Age-associated sleep-wake patterns are altered with Prdm13 signaling in the dorsomedial hypothalamus and dietary restriction in mice

  1. Shogo Tsuji
  2. Cynthia S Brace
  3. Ruiqing Yao
  4. Yoshitaka Tanie
  5. Hirobumi Tada
  6. Nicholas Rensing
  7. Seiya Mizuno
  8. Julio Almunia
  9. Yingyi Kong
  10. Kazuhiro Nakamura
  11. Noboru Ogiso
  12. Shinya Toyokuni
  13. Satoru Takahashi
  14. Michael Wong
  15. Shin-ichiro Imai
  16. Akiko Satoh

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Abstract

Old animals display significant alterations in sleep-wake patterns such as increases in sleep fragmentation and sleep propensity. Here we demonstrated that dorsomedial hypothalamus-specific PR-domain containing protein 13 -knockout (DMH- Prdm13 -KO) mice recapitulated age-associated sleep alterations such as sleep fragmentation and increased sleep attempts during sleep deprivation (SD). These phenotypes were further exacerbated during aging, with increased adiposity and decreased physical activity, resulting in shortened lifespan. Dietary restriction (DR), a well-known anti-aging intervention in diverse organisms, ameliorated age-associated sleep alterations, whereas these effects of DR were abrogated in DMH- Prdm13 -KO mice. Moreover, overexpression of Prdm13 in the DMH ameliorated sleep fragmentation and excessive sleepiness during SD in old mice. Therefore, maintaining Prdm13 signaling in the DMH might play an important role to control sleep-wake patterns during aging.

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

    In this manuscript, the authors report dorsomedial hypothalamus-specific PR-domain containing protein 13-knockout (DMH-Prdm13-KO) mice recapitulated age-associated sleep alterations such as sleep fragmentation and increased sleep attempts during sleep deprivation (SD). These phenotypes were further exacerbated during aging, with increased adiposity and decreased physical activity, resulting in shortened lifespan. Moreover, overexpression of Prdm13 in the DMH ameliorated sleep fragmentation and excessive sleepiness during SD in old mice. They identified maintaining Prdm13 signaling in the DMH might play an important role to control sleep-wake patterns during aging. These findings are interesting and novel and the evidence they provided looks solid.*

    We deeply appreciate that this reviewer found our findings are interesting and the evidence solid.

    *Major comments

    1. The author spent a lot of words on Sirt1 in the introduction. Since Sirt1 regulates Prdm13, is there a link between the two in age-related sleep changes? If so, you can add some results and discussion. *

    Thank you very much for raising this important issue. Our previous study demonstrated that a mouse model with high hypothalamic Sirt1 activity displays reduced number of transitions between wakefulness and NREM sleep (reference # 15), revealing that hypothalamic Sirt1, as well as Prdm13, is involved in the regulation of sleep fragmentation.However, sleep propensity was not altered in Sirt1-overexpressing transgenic mice (reference #13) and DMH-Prdm13-KO mice (Fig. 1). Based on these findings, we added the following sentence in the Results.

    On page 11, line 267-274

    "...... Similarly, a mouse model with high hypothalamic Sirt1 activity displays reduced number of transitions between wakefulness and NREM sleep15, revealing that hypothalamic Sirt1, as well as Prdm13, is involved in the regulation of sleep fragmentation. Sleep propensity was not altered in Sirt1-overexpressing transgenic mice13. Given that the level of hypothalamic Prdm13 and its function decline with age, age-associated sleep fragmentation could be promoted through the reduction of Prdm13/Sirt1 signaling in the DMH, but sleep propensity might be increased by other mechanisms. "

    • In Figure 2e, the author describes n=7-8 in the figure legend, but why do both groups on the column show eight data? Is there something wrong with the statistics? Please check the statistics in the article carefully. *

    We corrected n=7-8 to n=8 in the figure legend of Fig. 2e.

