The ALS-associated co-chaperone DNAJC7 mediates neuroprotection against proteotoxic stress by modulating HSF1 activity

Reviewer #1 (Public review):

Summary

Fleming et al. present the first, proteomics-based attempt to identify the possible mechanism of action of ALS-linked DNAJC7 molecular chaperone in pathology. Impressively, it is the first report of DNAJC7 interactome studies, using a suitable iPSC-derived lower motor neuron model. Using a co-immunoprecipitation approach the authors identified that the interactome of DNAJC7 is predominantly composed of proteins engaged in response to stress, but also that this interactome is enriched in RNA-binding proteins. The authors also created a DNAJC7 haploinsufficiency cellular model and show the resulting increased insolubility of HNRNPU protein which causes disruptions in its functionality as shown by analysis of its transcriptional targets. Finally, this study uses pharmacological agents to test the effect of decreased DNAJC7 expression on cell response to proteotoxic stress and finds evidence that DNAJC7 regulates the activation of Heat shock factor 1 (HSF1) protein upon stress conditions.

Strengths

(1)This study uses the best so far model to study the interactome and possible mechanism of action of DNAJC7 molecular chaperone in an iPSC-derived cellular model of motor neurons. Furthermore, the authors also looked into available transcriptome databases of ALS patient samples to further test whether their findings may yield relevance to pathology.

(2) The extent to which the authors are explicit about the sample sizes, protocols, and statistical tests used throughout this manuscript, should be applauded. This will help the whole field in their efforts to reliably replicate the results in this study.

Weaknesses

(1) The most significant caveat of interactome experiments inherently comes from the method of choice. It is possible that by using the co-purification approach of DNAJC7 IP the resulting pool of binding partners is depleted in proteins that interact with DNAJC7 weakly or transiently. An alternative approach presumably more sensitive towards weaker binders could use the TurboID-based proximity-labeling method.

(2) The authors mention in Results (and Figure 2D) that HNRNPA1 was identified as DNAJC7-interacting protein in their co-IP experiments, however, an identifier for this protein cannot be found in Figure 1C and Table S1 listing the proteomics results. Could the authors appropriately update Figure 1C and Table S1, or if HNRNPA1 wasn't really a hit then remove it from listed HNRNPs?

(3) No further validation of DNAJC7-interacting proteins from the heat-shock protein (HSP) family. Current validation of mass spectrometry-identified proteins comes from IP-western blots with antibodies against HSPs. It would be interesting to further inspect possible interactions of these proteins by inspecting co-localization with immunocytochemistry.

(4) Similarly, the observation of DNAJC7 haploinsufficiency causing an increase in HNRNPU insolubility could be also easily further confirmed by checking for the emergence of "puncta" under a fluorescence microscope, in addition to provided WB experiments from MN lysates.

(5) I would like to recommend the authors to also provide with this manuscript a complete dataset (possibly in the form of a table, presented similarly as Table S1) resulting from experiments presented in Figures 2F and S2D. The information on upregulated and downregulated targets in their DNAJC7 haploinsufficiency model would be a valuable resource for the field and enable further investigations.

Reviewer #2 (Public review):

Summary:

The manuscript titled "The ALS-associated co-chaperone DNAJC7 mediates neuroprotection against proteotoxic stress by modulating HSF1 activity" describes experiments carried out in iPS cells re-differentiated into motor neurons (iNeuons, MNs) seeking to assess the functions of the J protein DnaJC7 in proteostasis. This study also investigates how an ALS-associated mutant variant (R156X) alters DnaJC7 function.

The proteomic studies identify proteins interacting with DnaJC7. Using mRNA profiling in haplo-insufficient cells (+/R156X) compared to wild-type cells, the study seeks to identify pathways modulated by partial loss of DnaJC7 function. Studies in the DnaJC7 haplo-insufficient cells also indicate changes in the properties of ALS-associated proteins, such as HNRNPU and Matrin3 both of which are involved in the regulation of gene expression. The study also shows data indicating that DnaJC7 haploinsufficiency sensitizes cells to proteostatic stress induced by proteosome inhibition by MG132 and Hsp90 inhibition by Ganetespib. Lastly, the study investigates how DnaJC7 modulates the activity of the heat shock transcription factor (Hsf1) and thus the heat shock response.

