Abstract
The phosphorylation of synaptic proteins is a significant biochemical reaction that controls the sleep–wake cycle in mammals1,2,3. Protein phosphorylation in vivo is reversibly regulated by kinases and phosphatases. In this study, we investigate a pair of kinases and phosphatases that reciprocally regulate sleep duration. First, we perform a comprehensive screen of protein kinase A (PKA) and phosphoprotein phosphatase (PPP) family genes by generating 40 gene knockout mouse lines using prenatal and postnatal CRISPR targeting. We identify a regulatory subunit of PKA (Prkar2b), a regulatory subunit of protein phosphatase 1 (PP1; Pppr1r9b) and catalytic and regulatory subunits of calcineurin (also known as PP2B) (Ppp3ca and Ppp3r1) as sleep control genes. Using adeno-associated virus (AAV)-mediated stimulation of PKA and PP1–calcineurin activities, we show that PKA is a wake-promoting kinase, whereas PP1 and calcineurin function as sleep-promoting phosphatases. The importance of these phosphatases in sleep regulation is supported by the marked changes in sleep duration associated with their increased and decreased activities, ranging from approximately 17.3 h per day (PP1 expression) to 4.3 h per day (postnatal CRISPR targeting of calcineurin). Localization signals to the excitatory post-synapse are necessary for these phosphatases to exert their sleep-promoting effects. Furthermore, the wake-promoting effect of PKA localized to the excitatory post-synapse negated the sleep-promoting effect of PP1–calcineurin. These findings indicate that PKA and PP1–calcineurin have competing functions in sleep regulation at excitatory post-synapses.
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Data availability
The data supporting the findings of this study are available within the paper and the supplementary information files. Some of the gRNA sequences used in the triple-target CRISPR method were obtained from the mm10 CRISPR targets database (http://crispr.riken.jp/). Source data are provided with this paper.
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Acknowledgements
We thank all the laboratory members at the University of Tokyo and the RIKEN Center for Biosystems Dynamics Research (BDR), particularly, T. Miyawaki, S. Sato, K. Shimizu, Y. Nakano and K. Itayama for AAV preparations; S. S. Sugai for the CRISPR experiment; A. Shimokawa, S. Tomita, M. Kunimi and R. Inoue for assisting with sleep phenotyping; J. Garcon-Yoshida, G. N. Kanda, K. Kon, Y. Sugihara, N. Hori, E. Matsushita and Y. Uranyu for their assistance in animal experiments; members at LARGE, RIKEN BDR for their help with mouse ES cell production; and staff at Enago (www.enago.jp) for English language review. We thank Z. Molnar, R. Foster, V. Vyazovskiy, A. Jagannath, M. Hastings and R. Lucas for valuable comments and discussion. This work was supported by grants from Brain/MINDS JP21dm0207049, Science and Technology Platform Program for Advanced Biological Medicine JP21am0401011, AMED-CREST JP21gm0610006 (AMED/MEXT) (H.R.U.), Grant-in-Aid for Scientific Research (S) JP18H05270 (JSPS KAKENHI) (H.R.U.) and Scientific Research (C) JP23K05738 (JSPS KAKENHI) (K.L.O.), Grant-in-Aid for Transformative Research Areas (A) JP 24H02305 (JSPS KAKENHI) (K.L.O.), Grant-in-Aid for Early-Career Scientists JP19K16115 (JSPS KAKENHI) (D.T.), HFSP Research Grant Program RGP0019/2018 (HFSP) (H.R.U.), ERATO JPMJER2001 (JST) (H.R.U.), Q-LEAP JPMXS0120330644 (MEXT) (H.R.U.), Moonshot R&D JPMJMS2023 (JST) (R.G.Y) and an intramural Grant-in-Aid from the RIKEN BDR (H.R.U.).
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H.R.U., Y.W., S.C., D.T. and K.L.O. designed the study. Y.W., S.C., D.T., K.L.O., H.F., S.S., K.M. and R.G.Y. performed the sleep phenotype analyses. R.G.Y. performed EEG/EMG data analyses. Y.W., S.C., D.T. and S.Y. participated in AAV production. D.T., R.-i.O., S.S., M.K., M.U.T., H.U., C.H., H.K. and K.S. produced genetically modified mice. H.S., T.O. and K.H. performed synapse imaging. H.R.U., Y.W., S.C., D.T., R.G.Y. and K.L.O. wrote the manuscript. All authors discussed the results and commented on the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Sleep phenotypes of Prkar2b KO and PKI-expressing mice.