    • DMH is known as one of the major outputs of hypothalamus circadian system and is involved in the circadian regulation of sleep-wakefulness (J.Neurosci. 23, 10691-10702 ; Nat Neurosci 4:732-738). Does Prdm13 correlate with circadian rhythms? The author can add relevant content to the discussion *

    As per this reviewer's suggestion, we added the following sentence in the Discussion on page 20, line 500-508,

    "For instance, it would be of great interest to elucidate whether Prdm13 signaling in the DMH contributes to regulate the circadian system, since the DMH is known to be involved in the regulation of several circadian behaviors32,33. Although DMH-Prdm13-KO mice did not display abnormal period length compared with controls, further studies are needed to address this possibility."

    *Minor comments

    1. The immunohistochemical diagram in the paper is not representative enough, as shown in FIG. 2b and c. *

    We apologize that our presentation in Figs. 2a-c was confusing. Although Fig. 2b shows the numbers of cFos cells in the entire region of the DMH (summed up from three DMH regions), the images in Fig. 2c are from one of DMH regions for each condition. To avoid confusion, we revised the legend of Figs. 2a-c and the manuscript in the Results as follows:

    -In the figure legend of Figs. 2a-c

    "a, Total numbers of cFos+ cells ......... b,c, Images of DMH sections at bregma -1.67 mm ......."

    -In the Results on page 7, line 180

    "...... the hypothalamus, the DMH (summed up from bregma -1.67 to -1.91mm) showed a greater number of cFos+ cells during SD compared to SD-Cont (Fig. 2a-c, Supplementary Fig. 2a)..... "

    • In FIG. 5h, the authors showed that the effect of overexpression of Prdm13 was very obvious, but the expression range of the virus after injection was lacking. Is there a fluorescent gene such as GFP on the virus to directly see the expression of the virus in the brain? *

    Unfortunately, we do not hold extra samples to check the distribution of the virus after injection. However, we have established sufficient injection technique to target the DMH using the lentivirus system that we used in this study (Satoh et al Cell Metab 2013).

    • Were mice singly housed or housed in groups? *

    Most of the mice were housed in groups, except for the DR study. We added this information in the section Animal models of the Methods on page 41, line 935

    ".....RIKEN BRC. Most of the mice were housed in groups, except for the DR study. For the DR study ,..... "

    • The part of sleep analysis needs to be further refined. How can REM and NREM in mice be distinguished and according to what criteria? *

    We added the criteria to define NREM and REM in the section Sleep analysis of the Methods on page 42, line 995-998.

    ".......with visual examination. EEG periods dominated by higher amplitude delta wave activity with nuchal muscle atonia were scored as NREM sleep epochs. REM sleep consisted of periods of semi-uniform theta activity EEG with muscle atonia and/or muscle atonia with brief myoclonic twitches. Score was blinded ......"

    • The authors may consider adding more recent literature related to DMH and sleep, such as DOI: 10.1093/cercor/bhac258 * We incorporated this reference to the following sentence in the section Results on page 8, line 194.

    "........ Although DMH neurons are linked to sleep21, aging and longevity .... "

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

    Summary: In this study, Tsuji et al. demonstrate that Prdm13 signaling is involved in the regulation of sleep-wake pattern. They also identified Prdm13 as a transcription factor in the DMH neurons. Major comments:

    1. The evidence presented in Fig. 1 of age-related sleep fragmentation is potentially problematic. Although many previous studies have demonstrated fragmented sleep, especially fragmentation of NREM sleep, in aged mice compared to young mice, the data here do not suggest NREM fragmentation, because no change in the NREM bout duration was found. REM, on the other hand, may indeed have fragmentation during the dark phase, but REM only takes a small portion of the total sleep. Therefore, the conclusion that sleep is fragmented in old mice is not fully supported by Fig. 1. I noticed that the authors used 4-6 months old mice as the young group. Mice of this age can hardly be called "young". The females even start to have lowered fertility. This might be one of the reasons for the discrepancy between this and other studies. Repeating these experiments (and others involving the young group) with mice of more appropriate age (usually 2-3 months old) is recommended. Nonetheless, aging-caused sleep change is not new knowledge and has been reported repeatedly. This part of the results should be in the supplementary figures. *