Strengths:

The manuscript is well presented and most of the data is of high quality and convincing. The figures and supplementary figures are clear and easy to follow.

This study overall provides important new insights into a mostly underexplored molecular co-chaperone and its role in proteostasis. The proteomic and transcriptomic experiments certainly advance our understanding of DnaJC7. The MN model is well-suited for these studies addressing the role of DnaJC7, particularly regarding ALS. The haplo-insufficient MNs are also a suitable model to study a potential loss of function mechanism caused by (some) fALS-associated mutants in ALS, such as the R156X mutation used here.

Since so little is known about DnaJC7 function, the exploratory approaches applied here are particularly useful.

Weaknesses:

Without follow-up studies, however, e.g., with select interacting proteins, the study provides merely a descriptive list of possible interactions without mechanistic insights. Also, most interactions have not been extensively (only a few examples) validated by other methods or individual experiments.

A major limitation of the study in its current form is that none of the experimental approaches allow for assessing the specific functions of JC7. In the absence of specificity controls, e.g., other J proteins or HOP, which, like DnaJC7, contains TPR domains and can interact with Hsp70 and Hsp90, it remains unclear if the proposed functions of DnaJC7 are specific/unique or shared by other J proteins or molecular chaperones. Accordingly, it would be highly informative to add experiments to assess if some of the reported DnaJC7 protein-protein interactions and the transcriptional alterations in haplo-insufficient cells are DnaJC7specific or also occur with other J proteins or molecular chaperones. This seems particularly important to discern specific DnaJC7 functions from general effects caused by impaired proteostasis.

It would be informative to explore how cellular stress (e.g., MG132 treatment) alters DnaJC7 interactions with other proteins (J proteins, HOP), ideally in additional/comparative proteomic studies.
The mechanism underlying the proposed regulation of Hsf1 by DnaJC7 is not quite clear to me (Figures 4 A-I). There is no evidence of a direct physical interaction between DnJC7 and Hsf1 in the proteomic data or elsewhere. It seems plausible that Hsf1/HSR dysregulation in the haplo-insufficient cells might be due to rather indirect effects, e.g., increased protein misfolding. Also, additional data showing differential activation of Hsf1 in +/+ versus +/- cells would strengthen this part, e.g. showing differences in Hsf1 trimerization, Hsp70 interactions, nuclear localization, etc.

The manuscript might also benefit from considering the literature showing an unusually inactive HSR and Hsf1 activity in motor neurons (e.g. published by the Durham lab).

The correlation with transcriptomic data from ALS patients compared to neurotypical controls (Figures 4 L, M) suggesting a direct role of Hsf1/HSR seems unlikely at this point. In my view, the transcriptional dysregulation in ALS patients could be unrelated to Hsf1 dysregulation and caused by rather non-specific effects of neuronal decay in ALS.

Reviewer #3 (Public review):

Summary:

Fleming et al sought to better understand DNAJC7's function in motor neurons as mutations in this gene have been associated with amyotrophic lateral sclerosis (ALS). The research question is relevant and important. The authors use an induced pluripotent stem cell (iPSC) line to derive motor neurons (iMNs) finding that DNAJC7 interacts with RNA-binding proteins (RBP) in wild-type cells and a truncated mutant DNAJC7[R156*] disrupts the RBP, hnRNPU, by promoting its accumulation into insoluble fractions. Given that DNAJC7 is predicted to regulate stress responses, the authors then find that DNAJC7[R156*] expression sensitizes the iMNs to proteosomal stress by disrupting the expression of the key heat stress response regulator, HSF1. These findings support that loss-of-function mutations in DNAJC7 will indeed sensitize motor neurons to proteotoxic stress, potentially driving ALS. The association with RBPs, which routinely are found to be disrupted in ALS, is of interest and warrants further study.

Strengths:

(1) The research question is relevant and important. The authors provide interesting data that DNAJC7 mutations impact two important features in ALS, the dysregulation of RNA binding proteins and the sensitivity of motor neurons to proteotoxic stress.

