The ATF6β-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity

  1. Dinh Thi Nguyen
  2. Thuong Manh Le
  3. Tsuyoshi Hattori
  4. Mika Takarada-Iemata
  5. Hiroshi Ishii
  6. Jureepon Roboon
  7. Takashi Tamatani
  8. Takayuki Kannon
  9. Kazuyoshi Hosomichi
  10. Atsushi Tajima
  11. Shusuke Taniuchi
  12. Masato Miyake
  13. Seiichi Oyadomari
  14. Shunsuke Saito
  15. Kazutoshi Mori
  16. Osamu Hori

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Abstract

While ATF6α plays a central role in the endoplasmic reticulum (ER) stress response, the function of ATF6β is largely unknown. Here, we demonstrate that ATF6β is highly expressed in the hippocampus of the brain, and specifically regulates the expression of calreticulin, a molecular chaperone in the ER with a high Ca 2+ -binding capacity. Calreticulin expression was reduced to ~50% in the central nervous system of Atf6b −/− mice, and restored by ATF6β. Analysis using cultured hippocampal neurons revealed that ATF6β deficiency reduced Ca 2+ stores in the ER and enhanced ER stress-induced death, which was rescued by ATF6β, calreticulin, Ca 2+ -modulating reagents such as BAPTA-AM and 2-APB, and ER stress inhibitor salubrinal. In vivo , kainate-induced neuronal death was enhanced in hippocampi of Atf6b −/− and Calr +/− mice, and restored by 2-APB and salubrinal. These results suggest that the ATF6β-calreticulin axis plays a critical role in the neuronal survival by improving Ca 2+ homeostasis under ER stress.

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    Reply to the reviewers

    Reviewer 1

    We would like to thank you for the comments concerning our manuscript. We responded to each question, as described below. All the authors feel that our manuscript has been much improved by your comments.

    Minor comments:

    Q1. Fig 1 any male vs female mice differences in ATF6b expression?

    Response. We performed qPCR using several tissues from male and felame WT mice, and confirmed no significant differences in Atf6b mRNA levels between male and female mice. We put this result in Figure S1 C.

    Q2. Fig 2C. Please show molecular weight markers on blots

    Response. We put molecular weight markers in Fig.2C, as you suggested.

    Q3. Fig 2C. what are the doublet bands on calnexin?

    Response. Calnexin is sometimes shown as double bands in tissues such as kidney, liver and heart by western blotting (Zeng et al., PLoS One. 2009 Aug 26;4(8):e6787). Although the mechanism is unknown, it could be due to the post-translational modification such as phosphorylation (Wong et al, J Biol Chem. 1998 Jul 3;273(27):17227-35) or partial degradation although proteinase inhibitors are added in the lysis buffer. To my knowledge, alternative splicing is not likely to be the case.

    Q4. Fig 3. what are the ERSE sequences? several different binding sites are reported in literature.

    Response. We put the ERSE sequence in Materials and Methods and in the Figure legends for Figure 3 as “CCAATN9CCACG (Yoshida et al., 1998)”.,

    Q5. p8. What is meant by 5' Atf6b lacks 10 and 11?

    Response We corrected to “Atf6b transcript, which lacks exon 10 and 11, in these mice”.

    Discussion: Please clarify if anti-ATF6-beta antibodies were available for these studies.

    Response. We tried different anti-ATF6β antibodies to detect endogenous ATF6β in culture neurons by western blot. We successfully observed both full-length and N-terminal fragment (the active form) using the one from Biolegend (#853202) (Figure 1E in the new version). We replaced the result with FLAG antibody in HEK293T cells in the old version.

    Discussion: It is puzzling that ATF6a induces calreticulin more potently than ATF6b, but the calreticulin defect is selectively dependent on ATF6b. Could authors speculate on this paradox? It would be interesting to expand on differences between ATF6a and ATF6b function and phenotypes in Discussion in mouse and in people.

