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Cenpj Regulates Cilia Disassembly and Neurogenesis in the Developing Mouse Cortex

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This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. A link to any extended data will be provided when the final version is posted online. Research Articles: Development/Plasticity/Repair Cenpj regulates cilia disassembly and neurogenesis in the developing mouse cortex Wenyu Ding1,2, Qian Wu1,2, Le Sun1,2, Na Clara Pan1,2 and Xiaoqun Wang1,2,2 1State Key Laboratory of Brain and Cognitive Science, CAS Center for Excellence in Brain Science and Intelligence Technology (Shanghai), Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China 2University of Chinese Academy of Sciences, Beijing 100049, China 3Beijing Institute for Brain Disorders, Beijing 100069, China https://doi.org/10.1523/JNEUROSCI.1849-18.2018 Received: 20 July 2018 Revised: 19 December 2018 Accepted: 24 December 2018 Published: 9 January 2019 Author contributions: W.D., Q.W., and X.W. designed research; W.D., Q.W., L.S., N.C.P., and X.W. performed research; W.D., Q.W., L.S., N.C.P., and X.W. analyzed data; W.D., Q.W., L.S., N.C.P., and X.W. edited the paper; W.D., Q.W., and X.W. wrote the paper; Q.W. and X.W. wrote the first draft of the paper; X.W. contributed unpublished reagents/analytic tools. Conflict of Interest: The authors declare no competing financial interests. We gratefully acknowledge Dr. Bradley Yoder (University of Alabama at Birmingham) kindly shared and transferred the mouse strain to us. We thank Dr. Tian Xue (University of Science and Technology of China) for sharing ARPE19 cell line with us. We also thank Jianguo Zhang, Lei Sun and Can Peng of the Center for Biological Imaging, Institute of Biophysics, CAS for their help in making EM sample and taking EM images. We thank Dr. Arnold Kriegstein (University of California at San Francisco) and his lab members for their generous discussion and reagent support. This work was supported by the National Basic Research Program of China (2017YFA0103303, 2017YFA0102601); the Strategic Priority Research Program of the Chinese Academy of Sciences (grant number XDA16020601); National Natural Science Foundation of China (NSFC) (31371100) and Newton Advanced Fellowship (grant number NA140416) to X. W. Corresponding author: Xiaoqun Wang ([email protected]) Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.1849-18.2018 Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2019 the authors 1 Cenpj regulates cilia disassembly and neurogenesis in the developing 2 mouse cortex 3 Abbreviated Title: Cilia and neurogenesis regulated by Cenpj 4 Wenyu Ding1,2 *, Qian Wu1,2, *, Le Sun1,2, Na Clara Pan1,2 and Xiaoqun Wang1,2,3, # 5 6 1State Key Laboratory of Brain and Cognitive Science, CAS Center for Excellence in 7 Brain Science and Intelligence Technology (Shanghai), Institute of Biophysics, 8 Chinese Academy of Sciences, Beijing, 100101, China 9 2University of Chinese Academy of Sciences, Beijing 100049, China 10 3Beijing Institute for Brain Disorders, Beijing 100069, China 11 12 *These authors contributed equally to this work. 13 # Corresponding author: Xiaoqun Wang ([email protected]) 14 15 16 17 18 Number of pages: 49 19 The manuscript includes 9 figures and 6 movies. 20 Title 11 words˗Abbreviated Title 41 characters (including spaces); Abstract 185 21 words; Significance Statement 152 words; Introduction 593 words; Discussion 1323 22 words. 1 23 Author contributions 24 W. D., Q. W. and X. W. conceived the project, designed the experiments and wrote the 25 manuscript. W. D. and Q. W. conducted the experiments, including animal surgery, 26 tissue preparation, immunostaining, and imaging. N. C. P. cultured the cells. L. S. 27 performed RNA-seq data analysis and interpretation. All the authors edited and 28 proved the manuscript. 29 30 Acknowledgments 31 We gratefully acknowledge Dr. Bradley Yoder (University of Alabama at Birmingham) 32 kindly shared and transferred the mouse strain to us. We thank Dr. Tian Xue 33 (University of Science and Technology of China) for sharing ARPE19 cell line with 34 us. We also thank Jianguo Zhang, Lei Sun and Can Peng of the Center for Biological 35 Imaging, Institute of Biophysics, CAS for their help in making EM sample and taking 36 EM images. We thank Dr. Arnold Kriegstein (University of California at San 37 Francisco) and his lab members for their generous discussion and reagent support. 38 This work was supported by the National Basic Research Program of China 39 (2017YFA0103303, 2017YFA0102601); the Strategic Priority Research Program of 40 the Chinese Academy of Sciences (grant number XDA16020601); National Natural 41 Science Foundation of China (NSFC) (31371100) and Newton Advanced Fellowship 42 (grant number NA140416) to X. W. 43 Competing financial interests 44 The authors declare no competing financial interests. 