    We deeply appreciate this reviewer's comment. In accordance with this reviewer's suggestion, we carefully reconsidered the age of young mice. Most of published studies used mice at 2 to 4 months of age as the young group [2 to 4-month-old (7 studies), 4.6-month-old (1 study), 6-month-old (1 study), 2 to 6-month-old (1 study)]. Thus, to strictly use mice at 3-4 months of age as the young group, we excluded data of one cohort using mice at 6 months of age (2 mice each age group). Consistent with many previous studies, our revised data demonstrated that sleep fragmentation during NREM sleep is predominantly observed in old mice compared with young mice, particularly during the dark period. Based on these new results, we revised Fig.1, Suppl Fig.1, and all description related to Fig. 1 (manuscript on page 5-7, line 103-171). We would like to keep Fig. 1 as it is. Since most of the previous studies used males but not females, data from females are still lacking in the field (Campos-Beltran and Marshall, Pflugers. Arch., 473:841-851, 2021).

    • The sleep phenotypes in aged mice and in Prdm13-KO mice are clearly distinct from each other. In the old mice (Fig. 1), REM sleep is fragmented but the total amount remains unchanged, and NREM sleep is increased (both bout number and total amount), indicating there may be more REM-to-NREM transitions, which the authors should quantify. However, Fig. 3 shows in Prdm13-KO mice, there is no REM fragmentation. In fact, it even seems to stabilize REM. But NREM duration is shorted, and no change in the total NREM or REM sleep time. These results suggest that the sleep alterations caused by aging and Prdm13-KO might have some overlap but are mostly in parallel and likely through different mechanisms. Therefore, the rationale of connecting Prdm13 signaling to aging-caused sleep changes is questionable. Is there a developmental change of Prdm13 expression in DMH between young and old mice? The authors also showed that Prdm13-KO in old mice caused decrease in NREM duration but has no effect on REM sleep, but in normal old mice, it is REM, but not NREM that has a defect. Prdm13 overexpression also only mildly decreased NREM bout number without affecting the episode duration of either NREM or REM, which can hardly be interpreted as "ameliorating sleep fragmentation". To me, all these results just suggest parallel actions of Prdm13 and aging on sleep, with Prdm13 mostly affecting NREM sleep but aging mostly impairing REM sleep. *

    We deeply appreciate this reviewer's keen eyes. We carefully reassessed REM sleep data in Fig. 3. The revised data showed that whereas the duration of NREM episodes in DMH-Prdm13-KO mice during the dark period were significantly shorter compared to control group, the duration of REM episodes in the KO mice was not significantly altered. Therefore, after revising Fig. 1 and 3, our results showed that both aging and Prdm13-KO similarly affect the duration of NREM sleep episodes. These results suggest that sleep fragmentation, in particular, during NREM sleep, is commonly observed in old mice and DMH-Prdm13-KO mice. In addition to sleep fragmentation during NREM sleep, excessive sleepiness during SD was also commonly observed in old mice and DMH-Prdm13-KO mice. On the other hand, the effect of aging and Prdm13-KO on sleep propensity was distinct from each other. We think that age-associated sleep fragmentation could be promoted through Prdm13 signaling in the DMH, but sleep propensity might be increased by other mechanisms. We described these results and possibilities in the Results, and revised the Abstract as follows:

    On page 11, line 264-274

    "activity in DMH-Prdm13-KO mice (Fig. 3h, Supplementary Fig. 3f-h). Together, sleep fragmentation during NREM sleep and excessive sleepiness during SD are commonly observed in old mice and DMH-Prdm13-KO mice, but the effects of aging and Prdm13-KO on sleep propensity were distinct from each other.............. Given that the level of hypothalamic *Prdm13 *and its function decline with age16, age-associated sleep fragmentation could be promoted through the reduction of Prdm13/Sirt1 signaling in the DMH, but sleep propensity might be increased by other mechanisms."