(a–e) The genotyping of Prkacb (set1), Prkar1b (set1), Prkar2a (set1), Prkar2b (set1 and set2) KO mice qPCR was performed with primer pairs listed in SI Table 1 for the three target sites in a gene. The mouse was considered a KO mouse when the 0.5% criteria were met in either set. Each number represents each mouse used for the genotyping, and the magenta color indicates a KO-determined animal. wt: wild-type (f, g) Sleep/wake parameters (f) and sleep profiles (g) of Prkar2b KO (set2) mice, averaging over six days. A gRNA set (set 2) independent from Fig. 1b–f was used for KO mice production. The black broken line and the shaded area represent the mean and 1 s.d. range, respectively, of the wild-type mice wild-type C57BL/6N mice (n = 108). (h, i) Responses of wild-type C57BL/6N mice and Prkar2b KO mice to the cage change stimuli at ZT0 (h) or ZT12 (i). Total sleep duration of four hours just after the cage change was used for analysis. “Basal” represents the average of the sleep duration during the same time window over the previous three days. “Ratio” represents total sleep duration in cage change response divided by basal. (j) Sleep profiles measured by EEG/EMG recordings for Prkar2b KO (set 1) and wild-type C57BL/6N mice. (k) Sleep phenotypes measured by EEG/EMG recordings of Prkar2b KO (set 1) and WT C57BL/6N mice in the light phase (ZT0-12) or dark phase (ZT12-24). (l) Differences in transition probabilities (between wakefulness (W), NREM sleep (N), and REM sleep (R)) for Prkar2b KO mice. Magenta lines and broken blue lines indicate significantly (p < 0.05) higher and lower for the Prkar2b KO mice, respectively. (m) Sleep profiles measured by EEG/EMG recordings for PKI-expressing mice. (n) EEG/EMG recordings to determine the sleep phenotypes of PKI-expressing mice in the light phase (ZT0–12) or dark phase (ZT12–24). (o) Differences in transition probabilities (between wakefulness (W), NREM sleep (N), and REM sleep (R)) for PKI-expressing mice. Magenta lines and dashed blue lines represent significantly (p < 0.05) higher and lower values for PKI (WT) mice, respectively. The number of mice used in the analysis has been depicted as (n). Data are mean ± s.d., while shaded areas in the line plots represent mean ± s.e.m. The sample sizes for each experiment, the statistical methods used, and the statistical significance are summarized in SI Table 2. *p < 0.05, **p < 0.01, ***p < 0.001. ZT: zeitgeber time; WT: wild-type.
Extended Data Fig. 2 Expression of Prkar1 and Prkar2 mutants increased sleep.
(a) Schematic diagram of AAV-based PKA regulatory subunit expression. Gly to Asp mutation in the regulatory subunits used in this study blocks the binding of cAMP to the protein, resulting in continued inhibition of the catalytic subunits in the presence of cAMP. (b, c) Sleep/wake parameters (b) and sleep profiles (c) of mice expressing Prkar1a (WT) and its dominant-negative mutant (G325D) under the hSyn1 promoter, averaging over six days. (d, e) Sleep/wake parameters (d) and sleep profiles (e) of mice expressing Prkar1b WT and its dominant-negative mutant (G325D) under the hSyn1 promoter, averaging over six days. (f, g) Sleep/wake parameters (f) and sleep profiles (g) of mice expressing Prkar2a WT and its dominant-negative mutant (G335D) under the hSyn1 promoter, averaging over six days. (h, i) Sleep/wake parameters (h) and sleep profiles (i) of mice expressing Prkar2b WT and its dominant-negative mutant (G349D). The dosage of the AAVs was 4 × 1011 vg per mouse. The number of mice used in the analysis has been depicted as (n). Data are mean ± s.d., while shaded areas in the line plots represent mean ± s.e.m. The sample sizes for each experiment, the statistical methods used, and the statistical significance are summarized in SI Table 2. *p < 0.05, **p < 0.01, ***p < 0.001. AAV: adeno-associated virus; hSyn1: human synapsin 1; ZT: zeitgeber time; WT: wild-type.
