A splicing isoform of GPR56 mediates microglial synaptic refinement via phosphatidylserine binding
2020; Springer Nature; Volume: 39; Issue: 16 Linguagem: Inglês
10.15252/embj.2019104136
ISSN1460-2075
AutoresTao Li, Brian Chiou, Casey K. Gilman, Rong Luo, Tatsuhiro Koshi, Diankun Yu, Hayeon C Oak, Stefanie Giera, Erin Johnson‐Venkatesh, Allie K. Muthukumar, Beth Stevens, Hisashi Umemori, Xianhua Piao,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoArticle25 May 2020free access Source DataTransparent process A splicing isoform of GPR56 mediates microglial synaptic refinement via phosphatidylserine binding Tao Li orcid.org/0000-0002-9174-1271 Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Brian Chiou Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA Search for more papers by this author Casey K Gilman Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Rong Luo Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Tatsuhiro Koshi Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Diankun Yu Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA Search for more papers by this author Hayeon C Oak Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA Search for more papers by this author Stefanie Giera Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Erin Johnson-Venkatesh F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Allie K Muthukumar F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Beth Stevens F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, USA Search for more papers by this author Hisashi Umemori F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Xianhua Piao Corresponding Author [email protected] orcid.org/0000-0001-7540-6767 Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA Weill Institute for Neuroscience, University of California, San Francisco (UCSF), San Francisco, CA, USA Division of Neonatology, Department of Pediatrics, University of California, San Francisco (UCSF), San Francisco, CA, USA Newborn Brain Research Institute, University of California, San Francisco (UCSF), San Francisco, CA, USA Search for more papers by this author Tao Li orcid.org/0000-0002-9174-1271 Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Brian Chiou Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA Search for more papers by this author Casey K Gilman Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Rong Luo Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Tatsuhiro Koshi Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Diankun Yu Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA Search for more papers by this author Hayeon C Oak Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA Search for more papers by this author Stefanie Giera Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA Search for more papers by this author Erin Johnson-Venkatesh F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Allie K Muthukumar F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Beth Stevens F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, USA Search for more papers by this author Hisashi Umemori F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Xianhua Piao Corresponding Author [email protected] orcid.org/0000-0001-7540-6767 Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA Weill Institute for Neuroscience, University of California, San Francisco (UCSF), San Francisco, CA, USA Division of Neonatology, Department of Pediatrics, University of California, San Francisco (UCSF), San Francisco, CA, USA Newborn Brain Research Institute, University of California, San Francisco (UCSF), San Francisco, CA, USA Search for more papers by this author Author Information Tao Li1,2, Brian Chiou1, Casey K Gilman2, Rong Luo2, Tatsuhiro Koshi2, Diankun Yu1, Hayeon C Oak1, Stefanie Giera2, Erin Johnson-Venkatesh3, Allie K Muthukumar3, Beth Stevens3,4, Hisashi Umemori3 and Xianhua Piao *,1,2,3,5,6,7 1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco (UCSF), San Francisco, CA, USA 2Department of Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA 3F. M. Kirby Neurobiology Center, Children's Hospital, Harvard Medical School, Boston, MA, USA 4Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, USA 5Weill Institute for Neuroscience, University of California, San Francisco (UCSF), San Francisco, CA, USA 6Division of Neonatology, Department of Pediatrics, University of California, San Francisco (UCSF), San Francisco, CA, USA 7Newborn Brain Research Institute, University