    On page 2, line 45-46

    "Dietary restriction (DR), a well-known anti-aging intervention in diverse organisms, ameliorated age-associated sleep fragmentation and increased sleep attempts during SD, whereas these effects of DR were abrogated in DMH-Prdm13-KO mice."

    As this reviewer pointed out, the effect of Prdm13 overexpression on NREM sleep fragmentation seems to be moderate, but we still observed effects on excessive sleepiness during SD. Thus, we revised the manuscript related to Prdm13-overexpression study in the Abstract and Results as follows:

    On page 2, line 47-48

    "Moreover, overexpression of Prdm13 in the DMH ameliorated sleep fragmentation and excessive sleepiness during SD in old mice."

    On page 16, line 387-401

    "Overexpression of Prdm13 in the DMH partially affects age-associated sleep alterations

    ...... (Fig. 5h). The number of wakefulness and NREM sleep episodes in old Prdm13-OE mice were significantly lower, whereas duration of wakefulness in old Prdm13-OE mice tended to be longer than old control mice during the dark period with no change in the duration of NREM episodes (Fig. 5i,j). Intriguingly, .... Thus, the restoration of Prdm13 signaling in the DMH partially rescue age-associated sleep alterations, but its effect on sleep fragmentation is moderate."

    • What is the control manipulation for sleep deprivation? The authors need to clarify this in the Methods. Also, sleep deprivation has confounding effects including but not limited to stress, food deprivation (since food was removed during SD), human experimenter (since a gentle-touch method was used). Without proper controls for these variables, the authors should avoid concluding that the changes they saw at cellular level are due to sleep loss. *

    Thank you very much for this suggestion. We added detailed description for AL-SD (the control manipulation for SD) in the section SD study of the Materials as follows:

    On page 42-43, line 1014-1020

    "Mice for control manipulation (AL-SD) were also individually housed prior to the experiment without SD and food removal. We checked the level of blood glucose in the SD study, and found that the level of blood glucose was indistinguishable between SD and AL-SD groups (126±6 and 131±4 mg/dL, respectively), revealing that nutritional status is equal between these two groups."

    Identification of Prdm13+ cells using neuronal markers should be performed in addition to electrophysiological characterizations.

    We performed immunofluorescence using anti-MAP2 antibody and confirmed that most Prdm13+ cells are neurons. We added this new result in Suppl Fig. 2g.

    • Figs. 6 and 7 seem very disconnected from the main story. Identification of Prdm13 as a transcription factor is potentially interesting, but how does it account for its role in affecting sleep? The criteria of picking Cck, Grp and Pmch out of other candidate genes potentially regulated by Prdm13 and the rationale to investigate these genes seem unclear. More importantly, no evidence was shown regarding how Cck/Grp *

    Base on RNA-sequencing using DMH samples from DMH-Prdm13-KO and control mice, we got several candidate genes as downstream genes of Prdm13. After validating the candidate genes by qRT-PCR, Cck, Grp and Pmch were detected as top-hit genes. We thus further assessed these three genes in this study. Our result showed that Cckexpression in the hypothalamus significantly declines with age. Based on other literature, hypothalamic Cck seems to be involved in sleep control. Therefore, it is conceivable that Prdm13 controls age-associated sleep alterations via modulating Cck expression. However, as this reviewer pointed out, we are still lacking the evidence showing the role of Prdm13/Cck axis in age-associated sleep alterations. We now clearly described the limitation of our study in the Discussion on page 23, line 560-562.

    "However, the detailed molecular mechanisms by which Prdm13 in the DMH regulates age-associated sleep fragmentation and excessive sleepiness during SD still need to be elucidated in future study. "

    *Minor comments:

    1. Please note on the images of Fig. 2d what the green fluorescence was. It is very confusing as is, given that it's surrounded by quantifications of c-fos in the figure. *

    The label "Prdm13" was added in Fig. 2d.

    Please note use a different color for Prdm13 in several figure images (e.g., Fig. 2f, g, 7a,d, and Supplementary 2c). Yellow usually means overlap of red and green.