Extended Data Fig. 3 Detailed sleep phenotypes of Prkaca-expressing mice.
(a) Sleep phenotypes of the light phase (ZT0–12) or dark phase (ZT12–24) measured by EEG/EMG recordings for the Prkaca-expressing mice. (b) Differences in transition probabilities (between wakefulness (W), NREM sleep (N), and REM sleep (R)) between Prkaca (WT) and Prkaca (H88Q:W197R) mice under Camk2a promoter. Magenta lines and broken blue lines indicate significantly (p < 0.05) higher and lower values, respectively, for H88Q:W197R mice. (c) Schematic diagram of Prkaca expression without localized protein fusion. PSD95.FingR was excluded from the pAAV construct used in Fig. 2h. (d, e) Sleep/wake parameters (d) and sleep profiles (e) of mice expressing Prkaca kinase-dead (K73E:K169E) and active (H88Q:W197R) mutant under the Camk2a promoter, averaging over six days. (f) Representative immunohistochemistry images using layer 2/3 of the somatosensory cortex of mice injected with AAVs encoding PSD95.FingR-EGFP or with PBS. Gephyrin is a marker for inhibitory post-synapses and PSD95 for excitatory post-synapses. PSD95.FingR was expressed in conjunction with the FLAG sequence and was detected with FLAG antibodies. (g) Quantitative analysis of PSD95.FingR-EGFP localization based on immunohistochemistry images. The bar graph indicates the proportion of the observed PSD95.FingR (FLAG) signal localized to Gephyrin or PSD95. It was also confirmed that in a sample from a single independent mouse, the PSD95.FingR (FLAG) signal co-localized with PSD95. (h, i) Sleep/wake parameters (h) and sleep profiles (i) of PSD95.FingR-Prakca (K73E:K169E) mice (n = 6), averaging over six days. The black broken line and the shaded area represent the mean and one s.d. range, respectively, of the wild-type mice wild-type C57BL/6N mice (n = 108). (j) Expression level of PSD95.FingR-PRKACA in synaptosomal fraction. The dosages of the AAVs were 2.5 × 109 vg/mouse. The lane labeled “Control” contains synaptosomal fractions from control mice that do not express PSD95.FingR-PRKACA. The black arrow indicates the position of endogenous PRKACA (40.6 kDa). The white triangle marks the expected position of AAV-expressed PSD95.FingR-PRKACA (52.3 kDa). The asterisk represents an unknown band detected regardless of PSD95.FingR-Prkaca expression. For gel source data, see SI Fig. 1. (k) Sleep phenotypes of light phase (ZT0-12) or dark phase (ZT12-24) measured by EEG/EMG recordings for mice expressing PSD95.FingR-Prkaca. (l) Differences in transition probabilities (between wakefulness (W), NREM sleep (N), and REM sleep (R)) between PSD95.FingR-Prkaca inactive mutant (K73E:K169E) and its active mutant (H88Q:W197R). Magenta lines and broken blue lines indicate significantly (p < 0.05) higher and lower values for H88Q:W197R mice, respectively are. (m) Schematic diagram of pyramidal neuron-specific expression of PKA catalytic subunit using AAVs and Nex-Cre mice. The dosage of the AAVs was 5 × 1010 vg per mouse. (n, o) Sleep/wake parameters (n) and sleep profiles (o) of Nex-Cre mice expressing Prkaca kinase-dead mutant (K73E:K169E) and active mutant (H88Q:W197R) under the hSyn1 promoter, averaging over six days. The number of mice used in the analysis has been depicted as (n). Data are mean ± s.d., while shaded areas in the line plots represent mean ± s.e.m. The sample sizes for each experiment, the statistical methods used, and the statistical significance are summarized in SI Table 2. *p < 0.05, **p < 0.01, ***p < 0.001. ZT: zeitgeber time; WT: wild-type.
Extended Data Fig. 4 Sleep phenotypes of Ppp1r9b KO mice.
