This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Cellular/Molecular PKD1 promotes functional synapse formation coordinated with N-cadherin in hippocampus Cheng Cen1, Li-Da Luo1, Wen-Qi Li1, Gang Li1, Na-Xi Tian1, Ge Zheng1, Dong-Min Yin3, Yimin Zou4 and Yun Wang1,2 1Neuroscience Research Institute and Department of Neurobiology, School of Basic Medical Sciences, Key Laboratory for Neuroscience, Ministry of Education/National Health and Family Planning Commission, Peking University, Beijing 100191, China 2PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing 100871, China 3Key Laboratory of Brain Functional Genomics, Ministry of Education, Shanghai Key Laboratory of Brain Functional Genomics, School of Life Sciences, East China Normal University, Shanghai, China. 4Neurobiology Section, Biological Sciences Division, University of California, San Diego, La Jolla, California, USA. DOI: 10.1523/JNEUROSCI.1640-17.2017 Received: 13 June 2017 Revised: 11 October 2017 Accepted: 7 November 2017 Published: 13 November 2017 Author Contributions: Y.W. conceived and directed the project. C.C. conducted the electrophysiological studies, the animal behavior studies and analyzed the data. L.D.L. conducted the cell culture experiments, the biochemical experiments and analyzed the data. W.Q.L., G.L., N.X.T., G.Z and D.M.Y. conducted some of the experiments and analyzed the data. C.C., L.D.L., D.M.Y., Y.Z. and Y.W. discussed the data. C.C., L.D.L. and Y.W. wrote the manuscript. Conflict of Interest: The authors declare no competing financial interests. This work was supported by grants from the National Natural Science Foundation of China (81600989 to C.C., 31530028 and 31720103908 to Y.W.), the Ministry of Science and Technology of China (2014CB542204 and 2015BAI08B02 to Y.W.) and China Postdoctoral Science Foundation (2015M580025 to C.C.). C.C. was supported in part by the Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences. We thank Prof. Qihua He (Cell Analytical Laboratory, Medical and Healthy Analytical Center, Peking University) for technical support for imaging. Correspondence should be addressed to Yun Wang, Neuroscience Research Institute and Department of Neurobiology, School of Basic Medical Sciences, Key Laboratory for Neuroscience, Ministry of Education/ National Health and Family Planning Commission, Peking University, Beijing 100191, China. Email: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1640-17.2017 Alerts: Sign up at www.jneurosci.org/cgi/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 © 2017 the authors 1 PKD1 promotes functional synapse formation coordinated with 2 N-cadherin in hippocampus 3 Abbreviated tittle: PKD1, N-cadherin and synapse formation 4 Cheng Cen (዁〻)1*, Li-Da Luo (㖇ѭ䗮)1*, Wen-Qi Li (ᵾ᮷⩚)1, Gang Li (ᵾࡊ)1, 5 Na-Xi Tian (⭠㓣㾯)1, Ge Zheng (䜁ⅼ)1, Dong-Min Yin (⇧ь᭿)3, Yimin Zou (䛩⳺ 6 ≁)4, Yun Wang (⦻严)1,2 7 1Neuroscience Research Institute and Department of Neurobiology, School of Basic 8 Medical Sciences, Key Laboratory for Neuroscience, Ministry of Education/National 9 Health and Family Planning Commission, Peking University, Beijing 100191, China; 10 2PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing 11 100871, China˗ 12 3Key Laboratory of Brain Functional Genomics, Ministry of Education, Shanghai Key 13 Laboratory of Brain Functional Genomics, School of Life Sciences, East China 14 Normal University, Shanghai, China. 15 4Neurobiology Section, Biological Sciences Division, University of California, San 16 Diego, La Jolla, California, USA. 17 *C.C. and L.D.L. contributed equally to this work. 18 Correspondence should be addressed to Yun Wang, Neuroscience Research Institute 19 and Department of Neurobiology, School of Basic Medical Sciences, Key Laboratory 20 for Neuroscience, Ministry of Education/National Health and Family Planning 21 Commission, Peking University, Beijing 100191, China. Email: 22 [email protected] 1 23 Number of pages: 49 24 Number of figures: 10 25 Number of words: for Abstract, 183; for Introduction, 548; for Discussion, 756. 26 Author Contributions: Y.W. conceived and directed the project. C.C. conducted the 27 electrophysiological studies, the animal behavior studies and analyzed the data. L.D.L. 28 conducted the cell culture experiments, the biochemical experiments and analyzed the 29 data. W.Q.L., G.L., N.X.T., G.Z and D.M.Y. conducted some of the experiments and 30 analyzed the data. C.C., L.D.L., D.M.Y., Y.Z. and Y.W. discussed the data. C.C., 31 L.D.L. and Y.W. wrote the manuscript. 