    Since we have four-color images in Fig. 7, we consistently used yellow for Prdm13 throughout the main figures of the paper. At this moment, we would like to keep the current version of images, but we will revise images if the editor of affiliate journal requests this revision.

    • Please note the statistic test results on power spectrum graphs. *

    We added the statistic test results on power spectrum graphs in Figs. 1d, 4c, and 5d.

    • Inconsistency between the graphs in Fig. 3d and the description in the text. Fig. 3d suggests no change in Wake episode duration, significant decrease in Dark phase NREM and significant increase in Dark phase REM, whereas lines 224-227 in the main text state "The duration of wakefulness episodes ... was significantly shorter than control mice during the light period, and the duration of NREM sleep episodes ...was significantly longer ... during the dark period (Fig. 3d)". Which one is correct? Please check. *

    We apologize for this typo and unclear description. We revised the sentence regarding Fig. 3d as follows:

    On page 10, line 242-246

    "The duration of wakefulness episodes in DMH-Prdm13-KO mice was significantly shorter than control mice during the light period between ZT0 to ZT2. The duration of NREM sleep episodes in DMH-Prdm13-KO mice was significantly shorter than control mice during the dark period (Fig. 3d). These results indicate that DMH-Prdm13-KO mice showed mild sleep fragmentation compared with control mice."

    • Fig. 5f, Y-axis title should be EEG SWA. * We corrected it.
  5. Review Commons

    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

    Summary:

    In this study, Tsuji et al. demonstrate that Prdm13 signaling is involved in the regulation of sleep-wake pattern. They also identified Prdm13 as a transcription factor in the DMH neurons.

    Major comments:

    1. The evidence presented in Fig. 1 of age-related sleep fragmentation is potentially problematic. Although many previous studies have demonstrated fragmented sleep, especially fragmentation of NREM sleep, in aged mice compared to young mice, the data here do not suggest NREM fragmentation, because no change in the NREM bout duration was found. REM, on the other hand, may indeed have fragmentation during the dark phase, but REM only takes a small portion of the total sleep. Therefore, the conclusion that sleep is fragmented in old mice is not fully supported by Fig. 1. I noticed that the authors used 4-6 months old mice as the young group. Mice of this age can hardly be called "young". The females even start to have lowered fertility. This might be one of the reasons for the discrepancy between this and other studies. Repeating these experiments (and others involving the young group) with mice of more appropriate age (usually 2-3 months old) is recommended. Nonetheless, aging-caused sleep change is not new knowledge and has been reported repeatedly. This part of the results should be in the supplementary figures.
    2. The sleep phenotypes in aged mice and in Prdm13-KO mice are clearly distinct from each other. In the old mice (Fig. 1), REM sleep is fragmented but the total amount remains unchanged, and NREM sleep is increased (both bout number and total amount), indicating there may be more REM-to-NREM transitions, which the authors should quantify. However, Fig. 3 shows in Prdm13-KO mice, there is no REM fragmentation. In fact, it even seems to stabilize REM. But NREM duration is shorted, and no change in the total NREM or REM sleep time. These results suggest that the sleep alterations caused by aging and Prdm13-KO might have some overlap but are mostly in parallel and likely through different mechanisms. Therefore, the rationale of connecting Prdm13 signaling to aging-caused sleep changes is questionable. Is there a developmental change of Prdm13 expression in DMH between young and old mice? The authors also showed that Prdm13-KO in old mice caused decrease in NREM duration but has no effect on REM sleep, but in normal old mice, it is REM, but not NREM that has a defect. Prdm13 overexpression also only mildly decreased NREM bout number without affecting the episode duration of either NREM or REM, which can hardly be interpreted as "ameliorating sleep fragmentation". To me, all these results just suggest parallel actions of Prdm13 and aging on sleep, with Prdm13 mostly affecting NREM sleep but aging mostly impairing REM sleep.
    3. What is the control manipulation for sleep deprivation? The authors need to clarify this in the Methods. Also, sleep deprivation has confounding effects including but not limited to stress, food deprivation (since food was removed during SD), human experimenter (since a gentle-touch method was used). Without proper controls for these variables, the authors should avoid concluding that the changes they saw at cellular level are due to sleep loss.
    4. Identification of Prdm13+ cells using neuronal markers should be performed in addition to electrophysiological characterizations.
    5. Figs. 6 and 7 seem very disconnected from the main story. Identification of Prdm13 as a transcription factor is potentially interesting, but how does it account for its role in affecting sleep? The criteria of picking Cck, Grp and Pmch out of other candidate genes potentially regulated by Prdm13 and the rationale to investigate these genes seem unclear. More importantly, no evidence was shown regarding how Cck/Grp