of California, San Francisco (UCSF), San Francisco, CA, USA *Corresponding author. Tel: +1 415 502 3460; E-mail: [email protected] EMBO J (2020)39:e104136https://doi.org/10.15252/embj.2019104136 See also: G Peet et al (August 2020) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Developmental synaptic remodeling is important for the formation of precise neural circuitry, and its disruption has been linked to neurodevelopmental disorders such as autism and schizophrenia. Microglia prune synapses, but integration of this synapse pruning with overlapping and concurrent neurodevelopmental processes, remains elusive. Adhesion G protein-coupled receptor ADGRG1/GPR56 controls multiple aspects of brain development in a cell type-specific manner: In neural progenitor cells, GPR56 regulates cortical lamination, whereas in oligodendrocyte progenitor cells, GPR56 controls developmental myelination and myelin repair. Here, we show that microglial GPR56 maintains appropriate synaptic numbers in several brain regions in a time- and circuit-dependent fashion. Phosphatidylserine (PS) on presynaptic elements binds GPR56 in a domain-specific manner, and microglia-specific deletion of Gpr56 leads to increased synapses as a result of reduced microglial engulfment of PS+ presynaptic inputs. Remarkably, a particular alternatively spliced isoform of GPR56 is selectively required for microglia-mediated synaptic pruning. Our present data provide a ligand- and isoform-specific mechanism underlying microglial GPR56-mediated synapse pruning in the context of complex neurodevelopmental processes. Synopsis Phosphatidylserine serves an "eat-me" signal for apoptotic cells to be phagocytosed by macrophages. Here we reveal phosphatidylserine also flags synapses to be removed by microglia during early brain development. ADGRG1/GPR56 S4 isoform in microglia regulates circuit-specific synapse refinement by binding to phosphatidylserine on pre-synaptic elements. Synapses destined to be removed externalize phosphatidylserine to serve as an "eat-me" signal. Microglia prune phosphatidylserine-positive synapses. A splicing isoform of GPR56, the S4 variant, regulates microglia-mediated synaptic pruning by binding to phosphatidylserine. Deleting microglial Gpr56 results in excess synapses. Introduction Microglia, tissue-resident macrophages of the central nervous system (CNS), are important for synaptic development, both in promoting synapse formation (Parkhurst et al, 2013; Miyamoto et al, 2016) and in engulfing redundant synapses (Paolicelli et al, 2011; Schafer et al, 2012). Immune molecules such as classical complement components and receptors, CX3CL1/CX3CR1, MHC class I, and PirB have been implicated in developmental synaptic refinement (Stevens et al, 2007; Paolicelli et al, 2011; Schafer et al, 2012; Lee et al, 2014; Vainchtein et al, 2018; Djurisic et al, 2019) and in synapse loss in disease models (Hong et al, 2016; Sekar et al, 2016; Vasek et al, 2016; Bialas et al, 2017; Sellgren et al, 2019). Mammalian neurodevelopment involves a succession of overlapping processes beginning with neurogenesis and neuronal migration, which are concurrent with microglial infiltration and morphogenesis. Subsequently, neurite arborization sets the stage for synaptogenesis, circuit establishment, and refinement, as well as myelination. All of these processes entail cell–cell interactions; discovery of molecules involved in multiple processes in a cell type-specific fashion can inform our understanding about how overlapping and sequential programs of intercellular signaling events are coordinately regulated. The adhesion G protein-coupled receptor (aGPCR) ADGRG1/GPR56 controls several aspects of brain development in a cell type-specific manner by mediating cell–cell and cell–matrix interactions (Singer et al, 2013; Langenhan et al, 2016). During embryonic brain development, GPR56 is expressed in neural progenitor cells and migrating neurons, and interacts with its extracellular matrix (ECM) ligand collagen III to regulate cortical lamination (Luo et al, 2011; Jeong et al, 2012a,b). In later stages of brain development and throughout postnatal life, GPR56 is highly expressed in glial cells, including astrocytes, oligodendrocyte lineage cells, and microglia (Zhang et al, 2014; Bennett et al, 2016). We recently showed that oligodendrocyte precursor cell (OPC) GPR56 functions together with its microglia-produced ligand, tissue transglutaminase (TG2, gene symbol