Complement-mediated synapse elimination has emerged as an important process in both brain development and neurological diseases, but whether neurons express complement inhibitors that protect synapses against complement-mediated synapse elimination remains unknown. Here, we show that the sushi domain protein SRPX2 is a neuronally expressed complement inhibitor that regulates complement-dependent synapse elimination. SRPX2 directly binds to C1q and blocks its activity, and SRPX2−/Y mice show increased C3 deposition and microglial synapse engulfment. They also show a transient decrease in synapse numbers and increase in retinogeniculate axon segregation in the lateral geniculate nucleus. In the somatosensory cortex, SRPX2−/Y mice show decreased thalamocortical synapse numbers and increased spine pruning. C3−/−;SRPX2−/Y double-knockout mice exhibit phenotypes associated with C3−/− mice rather than SRPX2−/Y mice, which indicates that C3 is necessary for the effect of SRPX2 on synapse elimination. Together, these results show that SRPX2 protects synapses against complement-mediated elimination in both the thalamus and the cortex.
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.
Penzes, P., Cahill, M. E., Jones, K. A., VanLeeuwen, J.-E. & Woolfrey, K. M. Dendritic spine pathology in neuropsychiatric disorders. Nat. Neurosci. 14, 285–293 (2011).
Stevens, B. et al. The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178 (2007).
Schafer, D. P. et al. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron 74, 691–705 (2012).
Chu, Y. et al. Enhanced synaptic connectivity and epilepsy in C1q knockout mice. Proc. Natl Acad. Sci. USA 107, 7975–7980 (2010).
Hong, S. et al. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712–716 (2016).
Shi, Q. et al. Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice. Sci. Transl Med. 9, eaaf6295 (2017).
Lui, H. et al. Progranulin deficiency promotes circuit-specific synaptic pruning by microglia via complement activation. Cell 165, 921–935 (2016).
Simonetti, M. et al. Nuclear Calcium Signaling in Spinal Neurons Drives a Genomic Program Required for Persistent Inflammatory Pain. Neuron 77, 43–57 (2013).
Vasek, M. J. et al. A complement–microglial axis drives synapse loss during virus-induced memory impairment. Nature 534, 538–543 (2016).
Sekar, A. et al. Schizophrenia risk from complex variation of complement component 4. Nature 530, 177–183 (2016).
Ricklin, D., Hajishengallis, G., Yang, K. & Lambris, J. D. Complement—a key system for immune surveillance and homeostasis. Nat. Immunol. 11, 785–797 (2010).
Sia, G. M., Clem, R. L. & Huganir, R. L. The human language-associated gene SRPX2 regulates synapse formation and vocalization in mice. Science 342, 987–991 (2013).
Chen, X. S. et al. Next-generation DNA sequencing identifies novel gene variants and pathways involved in specific language impairment. Sci. Rep. 7, 46105 (2017).
Roll, P. et al. SRPX2 mutations in disorders of language cortex and cognition. Hum. Mol. Genet. 15, 1195–1207 (2006).
Schirwani, S., McConnell, V. & Willoughby, J., DDD Study & Balasubramanian, M. Exploring the association between SRPX2 variants and neurodevelopment: how causal is it? Gene 685, 50–54 (2018).
Soteros, B. M., Cong, Q., Palmer, C. R. & Sia, G.-M. Sociability and synapse subtype-specific defects in mice lacking SRPX2, a language-associated gene. PLoS ONE 13, e0199399 (2018).
Kirkitadze, M. D. & Barlow, P. N. Structure and flexibility of the multiple domain proteins that regulate complement activation. Immunol. Rev. 180, 146–161 (2001).
Håvik, B. et al. The complement control-related genes CSMD1 and CSMD2 associate to schizophrenia. Biol. Psychiatry 70, 35–42 (2011).
Shimizu, A. et al. A novel giant gene CSMD3 encoding a protein with CUB and sushi multiple domains: a candidate gene for benign adult familial myoclonic epilepsy on human chromosome 8q23.3-q24.1. Biochem. Biophys. Res. Commun. 309, 143–154 (2003).
Yu, Z.-L. et al. Febrile seizures are associated with mutation of seizure-related (SEZ) 6, a brain-specific gene. J. Neurosci. Res. 85, 166–172 (2007).
Stephan, A. H., Barres, B. A. & Stevens, B. The complement system: an unexpected role in synaptic pruning during development and disease. Annu. Rev. Neurosci. 35, 369–389 (2012).
