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Chemico-Genetic Discovery of Astrocytic Control of Inhibition in Vivo

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Chemico-Genetic Discovery of Astrocytic Control of Inhibition in Vivo Article Chemico-genetic discovery of astrocytic control of inhibition in vivo https://doi.org/10.1038/s41586-020-2926-0 Tetsuya Takano1 ✉, John T. Wallace1, Katherine T. Baldwin1, Alicia M. Purkey1, Akiyoshi Uezu1, Jamie L. Courtland2, Erik J. Soderblom1,3, Tomomi Shimogori4, Patricia F. Maness5,6, Received: 23 January 2020 Cagla Eroglu1,2 ✉ & Scott H. Soderling1,2 ✉ Accepted: 19 August 2020 Published online: xx xx xxxx Perisynaptic astrocytic processes are an integral part of central nervous system Check for updates synapses1,2; however, the molecular mechanisms that govern astrocyte–synapse adhesions and how astrocyte contacts control synapse formation and function are largely unknown. Here we use an in vivo chemico-genetic approach that applies a cell-surface fragment complementation strategy, Split-TurboID, and identify a proteome that is enriched at astrocyte–neuron junctions in vivo, which includes neuronal cell adhesion molecule (NRCAM). We fnd that NRCAM is expressed in cortical astrocytes, localizes to perisynaptic contacts and is required to restrict neuropil infltration by astrocytic processes. Furthermore, we show that astrocytic NRCAM interacts transcellularly with neuronal NRCAM coupled to gephyrin at inhibitory postsynapses. Depletion of astrocytic NRCAM reduces numbers of inhibitory synapses without altering glutamatergic synaptic density. Moreover, loss of astrocytic NRCAM markedly decreases inhibitory synaptic function, with minor efects on excitation. Thus, our results present a proteomic framework for how astrocytes interface with neurons and reveal how astrocytes control GABAergic synapse formation and function. The majority of central nervous system (CNS) synapses are ensheathed (Extended Data Fig. 1a), the biotinylation activity of Split 1-TurboID by tiny astrocytic processes1,2. These astrocytic contacts are an integral was higher than that of Split 2-TurboID (Extended Data Fig. 1b, lane 8). functional compartment of the tripartite synapse, which is defined as We therefore used Split 1-TurboID for the remainder of this study, and the combination of pre- and postsynaptic neuronal, and perisynaptic named the two molecules N-TurboID and C-TurboID. We also used a astrocytic, processes3. At the synapse, astrocytes control basal synaptic GRAPHIC-tagged full-length TurboID construct, TurboID-surface, to transmission, neuromodulation, ionic balance and neurotransmitter biotinylate astrocyte surface proteins (Extended Data Fig. 1a, bottom). clearance4–8. Furthermore, astrocyte and synapse development are In astrocyte–neuron co-cultures, astrocytes expressing TurboID- interdependent processes that are regulated by dynamic bidirectional surface under the control of the GfaABC1D promoter17 (Extended intercellular communication via secreted factors and cell adhesion Data Fig. 1c) exhibited biotinylation activity along their mem- molecules9–12. Historically, however, gaining molecular insights branes (Extended Data Fig. 1d). Moreover, the reconstituted activ- into perisynaptic astrocyte–neuron signalling has been hampered ity of Split-TurboID was found only at contact sites between neurons owing to the lack of biochemical methods for isolating this astrocytic and astrocytes (Extended Data Fig. 1c), but not when either of the compartment. halves were expressed alone (Extended Data Fig. 1d). To investi- To identify proteins at the extracellular clefts between astro- gate whether TurboID-surface or Split-TurboID biotinylates tripar- cytes and neurons, we developed a chemico-genetic in vivo BioID tite synapses in these cultures, astrocytes were co-transduced with (iBioID) approach, based on reconstituting the enzymatic activity of GfaABC1D-mCherry-CAAX to mark astrocyte membranes, and synapses a proximity-biotinylating enzyme, TurboID13, at astrocyte–neuron were labelled by immunostaining with pre- and postsynaptic makers junctions (Fig. 1a). Recent studies have shown that split biotinyla- (excitatory, VGLUT1 and HOMER 1; inhibitory, VGAT and gephyrin). tion constructs could recover enzymatic activity when they were in Both constructs mediated biotinylation that overlapped with astro- close proximity in the cell cytoplasm14,15. In this study, we used our cytic membranes and closely associated with excitatory and inhibi- glycosylphosphatidylinositol-anchored reconstitution-activated tory synaptic markers (Extended Data Fig. 2a–d), demonstrating the proteins highlight intercellular connections (GRAPHIC) strategy16 to functional reconstitution of TurboID transcellularly at perisynaptic direct N- and C-terminal TurboID fragments to the extracellular sur- astrocyte–neuron junctions in vitro. face of neurons and astrocytes (Fig. 1a, Extended Data Fig. 1a). Among To test their activity in vivo, the constructs were introduced into the two Split-TurboID construct pairs that we tested in HEK 293T cells mouse brain astrocytes and/or neurons via retro-orbital injections 1The Department of Cell Biology, Duke University Medical School, Durham, NC, USA. 2Department of Neurobiology, Duke University Medical School, Durham, NC, USA. 3Duke Proteomics and Metabolomics Shared Resource and Duke Center for Genomic and Computational Biology, Duke University Medical School, Durham, NC, USA. 4Molecular Mechanisms of Brain Development, Center for Brain Science (CBS), RIKEN, Saitama, Japan. 