Research Articles: Cellular/Molecular SPARCL1 promotes excitatory but not inhibitory synapse formation and function independent of neurexins and neuroligins https://doi.org/10.1523/JNEUROSCI.0454-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0454-20.2020 Received: 25 February 2020 Revised: 17 June 2020 Accepted: 12 July 2020 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 © 2020 the authors 1 2 3 4 SPARCL1 promotes excitatory but not inhibitory synapse formation and 5 function independent of neurexins and neuroligins 6 7 8 Kathlyn J. Gan1,# and Thomas C. Südhof1,2,# 9 10 1Department of Molecular and Cellular Physiology, 2Howard Hughes Medical Institute, 11 Stanford University School of Medicine, 265 Campus Drive, Stanford, CA 94305, USA 12 13 Short title: Mechanism of SPARCL1-induced synaptogenesis 14 15 Keywords: SPARC-like protein 1 (SPARCL1); synapse; synapse formation; NMDA 16 receptor; neurexin; neuroligin 17 18 Number of figures: 8 19 Number of tables: 1 20 Extended data: 8 Excel files 21 Number of words: Abstract = 228; Introduction = 681; Discussion = 1287 22 23 #Corresponding authors: [email protected] and [email protected] 24 25 The authors declare no competing financial interests. 26 ABSTRACT 27 Emerging evidence supports roles for secreted extracellular matrix proteins in boosting 28 synaptogenesis, synaptic transmission, and synaptic plasticity. SPARCL1 (a.k.a. Hevin), 29 a secreted non-neuronal protein, was reported to increase synaptogenesis by 30 simultaneously binding to presynaptic neurexin-1α and to postsynaptic neuroligin-1B, 31 thereby catalyzing formation of trans-synaptic neurexin/neuroligin complexes. However, 32 neurexins and neuroligins do not themselves mediate synaptogenesis, raising the 33 question of how SPARCL1 enhances synapse formation by binding to these molecules. 34 Moreover, it remained unclear whether SPARCL1 acts on all synapses containing 35 neurexins and neuroligins or only on a subset of synapses, and whether it enhances 36 synaptic transmission in addition to boosting synaptogenesis or induces silent synapses. 37 To explore these questions, we examined the synaptic effects of SPARCL1 and their 38 dependence on neurexins and neuroligins. Using mixed neuronal and glial cultures from 39 neonatal mouse cortex of both sexes, we show that SPARCL1 selectively increases 40 excitatory but not inhibitory synapse numbers, enhances excitatory but not inhibitory 41 synaptic transmission, and augments NMDA-receptor-mediated synaptic responses 42 more than AMPAR-mediated synaptic responses. None of these effects were mediated 43 by SPARCL1-binding to neurexins or neuroligins. Neurons from triple neurexin-1/2/3 or 44 from quadruple neuroligin-1/2/3/4 conditional knockout mice that lacked all neurexins or 45 all neuroligins were fully responsive to SPARCL1. Taken together, our results reveal that 46 SPARCL1 selectively boosts excitatory but not inhibitory synaptogenesis and synaptic 47 transmission by a novel mechanism that is independent of neurexins and neuroligins. 48 49 50 51 52 53 54 55 56 2 57 SIGNIFICANCE STATEMENT 58 Emerging evidence supports roles for extracellular matrix proteins in boosting synapse 59 formation and function. Previous studies demonstrated that SPARCL1, a secreted non- 60 neuronal protein, promotes synapse formation in rodent and human neurons. However, 61 it remained unclear whether SPARCL1 acts on all or on only a subset of synapses, 62 induces functional or largely inactive synapses, and generates synapses by bridging 63 presynaptic neurexins and postsynaptic neuroligins. Here, we report that SPARCL1 64 selectively induces excitatory synapses, increases their efficacy, and enhances their 65 NMDA receptor content. Moreover, using rigorous genetic manipulations, we show that 66 SPARCL1 does not require neurexins and neuroligins for its activity. Thus, SPARCL1 67 selectively boosts excitatory synaptogenesis and synaptic transmission by a novel 68 mechanism that is independent of neurexins and neuroligins. 