Research Articles: Cellular/Molecular The nebulin family LIM and SH3 proteins regulate postsynaptic development and function https://doi.org/10.1523/JNEUROSCI.0334-19.2019 Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.0334-19.2019 Received: 11 February 2019 Revised: 12 November 2019 Accepted: 15 November 2019 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2019 the authors 1 The nebulin family LIM and SH3 proteins regulate postsynaptic 2 development and function 3 4 Kenneth R Myers1*, Kuai Yu1, Joachim Kremerskothen2, Elke Butt3, and James Q. Zheng1,4* 5 1 Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322. 6 2 Medizinische Klinik D, Abteilung für Molekulare Nephrologie, Universitätsklinikum Münster, 7 Münster, Germany. 8 3Institute of Experimental Biomedicine II, University Medical Clinic of Wuerzburg, Wuerzburg, 9 Germany. 10 4 Department of Neurology and Center for Neurodegenerative Diseases, Emory University School 11 of Medicine, Atlanta, GA 30322 12 13 Abbreviated title: LASP regulation of dendrite development 14 Keywords: Actin cytoskeleton, synapse, dendrite, spines, neuronal development 15 Correspondence: Dr. James Zheng, Department of Cell Biology, Emory University School of 16 Medicine, Atlanta, GA 30322. Email: [email protected]; Dr. Kenneth Myers, 17 Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322. Email: 18 [email protected]. 19 Number of pages: 41 20 Number of figures: 10 1 21 Number of words: Abstract (150), Introduction (650), and Discussion (1,188) 22 Conflict of interest statement: The authors declare no competing financial interests. 23 Acknowledgements: This work is supported in part by research grants from National Institutes 24 of Health to JQZ (MH104632 and GM083889), and a Ruth L. Kirschstein NRSA Postdoctoral 25 Fellowship to KRM (NS092342). We thank Jinny Yoo and Jenny Mai for help with image analysis, 26 and Madeline Morgan for helpful discussions. The authors declare no competing financial 27 interests. 28 29 30 31 32 33 34 35 36 37 38 39 2 40 ABSTRACT 41 Neuronal dendrites have specialized actin-rich structures called dendritic spines that 42 receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines 43 during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic 44 morphology and stability are largely mediated by regulation of the actin cytoskeleton, however 45 the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the 46 nebulin family members LASP1 and LASP2 play an important role in the postsynaptic 47 development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 48 are enriched in dendritic spines, and their knockdown impairs spine development and synapse 49 formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine 50 stabilization. Importantly the actin-binding N-terminal LIM domain and nebulin repeats of 51 LASP2 are required for spine stability and dendritic arbor complexity. These findings identify 52 LASP1 and LASP2 as novel regulators of neuronal circuitry. 53 54 55 56 57 58 59 3 60 SIGNIFICANCE STATEMENT 61 Proper regulation of the actin cytoskeleton is essential for the structural stability of 62 dendrites and dendritic spines. Consequently, malformation of dendritic structures accompanies 63 numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are 64 best known for their role in regulating the stabilization and function of actin thin filaments in 65 muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse 66 and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly 67 expressed in the brain, little is currently known about their function in the nervous system. In this 68 study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation 69 and long-term maintenance of dendrites and dendritic spines. 70 71 72 73 74 75 76 77 78 79 80 4 81 INTRODUCTION 82 The structure of a neuron’s dendritic arbor is essential for neural circuit formation, 83 determining how synaptic inputs are received and integrated. In turn, presynaptic inputs 84 themselves can shape and stabilize the dendritic arbor (Parrish et al., 2007). The arbor is highly 85 dynamic during early development as numerous branches form, elongate, and retract (Scott 86 and Luo, 2001; Parrish et al., 2007; Koleske, 2013). However, as development proceeds 87 increased neuronal activity stabilizes individual branches, and less active branches retract (Van 88 Aelst and Cline, 2004; Cline and Haas, 2008). The majority of excitatory inputs are received