(a, b) The genotyping of Ppp1r9b KO mice. qPCR was performed with primer pairs listed in SI Table 1 for the three target sites in a gene. When 0.5% criteria were met in either set, the mouse was considered a KO mouse. Each number represents each mouse used for the genotyping, and magenta color indicates a KO-determined animal. (c) 24-hour sleep profiles of Ppp1r9b KO mice demonstrated in Fig. 3b.(d, e) Sleep/wake parameters of Ppp1r9b KO (set 2) mice, averaging over six days (d) and sleep profiles (e). KO mice were compared with 8-week-old WT C57BL/6N male mice. The black broken line, and the shaded area represent the mean and one s.d. range, respectively, of the WT mice. (f) Sleep phenotypes of first and second half day of Ppp1r9b KO mice and WT C57BL/6N mice indicated in Fig. 3c–e. (g) NREM power density in delta domain (1–4 Hz) and EEG power spectra of Ppp1r9b KO mice (n = 8) and WT C57BL/6N mice (n = 8) depicted in Fig. 3c–e. (h) Differences in transition probabilities (between wakefulness (W), NREM sleep (N), and REM sleep (R)) between WT C57BL/6N mice and Ppp1r9b KO mice. Magenta lines and broken blue lines indicate significantly (p < 0.05) higher and lower values for Ppp1r9b KO mice, respectively. The number of mice used in the analysis has been depicted as (n). Data are mean ± s.d., while shaded areas in the line plots represent mean ± s.e.m. The sample sizes for each experiment, the statistical methods used, and the statistical significance are summarized in SI Table 2. *p < 0.05, **p < 0.01, ***p < 0.001. KO: knock out; WT: wild-type; ZT: zeitgeber time.
Extended Data Fig. 5 Postsynaptic localization is critical for the sleep extension by PP1.
(a-f) Sleep parameters (a, c, e) and 24-hour sleep profile (b, d, f) of PSD95-fused PP1c-expressing mice measured by SSS, averaging over six days. (g) Schematic diagram of the AAV-PHP.eB for expression of PP1 catalytic subunits (PPP1CA, PPP1CB, PPP1CC) in mice brain. (h–m) Sleep parameters (h, j, l) and 24-hour sleep profiles (i, k, m) of mice expressing PP1 catalytic subunit, averaging over six days. The dosage of the AAVs was 4 × 1011 vg per mouse. The number of mice used in the analysis has been depicted as (n). Data are mean ± s.d., while shaded areas in the line plots represent mean ± s.e.m. The sample sizes for each experiment, the statistical methods used, and the statistical significance are summarized in SI Table 2. *p < 0.05, **p < 0.01, ***p < 0.001. WT: wild-type; ZT: zeitgeber time.
Extended Data Fig. 6 Detailed analysis of the sleep-promoting effect of PP1.
(a) Sleep phenotypes of the first and second half day of PSD95-fused PPP1CA H248K or T320A mutant shown in Fig. 3g–i. (b) NREM power density in the delta domain (1–4 Hz) and EEG power spectra of the mice expressing PSD95-PPP1CA H248K or T320A mutant has been shown in Fig. 3g–i. (c) Differences in transition probabilities (between wakefulness (W), NREM sleep (N), and REM sleep (R)) between mice expressing PSD95-fused PPP1CA H248K or T320A mutant. Magenta lines and broken blue lines indicate significantly (p < 0.05) higher and lower values for T320A mice, respectively. (d) Expression level of PSD95-PPP1CA in synaptosomal fraction. The dosages of the AAVs were 5.0 × 1010 vg/mouse. The lane labeled “Control” contains synaptosomal fractions from control mice that do not express PSD95-PPP1CA. The black arrow indicates the position of endogenous PPP1CA (37.5 kDa) or PSD95 (80.5 kDa). White triangle marks the expected position of AAV-expressed PSD95-PPP1CA (121.2 kDa). Asterisk denotes an unknown band detected regardless of PSD95-PPP1CA expression. For gel source data, see SI Fig. 1. (e–g) Sleep parameters of AAV-administrated mice expressing PPP1R1B(e), PPP1R2(f), and PPP1R14B (g), measured by SSS, averaging over six days. The dosages of the AAVs were 4 × 1011 vg/mouse. Blue, orange, and sand color represent WT, constitutively active mutant, and dominant, respectively. The black broken line and the shaded area represent the mean and one s.d. range, respectively, of the WT mice WT C57BL/6N mice (n = 101). (h) Responses of Ppp1r9b KO mice and WT C57BL/6N mice mentioned in Fig. 3c–e to the cage change stimuli. Total sleep duration of four hours just after the cage change was used for the analysis. “Basal” represents the average sleep duration during the same time window over the previous three days. “Ratio” represents total sleep duration in cage change response divided by basal. (i–k) Responses of mice expressing PSD95-fused PP1 catalytic subunits mentioned in Extended Data Fig. 5a–f to the cage change stimuli. The dosages of the AAVs were 4 × 1011 vg/mouse. The number of mice used in the analysis has been depicted as (n). Data are mean ± s.d., while shaded areas in the line plots represent mean ± s.e.m. The sample sizes for each experiment, the statistical methods used, and the statistical significance are summarized in SI Table 2. *p < 0.05, **p < 0.01, ***p < 0.001. ZT: zeitgeber time; WT: wild-type.