Tgm2), and an ECM component laminin to control developmental myelination and myelin repair (Ackerman et al, 2015; Giera et al, 2015, 2018). Consistent with these findings, germline homozygous loss-of-function mutations in GPR56 cause a compound human brain malformation whose phenotype includes aberrant cortical architecture and dysmyelination (Piao et al, 2004, 2005). This phenotype is recapitulated in genetic mouse models indicating conserved GPR56 function (Li et al, 2008; Giera et al, 2015). Recent studies showed that Gpr56 is only expressed in yolk sac-derived microglia but not in microglia-like cells engrafted from fetal liver- and bone marrow-derived hematopoietic stem cells, even after long-term adaptation in the CNS in vivo (Bennett et al, 2018; Cronk et al, 2018). Furthermore, Gpr56 expression is promptly lost in primary cultures of microglia (Bohlen et al, 2017; Gosselin et al, 2017). Thus, Gpr56 is one of few genes that defines the microglial lineage and requires both the appropriate ontogeny and environmental cues for its expression. Motivated by the concept that cell type-specific functions of GPR56 might coordinate multiple sequential and overlapping neurodevelopmental processes, we tested the hypothesis that microglial GPR56 mediates synapse refinement during postnatal life. Our study results uncover that an alternatively spliced isoform of GPR56 is required for microglia-mediated synapse refinement via binding to phosphatidylserine (PS). Results PS flags retinal ganglion cell (RGC) synaptic inputs for removal by microglia How do microglia discriminate which synapse to eliminate? PS is a phospholipid that largely resides on the inner leaflet of the plasma membrane under normal conditions (McLaughlin & Murray, 2005). PS externalization serves as an "eat-me" signal for clearance of apoptotic and stressed cells as well as outer segment membranes of retinal photoreceptors (Feng et al, 2002; Segawa et al, 2011; Neher et al, 2013; Tufail et al, 2017). Here, we hypothesize that PS flags synapses for removal. To test this hypothesis, we employed the mouse retinogeniculate system, a classic model to study developmental synaptic pruning (Shatz & Kirkwood, 1984; Schafer et al, 2012). During early postnatal stage, the axons of RGCs extend into the dorsal lateral geniculate nucleus (dLGN) and form excessive synaptic connections with relay neurons. These RGC synaptic inputs undergo pruning by microglia (Schafer et al, 2012). We performed sequential dual labeling with an RGC anterograde tracer—fluorescent cholera toxin B (CTB) and a PS marker—PSVue (Koulov et al, 2003; Smith et al, 2011), a small PS-binding molecule with superior tissue diffusion than pSIVA (an Annexin B12 derivative that binds PS) (Fig EV1). CTB was intravitreally injected into eyes to trace RGC inputs at P5, and PSVue was injected into the board area between the dLGN and hippocampus at P6 (Fig 1A). Six hours after PSVue injection, fresh mouse brains were sectioned and imaged under a confocal microscope (Fig 1B). We observed PSVue colocalizing with some RGC inputs in the dLGN of WT mice (Fig EV1B). In the developing dLGN, microglial engulfment of synaptic elements peaks at P5 and is dramatically decreased at P10 (Schafer et al, 2012). To investigate whether there are more PS+ synapses during the time point of peak synaptic pruning, we performed dual labeling of PSVue and CTB at P6 and P13. Indeed, we observed that nearly 10% of RGC inputs were PSVue-positive at P6, but only 3% were PSVue-positive at P13 (Fig 1C and D), coinciding with the rise and fall of microglia-mediated synaptic pruning from P5 to P10. To further confirm PS reside with synaptic structures, we performed immunohistochemistry (IHC) on P6 brain for vesicular glutamate transporter 2 (vGlut2), a presynaptic marker specific to RGC inputs in the dLGN (Land et al, 2004), and Homer1, a postsynaptic marker. Indeed, we observed colocalization of PSVue with vGlut2+ presynapses and Homer1+ postsynapses (Fig 1E and F). Click here to expand this figure. Figure EV1. RGC synaptic inputs were labeled by PSVue.RGC synaptic inputs were labeled by PSVue A. A diagram illustrating PS labeling by PSvue550 and RGC inputs anterograde tracing by CTB. CTB was intraoccularly injected 24 h prior to PSVue/pSIVA injection. B. Left panel shows well-diffused PSVue into dLGN. The yellow box indicates the region where the images were taken. Right panel shows RGC inputs colocalize with PSVue signal. The