    Response. In Discussion, we added sentences regarding a bit puzzling role for ATF6β in CRT expression in the CNS, as below. “All the data from RNA-sequence to the promoter analysis suggested that CRT expression was ATF6β-dependent in primary hippocampal neurons. However, overexpression of ATF6α and ATF6β both enhanced CRT promoter activity…”

    And we proposed a new scenario as below,

    “These results may raise a scenario that, in the CNS, expression of molecular chaperones in the ER is generally governed by ATF6α as previously described (Yamamoto et al., 2007) and that ATF6β functions as a booster if their levels are too low. However, expression of CRT is somewhat governed by ATF6β, and ATF6α functions as a booster. The underlying mechanism for this scenario is not clear yet, but neurons may require a high level of CRT expression even under normal condition, as described in Table S2, which may lead to the development of a unique biological system to constitutively produce CRT in neurons. Further studies are required to clarify the molecular basis how this unique system is constructed and regulated.”

    Reviewer 2

    We would like to thank you for the comments concerning our manuscript. We responded to each question, as described below. All the authors feel that our manuscript has been much improved by your comments.

    Major comments:

    Q1. The post-translational processing of ATF6beta must be demonstrated in hippocampal neurons and not in HEK293T cells in Figure 1E. The authors conclude on Page 6, line 18 that "these results suggest that ATF6beta functions in neurons" but it is not obvious how expression in HEK293T cells contributes to this conclusion in any way.

    Response. We performed western blot with different anti-ATF6β antibodies to detect endogenous ATF6β in culture neurons. We successfully observed both full-length and N-terminal fragment (the active form) from the one from Biolegend (#853202). We therefore replaced the result in HEK293T cells with the one in the hippocampal neurons (Figure 1E in new version).

    Q2. The hippocampal neurons are affected by the loss of ATF6β, even though the mice are not exposed to tunicamycin. Could the authors present evidence that there is physiological ER stress in hippocampal neurons? If not, why is ATF6beta required.

    Response Evidence suggests that neuronal activities including excitatory signals can cause physiological ER stress and induce the UPR at the distal dendrites in the hippocampal neurons (Murakami et al., Neuroscience. 2007 Apr 25;146(1):1-8, Saito et al., J Neurochem. 2018 Jan;144(1):35-49). Among the UPR branches, Ire1-XBP1 pathway has been reported to play an important role in this dendritic UPR and expression of BDNF in cell soma (Saito et al., 2018). Although the present study focuses on the role of ATF6β in the pathological ER stress which causes neuronal death, we believe that it will be intriguing to analyze its role of ATF6β in the physiological ER stress and in the local UPR machinery in neurons.

    Q3. In Figure 3, is there a specific reason why the authors do not mutate the ERSEs in the mouse CRT reporter, pCC1 and instead opt to analyze the huCRT reporter? Given that all the other observations in the manuscript are in mouse calreticulin, it is important to show that the ERSEs in the mouse calreticulin promoter are also regulated in an ATF6beta-dependent manner. Similar to the huCRT reporter, it is also crucial to examine if ATF6beta can regulate the mouse CRT promoter. This would provide an explanation for why calreticulin expression is not completely abolished in ATF6beta mutants.

    Response We added the data of the deletion mutant of mouse CRT promoter, pCC3, which has only 415bp, but still keeps both ERSE1 and 2 in it. pCC3 showed similar promoter activity to pCC1 (Figure 3 B) and huCRT (wt) (Figure 3 C) in both of WT and Atf6b-/- neurons. Because pCC5, which has 260bp but does not have ERSEs in it, lost completely CRT promoter activity (Waser et al., 1997), it is most likely that mouse and human CRT promoters are regulated in a similar manner via ERSEs.

    Q4. In Figure 5A and B, the density of Tubulin staining varies from panel to panel, and is much lower in ATF6beta mutants treated with Tg/Tm. Presumably this is because of cell death but this should be clarified in the main text. Additionally, it is unclear if the EthD-1 staining is nuclear localized. It would help if single channel images for Hoechst and EthD-1 were provided to visualize this.

    Response In Figure 5A and B, we added the statement for the reduction of Calcein-AM (A) and βIII tubulin (B) in the main text. We also added single channel images for Hoechst and EthD-1 in Figure S4 to confirm the nuclear localization of EthD-1.

    Q5. The literature reports that BAPTA-AM treatment itself could cause ER stress (e.g. PMID: 12531184). Here, the authors report the opposite effect. How could the authors reconcile the difference? The effects of BAPTA-AM and 2-APB must individually be examined in Figure 6C and not just in combination with Tm.

    Response. We added the data that BAPTA-AM and 2-APB alone did not cause neuronal death at the concentrations used in this study in Figure S6 B and in the main text.