Extended Data Fig. 7 Detailed sleep phenotypes of calcineurin postnatal KO mice.
(a) Sleep phenotypes of first and second half day of Tyr and Ppp3ca -targeted KO mice Fig. 4d–g. (b) Schematic representation of the postnatal CRISPR KO method by co-injecting AAVs for triple gRNAs and for SpCas9 into the wild-type C57BL/6N mice. The dosages of each AAV were 1 × 1012 vg/mouse. (c and d) Sleep/wake parameters (c) and sleep profiles (d) of Tyr and Ppp3ca -targeted mice at nine weeks, averaging over six days. (e) Schematic representation of postnatal double KO for Ppp3ca and Ppp3cb genes by injecting two sets of triple gRNA-expressing AAVs into H11-Cas9 knock-in mice, used in Fig. 4i–k. (f) Expression levels of PPP3CA, PPP3CB, PPP3R1 and alpha tubulin in Tyr and Ppp3ca/Ppp3cb -targeted KO mice’s brains used in Fig. 4i–k. Immunoblotting images indicate lower expression levels of PPP3CA, PPP3CB, and even PPP3R1 in Ppp3ca/Ppp3cb-targeted mice compared to Tyr-targeted mice. For gel source data, see SI Fig. 1. (g, h) Sleep phenotypes (g) and sleep profiles (h) of Tyr and Ppp3ca/Ppp3cb -targeted mice obtained from the EEG/EMG recordings. (i) Sleep phenotypes of first and second half day of Tyr and Ppp3ca/3cb -targeted KO mice. (j) NREM power density in delta domain (1–4 Hz) and EEG power spectra of Tyr- and Ppp3ca/Ppp3cb-targeted mice. (k) Estimated time constants for the increase (τi) and decrease (τd) of the NREM delta power of Tyr- and Ppp3ca/Ppp3cb-targeted mice. The number of mice used in the analysis has been depicted as (n). Data are mean ± s.d., while shaded areas in the line plots represent mean ± s.e.m. The sample sizes for each experiment, the statistical methods used, and the statistical significance are summarized in SI Table 2. *p < 0.05, **p < 0.01, ***p < 0.001. ZT: zeitgeber time.
Extended Data Fig. 8 Calcineurin catalytic subunits without postsynaptic localization do not increase sleep.
(a) (left) Schematic diagram of PP2Bc genes (PPP3CA, PPP3CB) expression without localized protein fusion. PSD95.FingR was removed from the pAAV construct shown in Fig. 5a. (right) Information on mutation sites used in this study. The loss-of-function mutation site has been highlighted in red, and the deletion regions for constitutive activation are represented by a red dotted line. AAVs were injected into 6-week-old male WT C57BL/6N mice at the dosage of 4 × 1011 vg/mouse. (b–e) Sleep parameters (b, d) and 24-hour sleep profiles (c, e) of mice expressing PP2B catalytic subunit, averaging over six days. (f) Schematic diagram of AAV-PHP.eB based expression of PSD95.FingR-fused PP2B catalytic subunits (PPP3CA, PPP3CB) in postsynaptic density. The overall scheme is the same as Fig. 5a. The dosages of the AAVs were 2.5 × 1010 vg/mouse (half dosage of Fig. 5a). (g–j) Sleep parameters (g, i) and 24-hour sleep profiles (h, j) of mice expressing PSD95.FingR-fused PP2B catalytic subunit, averaging over six days. The number of mice used in the analysis has been depicted as (n). Data are mean ± s.d., while shaded areas in the line plots represent mean ± s.e.m. The sample sizes for each experiment, the statistical methods used, and the statistical significance are summarized in SI Table 2. *p < 0.05, **p < 0.01, ***p < 0.001. WT: wild-type; ZT: zeitgeber time.