white box indicates the region of higher magnification image shown. C. A diagram showing pSIVA labeling and RGC inputs tracing by CTB. D. Left panel shows pSIVA accumulated in the border between hippocampus and LGN. Right panel shows minimal pSIVA colocalized with RGC inputs. Scale bar, 20 μm. Download figure Download PowerPoint Figure 1. PS flags RGC inputs for removal by microglia during early dLGN development A. A schematic drawing of experimental procedure where CTB488 is intraocularly injected, followed by intracranial injection of PSvue550 to dLGN border. B. A schematic diagram shows the timeline of procedures for ex vivo imaging and engulfment analysis. C. Top panel: Representative images show PS labeling in the WT dLGN at P6 and P13. RGC inputs were labeled with CTB488. Bottom panel: Enlarged images of the boxed region in top panels. Circles indicate PS+ RGC inputs. Arrows point to the enlarged PS+ RGC inputs in the insets. Scale bar, 5 μm. D. Quantification of the percentage of PS+ RGC inputs in total RGC inputs at P6 and P13. N = 4, ****P < 0.0001. Student's t-test. E. Co-labeling of vGlut2 and PSVue in the dLGN at P6. Circles indicate colocalized vGlut2 and PSVue. F. Co-labeling of Homer1 and PSVue in the dLGN at P6. Circles indicate colocalized Homer1 and PSVue. Scare bar, 5 μm. G. Orthogonal sections showed a triple-positive PS+/CTB488+/CD68+ RGC inputs inside of microglia. Scare bar, 5 μm. H. Orthogonal sections showed a triple-positive PS+/vGlut2+/CD68+ synapse inside of microglia. Scare bar, 5 μm. I. A representative image of microglia (upper-left) is surface rendered (bottom-left). The white arrow points to cells which might be apoptotic cells labeled by PSVue. RGC inputs and PSVue outside or inside of microglia are shown in the two right panels. Scare bar, 20 μm. J. Quantification of the percentage of PS+ and PS− RGC inputs outside of microglia in total inputs. N = 4. K. Quantification of the percentage of engulfed PS+ and PS− RGC inputs in total engulfed inputs. N = 4. Data information: Data are presented as mean ± SD. Download figure Download PowerPoint We next investigated whether microglia engulf PS+ RGC synaptic inputs by combining the RGC anterograde tracing, PS labeling, and microglial engulfment assays. RGC inputs were labeled with Alexa 488-conjugated CTB via intravitreal injection at P5, and PS exposed synapses were labeled by PSVue via intracranial injection at P6. Brains were harvested 24 hours later at P7 for analysis of microglial phagocytosis (Fig 1B). We first tested the hypothesis that microglia engulf PS+ presynaptic elements rather than free fluorescent dye by performing a negative control experiment using 5-carboxytetramethylrhodamine (5-TAMRA), the fluorophore component of PSVue that does not bind PS (Hanshaw & Smith, 2005). As shown in Fig EV2, we observed very sparse 5-TAMRA signals in microglia compared to PSVue, supporting the validity of our assay. In WT animals, we observed that PSVue colocalized with RGC inputs inside microglia (Fig EV2B). To examine whether RGC inputs were targeted to lysosome in microglia, we did additional staining against CD68, a lysosomal marker for microglia, and detected internalized PS+ RGC synaptic inputs colocalized with CD68 (Fig 1G). Moreover, vGlut2+ presynapses were also colocalized with CD68 and PSVue in microglia (Fig 1H). Next, we quantified the percentage of PS+ RGC inputs. We saw < 10% RGC inputs outside microglia were PS+ (Fig 1I and J), but ~73% RGC inputs inside microglia were PS+ (Fig 1K), suggesting microglia favorably engulf PS+ synaptic inputs, although they do also engulf PS− inputs to a lesser degree (Fig 1K). Taken together, these data demonstrated that exposed PS flags RGC presynapses for removal by microglia during the early development of dLGN. Click here to expand this figure. Figure EV2. Microglia engulf PSVue-labeled RGC inputs A. An image shows the PSVue signal after intracranial injection. Dotted areas indicate the dLGN. B. A representative image of microglia from PSVue treated dLGN. Nuclei were labeled with DAPI. The mid-panel shows a 3D surface rendered microglia (purple) with DAPI (blue) and engulfed inputs (green) and PSVue (red). The right panel shows engulfed RGC inputs and PSVue inside of microglia. The magnified insert shows PS+ RGC inputs. C. An image shows the 5-TAMRA signal after injection. Dotted areas indicate the dLGN. D. A representative image of microglia from 5-TAMRA treated dLGN. Very few 5-TAMRA