    Q6. The authors allude to "impairment of Ca2+ homeostasis in ATF6beta mutants" in Page 13 Line 2, but do not show any direct evidence in support of it. While treatment with BAPTA-AM and 2-APB is a start in that direction, it certainly does not demonstrate that under homeostatic conditions in vivo or in vitro there is any change in calcium flux in ATF6beta hippocampal neurons. To make the case that there is indeed perturbation of Ca2+ in ATF6beta mutant hippocampal neurons, the authors need to examine calcium flux and measure calcium indicators and how they are affected when ER stress is induced in these mutant cells.

    Response We added the data that the Ca2+ store in the ER was reduced and Ca2+ concentration in the cytosol increased in Atf6b-/- neurons both under normal and ER stress conditions in Figure 4C.

    Q7. The effect of 2-APB and salubrinal alone on hippocampal neurons need to be examined in Figure 9B-D to eliminate the possibility that these drugs are not enhancing cell survival under normal conditions in a parallel manner.

    Response We added the data that 2-APB and salubrinal alone did not cause neuronal death in the hippocampus in our model in Figure S8 C.

    Q8. The rationale for the examination of Fos, Fosb and Bdnf is poorly described (page 14, line 13) and the conclusions from this line of experimentation are rather weak. The results from Figure 9 to some extent serve to confirm in vivo the data seen in Figure 6C but by no means provide a mechanism for why ATF6beta mutants have perturbed calcium homeostasis (page 14, line 22).

    Response We agreed with your comments that the examination of Fos, Fosb and Bdnf is relatively weak. We, therefore, moved these data to supplementary information (Figure S8 A and B).

    Minor comments:

    Q1. Page 8, line 3: Their rationale for why ATF6beta 5'UTR sequences are seen in their RNA seq data is not clearly explained. This must be rewritten for clarity.

    Response In Atf6b-/- mice, exon 10 and 11 were deleted by homologous recombination. Therefore, 5’ part of Atf6b gene including exon 1-9 can be transcribed. We added the statement in Results, as below.

    “this may be due to the presence of the 5’ Atf6b transcript with exon 1-9 in these mice, in which exon 10 and 11 were deleted by homologous recombination.”

    Q2. Page 8, line 5, the authors write that besides Atf6β , CRT was the only UPR-regulated gene downregulated in Atf6β mutant mice. The authors need to state how they defined "UPR-regulated genes". There must be a list, which the authors do not cite.**

    Response. To avoid the possible confusion, we changed the term “UPR-regulated genes” to “ER stress-responsive genes”.

    Q3. Page 9, line 10: A reference is required for ERSEs.

    Response We added the reference for ERSEs, as you suggested.

    Q4. Page 10, line 6: The authors say "ATF6beta specifically induces CRT promoter activity". This is a confusing** **statement because "induction" is in response to stress, but the context here is homeostatic regulation since there is ostensibly no stress being induced. This distinction should be made and corrected here and throughout the manuscript.

    Response To avoid the confusion, we changed the sentence to “ATF6β specifically enhances CRT promoter activity”.

    Q5. Page 10, line 16: The use of "latter" here is confusing and it would help to restructure this sentence for clarity.

    Response To avoid the confusion, we changed the phrase to “under control condition and after stimulation with Tg (Figure 4A upper row) or Tm (Figure 4A lower row)

    Q6. Figure 9A is missing Y-axis labels.

    Response We changed Figure 9A (Figure S8 A in new version) and Figure Legends to clarify what each axis indicates.

    Reviewer 3

    We would like to thank you for the comments concerning our manuscript. We responded to each question, as described below. All the authors feel that our manuscript has been much improved by your comments.

    Major comments

    Comment #1. The authors show that overexpression of either Atf6a or Atf6b both increase Crt expression in Atf6b knockout cells. While it is clear that deletion of Atf6a does not basally reduce Crt levels, the overexpression experiment does lead to a question as to how Atf6b can specifically be involved in regulating Crt expression. In the discussion, the authors seem to propose that homo- and hetero-dimerization of ATf6a and Atf6b are required for the basal expression of Crt and that Atf6b serves as a 'booster' of ER chaperone expression. They explicitly state that "Atf6a and Atf6b are required to induce CRT expression". However, it remains unclear to me why in this case would Atf6a deletion not impair Crt expression? The authors address this by invoking a mechanism whereby hippocampal neurons are more reliant on Atf6b for Crt expression, but this doesn't really make sense to me. Ultimately, this point underscores the lack of clear mechanistic basis to explain how Atf6b selectively regulates Crt in the hippocampus. This needs to be better resolved through more experimentation. For example, a ChIP experiment monitoring the binding of ATF6b and ATF6a to the Crt promoter in hippocampal and control cells would go a long way towards addressing this issue.**