    Minor comments:

    1. Please note on the images of Fig. 2d what the green fluorescence was. It is very confusing as is, given that it's surrounded by quantifications of c-fos in the figure.
    2. Please note use a different color for Prdm13 in several figure images (e.g., Fig. 2f, g, 7a,d, and Supplementary 2c). Yellow usually means overlap of red and green.
    3. Please note the statistic test results on power spectrum graphs.
    4. Inconsistency between the graphs in Fig. 3d and the description in the text. Fig. 3d suggests no change in Wake episode duration, significant decrease in Dark phase NREM and significant increase in Dark phase REM, whereas lines 224-227 in the main text state "The duration of wakefulness episodes ... was significantly shorter than control mice during the light period, and the duration of NREM sleep episodes ...was significantly longer ... during the dark period (Fig. 3d)". Which one is correct? Please check.
    5. Fig. 5f, Y-axis title should be EEG SWA.

    Significance

    General assessment: There are discrepancies in the evidence presented, and the results were poorly organized. I found the main conclusions of the manuscript not very convincing and the causal links among Prdm13, aging and sleep alterations weak.

    Advance: The identification of DMH Prdm13 in regulating sleep is potentially interesting and of some novelty, but the underlying mechanism and its causal relationship with aging were not clearly elucidated.

    Audience: basic research

    My expertise: sleep, social behavior, hypothalamus, dopamine neuromodulation, neural circuit development, synaptic organization.

  6. Review Commons

    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

    In this manuscript, the authors report dorsomedial hypothalamus-specific PR-domain containing protein 13-knockout (DMH-Prdm13-KO) mice recapitulated age-associated sleep alterations such as sleep fragmentation and increased sleep attempts during sleep deprivation (SD). These phenotypes were further exacerbated during aging, with increased adiposity and decreased physical activity, resulting in shortened lifespan. Moreover, overexpression of Prdm13 in the DMH ameliorated sleep fragmentation and excessive sleepiness during SD in old mice. They identified maintaining Prdm13 signaling in the DMH might play an important role to control sleep-wake patterns during aging. These findings are interesting and novel and the evidence they provided looks solid.

    Major comments

    1. The author spent a lot of words on Sirt1 in the introduction. Since Sirt1 regulates Prdm13, is there a link between the two in age-related sleep changes? If so, you can add some results and discussion.
    2. In Figure 2e, the author describes n=7-8 in the figure legend, but why do both groups on the column show eight data? Is there something wrong with the statistics? Please check the statistics in the article carefully.
    3. DMH is known as one of the major outputs of hypothalamus circadian system and is involved in the circadian regulation of sleep-wakefulness (J.Neurosci. 23, 10691-10702 ; Nat Neurosci 4:732-738). Does Prdm13 correlate with circadian rhythms? The author can add relevant content to the discussion

    Minor comments

    1. The immunohistochemical diagram in the paper is not representative enough, as shown in FIG. 2b and c.
    2. In FIG. 5h, the authors showed that the effect of overexpression of Prdm13 was very obvious, but the expression range of the virus after injection was lacking. Is there a fluorescent gene such as GFP on the virus to directly see the expression of the virus in the brain?
    3. Were mice singly housed or housed in groups?
    4. The part of sleep analysis needs to be further refined. How can REM and NREM in mice be distinguished and according to what criteria?
    5. The authors may consider adding more recent literature related to DMH and sleep, such as DOI: 10.1093/cercor/bhac258

    Significance

    Akiko Satoh's 2015 article "Deficiency of Prdm13, a dorsomedial hypothalamus-enriched gene, mimics age-associated changes in sleep quality and adiposity "influenced the novelty of the study, but the authors went further in terms of details and mechanisms. The audience of the basic research will be influenced by this research.

