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Autistic-like behavior and cerebellar dysfunction in Bmal1 mutant mice ameliorated by mTORC1 inhibition

Molecular Psychiatry volume 28, pages 3727–3738 (2023)Cite this article

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Abstract

Although circadian and sleep disorders are frequently associated with autism spectrum disorders (ASD), it remains elusive whether clock gene disruption can lead to autistic-like phenotypes in animals. The essential clock gene Bmal1 has been associated with human sociability and its missense mutations are identified in ASD. Here we report that global Bmal1 deletion led to significant social impairments, excessive stereotyped and repetitive behaviors, as well as motor learning disabilities in mice, all of which resemble core behavioral deficits in ASD. Furthermore, aberrant cell density and immature morphology of dendritic spines were identified in the cerebellar Purkinje cells (PCs) of Bmal1 knockout (KO) mice. Electrophysiological recordings uncovered enhanced excitatory and inhibitory synaptic transmission and reduced firing rates in the PCs of Bmal1 KO mice. Differential expression of ASD- and ataxia-associated genes (Ntng2, Mfrp, Nr4a2, Thbs1, Atxn1, and Atxn3) and dysregulated pathways of translational control, including hyperactivated mammalian target of rapamycin complex 1 (mTORC1) signaling, were identified in the cerebellum of Bmal1 KO mice. Interestingly, the antidiabetic drug metformin reversed mTORC1 hyperactivation and alleviated major behavioral and PC deficits in Bmal1 KO mice. Importantly, conditional Bmal1 deletion only in cerebellar PCs was sufficient to recapitulate autistic-like behavioral and cellular changes akin to those identified in Bmal1 KO mice. Together, these results unveil a previously unidentified role for Bmal1 disruption in cerebellar dysfunction and autistic-like behaviors. Our findings provide experimental evidence supporting a putative role for dysregulation of circadian clock gene expression in the pathogenesis of ASD.

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Fig. 1: Bmal1 KO mice exhibit deficits in social interaction, excessive repetitive behaviors, and stereotypy.
Fig. 2: Bmal1 KO mice exhibit motor learning deficits and pervasive pathological changes in the cerebellum.
Fig. 3: Aberrant synaptic transmission and firing activities in Purkinje cells (PCs) of Bmal1 KO mice.
Fig. 4: Altered translational landscape and dysregulated translational control pathways in the cerebellum of Bmal1 KO mice.
Fig. 5: Metformin ameliorates behavioral deficits and reverses cerebellar mTORC1 hyperactivation in Bmal1 KO mice.
Fig. 6: Conditional deletion of Bmal1 in cerebellar Purkinje cells leads to autistic-like behavioral and cellular changes in Bmal1flx/flx: L7-Cre mice.

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Data availability

RNA-Seq and Ribo-Seq datasets that supported this study (Figs. S4 and 4) were deposited publicly in the Gene Expression Omnibus under accession code GSE196765. The published article includes all datasets generated or analysed during this study.

References

  1. Herzog ED, Hermanstyne T, Smyllie NJ, Hastings MH. Regulating the Suprachiasmatic Nucleus (SCN) Circadian Clockwork: Interplay between Cell-Autonomous and Circuit-Level Mechanisms. Cold Spring Harb Perspect Biol. 2017;9:a027706.

    PubMed  PubMed Central  Google Scholar 

  2. Takahashi JS, Hong HK, Ko CH, McDearmon EL. The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat Rev Genet. 2008;9:764–75.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Rosbash M. The implications of multiple circadian clock origins. PLoS Biol. 2009;7:e62.

    PubMed  Google Scholar 

  4. Bunger MK, Wilsbacher LD, Moran SM, Clendenin C, Radcliffe LA, Hogenesch JB, et al. Mop3 is an essential component of the master circadian pacemaker in mammals. Cell. 2000;103:1009–17.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Lai MC, Lombardo MV, Baron-Cohen S. Autism. Lancet. 2014;383:896–910.

    PubMed  Google Scholar 

  6. Lorsung E, Karthikeyan R, Cao R. Biological timing and neurodevelopmental disorders: a role for circadian dysfunction in autism spectrum disorders. Front Neurosci. 2021;15:642745.

    PubMed  PubMed Central  Google Scholar 

  7. Nicholas B, Rudrasingham V, Nash S, Kirov G, Owen MJ, Wimpory DC. Association of Per1 and Npas2 with autistic disorder: support for the clock genes/social timing hypothesis. Mol Psychiatry. 2007;12:581–92.