(2) The authors provide solid data to support their findings and the assays are appropriate.

Weaknesses:

(1) The authors rely on a single iPSC line throughout the text, using the same line to make the mutation-carrying cells. iPSCs are highly variable and at minimum 3 lines, typically 5 lines, should be used to define consistent findings. This work would be greatly strengthened if 3 or more lines were used to confirm consistent effects. This is particularly concerning given that iPSCs were differentiated using growth factors versus genetic induction. Growth-factor-based differentiations are more variable.

(2) The authors argue that HSF1 and its targets are downregulated in sporadic ALS and mutant C9orf72 ALS. The first concern is that these transcriptomics data were derived from cortical tissue which does not contain motor neurons (Pineda et al. 2024 Cell 187: 1971-1989.e1916). The second concern is that the inclusion of C9orf72 mutant tissue is not well justified as (1) this mutation is associated with an upregulation of HSF1 and its targets in patients (Mordes et al, Acta Neuropathol Commun 2018 6(1):55; Lee et al Neuron 2023 111(9):1381-1390) and (2) the C9orf72 mutation is associated with a ALS/FTD spectrum disorder defined by TDP-43 pathology. Disease mechanisms associated with this spectrum disorder may not overlap with traditional ALS which is typically defined by SOD1 pathology.

(3) As a whole, the findings are mechanistically disjointed, and additional experiments or discussion would help to connect the dots a bit more.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary

Fleming et al. present the first, proteomics-based attempt to identify the possible mechanism of action of ALS-linked DNAJC7 molecular chaperone in pathology. Impressively, it is the first report of DNAJC7 interactome studies, using a suitable iPSC-derived lower motor neuron model. Using a co-immunoprecipitation approach the authors identified that the interactome of DNAJC7 is predominantly composed of proteins engaged in response to stress, but also that this interactome is enriched in RNA-binding proteins. The authors also created a DNAJC7 haploinsufficiency cellular model and show the resulting increased insolubility of HNRNPU protein which causes disruptions in its functionality as shown by analysis of its transcriptional targets. Finally, this study uses pharmacological agents to test the effect of decreased DNAJC7 expression on cell response to proteotoxic stress and finds evidence that DNAJC7 regulates the activation of Heat shock factor 1 (HSF1) protein upon stress conditions.

Strengths

(1) This study uses the best so far model to study the interactome and possible mechanism of action of DNAJC7 molecular chaperone in an iPSC-derived cellular model of motor neurons. Furthermore, the authors also looked into available transcriptome databases of ALS patient samples to further test whether their findings may yield relevance to pathology.

(2) The extent to which the authors are explicit about the sample sizes, protocols, and statistical tests used throughout this manuscript, should be applauded. This will help the whole field in their efforts to reliably replicate the results in this study.

We thank the reviewer for highlighting the strengths of our study.

Weaknesses

(1) The most significant caveat of interactome experiments inherently comes from the method of choice. It is possible that by using the co-purification approach of DNAJC7 IP the resulting pool of binding partners is depleted in proteins that interact with DNAJC7 weakly or transiently. An alternative approach presumably more sensitive towards weaker binders could use the TurboID-based proximity-labeling method.

The reviewer raises a valid point that TurboID-based proximity biotinylation could be a more sensitive approach for identifying DNAJC7 protein-protein interactions compared to IP-MS. We agree that this strategy could be better suited to detect weak or transient interactions, and we have previously used it to characterize protein nanoenvironments and interactomes in vitro and in vivo (Wang et al. Mol Psychiatry 2024, Quan et al. mBio 2024). However, proximity biotinylation also has significant limitations, such as potential artifacts due to overexpression and high background levels. We selected the IP-MS approach to identify DNAJC7 binding partners in neurons without the need of genetically modifying or over-expressing DNAJC7.

(2) The authors mention in Results (and Figure 2D) that HNRNPA1 was identified as DNAJC7-interacting protein in their co-IP experiments, however, an identifier for this protein cannot be found in Figure 1C and Table S1 listing the proteomics results. Could the authors appropriately update Figure 1C and Table S1, or if HNRNPA1 wasn't really a hit then remove it from listed HNRNPs?