  7. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7267268.

    This review has been formulated based on my brief comment posted on Pubpeer.

    https://pubpeer.com/publications/030D11C6AA3F20092C564B6599BFE1#

     

    In this preprint, Tsuji et al. deal with an interesting topic of aging and sleep and also present a great deal of data. However, in the current version, there seem clear discrepancies between the data presented and the authors' claims. I also found it very difficult to draw a logical conclusion from the data presented in this study due to inconsistent results and issues with experimental design. 

     

    Major concerns:

     

    1.   No clear evidence that DMH-Prdm13-KO mice recapitulate the age-associated alterations of sleep observed in the wild-type mice

    Tsuji et al. generated a dorsomedial hypothalamus (DMH)-specific Prdm13 KO mice and measured various sleep indices to test the hypothesis that the Prdm13 function in the DMH may be causally involved in age-associated sleep alterations. However, although the authors claim that they found a commonality in sleep phenotypes between DMH-Prdm13-KO mice and aged C57BL/6J mice, the presented data do not support the claim: Specifically, the data in Fig. 3 show that sleep measures of DMH-Prdm13-KO mice were mostly similar to those of controls. Looking more closely, DMH-Prdm13-KO mice showed significantly decreased episode duration of N-REM sleep in the dark phase and significantly increased episode duration of REM sleep in the dark phase (Fig. 3c, d, h, and g), which are completely different from the age-associated changes of sleep measures observed in C57BL/6J mice (Fig. 1a, b, c, and d).

    The same problem can be seen in the comparison between the sleep phenotypes of aged C57BL/6J mice and aged DMH-Prdm13-KO mice: Although aged DMH-Prdm13-KO mice exhibited changes in some of the sleep indices compared to their age-matched controls such as significantly increased wakefulness bouts and N-REM sleep bouts in the light phase, and significantly decreased wakefulness episode duration in the light phase and N-REM sleep episode duration in the dark phase (Fig. 4a, b, and c, Supplementary Fig. 4e). However, again, these changes have no commonality to the age-associated changes in sleep measures observed in C57BL/6J mice (Fig. 1a, b, c, and d).

    Thus, because both young and aged DMH-Prdm13-KO mice did not share changes in sleep parameters with aged C57BL/6J mice, the authors' key assertion that the DMH-Prdm13-KO mice recapitulated age-associated sleep alterations is not supported by actual data.

     

    2.    Problematic experimental design of sleep deprivation (SD) assay

    The only consistent phenotype among old C57BL/6J mice, young DMH-Prdm13-KO mice, and old DMH-Prdm13-KO mice seems the increased number of sleep attempts shown in Fig. 1g, 3e, and 4d. However, given the design of the authors' SD experiments, it is highly doubtful that this increase in sleep attempts specifically reflects an increase in sleepiness as the authors interpret.

    According to the description in the methods section, the authors first removed food from cages and then kept mice awake for 6 hours by stimulating them with long Q-tips, simultaneously counting sleep attempts relying only on behavioral observation. However, in this experimental design, changes in the number of sleep attempts could be caused by various sensory and emotional changes, such as habituation or hypersensitivity to Q-tip stimulation and the presence of the experimenter, and changes in stress and anxiety levels. In addition, as shown by Đukanović et al, mice deprived of sleep are also deprived of food, even when they have access to food (Dukanovic et al., 2022). In the present study, the authors completely deprived the mice of food during the 6 hours of SD manipulation, which must have caused a greater effect of food deprivation on the mice.