2 45 Abstract 46 Primary cilia are microtubule-based protuberances that project from the eukaryotic 47 cell body to sense the extracellular environment. Ciliogenesis is closely correlated to 48 the cell cycle, and defects of cilia are related to human systemic diseases, such as 49 primary ciliary dyskinesia. However, the role of ciliogenesis in cortical development 50 remains unclear. Here, we demonstrate that Cenpj, a protein that is required for 51 centriole biogenesis, plays a role in regulating cilium disassemblyin vivo. Depletion 52 of Cenpj in neural progenitor cells results in long cilia and abnormal cilia disassembly. 53 Radial glial cells (RG cells) with Cenpj depletion exhibit uncompleted cell division, 54 reduced cell proliferation and increased cell apoptosis in the developing mouse 55 cerebrum cortex, leading to microcephaly. In addition, Cenpj depletion causes long 56 and thin primary cilia and motile cilia in adult neural stem cells and reduced cell 57 proliferation in the subventricular zone (SVZ). Furthermore, we show that Cenpj 58 regulates cilia disassembly and neurogenesis through Kif2a, a plus-end-directed motor 59 protein. These data, collected from mice of both sexes, provide insights into how 60 ciliogenesis plays roles in cortical development and primary microcephaly induced by 61 Cenpj mutations in humans. 62 63 64 65 66 3 67 Significance Statement 68 Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental 69 disorder showing the major symptoms of reduction of circumference of the head, 70 brain volume and cortex thickness with normal brain architecture in birth. We used 71 conditional Cenpj deletion mice and found that neural progenitor cells exhibited long 72 primary cilia and abnormal cilium appendages. The defective cilium disassembly 73 caused by Cenpj depletion might correlate to reduced cell proliferation, uncompleted 74 cell division, cell apoptosis, and microcephaly in mice. Cenpj also regulates cilium 75 structure of adult neural stem cells and adult neurogenesis in mice. Additionally, our 76 results illustrated that Cenpj regulated cilia disassembly and neurogenesis through 77 Kif2a, indicating that primary cilia dynamic plays a crucial role in neural progenitor 78 cell mitosis and adult neurogenesis. 79 80 81 82 83 84 85 86 87 88 4 89 Introduction 90 Centrosomes are the organelles at the poles of the spindle that can persist into 91 interphase as microtubule organizing centers (MTOCs). The core is a ninefold 92 symmetrical centriole, and their ability to nucleate cytoplasmic microtubules (MTs) is 93 a property of the surrounding pericentriolar material (PCM). The centriole has a dual 94 life, existing not only as the core of the centrosome but also as the basal body for 95 primary cilia assembly. As a result, the structure and function of the centriole, the 96 centrosome, and the cilia have an impact on many aspects of development and 97 physiology. The primary cilium, a continuation and extension of microtubule doublets 98 from the basal body structure, is localized to the apical region of a radial glial cell 99 (RG cell). In the G0 quiescent phase, the RG cell processes a primary cilium out of 100 the ventricular endfoot surface. Upon entry into the cell cycle, the primary cilium 101 disassembles quickly to release the centrioles to form a bipolar spindle (Kobayashi 102 and Dynlacht, 2011; Nigg and Stearns, 2011). Cilium assembly and disassembly are 103 highly correlated to the cell cycle and centrosome (Pugacheva et al., 2007; Qin et al., 104 2007; Robert et al., 2007). The dynamics of primary cilia, unique in their ability to 105 function as sensors and conveyors of critical signals in a complex environment, may 106 have a supervisory role in the neurogenesis of RG cells. 107 Mutations of Cenpj or CPAP (a centrosomal-P4.1-associated protein) cause 108 microcephaly, dwarfism, low birthweight, and intellectual disability (Al-Dosari et al., 109 2010), which are thought to arise from a decline in neural progenitor cells (NPCs) 110 during development (Insolera et al., 2014b). Depletion of Cenpj causes severe defects 5 111 in centriole duplication and increases the population of monopolar, asymmetric 112 bipolar, and multipolar spindles in mitotic cells (Cho et al., 2006b; Kitagawa et al., 113 2011a; Kohlmaier et al., 2009; Schmidt et al., 2009; Tang et al., 2009a). In addition, 114 Cenpj acts as a scaffold for the cilium disassembly complex (CDC), which includes 115 Nde1, Aurora A, and OFD1, that is recruited to the ciliary base for timely cilium 116 disassembly(Gabriel et al., 2016). Mouse embryos with complete elimination of 117 Cenpj present prolonged mitosis and a widespread, p53-dependent cell death 118 phenotype, resulting in a reduction in RG cells and other types of neural progenitor 119 cells (Bazzi and Anderson, 2014; Garcez et al., 2015; Insolera et al., 2014b; McIntyre 120 et al., 2012).
