Peripheral immune system modulates Purkinje cell degeneration in Niemann–Pick disease type C1

We demonstrate that peripheral immune cells are involved in the cerebral neuronal degeneration in Niemann–Pick disease type C1. Our work can contribute to a novel therapeutic strategy that can potentially be combined with the ongoing lipid reduction therapeutic efforts.

The present study entitled "Peripheral immune system modulates Purkinje cell degeneration in Niemann-Pick disease type C1" by Yasuda and coworkers is in interesting study building up on the existing literature. The authors validate existing observations, the lack of effect of Csfr1 inhibition on survival, the transcriptomic changes in myeloid cells following the loss of Npc1. The authors also shows that the timing of the bone marrow transplant impact the outcome of the treatment. More importantly the present work investigates the potential role of lymphocytes in the disease and most notably Treg that have received little attention in NPC. The authors point that targeting T cell biology might be a promising avenue in NPC1 and this deserves further investigations. It is surprising that the authors performed the transcriptomic analysis on the monocytes rather than the T cells but this opens the way for more in depth studies of these cells in the future. It is worth mentioning that the authors studied separately the effect in males and female animals. This is especially relevant to NPC1 where a gender difference in the survival, of mice, has been reported by independent groups. Overall, the manuscript is well written, the experiments well designed, and the results clearly presented.
While good and fitting to the target journal I have a few comments and suggestions: -The BBB integrity in the same mouse model was previously evaluated using a different experimental setting and no leakage was observed. Could the discrepancy in the results comes from the sensitivity of the method used? -The neonatal BM transplant is effective while similar transplantation in older animal is not. This is an interesting observation that should be discussed further especially the discrepancies of results depending on the age of the animals at transplant. What could be the reasons of such defenses should be elaborated? -Several studies using different methodological approaches (https://academic.oup.com/hmg/article/27/12/2076/4956805 ; https://onlinelibrary.wiley.com/doi/full/10.1111/jnc.14483 ; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432835/ ) failed to identify infiltrating immune cells in the cerebellum. The discrepancy between the authors results and the literature should be discussed.
-The lack of effect of PLX3397 treatment has previously been reported (https://doi.org/10.3390/ijms21155368 ) the results were therefore to be expected. In the same study the similarities with the "DAM" signatures from other mouse model were evaluated and discussed. -Newton and coworker (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349795/ ) evaluated the effect of FTY720 in NPC. In this study the authors suggested it may be a promising avenue in NPC. Based on your results lymphodepletion would exacerbate the disease. These maybe conflicting observations should be discussed? -From the genes up regulated in the monocytes and suggested to be involved in the pathology several have already been studied and could be included in the discussion or at least referenced: (https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.107.701276 ; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877698/ ; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935239/ ) -TLR4 was found constitutively activated in NPC (https://www.jneurosci.org/content/27/8/1879 ) it is surprising not to find any signature of this mechanism in your analysis. Do you have any hypothesis regarding this? -It might me more accurate to describe he rotarod test as a motor coordination test rather than a behavior test.
-The reference for g:Profiler is missing (https://pubmed.ncbi.nlm.nih.gov/27098042/ ) -The reference for EdgeR is missing (https://pubmed.ncbi.nlm.nih.gov/19910308/ ) -Several reports, including in NPC, have shown that Gapdh shouldn't be used as internal control for myeloid cells (e.g. :https://bmcimmunol.biomedcentral.com/articles/10. 1186/1471-2172-11-21 ). Why were the genes expression normalized to Gapdh? This has to be justified or additional experiments demonstrating that Gapdh has a stable expression between control and mutant cells should be performed or at leave evaluated in the transcriptomic data.

