necroptosis. As emphasized in the description for Fig. 1b in Line 91, axonal degeneration was restricted to the hilus of dentate gyrus (revealed by Fluoro Jade C staining). However, synaptic transmission (Fig. 4a-f, Fig. 6a-e) and plasticity (Fig. 8c,d) were tested for CA3-CA1 projection, instead of DG-CA3 projection. Moreover, cellular senescence, as detected by SA-beta-gal in Fig. S11, was not in granule cells or hilar cells at the dentate gyrus.
   4. Axonal tracts for DG-CA3 projection were from granule cells at DG. However, pMLKL was found to be increased in hilar cells. In contrast, the authors concluded that pMLKL in granule cells at DG did not exhibit difference during aging (Line 115). The fact is pMLKL can be easily visualized in many cells including granule cells in adult mice that were not aged (Fig.2a, Fig. S1). Moreover, the signal of pMLKL in granule cells can be seen to be increased in aged mice, although they overlapped DAPI on it. These facts lead to a doubt that their immunostaining of pMLKL was not specific, or they did not analyze the signal accurately.
   5. The pattern of non-pNF staining in Fig. 1c is not consistent with that in Fig. 3d, Fig. 5a.

   Minor comments:

   1. For Fig. 3d,e and Fig. S4, GFAP staining is also suggested since astrocytes are the other glia that are easily reactive to inflammatory pathogenic conditions.
   2. Why was there no colocalization of pMLKL with NF in degenerating axonal tracts?
   3. Fig. 3a showed no hilar cells stained for pMLKL in aged mice, which is different from that shown in Fig. 2b.
   4. Images in Fig. S4 lack labels.
   5. Fig. S6, pRIPK3 staining pattern is different from that of pMLKL. They were not activated in the same cells?
   6. Fig. S7, pMLKL staining pattern is different from that in Fig. 2a,f?
   7. Resolution for signaling pathway annotations in Fig. 8, Fig. S12, Fig. S13, and Fig. S14, is too low.
   8. The titles for Table S1 and S2 should be on the top of the tables.

   Significance

   The finding that systemic administration of GSK'872 improved synaptic plasticity and mouse performances in water maze is exciting, indicating a potential medicine for brain rejuvenation.

   Read the original source
6. 

   Version published to 10.1101/2021.11.10.468052v2 on bioRxiv

   Apr 18, 2022
7. 

   Version published to 10.1101/2021.11.10.468052v1 on bioRxiv

   Nov 11, 2021