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    Mahmood et al. Orphanet Journal of Rare Diseases 2011, 6:39 http://www.ojrd.com/content/6/1/39 REVIEW Open Access Autosomal recessive primary microcephaly (MCPH): clinical manifestations, genetic heterogeneity and mutation continuum Saqib Mahmood1, Wasim Ahmad2 and Muhammad J Hassan3* Abstract Autosomal Recessive Primary Microcephaly (MCPH) is a rare disorder of neurogenic mitosis characterized by reduced head circumference at birth with variable degree of mental retardation. In MCPH patients, brain size reduced to almost one-third of its original volume due to reduced number of generated cerebral cortical neurons during embryonic neurogensis. So far, seven genetic loci (MCPH1-7) for this condition have been mapped with seven corresponding genes (MCPH1, WDR62, CDK5RAP2, CEP152, ASPM, CENPJ, and STIL) identified from different world populations. Contribution of ASPM and WDR62 gene mutations in MCPH World wide is more than 50%. By and large, primary microcephaly patients are phenotypically indistinguishable, however, recent studies in patients with mutations in MCPH1, WDR62 and ASPM genes showed a broader clinical and/or cellular phenotype. It has been proposed that mutations in MCPH genes can cause the disease phenotype by disturbing: 1) orientation of mitotic spindles, 2) chromosome condensation mechanism during embryonic neurogenesis, 3) DNA damage- response signaling, 4) transcriptional regulations and microtubule dynamics, 5) certain unknown centrosomal mechanisms that control the number of neurons generated by neural precursor cells. Recent discoveries of mammalian models for MCPH have open up horizons for researchers to add more knowledge regarding the etiology and pathophysiology of MCPH. High incidence of MCPH in Pakistani population reflects the most probable involvement of consanguinity.
  • Identification and Characterization of Genes Essential for Human Brain Development

    Identification and Characterization of Genes Essential for Human Brain Development

    Identification and Characterization of Genes Essential for Human Brain Development The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Ganesh, Vijay S. 2012. Identification and Characterization of Genes Essential for Human Brain Development. Doctoral dissertation, Harvard University. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:9773743 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Copyright © 2012 by Vijay S. Ganesh All rights reserved. Dissertation Advisor: Dr. Christopher A. Walsh Author: Vijay S. Ganesh Identification and Characterization of Genes Essential for Human Brain Development Abstract The human brain is a network of ninety billion neurons that allows for many of the behavioral adaptations considered unique to our species. One-fifth of these neurons are layered in an epithelial sheet known as the cerebral cortex, which is exquisitely folded into convolutions called gyri. Defects in neuronal number clinically present with microcephaly (Greek for “small head”), and in inherited cases these defects can be linked to mutations that identify genes essential for neural progenitor proliferation. Most microcephaly genes are characterized to play a role in the centrosome, however rarer presentations of microcephaly have identified different mechanisms. Charged multivesicular body protein/Chromatin modifying protein 1A (CHMP1A) is a member of the ESCRT-III endosomal sorting complex, but is also suggested to localize to the nuclear matrix and regulate chromatin.