    Response. In Discussion, we first made the point clearer that CRT expression is ATF6β-dependent, while those of other molecular chaperones in the ER are ATF6α-dependent. Then, we raised a scenario that, in the CNS, expression of molecular chaperones in the ER is generally governed by ATF6α as previously described (Yamamoto et al., 2007) and ATF6β functions as a booster if their levels are too low. However, expression of CRT is somewhat governed by ATF6β, and ATF6α functions as a booster. We also wrote the limitation of the current study and requirement of the further study to clarify the molecular basis of the unique system to ensure CRT expression in neurons.

    Comment #2. The importance of ATF6b for protecting against insults needs to be better described. For example, the authors should show that overexpression of ATF6b protects against ER stress induced neuronal toxicity in cell culture and in vivo kainate induced neuronal toxicity. Similarly, the authors should evaluate how overexpression of ATF6a protects against these insults to better define the specific dependence of hippocampal neurons on ATF6b. The authors do show that overexpression of ATF6b can rescue the reduced Crt observed in Atf6b-deleted neurons, but the protection should similarly be demonstrated.**

    Response. We performed rescuing experiments to see both of ATF6β and ATF6α overexpression improve cell viability of Atf6b-/- neurons under ER stress. Interesting. ATF6β, but not ATF6α, rescued Atf6b-/- neurons. In Discussion, we raised the possible reasons as below.

    “The lack of rescuing effect of ATF6α may be due to the fact that this molecule enhances the expression of different genes including cell death-related molecule CHOP in addition to molecular chaperons in the ER (Yoshida et al., 2000).”

    Comment #3. Similar to #2, the authors should show that the potential for ATF6b (and ATF6a) overexpression to protect against different insults is impaired in Crt+/- neurons. The authors demonstrate that Crt-depletion increases sensitivity to toxic insults. This would go a long way to demonstrate the importance of the proposed ATF6b-CRT signaling axis in regulating neuronal survival in response to pathologic insults.**

    Response. Unfortunately, right now, the breeding of Calr+/- mice is not in good condition. Although we are increasing the number of mice used for breeding, we have to wait pregnancies to get embryos for isolating neurons from hippocampus. Once we get enough number of mice, we would try the rescuing experiment of Calr+/- hippocampal neurons with ATF6β and ATF6α. However, we also think rescuing experiments of Atf6b-/- neurons by ATF6β, ATF6α, and CRT may be enough in this paper.

    Comment #4. When reporting the RNAseq data, the authors should use the q-value (i.e., FDR) instead of the p-value. This will likely affect the number of genes reported in Table 1, but it is the appropriate statistical test for this type of data.**

    Response. As you suggested, we replace Table1 with a new list which was filtered with the q-value. However, some important and consistent information were obtained from the list filtered with the p-value, we keep it as Table S1 in the supplementary information.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors define the functional importance of ATF6b in the hippocampus. They show that ATF6b is highly expressed in the hippocampus relative to other tissues. They demonstrate that deletion or depletion of ATF6b in cultured hippocampal neurons enhances ER stress induced death. Similarly, Atf6b-/- mice show increased sensitivity to kainate induced neuronal death. These results reveal an important role for ATF6b in regulating hippocampal survival in response to pathologic insults. To define a molecular basis for this protection, the authors utilized RNAseq to identify the lectin chaperone calreticulin (Crt) as a gene whose expression is basally reduced in cultured hippocampal neurons where Atf6b is deleted. They show the re-overexpression of Atf6b (or Atf6a) both restore Crt levels in these neurons, underscoring the importance of Atf6 in regulating basal Crt levels. They go on to demonstrate that loss of Atf6b impairs ER stress-dependent increases in Crt, while minimally impacting other Atf6 target genes, again highlighting the importance of Atf6b for Crtregulation. Importantly, overexpression of Crt rescues the increased ER stress-induced toxicity observed in Atf6b knockout neurons, indicating that a primary mechanism by which Atf6b regulates neuronal survival in response to ER stress is through increased Crt expression. Consistent with this, mimicking the 50% reduction in Crt observed in Atf6b knockout neurons using Crt+/- mice showed similar sensitivity to kainate induced neuronal death. Collectively, these results describe an Atf6-Crt axis that is important for regulating neuronal survival in response to pathologic insults.