5Department of Biochemistry, University of North Carolina School of Medicine, Chapel Hill, NC, USA. 6Department of Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC, USA. ✉e-mail: [email protected]; [email protected]; [email protected] Nature | www.nature.com | 1 Article a b Cell-type-specic AAVs AAV PHP.eB-hSynI-N-TurboID Astrocyte Presynapse ITR hSynI N-TurboID GPI WPRE pA ITR Biotinylation Split-TurboID AAV PHP.eB-GfaABC1D-C-TurboID TurboID-HA ITR GfaABC1D C-TurboID GPI WPRE pA ITR AAV injection Biotin injection Postsynapse Analysis P21 P42 P48 hSynI-eGFP GfaABC1D-mCherry–CAAX c (neuron) (astrocyte) Streptavidin Merge Control TurboID-surface Split-TurboID 5 μm def ) TurboID-surface Split-TurboID TurboID-surface Split-TurboID 2 100 P < 0.001 m PSD95 Gephyrin μ VGLUT1 VGAT 80 P < 0.05 Streptavidin Streptavidin 60 P < 0.001 VGLUT1 40 PSD95 P < 0.001 VGAT STED 20 STED Gephyrin Other (% puncta per 100 0 Streptavidin colocalization 1 μm 1 μm Split-TurboID TurboID-surface Fig. 1 | Identification of the astrocyte–neuron synaptic cleft proteome neuronal eGFP and astrocyte mCherry–CAAX. d, e, Three-colour STED images using in vivo Split-TurboID. a, Schematic of the Split-surface iBioID showing biotinylated proteins adjacent to excitatory synaptic markers PSD95 approach. b, Outline of Split-TurboID method using cell-type-specific AAVs. and VGLUT1 (d), and inhibitory synaptic markers gephyrin and VGAT (e). f, The ITR, inverted terminal repeats; hSyn1, human synapsin 1 promoter; GPI, ratio of biotinylated proteins that colocalized with VGLUT1, PSD95, VGAT or glycosylphosphatidylinositol; WPRE, woodchuck hepatitis virus post- gephyrin. n = 15 cells per each condition from 3 mice. n = 3 biological repeats. transcriptional regulatory element; pA, polyadenylation. c, Confocal images of Student’s paired t-test, comparing TurboID-surface and Split-TurboID. Data are cortical expression of Split-TurboID or TurboID-surface coexpressed with mean ± s.e.m. of adeno-associated viruses (AAVs)18 at postnatal day (P)21 (Fig. 1b, purified and analysed by quantitative high-resolution liquid chro- Extended Data Fig. 3a–c) and the mice were given subcutaneous biotin matography–tandem mass spectrometry (LC–MS) (Fig. 2a). When injections starting at P42 for 7 days (Fig. 1b)19. Biotinylated proteins were combined, the Split-TurboID and astrocyte-specific TurboID-surface detected by immunoblotting and immunohistochemistry (Extended datasets identified 776,376 peptides corresponding to 3,171 distinct Data Fig. 4a–d) both for astrocyte-specific TurboID-surface and recon- proteins (Extended Data Fig. 4g). After three independent experiments stituted Split-TurboID constructs. However, when Split-TurboID frag- and following removal of known contaminants19, 173 and 178 proteins ments were expressed alone, no biotinylation was observed (Extended were found to be significantly enriched (1.5. fold) in Split-TurboID and Data Fig. 4a–c). These results show that the TurboID-surface and astrocyte-specific TurboID-surface fractions, respectively, compared Split-TurboID constructs generate extracellular biotinylation in vivo. with soluble TurboID control (Extended Data Fig. 4g–i, Supplementary To confirm that biotinylated proteins localize to neuron–astro- Tables 1, 2). This enrichment approach is stringent, and thus may not cyte contacts, we labelled neurons with eGFP (using AAV PHP. identify all astrocytic proteins that are present at perisynaptic pro- eB-hSynI-eGFP) and astrocyte membranes with mCherry–CAAX cesses, as it selects only those that are overrepresented at synapses (using AAV PHP.eB-GfaBC1D-mCherry-CAAX) and co-injected either compared with other compartments. astrocyte-specific TurboID-surface or Split-TurboID-expressing A total of 118 proteins were common between the two datasets, yield- viruses. In both conditions, biotinylated proteins were located at the ing a high-confidence tri-partite synapse proteome (Fig. 2b, Extended contacts between astrocytic and neuronal processes (Fig. 1c). Using Data Fig. 4g–i, Supplementary Table 3). This list includes known tripar- super-resolution stimulated emission depletion (STED) microscopy, tite synapse proteins
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