    CAS  PubMed  Google Scholar 

  8. Iossifov I, O’Roak BJ, Sanders SJ, Ronemus M, Krumm N, Levy D, et al. The contribution of de novo coding mutations to autism spectrum disorder. Nature. 2014;515:216–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Yang Z, Matsumoto A, Nakayama K, Jimbo EF, Kojima K, Nagata K, et al. Circadian-relevant genes are highly polymorphic in autism spectrum disorder patients. Brain Dev. 2016;38:91–99.

    CAS  PubMed  Google Scholar 

  10. Abrahams BS, Arking DE, Campbell DB, Mefford HC, Morrow EM, Weiss LA, et al. SFARI Gene 2.0: a community-driven knowledge base for the autism spectrum disorders (ASDs). Mol Autism. 2013;4:36.

    PubMed  PubMed Central  Google Scholar 

  11. Bralten J, Mota NR, Klemann C, De Witte W, Laing E, Collier DA, et al. Genetic underpinnings of sociability in the general population. Neuropsychopharmacology. 2021;46:1627–34.

    PubMed  PubMed Central  Google Scholar 

  12. Lipton JO, Yuan ED, Boyle LM, Ebrahimi-Fakhari D, Kwiatkowski E, Nathan A, et al. The Circadian protein BMAL1 regulates translation in response to S6K1-mediated phosphorylation. Cell. 2015;161:1138–51.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Kelleher RJ 3rd, Bear MF. The autistic neuron: troubled translation? Cell. 2008;135:401–6.

    CAS  PubMed  Google Scholar 

  14. Gkogkas CG, Khoutorsky A, Ran I, Rampakakis E, Nevarko T, Weatherill DB, et al. Autism-related deficits via dysregulated eIF4E-dependent translational control. Nature. 2013;493:371–7.

    CAS  PubMed  Google Scholar 

  15. Santini E, Huynh TN, MacAskill AF, Carter AG, Pierre P, Ruggero D, et al. Exaggerated translation causes synaptic and behavioural aberrations associated with autism. Nature. 2013;493:411–5.

    CAS  PubMed  Google Scholar 

  16. Hering H, Sheng M. Dendritic spines: structure, dynamics and regulation. Nat Rev Neurosci. 2001;2:880–8.

    CAS  PubMed  Google Scholar 

  17. Cao R, Gkogkas CG, de Zavalia N, Blum ID, Yanagiya A, Tsukumo Y, et al. Light-regulated translational control of circadian behavior by eIF4E phosphorylation. Nat Neurosci. 2015;18:855–62.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Amorim IS, Kedia S, Kouloulia S, Simbriger K, Gantois I, Jafarnejad SM, et al. Loss of eIF4E phosphorylation engenders depression-like behaviors via selective mRNA translation. J Neurosci. 2018;38:2118–33.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Gantois I, Khoutorsky A, Popic J, Aguilar-Valles A, Freemantle E, Cao R, et al. Metformin ameliorates core deficits in a mouse model of fragile X syndrome. Nat Med. 2017;23:674–7.

    CAS  PubMed  Google Scholar 

  20. Dziuk MA, Gidley Larson JC, Apostu A, Mahone EM, Denckla MB, Mostofsky SH. Dyspraxia in autism: association with motor, social, and communicative deficits. Dev Med Child Neurol. 2007;49:734–9.

    CAS  PubMed  Google Scholar 

  21. Whitney ER, Kemper TL, Rosene DL, Bauman ML, Blatt GJ. Calbindin-D28k is a more reliable marker of human Purkinje cells than standard Nissl stains: a stereological experiment. J Neurosci Methods. 2008;168:42–47.

    CAS  PubMed  Google Scholar 

  22. De Zeeuw CI, Hoebeek FE, Bosman LW, Schonewille M, Witter L, Koekkoek SK. Spatiotemporal firing patterns in the cerebellum. Nat Rev Neurosci. 2011;12:327–44.

    PubMed  Google Scholar 

  23. Kawamura Y, Nakayama H, Hashimoto K, Sakimura K, Kitamura K, Kano M. Spike timing-dependent selective strengthening of single climbing fibre inputs to Purkinje cells during cerebellar development. Nat Commun. 2013;4:2732.

