Research Article: New Research | Development Heparan sulfated Glypican-4 is released from astrocytes by proteolytic shedding and GPI- anchor cleavage mechanisms https://doi.org/10.1523/ENEURO.0069-21.2021 Cite as: eNeuro 2021; 10.1523/ENEURO.0069-21.2021 Received: 17 February 2021 Revised: 9 July 2021 Accepted: 15 July 2021 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.eneuro.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 Huang and Park This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 1. Manuscript Title: Heparan sulfated Glypican-4 is released from astrocytes by proteolytic 2 shedding and GPI-anchor cleavage mechanisms 3 4 2. Abbreviated Title: GPC4 is released from astrocytes largely by proteolytic shedding 5 6 3. Authors: 7 Kevin Huang1, 2, Sungjin Park1 8 1 University of Utah School of Medicine, Department of Neurobiology, Salt Lake City, UT 84112 9 2 University of Utah, Interdepartmental Program in Neuroscience, Salt Lake City, UT 84112 10 11 4. Author Contributions: KH and SP designed research; KH performed research; KH and SP 12 analyzed data; KH and SP wrote the paper. 13 14 5. Correspondence should be addressed to: 15 Sungjin Park, PhD 16 Department of Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake 17 City, UT 84112, USA. Tel: +1-801-581-6106 18 E-mail: [email protected] 19 20 6. Number of Figures: 7 2410. Number of words for Significance 21 7. Number of Tables: 2 25Statement: 98 22 8. Number of Multimedia: 0 2611. Number of words for Introduction: 705 23 9. Number of words for Abstract: 168 2712. Number of words for Discussion: 1,212 28 29 13. Acknowledgements: We thank Joosang Park for maintaining the mouse colony and 30 experimental assistance; Williams, Shepherd, Sundquist labs for materials, equipment, and 31 advice; U of U Core facilities. 32 33 14. Conflict of Interest: Authors report no conflict of interest 34 35 15. Funding Sources: This work was supported by NIH grant R01NS102444 to S.P. 36 1 37 Abstract 38 Astrocytes provide neurons with diffusible factors that promote synapse formation and 39 maturation. In particular, glypican-4/GPC4 released from astrocytes promotes the maturation of 40 excitatory synapses. Unlike other secreted factors, GPC4 contains the C-terminal GPI- 41 anchorage signal. However, the mechanism by which membrane-tethered GPC4 is released 42 from astrocytes is unknown. Using mouse primary astrocyte cultures and a quantitative 43 luciferase-based release assay, we show that GPC4 is expressed on the astrocyte surface via a 44 GPI-anchorage. Soluble GPC4 is robustly released from the astrocytes largely by proteolytic 45 shedding and, to a lesser extent, by GPI-anchor cleavage, but not by vesicular release. 46 Pharmacological, overexpression, and loss of function screens showed that ADAM9 in part 47 mediates the release of GPC4 from astrocytes. The released GPC4 contains the heparan 48 sulfate side chain, suggesting that these release mechanisms provide the active form that 49 promotes synapse maturation and function. Overall, our studies identified the release 50 mechanisms and the major releasing enzyme of GPC4 in astrocytes and will provide insights 51 into understanding how astrocytes regulate synapse formation and maturation. 2 52 Significance Statement 53 Astrocyte-derived diffusible factors regulate synapse development and function. However, the 54 regulatory mechanism underlying the release of astrocyte-derived factors is poorly understood. 55 Noting that, unlike many other secreted factors, glypican-4/GPC4 is GPI-anchored, we 56 characterized the release mechanism of GPI-anchored GPC4 from astrocytes and identified the 57 releasing enzyme. Heparan sulfated GPC4 is robustly released from the astrocytes largely by 58 proteolytic shedding. In particular, ADAM9 in part mediates the release of GPC4 from 59 astrocytes. Our study provides an enzymatic mechanism for releasing GPC4 from astrocytes 60 and will provide a novel opportunity to understand the regulatory mechanism of neuron-glia 61 communication for synaptogenesis. 3 62 Introduction 63 Surface shedding is a mechanism by which specific surface proteins are cleaved and an 64 ectodomain is released into the extracellular space. The shedding or release of surface proteins 65 have been found to play important roles in various biological functions (Lichtenthaler et al., 66 2018) . Ectodomain shedding can act to stop activity of proteins at the membrane, such as 67 removing cell surface receptors or cell adhesion proteins, as in the case of N-cadherin (Reiss et 68 al., 2005). Alternatively, shedding can release an active protein from the cell surface to act as a 69 long range, diffusible signaling factor such as syndecan-1 in FGF-2 activation during wound 70 healing (Kato et al., 1998). In