Reviewer #3 (Comments to the Authors (Required)):
This is an interesting manuscript that reports on the significance of peripheral immune cells on CNS neurodegeneration in a mouse model of Niemann-Pick disease type C1 (NPC1), which is a lysosomal storage disorder among others characterized by progressive neuronal degeneration. Manipulation of the immune system may have perspectives as a novel therapeutic adjacent to current lipid reductions. The manuscript describes how bone marrow-derived CD4-positive cells transplantation partly quite dramatically ameliorates Purkinje cell degeneration and restore functions in NPC1 mice. Interestingly mature lymphocytes, next to transplanted cells, also have neuroprotective effects. On the reverse depletion of T cells promoted neurodegeneration. Circulatory monocytes also promoted neurodegeneration. Overall, the study suggests a protective role of the specific immune system and the reverse of the innate ditto.
The authors should take these comments into consideration: Protocol: What is meant by BBB breaching? Blood-brain barrier? Please explain A concern is the illustrations underpinning the FACS and survival data: Morphological evidence is provided for microglia infiltration; but there is not much evidence for lymphocyte infiltration or macrophage infiltration. The demonstration of perivascular cells does not really convincingly show direct interaction between Purkinje cells and cells of the immune system. Please explain The comments on the blood-brain barrier in the supplementary are not sufficient. There must be a better provision of the criteria for BBB breaching and the broad staining by Evans Blue is almost too good. The animals must be very sick if this is true BBB breaching; does this lead to labeling of Purkinje cells? Does this breaching lead to extravasation and presence of immunoglobulins in Purkinje cells? Thank you very much for your review and comments for our manuscript. The comments raised by the reviewers were very helpful to improve our manuscript. According to their comments, we newly performed several experiments and revised the manuscript to meet the comments. According to your request, we prepared our point-by-point responses to the reviewers' comments, which were raised in the reviewing process of Life Science Alliance.
The changes made in the manuscript and our point-by-point responses are provided below: This comparison would add significance and mechanism to the lack of effect in male mice from BM-transplantation, Rag1-KO, and CD4+ cell infusion. However, if male mice also show BBB immune infiltration -authors should speculate on a sex-specific mechanism that explains this discrepancy in the text.
We will deeply appreciate the reviewer's excellent comment to improve our manuscript.
We have investigated the BBB leakage and infiltration of KuO-positive immune cells in both female and male mice and have not found any sex differences. All images shown in

Results section
We did not find any sex differences in these experiments.
We newly carried out bone marrow transplantation using npc1−/−_KuO-transgenic mice as a donor. As shown in the revised Supplemental figure 1 S-Z, we found an invasion of the KuO-CD11b-double-positive monocyte-derived macrophages not only into the perivascular zone of the interlobular space, but also into the perivascular zones of molecular layer and Purkinje cell layer of the cerebellum. We speculated that these results strengthen our concept that peripheral immune cells can be involved in the modulation of the neurodegenerative process of NPC1. Accordingly, we have revised our manuscript to provide the new data as shown below: blood vessels (anti-Glut-1, gray). An enlarged images of panel U, W, and Y are indicated in panels V, X, and Z, respectively. Z-stacks were captured at 1.00-μm intervals by using a confocal laser-scanning microscope. Note that the KuO + BM-derived cells (red) were CD11b + macrophages and localized perivascular zones of the interlobular space (indicated by white arrowhead in panels U and W), the ML (blue arrowhead in panel W), and the PCL of the cerebellum (blue arrowhead in panel Y). Scale bars in (U): 50μm (applicable to panels W and Y) and (V): 10μm (applicable to panels X and Z). Page 24, lines 477-480 in the revised manuscript;

Discussion section
We speculated that the BM-derived macrophages found in the cerebellar perivascular zones We will agree totally to the reviewer's suggestion that a sex-specific mechanism should be discussed precisely to explain the discrepancy in the BM-transplantation,

Rag1-KO, and CD4+ cell infusion experiments.
We have carefully read several papers, which showed the effects of sex hormones on adaptive immune cells. In males, androgens show a suppressive effect on lymphopoiesis in early development. While females are known to have higher numbers of total and activated CD4-positive T cells than males, estrogen and progesterone have ability to augment expression of Foxp3 in CD4-positive cells in mice and humans. We speculated these effects can explain the gender-specific neuronal protection and degeneration observed in our present study. And, we will greatly appreciate the reviewer if he/she could check whether the revised manuscript could discuss the gender-specific effects precisely: Page 20, lines 407-417 in the revised manuscript;

Discussion section
Previous reports showed the effects of sex hormones on adaptive immune cells (Dodd & Menon, 2022;Klein & Flanagan, 2016). Deprivation of androgen and knockout of androgen receptors in mice resulted in thymic enlargement, indicating that androgens have a suppressive effect on lymphopoiesis in early development (Dodd & Menon, 2022;Lai et al., 2012;Roden et al., 2004).

2) In regard to RNA-seq performed on peripheral monocytes, it would also add critical insight into this sex-specific response if male monocytes were also analyzed -which is unfortunately lacking here. For the female samples analyzed, did the authors perform a background correction for Gene Ontology analysis? Given that the gene expression signature in these cells will be heavily enriched for immune related processes, it is critical to correct the DEG enrichment according to the background gene-set detected in these cells. A different GO tool such as ShinyGO should be used to include a background correction.
We are grateful to the reviewer for his/her helpful comment. According to the reviewer's comment, we carried out Gene Ontology analysis using a different tool ShinyGO. Several important biological pathways were enriched by the ShinyGO analysis, including steroid biosynthesis, cholesterol metabolism, and cytokine-cytokine receptor interaction. We have revised our manuscript to provide the data in Table S2 based on our idea described below: We speculated that the GO data obtained from g:Profiler analysis that have been provided in Table S1 in the previously submitted manuscript may also be informative for the readers, since this analysis highlighted several critical processes including cell migration, inflammatory response, and the regulation of cytokine production, that well explain our concept that the invasion of peripheral monocytes/macrophages into the cerebellum can be an important process to induce neuronal degeneration. Accordingly, we have revised our manuscript to add the new data from ShinyGO analysis as shown below: Page 15, lines 298-302 in the revised manuscript;

Materials and methods section
The GO analyses were performed through webtools g:Profiler

Minor Comments
Page 18, paragraph 2 -"NCP1" should read "NPC1" We appreciate the reviewer's comment. Accordingly, we have revised the description that reviewer pointed out as shown below: Page 21, line 423 in the revised manuscript; Discussion section NPC1 patients.