    PubMed  Google Scholar 

  24. Wardlaw SM, Phan TX, Saraf A, Chen X, Storm DR. Genetic disruption of the core circadian clock impairs hippocampus-dependent memory. Learn Mem. 2014;21:417–23.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Snider KH, Dziema H, Aten S, Loeser J, Norona FE, Hoyt K, et al. Modulation of learning and memory by the targeted deletion of the circadian clock gene Bmal1 in forebrain circuits. Behav Brain Res. 2016;308:222–35.

    CAS  Google Scholar 

  26. Musiek ES, Lim MM, Yang G, Bauer AQ, Qi L, Lee Y, et al. Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration. J Clin Invest. 2013;123:5389–5400.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Crawford JD, Chandley MJ, Szebeni K, Szebeni A, Waters B, Ordway GA. Elevated GFAP protein in anterior cingulate cortical white matter in males with autism spectrum disorder. Autism Res. 2015;8:649–57.

    PubMed  Google Scholar 

  28. Laurence JA, Fatemi SH. Glial fibrillary acidic protein is elevated in superior frontal, parietal and cerebellar cortices of autistic subjects. Cerebellum. 2005;4:206–10.

    CAS  PubMed  Google Scholar 

  29. Khan S, Gramfort A, Shetty NR, Kitzbichler MG, Ganesan S, Moran JM, et al. Local and long-range functional connectivity is reduced in concert in autism spectrum disorders. Proc Natl Acad Sci USA. 2013;110:3107–12.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Hickman RA, O’Shea SA, Mehler MF, Chung WK. Neurogenetic disorders across the lifespan: from aberrant development to degeneration. Nat Rev Neurol. 2022;18:117–24.

    PubMed  PubMed Central  Google Scholar 

  31. Wang SS, Kloth AD, Badura A. The cerebellum, sensitive periods, and autism. Neuron. 2014;83:518–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Bauman ML, Kemper TL. The neuropathology of the autism spectrum disorders: what have we learned? Novartis Found Symp. 2003;251:112–22.

    PubMed  Google Scholar 

  33. Zhou H, Lin Z, Voges K, Ju C, Gao Z, Bosman LW, et al. Cerebellar modules operate at different frequencies. Elife. 2014;3:e02536.

    PubMed  PubMed Central  Google Scholar 

  34. Yang YM, Arsenault J, Bah A, Krzeminski M, Fekete A, Chao OY, et al. Identification of a molecular locus for normalizing dysregulated GABA release from interneurons in the Fragile X brain. Mol Psychiatry. 2020;25:2017–35.

    CAS  PubMed  Google Scholar 

  35. Kondratov RV, Kondratova AA, Gorbacheva VY, Vykhovanets OV, Antoch MP. Early aging and age-related pathologies in mice deficient in BMAL1, the core component of the circadian clock. Genes Dev. 2006;20:1868–73.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Leliavski A, Shostak A, Husse J, Oster H. Impaired glucocorticoid production and response to stress in Arntl-deficient male mice. Endocrinology. 2014;155:133–42.

    PubMed  Google Scholar 

  37. Monyak RE, Emerson D, Schoenfeld BP, Zheng X, Chambers DB, Rosenfelt C, et al. Insulin signaling misregulation underlies circadian and cognitive deficits in a Drosophila fragile X model. Mol Psychiatry. 2017;22:1140–8.

    CAS  PubMed  Google Scholar 

  38. Dy ABC, Tassone F, Eldeeb M, Salcedo-Arellano MJ, Tartaglia N, Hagerman R. Metformin as targeted treatment in fragile X syndrome. Clin Genet. 2018;93:216–22.

    CAS  PubMed  Google Scholar 

  39. Biag HMB, Potter LA, Wilkins V, Afzal S, Rosvall A, Salcedo-Arellano MJ, et al. Metformin treatment in young children with fragile X syndrome. Mol Genet Genom Med. 2019;7:e956.

    Google Scholar 

  40. Ramanathan C, Kathale ND, Liu D, Lee C, Freeman DA, Hogenesch JB, et al. mTOR signaling regulates central and peripheral circadian clock function. PLoS Genet. 2018;14:e1007369.

    PubMed  PubMed Central  Google Scholar 

  41. Cao R, Robinson B, Xu H, Gkogkas C, Khoutorsky A, Alain T, et al. Translational control of entrainment and synchrony of the suprachiasmatic circadian clock by mTOR/4E-BP1 signaling. Neuron. 2013;79:712–24.