this context, shedding can allow for temporally and spatially 71 regulated release or activity of a biomolecule. In other cases, shedding acts as a molecular 72 switch, changing the signaling function of the protein from one role while anchored to the 73 surface membrane to another when shed. For example, membrane-anchored TNF- can 74 activate TNFR2 upon direct cell to cell contact to activate anti-inflammatory pathways, however 75 when shed, TNF- acts as a paracrine signal to drive proinflammatory TNFR1 signaling (Grell et 76 al., 1995). In other cases, released proteins can change the signaling functions of related 77 receptor complexes, such as when released soluble CNTFR converts the LIF receptor 78 complex and transduces CNTF signaling in the cells that are not normally responsive to CNTF 79 (Davis et al., 1993). 80 The release of surface proteins also been shown to play important roles in synapse 81 development (Lichtenthaler et al., 2018). For example, Nogo-66 receptor ectodomain shedding 82 drives excitatory synapse formation in vivo (Sanz et al., 2018). Proteolytic shedding of cell 83 adhesion molecules causes the structural and functional modifications of synapses (Bajor and 84 Kaczmarek, 2013; Nagappan-Chettiar et al., 2017). Glypican-4/GPC4 has been identified as a 85 cell surface protein sufficient to drive neuronal synaptogenesis in vitro (Allen et al., 2012). GPC4 86 is a GPI-anchored protein (Filmus et al., 2008) and highly expressed in astrocytes (Cahoy et al., 4 87 2008; Zhang et al., 2014). Notably, GPC4 expressed in astrocytes are released from the cell 88 and the released GPC4 facilitates synapse maturation in vitro (Allen et al., 2012). While GPC4’s 89 downstream mechanism of action of signaling through RPTPs been characterized (Farhy- 90 Tselnicker et al., 2017), little is known about how GPC4 is shed or released from astrocytes. A 91 better understanding of the release mechanism of GPC4 offers insights into the synaptogenic 92 role of astrocytes in neuronal development and could lead to novel therapeutic targets for 93 synaptopathic disorders such as autism, schizophrenia, Down syndrome, and epilepsy (Dowling 94 and Allen, 2018; Wilson and Newell-Litwa, 2018). 95 GPI-anchored proteins can be released from the producing cells via multiple 96 mechanisms, which include proteolytic shedding, GPI-anchor cleavage, and vesicular release 97 (Muller, 2018). There are GPI-anchored proteins which are shed through proteolytic 98 mechanisms such as metalloprotease-mediated shedding of Prion protein and UL16 binding 99 proteins, MHC class I-related molecules (Fernandez-Messina et al., 2010; Linsenmeier et al., 100 2018). GPI-anchored proteins can also be shed though a lipase-based cleavage mechanism 101 (Park et al., 2013; van Veen et al., 2017). In this context, unlike proteolytic shedding which 102 releases an ectodomain fragment, an entire protein is released from the cell. GPI-anchored 103 proteins have also been shown to associate with a vesicular membrane or lipid particle while 104 becoming separated from the cell (Muller, 2018). 105 The mechanism by which GPC4 is released has implications to the role GPC4 plays in 106 downstream signaling. GPC4 is post-translationally modified with heparan sulfate side chains at 107 residues near the C-terminus of the protein. These heparan sulfate side chains are necessary 108 for the known synaptogenic signaling functions of GPC4 (Allen et al., 2012; Condomitti et al., 109 2018). Thus, identification of the released form of GPC4 is critical to understand how astrocyte- 110 derived GPC4 regulates synaptogenesis. 111 Here, we show that GPC4 is released from the cell surface of astrocytes in vitro. We 112 observed that GPC4 is released predominantly via proteolytic shedding, and to a lesser extent 5 113 by GPI-anchor cleavage, but not by vesicular release. The released GPC4 contains heparan 114 sulfate attachment, suggesting that both release mechanisms preserve the synaptogenic activity 115 of GPC4 in the extracellular space. We also identify ADAM9 as a proteolytic sheddase for 116 GPC4. Overall, these observations show that astrocytes release heparan sulfate containing 117 GPC4 by multiple mechanisms. 118 119 Materials and Methods: 120 Animals 121 All animal care and experiments were conducted in accordance with NIH guidelines and 122 the IACUC committee of the University of Utah (protocol no. 21-02004). C57Bl6/J mouse lines 123 were maintained under the normal housing conditions with food and water available ad libitum 124 and 12h light/dark cycle in a dedicated facility. All primary astrocyte cultures were generated 125 using neonatal wild-type mice of either sex. 126 Statistical Analyses 127 Statistics were performed using Graphpad Prism software version 9.