    Overall the experiments are interesting and provide new insights into the importance of Atf6b in neuronal survival. Notably, the evidence showing that loss of Atf6b increases hippocampal neuron sensitivity to ER stress and kainate induced toxicity are compelling. Any results describing the biological function of Atf6b are interesting, considering how little we know about this ER stress sensing protein. That being said, I have some concerns about the work described that require addressing before publication. Notably, I think more work needs to be done to define the molecular basis for the specific dependence of Crt expression on ATF6b in hippocampal neurons. Further, the authors need to do more experiments to demonstrate the specific importance of ATF6b signaling in the context of ER stress and in vivo neuronal death. I outline these various concerns below:

    Comment #1. The authors show that overexpression of either Atf6a or Atf6b both increase Crt expression in Atf6b knockout cells. While it is clear that deletion of Atf6a does not basally reduce Crt levels, the overexpression experiment does lead to a question as to how Atf6b can specifically be involved in regulating Crt expression. In the discussion, the authors seem to propose that homo- and hetero-dimerization of ATf6a and Atf6b are required for the basal expression of Crt and that Atf6b serves as a 'booster' of ER chaperone expression. They explicitly state that "Atf6a and Atf6b are required to induce CRT expression". However, it remains unclear to me why in this case would Atf6a deletion not impair Crt expression? The authors address this by invoking a mechanism whereby hippocampal neurons are more reliant on Atf6b for Crt expression, but this doesn't really make sense to me. Ultimately, this point underscores the lack of clear mechanistic basis to explain how Atf6b selectively regulates Crt in the hippocampus. This needs to be better resolved through more experimentation. For example, a ChIP experiment monitoring the binding of ATF6b and ATF6a to the Crt promoter in hippocampal and control cells would go a long way towards addressing this issue.

    Comment #2. The importance of ATF6b for protecting against insults needs to be better described. For example, the authors should show that overexpression of ATF6b protects against ER stress induced neuronal toxicity in cell culture and in vivo kainate induced neuronal toxicity. Similarly, the authors should evaluate how overexpression of ATF6a protects against these insults to better define the specific dependence of hippocampal neurons on ATF6b. The authors do show that overexpression of ATF6b can rescue the reduced Crt observed in Atf6b-deleted neurons, but the protection should similarly be demonstrated.

    Comment #3. Similar to #2, the authors should show that the potential for ATF6b (and ATF6a) overexpression to protect against different insults is impaired in Crt+/- neurons. The authors demonstrate that Crt-depletion increases sensitivity to toxic insults. This would go a long way to demonstrate the importance of the proposed ATF6b-CRT signaling axis in regulating neuronal survival in response to pathologic insults.

    Comment #4. When reporting the RNAseq data, the authors should use the q-value (i.e., FDR) instead of the p-value. This will likely affect the number of genes reported in Table 1, but it is the appropriate statistical test for this type of data.

    Significance

    This manuscript provides new context for understanding the functional relationship between Atf6a and the less-studied Atf6b in regulating neuronal survival. As with other studies focused on the relationship between these two ATF6 isoforms, this study demonstrates that these transcriptional programs integrate to coordinate a tissue-specific response to ER stress. Intriguingly, these studies indicate that ATF6b has a specific role in regulating the ER lectin chaperone CRT and that this ATF6b-CRT axis uniquely regulates neuronal survival in response to ER stress. While additional experiments are required to support this claim, the work described herein is a nice addition to our evolving understanding of the importance of ATF6b in regulating ER and cellular physiology during pathologic insults.

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    Referee #2

    Evidence, reproducibility and clarity

    Nguyen and colleagues provide evidence that ATF6-beta selectively induces calreticulin expression in mouse hippocampal neurons to protect these neurons from ER stress-inducing toxins. This is a well-written and well-organized report that provides functional information about ATF6-beta, a poorly studied homolog of the ATF6-alpha Unfolded Protein Response regulator. The report suggests that ATF6-beta has a previously unknown and important function in helping brain neurons survive ER stress by regulating calreticulin.