    Therefore, it is difficult to interpret the cFos expression in Prdm13+ cells shown in Fig. 2 or Fig. 7 as evidence that Prdm13+ cells were activated by sleep deprivation. As mentioned above, there are various alternative possibilities; Prdm13+ cells might have been activated by Q-tip stimulation itself, stress, changes in emotional states due to repeated tactile stimulation, changes in internal states due to lack of food, a complex combination of these factors. Similarly, it is not possible to conclude from the presented data that changes in the number of sleep attempts directly reflect sleep-related changes. Indeed, despite the significant increase in the number of sleep attempts, there was no alteration in EEG SWA after SD in young DMH-Prdm13-KO animals (Fig. 3e, f).

    In addition to the above problems, the authors' sleep attempt counts rely solely on subjective behavioral observations and lack the use of EEG/EMG measurements that would have allowed more objective determinations of sleep onset. Moreover, there is no description of the strength and location of the Q-tip stimuli, as well as the time between the presentation of the Q-tip and the judgment of whether the mouse reacted or not. Although the authors wrote that they performed their SD procedure as previously described by Franken et al., the cited study actually performed a very different SD procedure, in which rats were kept awakened by presenting novel objects, acoustic stimuli, and/or gentle tactile stimuli with having undisturbed access to food, and EEG/EMG measurements were also used to determine sleep onsets (Franken et al., 1991). Such an inappropriate citation and inadequate description of the method raise a concern that the changes in the number of sleep attempts that the authors report in this study may have been partly due to artifacts caused by the authors' arbitrary and variable experimental procedure.

     

    More minor points:

     

    -The authors show that many sleep measures changed after dietary restriction (DR) (Fig. 5), but these could easily be indirect or non-specific effects on sleep measures and not reflect changes in sleep per se. Indeed, the authors discuss that the further reduced EEG power during N-REM and REM sleep in old-DR mice (Fig. 5d) might be due to lower body temperature and not necessarily reflect changes in sleep pressure. The authors should use the same level of caution on the possibility of such indirect or non-specific effects throughout the manuscript.

     

    -The authors extracted more than 20 sleep indices from the same EEG traces and performed statistical comparisons of each index individually (Figures 1, 3-5, Supplementary figures 1, 3-5). In such cases, the multiplicity of hypothesis testing becomes an issue. For example, when 20 hypotheses are simultaneously tested at an alpha level of 0.05, the probability that at least one non-significant trait is determined to be significant by chance is more than 64% (actual alpha level > 0.64). The p-values should be appropriately adjusted for the multiple hypothesis testing.

     

    -The authors previously reported that DMH-specific Prdm13 knockdown causes low delta power in N-REM sleep (Satoh et al., 2015), but do not discuss whether this was reproduced by the DMH-specific Prdm13 KO in the current study. This is a very important point and should be discussed explicitly.

     

    -The authors counted and compared the number of cFos+, CCK+, GRP+ cells, in Fig. 2a, and Supplementary Fig. 7b, d. However, the number of positive cells is greatly influenced by the difference in the number of total cells examined. The percentage of positive cells in the total number of examined cells should be reported to ensure fair comparisons.

     

    -The authors suggest that Prdm13+ cells are "electrically active cells such as a neuron" using a whole-cell patch technique (Supplementary Fig. 2d-f). However, immunostaining with neuronal markers such as NeuN (and glial markers) should be more informative to estimate the identities of the Prdm13+ cell population.

      

    References:

    Dukanovic, N., La Spada, F., Emmenegger, Y., Niederhauser, G., Preitner, F., and Franken, P. (2022). Depriving Mice of Sleep also Deprives of Food. Clocks & sleep 4, 37-51.

    Franken, P., Dijk, D.J., Tobler, I., and Borbely, A.A. (1991). Sleep deprivation in rats: effects on EEG power spectra, vigilance states, and cortical temperature. Am J Physiol 261, R198-208.

    Satoh, A., Brace, C.S., Rensing, N., and Imai, S. (2015). Deficiency of Prdm13, a dorsomedial hypothalamus-enriched gene, mimics age-associated changes in sleep quality and adiposity. Aging Cell 14, 209-218.

  8. Version published to 10.1101/2022.09.26.509442v1 on bioRxiv