at 89 glutamatergic synapses, which are formed between presynaptic axonal terminals and 90 specialized postsynaptic structures called dendritic spines (Nimchinsky et al., 2002; Bourne and 91 Harris, 2008; Rochefort and Konnerth, 2012). Spines are small membranous protrusions that 92 form on dendrites and function as the sites of signal integration and transduction downstream 93 of glutamate receptor activity (Chen and Sabatini, 2012). Dendritic spines are highly plastic, and 94 changes in their morphology underlie synapse formation and modification during learning and 95 memory (Segal, 2005; Bosch and Hayashi, 2012; Lai and Ip, 2013). The growth of the dendritic 96 arbor and dendritic spines are therefore closely linked, and both are essential for the 97 development and function of neuronal circuits (Koleske, 2013). However, as neurons mature, 98 the dendritic arbor typically stabilizes while spines retain a reduced level of plasticity. 99 Changes in spine morphology are largely driven by remodeling of the actin cytoskeleton 100 (Hotulainen and Hoogenraad, 2010; Lei et al., 2016). Actin filaments are remodeled by 101 regulatory proteins that arrange actin into distinct networks with different functions. These 102 regulatory proteins modify actin structures by crosslinking, severing, end capping, and bundling 5 103 actin filaments. The LIM and SH3 domain-containing proteins, LASP1 and LASP2, are small 104 multi-domain actin-binding proteins that have been implicated in the stabilization of actin 105 filaments (Grunewald and Butt, 2008; Pappas et al., 2011). Consequently, LASP1 and LASP2 106 localize to numerous sites of dynamic actin assembly and disassembly including; focal 107 adhesions, membrane ruffles, lamellipodia, and pseudopodia (Chew et al., 2002b). In non- 108 neuronal cells, both LASP proteins have been shown to regulate cell adhesion and cell migration 109 (Lin et al., 2004; Bliss et al., 2013). LASP1 and LASP2 are highly homologous, and are 110 differentiated from the other members of the Nebulin family of actin binding proteins by their 111 small size and the presence of an N-terminal LIM domain (Pappas et al., 2011). LIM domains 112 participate in a wide variety of cellular functions including the regulation of gene expression, 113 signal transduction, cell adhesion, and cell motility (Kadrmas and Beckerle, 2004). In addition, 114 LASP1 and LASP2 feature a C-terminal SRC homology region 3 (SH3) domain, a flexible non- 115 structured linker region, and either two or three 35-amino acid actin-binding nebulin-like 116 repeats, respectively (Grunewald and Butt, 2008). This unique domain architecture enables the 117 LASP proteins to serve as signaling hubs, linking numerous proteins to the actin cytoskeleton 118 (Orth et al., 2015). Studies have shown that both proteins are highly expressed in the central 119 nervous system, and that LASP1 is concentrated in the postsynaptic density (Phillips et al., 120 2004; Terasaki et al., 2004). In addition, LASP1 has been identified as a risk factor for both 121 autism and schizophrenia (Stone et al., 2007; Joo et al., 2013). However, little is currently 122 known about the physiological role of either LASP1 or LASP2 in the nervous system. 123 In this study, we present evidence that LASP1 and LASP2 play an important role in 124 dendritic spine development and stabilization, respectively. Furthermore, we demonstrate that 6 125 LASP2, but not LASP1, promotes dendritic complexity by stabilizing dendritic branches. LASP2, 126 but not LASP1 has been identified as an actin bundling protein (Chew et al., 2002a; Zieseniss et 127 al., 2008), and here we identify a role for the actin-binding LIM domain and Nebulin repeats in 128 LASP2-mediated dendritic stabilization. Our findings demonstrate an important new role for 129 nebulin family members in the stabilization of dendritic spines and dendritic arbor, and in 130 regulating synapse formation. 131 7 132 METHODS 133 Antibodies 134 The following commercial antibodies were used: rabbit anti-MAP2 (AB5622; EMD 135 Millipore; 1:1000), rabbit anti-RFP (600-401-279; Rockland; 1:1000), rabbit anti-GFP (A-11122; 136 Invitrogen; 1:1,000), mouse anti-PSD95 (MA1-045; ThermoFisher; 1:1,000), and mouse anti-SV2 137 (DSHB; 1:1000). To examine LASP localization, we used total-LASP antibody (recognizes both 138 LASP1 and LASP2) which was generated by immunizing rabbit with full-length GST-LASP1 (Butt 139 et al.,