    The study shows that addition of BAPTA, 2-APB, or salubrinal significantly improves neuronal survival in ATF6-/- explants and mice brains in response to ER toxins. But, prior study (PMID: 15705855) used salubrinal at much higher concentration 75uM with little effect at the 5uM dose used in the current study. Evidence should be provided that these drugs are specifically inhibiting ER stress or off-target mechanisms should be discussed in their experimental models.

    Minor comments:

    Fig 1 any male vs female mice differences in ATF6b expression?

    Fig 2C. Please show molecular weight markers on blots

    Fig 2C. what are the doublet bands on calnexin?

    Fig 3. what are the ERSE sequences? several different binding sites are reported in literature.

    p8. What is meant by 5' Atf6b lacks 10 and 11?

    Discussion: Please clarify if anti-ATF6-beta antibodies were available for these studies.

    Discussion: It is puzzling that ATF6a induces calreticulin more potently than ATF6b, but the calreticulin defect is selectively dependent on ATF6b. Could authors speculate on this paradox? It would be interesting to expand on differences between ATF6a and ATF6b function and phenotypes in Discussion in mouse and in people.

    Significance

    ATF6-beta is homolog of ATF6-alpha and assumed to function like ATF6-alpha. This report describes a selective function of ATF6-beta in inducing calreticulin in mouse neurons during ER stress. This suggests ATF6-beta has some different functions than ATF6-alpha in the mouse hippocampal neurons.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    Unfolded Protein Response (UPR) refers to homeostatic signaling pathways that play protective roles in various cell types. This work by Nguyen et al focuses on the UPR-mediator ATF6. In mammals, there are two isoforms of ATF6, alpha and beta. Nguyen et al show that the expression of the ATF6beta isoform is higher in hippocampal neurons whereas the ATF6alpha isoform is more evenly distributed across various neuronal subtypes. By performing gene expression profiling in mouse brain samples, they identify the ER chaperone calreticulin (CRT) as being significantly downregulated in ATF6beta null mutants. They further validate this observation by comparing hippocampi from ATF6alpha and ATF6beta null mice, where CRT is lowered in the latter but not the former. They identify and mutate putative ER stress response elements (ERSE) in the CRT promoter region to show that expression of CRT can be regulated by both ATF6alpha and beta. They demonstrate that treatment of hippocampal neurons with ER stress inducing chemicals leads to induction of CRT, which is suppressed in ATF6beta mutants. Such treatment also leads to cell death, which is exacerbated in ATF6beta mutants but rescued by ectopic expression of CRT. They also extend these observations to cell death induced by treatment with the glutamate receptor agonist, kainate, which was also exacerbated in ATF6beta mutants, but was rescued by counter treatment with ER stress inhibitors. Together, their data suggest a protective role for ATF6beta in hippocampal neurons in the context of ER stress.

    Major comments

    The primary advantage of this work is that much of it was done in vivo in mice, providing immediate context for the role of ATF6β under physiological conditions. They identify a specific region of the brain that requires ATF6beta. On the other hand, the ATF6-CRT signaling axis reported here had been established previously, and therefore, this study brings limited conceptual advances regarding the signaling mechanism itself (see Significance section below).

    Overall, the authors' data support their primary claim that ATF6β has a neuroprotective role in the context of ER stress. The data presented are clear and convincing, and their methods appear rigorous. The manuscript could be further improved if the authors could provide sufficient rationale for some of their experiments, which are discussed below.