69 3 70 INTRODUCTION 71 During development and throughout life, synapses are formed by a multi-component 72 process that is controlled by synaptic organizer molecules. Synapse formation requires 73 the initial establishment of contacts between an axon and a target cell, assembly of the 74 presynaptic release machinery and the postsynaptic receptor apparatus, and activity- 75 dependent specification of synapse properties (Südhof, 2018). Synapse formation is 76 guided by two types of organizer molecules: trans-synaptic adhesion molecules and 77 secreted factors (Ferrer-Ferrer and Dityatev, 2018; Yuzaki, 2018). Among adhesion 78 molecules, neurexins and their ligands, including neuroligins, orchestrate synapse 79 specification by mediating bidirectional, trans-cellular signaling (Südhof, 2017; Cao and 80 Tabuchi, 2017; Katzman and Alberini, 2018; Wang et al., 2019). Secreted factors act by 81 several mechanisms, for example by enhancing the recruitment of presynaptic active 82 zone proteins and postsynaptic scaffolding proteins (Wnts; Sahores et al., 2010; Cerpa 83 et al., 2011) or by connecting pre- and postsynaptic adhesion molecules (cerebellins; 84 Yuzaki, 2018). Extracellular matrix (ECM) proteins, reportedly secreted by glia (such as 85 thrombospondins and glypicans), may bridge pre- and postsynaptic structures and cluster 86 ionotropic glutamate receptors at postsynaptic sites (Christopherson et al., 2005; Allen et 87 al, 2012). Although an important role in synaptogenesis is emerging for several proteins 88 traditionally associated with the ECM, the underlying molecular mechanisms remain 89 unknown. 90 The present study examines SPARCL1 (‘secreted protein acidic and rich in cysteine-like 91 1’, a.k.a. Hevin), an ECM protein that induces synapse formation in rodent and human 92 neurons (Kucukdereli et al., 2011; Singh et al., 2016; Gan and Südhof, 2019). SPARCL1 93 belongs to the SPARC family of extracellular glycoproteins (Brekken and Sage, 2001). 94 SPARCL1 contains an N-terminal acidic domain, a central cysteine-rich follistatin domain, 95 and a C-terminal calcium-binding domain (Brekken and Sage, 2000). Little is known about 96 how SPARCL1 is secreted and processed. Like other ECM proteins, SPARCL1 is more 97 abundant during embryonic development than during adulthood, but its expression is 98 upregulated in response to trauma or disease (Lively and Brown, 2008 a,b). 4 99 In brain, SPARCL1 is localized to postsynaptic membranes and perisynaptic glial 100 processes in the cerebral cortex and the cerebellum (Lively et al., 2007), raising the 101 possibility that SPARCL1 regulates synapse formation. Indeed, Kucukdereli et al. (2011) 102 showed that SPARCL1 induced synapses in mouse retinal ganglion cells. Surprisingly, 103 however, these synapses were morphologically normal but did not appear to be 104 functional. In contrast, we found that SPARCL1 promotes formation of active synapses in 105 human excitatory neurons derived from embryonic stem cells (Gan and Südhof, 2019). 106 Moreover, SPARCL1 reportedly enhanced synapse formation by simultaneously binding 107 to presynaptic neurexin-1α (Nrxn1α) and postsynaptic neuroligin-1B (Nlgn1B) to form a 108 trans-synaptic complex (Singh et al., 2016). This finding was puzzling because Nrxn1α 109 forms tight complexes with all neuroligins in the absence of other factors (Boucard et al., 110 2005; Chih et al., 2006), and because biophysical studies failed to detect binding of 111 SPARCL1 to Nlgn1 (Elegheert et al., 2017). Moreover, deletion of all neuroligins 112 (Varoqueaux et al., 2006; Jiang et al., 2017; Chandra et al., 2017) or all neurexins (Missler 113 et al., 2003; Chen et al., 2017) did not decrease excitatory synapses numbers but 114 impaired synaptic transmission. Given that neurexins and neuroligins are apparently not 115 required for establishing initial synaptic contacts, in remained unclear how SPARCL1 116 could form non-functional excitatory synapses by binding to these adhesion molecules. 117 In the present study, we examined how SPARCL1 potently boosts synaptogenesis, 118 whether it acts selectively on subsets of synapses, and what role neurexins and/or 119 neuroligins play in its activity. Because key prior studies of SPARCL1-induced 120 synaptogenesis were conducted in mouse neurons (Kucukdereli et al., 2011; Singh et al., 121 2016), we performed our experiments in mixed cultures of mouse cortical neurons and 122 glia. We confirm that SPARCL1 induces excitatory synapse formation; however, in 123 contrast with previous studies, we show that SPARCL1 also enhances neurotransmission 124 by differentially elevating AMPA-receptor- (AMPAR-) and NMDA-receptor- (NMDAR-) 125 mediated synaptic responses. SPARCL1 exerts a much larger effect on NMDARs, 126 increasing the NMDAR/AMPAR ratio. Importantly, using cultures from conditional 127 knockout mice, we determined that neurexins and neuroligins do not mediate any of these 128 effects. Thus, SPARCL1 selectively activates excitatory synapse formation and signaling 129 to boost synaptic connectivity by a neurexin- and neuroligin-independent mechanism. 5 130 MATERIALS AND METHODS 131 Experimental design and statistical analyses.