Responses to the comments by Reviewer #2
-The BBB integrity in the same mouse model was previously evaluated using a different experimental setting and no leakage was observed. Could the discrepancy in the results comes from the sensitivity of the method used?
We are grateful to the reviewer for his/her helpful comments to improve our manuscript.
We have carefully read the paper by Cougnoux  Breaching of the BBB was confirmed histologically by a leakage of peripherally injected Evans blue dye and sulfo-NHS-biotin molecules into the parenchyma of the cerebellum of NPC1 mice (Fig. S1 A'−D'), that could not be detected by a biochemical method in a previous work (Cougnoux et al., 2018a).

References revised in the revised manuscript:
The report by   indicated that brain-resident microglia were already activated at pre-symptomatic stage, 7-day-old, while the Calbindin-positive Purkinje cells were preserved intact. Accordingly, we speculated that such microglial state switch may lead to production of inflammatory cytokines and chemokines that may stimulate and recruit peripheral immune cells. We also speculated that a lethal irradiation in their transplantation study might have affected the inflammatory status of peripheral immune cells. Accordingly, we have added several sentences to discuss these important points, as shown below. And, we will greatly appreciate the reviewer if he/she could check whether the revised manuscript could discuss the discrepancy of the neuroprotective effect precisely: Page 18, lines 368-374 in the revised manuscript;

Discussion section
A previous study has found no change in motor activity, weight loss, or survival in BM-derived cell transplantation at 3−4-wk-old (Cougnoux et al., 2018a). Based on the finding that the brain-resident microglia were activated at 7-day-old (Colombo et al., 2021), we speculated that the age of recipients and/or lethal irradiation in their transplantation experiment might have affected the inflammatory state of peripheral immune cells.
-Several studies using different methodological approaches We will deeply appreciate the reviewer's excellent comment. We will agree totally to the comment that the discrepancy between our study and the literatures should be discussed.
We have carefully read the following three papers.

Discussion section
Microglial cells were depleted partially by the gene ablation of Csf-1 ( Fig. 7 and 8) or the oral administration of PLX3397 ( Fig. 9 and 10); however, unexpectedly, the Purkinje cell degeneration was not affected in these mouse models. The results are consistent with a previous report .
In the report by Cougnoux (Newton et al., 2017). This report provided a potential therapeutic opportunity to treat the patients with NPC. However, FTY720/fingolimod acts as a modulator of sphingosine-1-phosphate receptor to prevent the egress of lymphocytes from secondary lymphoid organs and is frequently used for the treatment of MS (Dumitrescu et al., 2023;McGinley & Cohen, 2021). The neuroprotective property of FTY720/fingolimod should be carefully evaluated in NPC1 mutant animals.
-From the genes up regulated in the monocytes and suggested to be involved in the pathology several have already been studied and could be included in the discussion or at least referenced: We are grateful to the reviewer for his/her helpful comments to improve our manuscript.
Accordingly, we have added the following sentence as reviewer pointed out: Page 25, lines 501-502 in the revised manuscript;

Discussion section
Similar results have been reported previously (Cologna et al., 2014;Cougnoux et al., 2019;Welch et al., 2007). -It might me more accurate to describe the rotarod test as a motor coordination test rather than a behavior test.
We appreciate the reviewer's helpful comments to improve our manuscript. We will agree to the reviewer's suggestion that it may be more accurate to describe the rotarod test as a motor coordination test rather than a behavior test. Accordingly, we have revised the description that reviewer pointed out as shown below: Page 30, line 631 in the revised manuscript;

Materials and methods section
Motor coordination test -The reference for g:Profiler is missing (https://pubmed.ncbi.nlm.nih.gov/27098042/ ) We appreciate the reviewer's helpful comments to improve our manuscript. Accordingly, we have revised our manuscript to add the new reference that reviewer pointed out as shown below: Page 34, lines 717-718 in the revised manuscript; Materials and methods section

21
The GO analyses were performed through webtools g:Profiler We appreciate the reviewer's helpful comments to improve our manuscript. Accordingly, we have revised our manuscript to add the new reference that reviewer pointed out as shown below: Page 34, lines 715-717 in the revised manuscript;

