Sleep–wake patterns are altered with age, Prdm13 signaling in the DMH, and diet restriction in mice

This study found that age-associated sleep changes are ameliorated by DR in the presence of Prdm13 signaling in the DMH, suggesting Prdm13+ DMH neurons will be of great interest to explore a potential intervention on age-associated sleep changes.

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.

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On page 12, line 308-318 "...... Similarly, a mouse model with high hypothalamic Sirt1 activity displays reduced number of transitions between wakefulness and NREM sleep 15 , 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 mice 13 . 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. " 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.
We corrected n=7-8 to n=8 in the figure legend of Fig. 2e. 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 As per this reviewer's suggestion, we added the following sentence in the Discussion on page 22, line 614-618, "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 behaviors 32,33 . Although DMH-Prdm13-KO mice did not display abnormal period length compared with controls, further studies are needed to address this possibility."  Fig. 2a-c, Supplementary Fig. 2a) 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-monthold (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 109-180). We would like to keep Fig. 1 as it is. Since most of the previous studies used males but not females, data from Full Revision females are still lacking in the field (Campos-Beltran and Marshall, Pflugers. Arch., 473:841-851, 2021).
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 agingcaused 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 new Fig. 4. 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  4, 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. Additionally, we have recently conducted a chemogenetic (DREADD) experiment to test whether inhibition of Prdm13+ DMH neurons recapitulates phenotypes of aged mice during SD, since Prdm13+ DMH neurons get activated during SD, compared with a normal sleep state, in young mice but not old mice. Chemogenetic inhibition of Prdm13+ DMH neurons was able to recapitulate excessive sleepiness during SD, which is one of key age-associated sleep alterations. This new finding significantly strengthens our conclusion that Prdm13 in the DMH neurons plays an important role in the regulation of age-associated sleep alterations (new data shown in Fig. 3). 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 12, line 308-318 "activity in DMH-Prdm13-KO mice (Fig. 4h, Supplementary Fig. 4f-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 .... Given that the level of hypothalamic Prdm13 and its function decline with age 16 , 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."

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On page 2, line 49 "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." We also added new Figures (Fig. 2h, Supplementary Fig. 2h, Fig. 3a-e, Supplementary  Fig. 3) and description related to new data from chemogenetic study in the Abstract, Introduction, Results, Discussion, and Materials and Methods. On page 2, line 39-44, "... Here, we demonstrated that PR-domain containing protein 13 (Prdm13) + neurons in the dorsomedial hypothalamus (DMH) are activated during sleep deprivation (SD) in young mice but not old mice. Chemogenetic inhibition of Prdm13+ neurons in the dorsomedial hypothalamus (DMH) in young mice promotes increase in sleep attempts during sleep deprivation (SD), suggesting its involvement in sleep control. Furthermore, DMH-specific Prdm13-knockout ...." On page 4, line 100-102, "... Chemogenetic inhibition of Prdm13+ neurons in the DMH in young mice promotes increase in sleep attempts during SD, suggesting its involvement in sleep control.... " On page 9-10, line 228-254, "........sleep loss during SD in young mice. On the other hand, the percentage of cFos+ cells in Prdm13+ DMH neurons during SD did not differ from its percentage during SD-Cont in old mice (Fig. 2h, Supplementary Fig. 2h). These results indicate that normal neuronal activation of Prdm13+ DMH neurons to SD is impaired with aging. Chemogenetic inhibition of Prdm13+ DMH neurons induces excessive sleepiness during.. Next, using Prdm13-CreERT2 mice and the inhibitory designer receptor exclusively activated by designer drug (DREADD) hM4Di, ..... was comparable between groups. " On page 24, line 671-674, ".....Chemogenetic manipulation of Prdm13+ neurons in the DMH reveals that the inhibition of this neuronal population promotes excessive sleepiness during SD, demonstrating the direct involvement of Prdm13+ DMH neurons in controlling sleep-wake patterns through neuronal activity......" 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 51 "Moreover, overexpression of Prdm13 in the DMH ameliorated sleep fragmentation and increased sleep attempts during SD in old mice." On page 17-18, line 466-491 "Overexpression of Prdm13 in the DMH partially affects age-associated sleep alterations ...... (Fig. 6h). 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