32 Acknowledgments: This work was supported by grants from the National Natural 33 Science Foundation of China (81600989 to C.C., 31530028 and 31720103908 to 34 Y.W.), the Ministry of Science and Technology of China (2014CB542204 and 35 2015BAI08B02 to Y.W.) and China Postdoctoral Science Foundation (2015M580025 36 to C.C.). C.C. was supported in part by the Postdoctoral Fellowship of 37 Peking-Tsinghua Center for Life Sciences. We thank Prof. Qihua He (Cell Analytical 38 Laboratory, Medical and Healthy Analytical Center, Peking University) for technical 39 support for imaging. 40 Competing Interests: The authors declare no competing financial interests. 41 2 42 Abstract 43 Functional synapse formation is critical for the wiring of neural circuits in the 44 developing brain. The cell adhesion molecule N-cadherin plays important roles in 45 target recognition and synaptogenesis. However, the molecular mechanisms that 46 regulate the localization of N-cadherin and the subsequent effects remain poorly 47 understood. Here, we show that protein kinase D1 (PKD1) directly binds to 48 N-cadherin at amino acid residues 836-871 and phosphorylates it at Ser 869, 871, 872, 49 thereby increasing the surface localization of N-cadherin and promoting functional 50 synapse formation in primary cultured hippocampal neurons obtained from embryonic 51 day 18 rat embryos of either sex. Intriguingly, neuronal activity enhances the 52 interactions between N-cadherin and PKD1, which are critical for the 53 activity-dependent growth of dendritic spines. Accordingly, either disruption the 54 binding between N-cadherin and PKD1 or preventing the phosphorylation of 55 N-cadherin by PKD1 in the hippocampal CA1 region of male rat leads to the 56 reduction in synapse number and impairment of long-term potentiation (LTP). 57 Together, this study demonstrates a novel mechanism of PKD1 regulating the surface 58 localization of N-cadherin and suggests that the PKD1-N-cadherin interaction is 59 critical for synapse formation and function. 60 61 Significance Statement 62 Defects in synapse formation and function lead to various neurological diseases, while 63 the mechanisms underlying the regulation of synapse development are far from clear. 3 64 Our results suggest that protein kinase D1 (PKD1) functions upstream of N-cadherin, 65 a classical synaptic adhesion molecule, to promote functional synapse formation. 66 Notably, we identified a crucial binding fragment to PKD1 at C-terminus of 67 N-cadherin, and this fragment also contains PKD1 phosphorylation sites. Through this 68 interaction, PKD1 enhances the stability of N-cadherin on cell membrane and 69 promotes synapse morphogenesis and synaptic plasticity in an activity-dependent 70 manner. Our study reveals the role of PKD1 and the potential downstream mechanism 71 in synapse development, and contributes to the research for neurodevelopment and the 72 therapy for neurological diseases. 73 74 Introduction 75 Brain function relies on the establishment and organization of proper neuronal 76 circuitry consisting of a vast but sophisticated interconnected network of synapses. 77 Synapses are specialized asymmetrical connections between neurons that function in 78 information processing and integration. Synapse formation, elimination and 79 remodeling are essential for the development of neural circuits and cognitive 80 functions such as learning and memory (Kandel et al., 2014). Cell adhesion molecules 81 (CAMs), which can align the presynaptic active zone and the postsynaptic density 82 across the synaptic cleft, have been intensively studied for their important roles in 83 neurite outgrowth, neuronal polarity, neuronal migration, synapse formation and 84 synaptic plasticity both in the developing and mature brain (Togashi et al., 2002; 85 Arikkath and Reichardt, 2008; Seong et al., 2015). 4 86 N-cadherin, one of the important CAMs in nervous system, is a type I classical 87 cadherin that is widely expressed in both pre- and postsynaptic membranes of 88 excitatory neurons in mammals (Arikkath and Reichardt, 2008). N-cadherin mediates 89 calcium-dependent, homophilic interactions across the synaptic cleft, and plays 90 crucial roles in dendrite morphogenesis, synapse formation, synaptic plasticity and 91 neural disorders associated with autism, bipolar disease, schizophrenia and 92 Alzheimer’s disease (Schrick et al., 2007; Bacchelli et al., 2014; Tucci et al., 2014; 93 Uribe-Arias et al., 2016). Although the functions of N-cadherin in neurons have been 94 extensively investigated, the post-translational modification and regulation of the 95 membrane localization of N-cadherin have been rarely studied. 96 Protein kinase D (PKD) is a serine/threonine