We apologize for the confusion. HNRNPA1 was pulled down exclusively with DNAJC7 in 2/3 independent experiments and was initially included in our list of targets. However, in our final and most stringent analysis we only considered proteins that appeared in 3/3 experiments and thus HNRNPA1 was filtered out of Figure 1C and Table S1. We will therefore remove it from Figure 2D in the revised manuscript.

(3) No further validation of DNAJC7-interacting proteins from the heat-shock protein (HSP) family. Current validation of mass spectrometry-identified proteins comes from IP-western blots with antibodies against HSPs. It would be interesting to further inspect possible interactions of these proteins by inspecting co-localization with immunocytochemistry.

As the reviewer points out we did in fact validate the interaction of DNAJC7 with HSP90 and HSP70 (HSP90AB1 and HSPA1A) by IP-WB as shown in Fig 1F. We agree that examining co-localization of these proteins by immunocytochemistry (ICC) would be important to investigate. However, we have been unable to do this due to technical limitations. Specifically, we have tried to perform ICC using 6 commercially available DNAJC7 antibodies and have so far been unsuccessful. In our hands the DNAJC7 ICC signal appears to be non-specific as it is not reduced when using DNAJC7 knockout and knockdown cells as controls.

(4) Similarly, the observation of DNAJC7 haploinsufficiency causing an increase in HNRNPU insolubility could be also easily further confirmed by checking for the emergence of "puncta" under a fluorescence microscope, in addition to provided WB experiments from MN lysates.

This is a good suggestion, and we can assess the emergence of HNRNPU "puncta" by ICC in DNAJC7 mutant iPSC-derived neurons and/or postmortem sporadic ALS patient tissue.

(5) I would like to recommend the authors to also provide with this manuscript a complete dataset (possibly in the form of a table, presented similarly as Table S1) resulting from experiments presented in Figures 2F and S2D. The information on upregulated and downregulated targets in their DNAJC7 haploinsufficiency model would be a valuable resource for the field and enable further investigations.

This is a good suggestion and in the revised version we will provide in Table S2 the dataset presented in Figs. 2F and S2D.

Reviewer #2 (Public review):

Summary:

The manuscript titled "The ALS-associated co-chaperone DNAJC7 mediates neuroprotection against proteotoxic stress by modulating HSF1 activity" describes experiments carried out in iPS cells re-differentiated into motor neurons (iNeuons, MNs) seeking to assess the functions of the J protein DnaJC7 in proteostasis. This study also investigates how an ALS-associated mutant variant (R156X) alters DnaJC7 function. The proteomic studies identify proteins interacting with DnaJC7. Using mRNA profiling in haplo-insufficient cells (+/R156X) compared to wild-type cells, the study seeks to identify pathways modulated by partial loss of DnaJC7 function. Studies in the DnaJC7 haplo-insufficient cells also indicate changes in the properties of ALS-associated proteins, such as HNRNPU and Matrin3 both of which are involved in the regulation of gene expression. The study also shows data indicating that DnaJC7 haploinsufficiency sensitizes cells to proteostatic stress induced by proteosome inhibition by MG132 and Hsp90 inhibition by Ganetespib. Lastly, the study investigates how DnaJC7 modulates the activity of the heat shock transcription factor (Hsf1) and thus the heat shock response.

Strengths
(1) The manuscript is well presented and most of the data is of high quality and convincing. The figures and supplementary figures are clear and easy to follow.

(2) This study overall provides important new insights into a mostly underexplored molecular co-chaperone and its role in proteostasis. The proteomic and transcriptomic experiments certainly advance our understanding of DnaJC7. The MN model is well-suited for these studies addressing the role of DnaJC7, particularly regarding ALS. The haplo-insufficient MNs are also a suitable model to study a potential loss of function mechanism caused by (some) fALS-associated mutants in ALS, such as the R156X mutation used here.

(3) Since so little is known about DnaJC7 function, the exploratory approaches applied here are particularly useful.

We thank the reviewer for highlighting the strengths of our study.