    CAS  PubMed  Google Scholar 

  42. Cao R, Anderson FE, Jung YJ, Dziema H, Obrietan K. Circadian regulation of mammalian target of rapamycin signaling in the mouse suprachiasmatic nucleus. Neuroscience. 2011;181:79–88.

    CAS  PubMed  Google Scholar 

  43. Lipton JO, Sahin M. The neurology of mTOR. Neuron. 2014;84:275–91.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Tsai PT, Hull C, Chu Y, Greene-Colozzi E, Sadowski AR, Leech JM, et al. Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice. Nature. 2012;488:647–51.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Bhattacharya A, Kaphzan H, Alvarez-Dieppa AC, Murphy JP, Pierre P, Klann E. Genetic removal of p70 S6 kinase 1 corrects molecular, synaptic, and behavioral phenotypes in fragile X syndrome mice. Neuron. 2012;76:325–37.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Gkogkas CG, Khoutorsky A, Cao R, Jafarnejad SM, Prager-Khoutorsky M, Giannakas N, et al. Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndrome-like phenotypes. Cell Rep. 2014;9:1742–55.

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank S. Fatemi and I. Gantois for critical reading of the manuscript. The work was supported by grants from NIH (NS118026 and DK109714 to RC; MH119355 to AA and PK.; NS097312, DA048822 to AA; NS022920 and NS045667 to HTO; GM114142 and CA217297 to VXJ), from the Winston and Maxine Wallin Neuroscience Discovery Fund, from the University of Minnesota Medical School Foundation and from Whiteside Institute for Clinical Research to RC.

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Author notes

  1. These authors contributed equally: Dong Liu, Carmen Nanclares.

Authors and Affiliations

  1. Department of Biomedical Sciences, University of Minnesota Medical School, Duluth, MN, 55812, USA

    Dong Liu, Ethan Lorsung, Nam Le, Salil Saurav Pathak, Jin Li & Ruifeng Cao

  2. Department of Neuroscience, University of Minnesota Medical School, Minneapolis, MN, 55455, USA

    Carmen Nanclares, Jonathan C. Gewirtz, Paulo Kofuji, Alfonso Araque & Ruifeng Cao

  3. Patrick Wild Centre, University of Edinburgh, Edinburgh, EH8 9XD, UK

    Konstanze Simbriger, Inês Silva Amorim, Kleanthi Chalkiadaki & Christos G. Gkogkas

  4. Simons Initiative for the Developing Brain, University of Edinburgh, Edinburgh, EH8 9XD, UK

    Konstanze Simbriger, Inês Silva Amorim, Kleanthi Chalkiadaki & Christos G. Gkogkas

  5. Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, EH8 9XD, UK

    Konstanze Simbriger, Inês Silva Amorim, Kleanthi Chalkiadaki & Christos G. Gkogkas

  6. Department of Molecular Medicine, The University of Texas Health San Antonio, San Antonio, TX, 78229, USA

    Kun Fang & Victor X. Jin

  7. Department of Psychology, University of Minnesota, Minneapolis, MN, 55455, USA

    Jonathan C. Gewirtz

  8. Department of Psychology, Arizona State University, Tempe, AZ, 85287, USA

    Jonathan C. Gewirtz

  9. Institute for Translational Neuroscience, University of Minnesota, Minneapolis, MN, USA

    Harry T. Orr

  10. Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA

    Harry T. Orr

  11. Biomedical Research Institute, Foundation for Research and Technology-Hellas, University Campus, 45110, Ioannina, Greece

    Christos G. Gkogkas

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Contributions

DL, CN, PK, AA, HTO, CGG, and RC designed research; DL, CN, KS, EL, ISA, KC, SP, and RC performed research; DL, CN, JL, KF, CGG, and RC analyzed data; DL, CN, EL, JCG, VXJ, PK, AA, HTO, CGG and RC wrote the manuscript.

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Correspondence to Christos G. Gkogkas or Ruifeng Cao.

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Liu, D., Nanclares, C., Simbriger, K. et al. Autistic-like behavior and cerebellar dysfunction in Bmal1 mutant mice ameliorated by mTORC1 inhibition. Mol Psychiatry 28, 3727–3738 (2023). https://doi.org/10.1038/s41380-022-01499-6

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