Materials and methods section
Gene transcripts were quantified by using a tool salmon (Patro et al., 2017) and were analyzed by EdgeR (Robinson et al., 2010). experiments demonstrating that Gapdh has a stable expression between control and mutant cells should be performed or at leave evaluated in the transcriptomic data.
We will deeply appreciate the reviewer's excellent comment to improve our manuscript.
We agree to the reviewer's suggestion that normalization of gene expression to Gapdh should be justified. We have carefully read the paper by Piehler et al. (Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes; BMC Immunology, 2010, 11, 21). This study demonstrated that Gapdh is not stable upon stimulation with LPS in human peripheral monocytes. The authors have described in the Conclusion section as shown below: The importance of our findings is highlighted by the fact that a review of the literature on gene expression in LPS-stimulated monocytes of the last years exhibited that the large majority of the published RT-qPCR results were normalized to a single gene, mainly GAPDH or ACTB.
Due to the absence of universal reference genes, however, the state-of-the-art evaluation of reference gene stability has to be documented for each experimental setup and tailored to every activation process.
Accordingly, as the reviewer also pointed out, we have evaluated our transcriptomic data and found no significant difference in the expression level of Gapdh between control and NPC1. And also, we speculated that citation of several references that show RT-qPCR data of NPC1 mice, which are normalized to Gapdh, can be helpful for the readers to evaluate our present data. Accordingly, we have revised our manuscript to provide previous RT-qPCR data in NPC1 mice as shown below: Page 15, lines 305-308 in the revised manuscript;

Results section
The data are expressed as mean values relative to the housekeeping gene (gapdh) that has been used as a reference gene in previous works in NPC1 mice (Cluzeau et al., 2012;Cologna et al., 2014), and they are normalized to the npc1+/+_5wk group.

Responses to the comments by Reviewer #3
Protocol: What is meant by BBB breaching? Blood-brain barrier? Please explain We are grateful to the reviewer for his/her helpful comments to improve our manuscript.
We agree to the reviewer's comment that BBB should be explained precisely. Accordingly, we have revised the description that reviewer pointed out as shown below: Page 29, line 602 in the revised manuscript

Blood-brain barrier breaching
A concern is the illustrations underpinning the FACS and survival data: Morphological evidence is provided for microglia infiltration; but there is not much evidence for lymphocyte infiltration or macrophage infiltration. The demonstration of perivascular cells does not really convincingly show direct interaction between Purkinje cells and cells of the immune system. Please explain We will appreciate the reviewer's excellent comment to improve our manuscript.
According to the reviewer's comment, we have provided a representative picture of gating A prepare samples for flow cytometry and single cell transcriptome analysis. We also speculated that a small number of infiltrating cells in NPC1 were difficult to detect when compared with those in experimental autoimmune encephalomyelitis model  or mucolipidosis type IV model . Moreover, in our present study, the histological detection of infiltrating CD4+ lymphocytes and Foxp3-GFP+ Treg cells was difficult because the cerebellum of NPC1 mice had a weak and broad autofluorescence. Accordingly, we have revised the description as shown below: Page 23, lines 456-459 in the revised manuscript;

Discussion section
Previous works failed to detect infiltrating cells in the cerebella of NPC1 mice, possibly due to an increased sensitivity of NPC1 cells to the enzymatic dissociation required for flow cytometry and single cell transcriptome analyses Cougnoux et al., 2018a.
We newly carried out bone marrow transplantation using npc1−/−_KuO-transgenic mice as a donor. As shown in the revised Supplemental figure 1 S-Z, we found an invasion of the KuO-CD11b-double-positive monocyte-derived macrophages not only into the perivascular zone of the interlobular space, but also into the perivascular zones of molecular layer and Purkinje cell layer of the cerebellum. We speculated that these results strengthen our concept that peripheral immune cells can be involved in the modulation of the neurodegenerative process of NPC1. Accordingly, we have revised our manuscript to provide the new data as shown below: The comments on the blood-brain barrier in the supplementary are not sufficient. There must be a better provision of the criteria for BBB breaching and the broad staining by Evans Blue is almost too good. The animals must be very sick if this is true BBB breaching; does this lead to labeling of Purkinje cells? Does this breaching lead to extravasation and presence of immunoglobulins in Purkinje cells?

Revised
We will appreciate the reviewer's excellent comment to improve our manuscript. We will totally agree to the reviewer's suggestion that a better provision of the criteria for BBB breaching including the broad staining of extravasated dye is necessary. As shown in the revised Supplemental figure 1 G' and H', we have provided a whole brain picture that shows extravasation of sulfo-NHS-biotin in several brain areas. Accordingly, we have revised our manuscript to provide the new data as shown below: Thank you for submitting your revised manuscript entitled "Peripheral immune system modulates Purkinje cell degeneration in Niemann-Pick disease type C1". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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