Extended Data Fig. 9 Detailed sleep phenotypes of mice expressing PSD-localized calcineurin.
(a–d) Responses of mice expressing PSD95.FingR-fused PP2B catalytic subunit (PPP3CA and PPP3CB) to the cage change stimuli at ZT0 (a, b) or ZT12 (c, d) (n = 6 each). Total sleep duration of four hours just after the cage change was used for analysis. “Basal” represents the average sleep duration during the same time window over the previous three days. “Ratio” represents total sleep duration in cage change response divided by basal. (e, f) All day sleep phenotypes (e), sleep profiles (f) of mice expressing PSD95.FingR-PPP3CA 1–389 or 1-389 (H151Q) downstream of the Synapsin-1 promoter. 1–389, constitutive active deletion mutant; 1-389(H151Q), deletion mutant with inactive mutation. The dosages of the AAVs were 5.0 × 1010 vg/mouse. (g) Sleep phenotypes of first and second half day of mice expressing PSD95.FingR-PPP3CA. (h, i) NREM power density in delta domain (1–4 Hz) and EEG power spectra (h) and estimated time constants for the increase (τi) and decrease (τd) of the NREM delta power (i) of mice expressing PSD95.FingR-PPP3CA. (j) Sleep profiles (g) measured by EEG/EMG recordings for mice expressing PSD95.FingR-PPP3CA 1–389 and 1–389(H151Q) under Camk2a promoter, mentioned in Fig. 5f–h. (k) Sleep phenotypes of first and second half day of mice expressing PSD95.FingR-PPP3CA under Camk2a promoter. Data are mean ± s.d., while shaded areas in the line plots represent mean ± s.e.m. The sample sizes for each experiment, the statistical methods used, and the statistical significance are summarized in SI Table 2. *p < 0.05, **p < 0.01, ***p < 0.001. ZT: zeitgeber time.
Extended Data Fig. 10 Behavioral motion analysis.
Behavioral analysis of mice performed using video data during EEG/EMG recording sessions. Each figure represents the normalized values of motion value over three days for mice expressing P(Camk2a)-Prkaca (EEG/EMG data shown in Fig. 2d–g), mice expressing P(Camk2a)-PSD95.FingR-Prkaca (EEG/EMG data shown in Fig. 2k–n), AAV-CRISPR KO mice for Ppp3ca (EEG/EMG data shown in Fig. 4d–g) and mice expressing P(Camk2a)-PSD95.FingR-PPP3CA (EEG/EMG data shown in Fig. 5f–i), normalized by each mouse’s wake duration. Data are mean ± s.d. The sample sizes for each experiment, the statistical methods used, and the statistical significance are summarized in SI Table 2. *p < 0.05, **p < 0.01, ***p < 0.001.
Supplementary information
Supplementary Information
Supplementary Discussion, Supplementary Figs. 1 and 2 and Supplementary References.
Supplementary Table 1
List of oligonucleotide sequences used for genotyping qPCR. Listed primers were used for the KO confirmation of PKA and phosphatase genes. The gRNA numbers 1–3 correspond to set 1 and 4–6 correspond to set 2.
Supplementary Table 2
Summary of the statistical tests conducted in this study. A summary of each statistical result obtained during the statistical analysis processes. The finally adopted outcomes are coloured. Results from statistical methods that were not adopted are also shown.
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Wang, Y., Cao, S., Tone, D. et al. Postsynaptic competition between calcineurin and PKA regulates mammalian sleep–wake cycles. Nature (2024). https://doi.org/10.1038/s41586-024-08132-2
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DOI: https://doi.org/10.1038/s41586-024-08132-2