puncta were observed. The middle panel shows a surface rendered microglia with engulfed inputs (green) and 5-TAMRA (red). The right panel shows engulfed RGC inputs and 5-TAMRA. The insert shows RGC inputs do not overlap with 5-TAMRA. E. Quantification of engulfed PSVue and 5-TAMRA dyes by microglia. N = 4 (PSVue), N = 3 (5-TAMRA), **P = 0.001. F. Quantification of engulfed PSVue-positive or 5-TAMRA-positive RGC inputs by microglia. N = 4 (PSVue), N = 3 (5-TAMRA), **P = 0.006. Data information: Student's t-test. Data are presented as mean ± SD. Download figure Download PowerPoint GPR56 binds to phosphatidylserine Gpr56 transcript increases in microglia from embryonic stage and reaches a high level between P3-P6, a period of active microglia-mediated synaptic pruning (Appendix Fig S1; Matcovitch-Natan et al, 2016). Considering the facts that Gpr56 defines true yolk sac-derived microglia (Matcovitch-Natan et al, 2016; Bennett et al, 2018; Cronk et al, 2018) and that BAI1/ADGRB1, another aGPCR family member, recognizes PS (Park et al, 2007), we hypothesized that microglial GPR56 regulates developmental synaptic pruning by binding to PS. GPR56 contains an extensive N-terminal fragment (NTF) followed by a classical seven-transmembrane region and a cytoplasmic tail (Fig 2A; Folts et al, 2019). Within the long NTF, there are two functional domains, termed pentraxin/laminin/neurexin/sex-hormone-binding-globulin-like (PLL) and GPCR autoproteolysis inducing (GAIN) domains (Araç et al, 2012; Salzman et al, 2016). We engineered recombinant proteins of human immunoglobulin Fc (hFc)-tagged full-length NTF (NTF-hFc) and GAIN-hFc (Fig 2B). Figure 2. GPR56 binds to PS A. A schematic drawing of GPR56 protein structure, with a N-terminal fragment (NTF), a seven-transmembrane domain (7-TM) and a C-terminal fragment (CTF). B. A diagram shows the hFc tag was added to the c-terminal of GPR56-NTF (NTF-hFC) or GAIN domain (GAIN-hFc). C. A flowchart showing the experimental design of flow cytometry analysis. Briefly, Ba/F3 cells are treated with A23187 to externalize PS. For the binding assay, Alexa Fluor 647-conjugated hFc, GAIN-hFc, or NTF-hFc were incubated with PS-externalized Ba/F3 cells. For the competition assay, Alexa Fluor 647-conjugated hFc, GAIN-hFc, or NTF-hFc were used to compete with FITC-conjugated Annexin V binding. D. The binding experiment using flow cytometry show that only the GAIN domain binds to PS, similar as Annexin V binding. E. In the competition experiment, AF647 channel shows that GAIN domain binds 60.1% of the PS+ Ba/F3 cells (left). Correspondingly, FITC channel reveals 39.7% Annexin V binding to PS+ Ba/F3 cells (right). F. A diagram of membrane lipid strips spotted with fifteen different lipids. DAG, diacylglycerol; PA, phosphatidic acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol; PI, phosphatidylinositol. G. Direct binding of hFc-tagged GPR56 GAIN proteins to PS and other phospholipids. HFc was used as a negative control, and GAS6 as a positive control. Download figure Download PowerPoint We investigated whether GPR56 binds PS on cells using flow cytometry analyses of Ba/F3, an IL-3 dependent murine pro-B cell line that externalizes PS upon calcium ionophore A23187 treatment (Appendix Fig S2; Suzuki et al, 2010). We first performed a direct binding assay using Alexa Fluor 647 (AF647)-labeled full-length NTF, GAIN domain, or hFc (Fig 2C). FITC-conjugated Annexin V, a known PS-binding protein (Koopman et al, 1994), served as a positive control. Unexpectedly, we found that only the GAIN domain, not the full-length NTF, bound PS (Fig 2D). To further verify this observation, we performed a competition assay, in which labeled full-length NTF, GAIN domain, or hFc was used to displace Annexin V binding (Fig 2C). Indeed, we confirmed that the GAIN domain, but not full-length NTF, competed with Annexin V for binding to PS (Fig 2E). The PLL and GAIN domains are constrained by an interdomain disulfide bond at two cysteine residues C121 and C177 (Salzman et al, 2016). It is conceivable that the PLL domain blocks GAIN domain binding to PS in the full-length NTF. To test whether GPR56 GAIN domain binds to other phospholipids besides PS, we performed a protein–lipid overlay experiment using membrane lipid strips, as previously described (Park et al, 2007). GAS6, a known PS-binding protein, was used as a positive control. Interestingly, we found GAIN domain has a similar lipid-binding profile as GAS6, except