    1. The post-translational processing of ATF6beta must be demonstrated in hippocampal neurons and not in HEK293T cells in Figure 1E. The authors conclude on Page 6, line 18 that "these results suggest that ATF6beta functions in neurons" but it is not obvious how expression in HEK293T cells contributes to this conclusion in any way.
    2. The hippocampal neurons are affected by the loss of ATF6β, even though the mice are not exposed to tunicamycin. Could the authors present evidence that there is physiological ER stress in hippocampal neurons? If not, why is ATF6beta required.
    3. In Figure 3, is there a specific reason why the authors do not mutate the ERSEs in the mouse CRT reporter, pCC1 and instead opt to analyze the huCRT reporter? Given that all the other observations in the manuscript are in mouse calreticulin, it is important to show that the ERSEs in the mouse calreticulin promoter are also regulated in an ATF6beta-dependent manner. Similar to the huCRT reporter, it is also crucial to examine if ATF6beta can regulate the mouse CRT promoter. This would provide an explanation for why calreticulin expression is not completely abolished in ATF6beta mutants.
    4. In Figure 5A and B, the density of Tubulin staining varies from panel to panel, and is much lower in ATF6beta mutants treated with Tg/Tm. Presumably this is because of cell death but this should be clarified in the main text. Additionally, it is unclear if the EthD-1 staining is nuclear localized. It would help if single channel images for Hoechst and EthD-1 were provided to visualize this.
    5. The literature reports that BAPTA-AM treatment itself could cause ER stress (e.g. PMID: 12531184). Here, the authors report the opposite effect. How could the authors reconcile the difference? The effects of BAPTA-AM and 2-APB must individually be examined in Figure 6C and not just in combination with Tm.
    6. The authors allude to "impairment of Ca2+ homeostasis in ATF6beta mutants" in Page 13 Line 2, but do not show any direct evidence in support of it. While treatment with BAPTA-AM and 2-APB is a start in that direction, it certainly does not demonstrate that under homeostatic conditions in vivo or in vitro there is any change in calcium flux in ATF6beta hippocampal neurons. To make the case that there is indeed perturbation of Ca2+ in ATF6beta mutant hippocampal neurons, the authors need to examine calcium flux and measure calcium indicators and how they are affected when ER stress is induced in these mutant cells.
    7. The effect of 2-APB and salubrinal alone on hippocampal neurons need to be examined in Figure 9B-D to eliminate the possibility that these drugs are not enhancing cell survival under normal conditions in a parallel manner.
    8. The rationale for the examination of Fos, Fosb and Bdnf is poorly described (page 14, line 13) and the conclusions from this line of experimentation are rather weak. The results from Figure 9 to some extent serve to confirm in vivo the data seen in Figure 6C but by no means provide a mechanism for why ATF6beta mutants have perturbed calcium homeostasis (page 14, line 22).

    Minor comments

    1. Page 8, line 3: Their rationale for why ATF6beta 5'UTR sequences are seen in their RNA seq data is not clearly explained. This must be rewritten for clarity.
    2. Page 8, line 5, the authors write that besides Atf6β , CRT was the only UPR-regulated gene downregulated in Atf6β mutant mice. The authors need to state how they defined "UPR-regulated genes". There must be a list, which the authors do not cite.
    3. Page 9, line 10: A reference is required for ERSEs.
    4. Page 10, line 6: The authors say "ATF6beta specifically induces CRT promoter activity". This is a confusing statement because "induction" is in response to stress, but the context here is homeostatic regulation since there is ostensibly no stress being induced. This distinction should be made and corrected here and throughout the manuscript.
    5. Page 10, line 16: The use of "latter" here is confusing and it would help to restructure this sentence for clarity.
    6. Figure 9A is missing Y-axis labels.

    Significance

    The authors summarize their major findings of the study (at the beginning of the Discussion) as ATF6β being required for CRT induction in the hippocampus, and that this ATF6β -CRT axis is important for the survival of hippocampal neurons. The idea that ATF6 induces CRT had been previously shown by others (PMID 9837962), and therefore, this is not the major new discovery of this study. In addition, the ATF6-calreticulin axis having a cell protective role had been reported in other biological contexts (e.g. PMID: 32905769), so that concept is also not a novel concept presented in this work. Similarly, the role of UPR in glutamate receptor agonist-induced neuronal cell death had been shown previously (the authors cite Kitao e tal., 2001; Sokka et al., 2007; Kezuka et al., 2016), so this link is not the major novel discovery revealed by this study. Instead, this study reports that ATF6β KO mice have specific phenotypes in hippocampal neurons, which had not been reported previously. Furthermore, this manuscript reports detailed information regarding Atf6β's downstream target genes in this tissue. In summary, this study's finding that ATF6 regulates CRT is confirmatory, rather than bringing new conceptual advances. The merit of this study is in the identification of the hippocampus as the organ that specifically requires ATF6beta. While the findings here may not appeal to a broader audience interested in UPR signaling mechanisms, it may draw interest from those who study hippocampal neuron physiology.

    For the editor's reference, this reviewer's field of expertise is in UPR signaling mechanisms in animal models

  5. Version published to 10.1101/2021.02.01.429116 on bioRxiv