Weaknesses

(1) Without follow-up studies, however, e.g., with select interacting proteins, the study provides merely a descriptive list of possible interactions without mechanistic insights. Also, most interactions have not been extensively (only a few examples) validated by other methods or individual experiments.

We appreciate the reviewers concern and agree that there are several intriguing DNAJC7 interactors worth studying further, that is why we wanted to share this resource with the broader community as quickly as possible. As the first study focused on DNAJC7 and its link to ALS we could not possibly investigate multiple potential interactors and focused on two: HNRNPU and HSP70/HSP90, associated with RNA metabolism and stress response respectively, as these are two pathways have previously been implicated in ALS pathogenesis. We do provide validation of these interactions and some mechanistic insight into how DNAJC7 haploinsufficiency impairs their function.

A major limitation of the study in its current form is that none of the experimental approaches allow for assessing the specific functions of JC7. In the absence of specificity controls, e.g., other J proteins or HOP, which, like DnaJC7, contains TPR domains and can interact with Hsp70 and Hsp90, it remains unclear if the proposed functions of DnaJC7 are specific/unique or shared by other J proteins or molecular chaperones. Accordingly, it would be highly informative to add experiments to assess if some of the reported DnaJC7 protein-protein interactions and the transcriptional alterations in haplo-insufficient cells are DnaJC7specific or also occur with other J proteins or molecular chaperones. This seems particularly important to discern specific DnaJC7 functions from general effects caused by impaired proteostasis.

We agree with the reviewer that is a very interesting question, as for example mutations in DNAJC6 can cause rare forms of Parkinson’s Disease1. However, addressing the functional overlap of DNAJC7 with other J proteins such as DNAJC6 would require substantial time and resources and is out of scope of the current manuscript.

It would be informative to explore how cellular stress (e.g., MG132 treatment) alters DnaJC7 interactions with other proteins (J proteins, HOP), ideally in additional/comparative proteomic studies. The mechanism underlying the proposed regulation of Hsf1 by DnaJC7 is not quite clear to me (Figures 4 A-I). There is no evidence of a direct physical interaction between DnJC7 and Hsf1 in the proteomic data or elsewhere. It seems plausible that Hsf1/HSR dysregulation in the haplo-insufficient cells might be due to rather indirect effects, e.g., increased protein misfolding. Also, additional data showing differential activation of Hsf1 in +/+ versus +/- cells would strengthen this part, e.g. showing differences in Hsf1 trimerization, Hsp70 interactions, nuclear localization, etc.

The reviewer makes two good points here. Firstly, we do agree we should provide additional data to better understand the differential activation of HSF1 in DNACJ7 heterozygous neurons and we will focus on this question during the revision. We also agree that the mechanism underlying the regulation of HSF1 by DNAJC7 is not well defined and we acknowledge it could be indirect. Of note, HSF1 activation is regulated by HSP70, of which DNAJC7 is a co-chaperone. We will attempt to define this mechanism better during the revision.

The manuscript might also benefit from considering the literature showing an unusually inactive HSR and Hsf1 activity in motor neurons (e.g. published by the Durham lab).

Yes—we did in fact note this in our discussion: “At the same time, mouse MNs have previously been shown to maintain a high threshold of induction of the HSF1-mediated stress response relative to other cell types including glial cells, with the suggestion that this contributes to their vulnerability to stress signals such as insoluble proteins.” We will further consider how our findings are in line with those of Durham et al., in the revised discussion.

The correlation with transcriptomic data from ALS patients compared to neurotypical controls (Figures 4 L, M) suggesting a direct role of Hsf1/HSR seems unlikely at this point. In my view, the transcriptional dysregulation in ALS patients could be unrelated to Hsf1 dysregulation and caused by rather non-specific effects of neuronal decay in ALS.

This is a very reasonable concern. We acknowledge that the HSF1 effects in patients could be driven by multiple other factors including C9-DPRs etc. However, the point of this analysis is not to claim that DNAJC7 is the cause; but rather to highlight the importance of the HSF1 pathway, which we identified as being mis-regulated in DNAJC7 mutant neurons, as broadly relevant in sporadic and other forms of genetic ALS.