cardiolipin (Fig 2F and G). In this in vitro membrane-based assay, GPR56 GAIN domain was able to bind phosphatidic acid (PA), phosphatidylserine (PS), cardiolipin, PI(4)P, PI(4,5)P2, and PI(3,4,5)P3. Given that PA, PI(4)P, PI(4,5)P2, and PI(3,4,5)P3 are normally present on the inner leaflet of the cell plasma membrane (Ingolfsson et al, 2014), and cardiolipin is a phospholipid almost exclusively located in the inner mitochondrial membrane (Paradies et al, 2014), GAIN domain binding to those lipids carries unclear biological relevance in the context of our current study. GPR56 S4 variant is dispensable for cortical development and CNS myelination but is essential for microglia-mediated synaptic refinement S4 is an alternatively spliced GPR56 isoform that initiates at an alternative ATG start codon in exon 4, resulting in a GPR56 variant that contains only the GAIN domain in its extracellular region, in both humans and mice (Fig 3A; Kim et al, 2010; Salzman et al, 2016). We published the cortical phenotype of the germline Gpr56 gene-targeted mice (Li et al, 2008), before the discovery of Gpr56 splicing variants. At that time, our data suggested that this line represented a null allele in regard to cortical phenotype. Subsequently, it was characterized as a hypomorphic allele, represented by selective expression of the Gpr56 S4 variant (Fig 3B, Appendix Fig S3; Salzman et al, 2016). In the present report, this genetic model is referred to as Gpr56 S4 (Fig 3C). To extend our investigation of Gpr56 biology, we deleted Gpr56 exons 4-6, yielding a global null mutant termed as Gpr56 null, lacking both full-length GPR56 and its S4 variant (Fig 3C). Based on the fact that the PLL domain binds collagen III (Luo et al, 2012), which is the relevant GPR56 ligand in the developing cerebral cortex, we speculated that the S4 isoform would not be required for cerebral cortical lamination. Indeed, we observed a comparable cortical ectopia size and distribution between Gpr56 S4 and Gpr56 null mice (Fig 3D–F). GPR56 also drives myelination and myelin repair via the PLL domain and TG2 interaction (Singer et al, 2013; Giera et al, 2015, 2018). As we expected, we observed comparable decreased myelination in Gpr56 null and Gpr56 S4 mice (Fig 3G and H), which suggested that the GPR56 S4 isoform is dispensable in OPC GPR56 regulation of CNS myelination. Figure 3. GPR56 S4 variant is required for synaptic refinement in the dLGN A. A diagram showing Gpr56 WT, null, and S4 transcripts. Solid boxes indicating exons that are transcribed. Arrowheads indicate the translation start sites. B. Gpr56 transcript levels in isolated microglia from Gpr56 S4 mice. N = 3, **P < 0.01 by Student's t-test. C. A table showing the expression status of Gpr56 WT and S4 variants in microglia of different transgenic mice. D. Representative images of Nissl staining of Gpr56 null and Gpr56 S4 E16.5 neocortex. Arrows indicate cortical ectopias that are shown in insets. E. Quantification of ectopia size per section. N = 6, P = 0.64 by Student's t-test. F. The distribution of ectopia from rostral to caudal cortex. N = 3, F (9, 81) = 0.65, P = 0.75 by two-way ANOVA with Bonferroni's post hoc test. G. Myelin basic protein (MBP) staining of corpus callosum in P28 controls, Gpr56 null and Gpr56 S4 mice. Scale bar, 100 μm. H. Quantification of MBP intensity in corpus callosum. N = 5, ***P < 0.001 by one-way ANOVA with Tukey's post hoc test. I. Gpr56 transcript levels in WT microglia at P5 and P14. N = 4, **P < 0.01, two-way ANOVA with Sidak's multiple comparisons test. J. An overview of vGlut2 and Homer1 staining in dLGN at P10. The yellow outline indicates the dLGN core, and the dotted boxes show where synapses are quantified. Scale bar, 200 μm. K. Representative images of vGlut2/Homer1 staining in the dLGN of control, Gpr56 null, and Gpr56 S4 brains at P10. Arrows pointing to the enlarged synapse in the insets. Scale bar, 5 μm. L. Relative vGlut2/Homer1 synapse density in dLGN. N = 10 (Ctrl), N = 6 (Gpr56 null), N = 3 (Gpr56 S4). **P < 0.01, ***P < 0.001, one-way ANOVA with Tukey's post hoc test. M. A schematic representation of the in vivo engulfment assay. CTB are injected into both eyes at P4, and anterogradely trace RGC projections to the dLGN. After 24 h, the brains are collected and analyzed at P5. N. Representative images and surface rendered microglia (green) in which CTB+(red) RGC inputs were engulfed. Scale b
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