Reviewer #3 (Public review):

Summary:

Fleming et al sought to better understand DNAJC7's function in motor neurons as mutations in this gene have been associated with amyotrophic lateral sclerosis (ALS). The research question is relevant and important. The authors use an induced pluripotent stem cell (iPSC) line to derive motor neurons (iMNs) finding that DNAJC7 interacts with RNA-binding proteins (RBP) in wild-type cells and a truncated mutant DNAJC7[R156*] disrupts the RBP, hnRNPU, by promoting its accumulation into insoluble fractions. Given that DNAJC7 is predicted to regulate stress responses, the authors then find that DNAJC7[R156*] expression sensitizes the iMNs to proteosomal stress by disrupting the expression of the key heat stress response regulator, HSF1. These findings support that loss-of-function mutations in DNAJC7 will indeed sensitize motor neurons to proteotoxic stress, potentially driving ALS. The association with RBPs, which routinely are found to be disrupted in ALS, is of interest and warrants further study.

Strengths

(1) The research question is relevant and important. The authors provide interesting data that DNAJC7 mutations impact two important features in ALS, the dysregulation of RNA binding proteins and the sensitivity of motor neurons to proteotoxic stress.

(2) The authors provide solid data to support their findings and the assays are appropriate.

We thank the reviewer for highlighting the strengths of our study.

Weaknesses

(1) The authors rely on a single iPSC line throughout the text, using the same line to make the mutation-carrying cells. iPSCs are highly variable and at minimum 3 lines, typically 5 lines, should be used to define consistent findings. This work would be greatly strengthened if 3 or more lines were used to confirm consistent effects. This is particularly concerning given that iPSCs were differentiated using growth factors versus genetic induction. Growth-factor-based differentiations are more variable.

We will substantiate the major findings by the use of additional models and genetic backgrounds during the revision. However, our experiments utilize isogenic controls and extensive quality control assays (on-target, off target analysis, whole genome sequencing, karyotype etc.) to ensure that our isogenic lines are genomically identical --other than the DNAJC7 mutation-- and thus any phenotypes are likely caused by mutant DNAJC7 itself.

(2) The authors argue that HSF1 and its targets are downregulated in sporadic ALS and mutant C9orf72 ALS. The first concern is that these transcriptomics data were derived from cortical tissue which does not contain motor neurons (Pineda et al. 2024 Cell 187: 1971-1989.e1916). The second concern is that the inclusion of C9orf72 mutant tissue is not well justified as (1) this mutation is associated with an upregulation of HSF1 and its targets in patients (Mordes et al, Acta Neuropathol Commun 2018 6(1):55; Lee et al Neuron 2023 111(9):1381-1390) and (2) the C9orf72 mutation is associated with a ALS/FTD spectrum disorder defined by TDP-43 pathology. Disease mechanisms associated with this spectrum disorder may not overlap with traditional ALS which is typically defined by SOD1 pathology.

SOD1 pathology represents only a small fraction (<2%) of all ALS patients and is therefore not traditional ALS. The majority (<97%) of sporadic and familial ALS cases (including C9orf72 but excluding SOD1 and FUS cases) are uniformly characterized by TDP-43 pathology. Nevertheless, we do agree that it would be better to assess spinal cord data but unfortunately such single cell datasets form ALS patients do not currently exist. We acknowledge that the HSF1 effects in patients could be driven by multiple other factors including C9-DPRs etc. However, the point of this analysis is not to claim that DNAJC7 is the cause; but rather to highlight the importance of the HSF1 pathway, which we identified as being mis-regulated in DNAJC7 mutant neuron, as being broadly relevant in sporadic and other forms of genetic ALS.

(3) As a whole, the findings are mechanistically disjointed, and additional experiments or discussion would help to connect the dots a bit more.

We will revise the manuscript with additional experiments and discussion to better connect the dots.

Citations

(1) Kurian, M. A. & Abela, L. in GeneReviews(®) (eds M. P. Adam et al.) (University of Washington, Seattle Copyright © 1993-2025, University of Washington, Seattle. GeneReviews is a registered trademark of the University of Washington, Seattle. All rights reserved., 1993).