Bally, I. et al. Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites. Proc. Natl Acad. Sci. USA 110, 8650–8655 (2013).
Holmquist, E., Okroj, M., Nodin, B., Jirström, K. & Blom, A. M. Sushi domain-containing protein 4 (SUSD4) inhibits complement by disrupting the formation of the classical C3 convertase. FASEB J. 27, 2355–2366 (2013).
Kölm, R. et al. Von Willebrand factor interacts with surface-bound C1q and induces platelet rolling. J. Immunol. 197, 3669–3679 (2016).
Anwer, M. et al. Sushi repeat-containing protein X-linked 2—a novel phylogenetically conserved hypothalamo–pituitary protein. J. Comp. Neurol. 526, 1806–1819 (2018).
Krasemann, S. et al. The TREM2–APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity 47, 566–581.e9 (2017).
Chen, C. & Regehr, W. G. Developmental remodeling of the retinogeniculate synapse. Neuron 28, 955–966 (2000).
Lehrman, E. K. et al. CD47 protects synapses from excess microglia-mediated pruning during development. Neuron 100, 120–134.e6 (2018).
Bialas, A. R. & Stevens, B. TGF-β signaling regulates neuronal C1q expression and developmental synaptic refinement. Nat. Neurosci. 16, 1773–1782 (2013).
Petersen, C. C. H. The functional organization of the barrel cortex. Neuron 56, 339–355 (2007).
Petreanu, L., Mao, T., Sternson, S. M. & Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature 457, 1142–1145 (2009).
Oberlaender, M. et al. Cell type-specific three-dimensional structure of thalamocortical circuits in a column of rat vibrissal cortex. Cereb. Cortex 22, 2375–2391 (2012).
Bian, W.-J., Miao, W.-Y., He, S.-J., Qiu, Z. & Yu, X. Coordinated spine pruning and maturation mediated by inter-spine competition for cadherin/catenin complexes. Cell 162, 808–822 (2015).
Kraus, D. M. et al. CSMD1 is a novel multiple domain complement-regulatory protein highly expressed in the central nervous system and epithelial tissues. J. Immunol. 176, 4419–4430 (2006).
Escudero-Esparza, A., Kalchishkova, N., Kurbasic, E., Jiang, W. G. & Blom, A. M. The novel complement inhibitor human CUB and Sushi multiple domains 1 (CSMD1) protein promotes factor I-mediated degradation of C4b and C3b and inhibits the membrane attack complex assembly. FASEB J. 27, 5083–5093 (2013).
Huberman, A. D., Feller, M. B. & Chapman, B. Mechanisms underlying development of visual maps and receptive fields. Annu. Rev. Neurosci. 31, 479–509 (2008).
Bjartmar, L. et al. Neuronal pentraxins mediate synaptic refinement in the developing visual system. J. Neurosci. 26, 6269–6281 (2006).
Corriveau, R. A., Huh, G. S. & Shatz, C. J. Regulation of class I MHC gene expression in the developing and mature CNS by neural activity. Neuron 21, 505–520 (1998).
Huh, G. S. et al. Functional requirement for class I MHC in CNS development and plasticity. Science 290, 2155–2159 (2000).
Royer-Zemmour, B. et al. Epileptic and developmental disorders of the speech cortex: ligand/receptor interaction of wild-type and mutant SRPX2 with the plasminogen activator receptor uPAR. Hum. Mol. Genet. 17, 3617–3630 (2008).
Salmi, M. et al. Tubacin prevents neuronal migration defects and epileptic activity caused by rat Srpx2 silencing in utero. Brain 136, 2457–2473 (2013).
Huttenlocher, P. R. Synaptic density in human frontal cortex—developmental changes and effects of aging. Brain Res. 163, 195–205 (1979).
Huttenlocher, P. R. & Dabholkar, A. S. Regional differences in synaptogenesis in human cerebral cortex. J. Comp. Neurol. 387, 167–178 (1997).
Petanjek, Z. et al. Extraordinary neoteny of synaptic spines in the human prefrontal cortex. Proc. Natl Acad. Sci. USA 108, 13281–13286 (2011).
Rakic, P., Bourgeois, J. P., Eckenhoff, M. F., Zecevic, N. & Goldman-Rakic, P. S. Concurrent overproduction of synapses in diverse regions of the primate cerebral cortex. Science 232, 232–235 (1986).
Pinto, J. G. A., Jones, D. A. & Murphy, K. M. Comparing development of synaptic proteins in rat visual, somatosensory, and frontal cortex. Front. Neural Circuits 7, 97 (2013).
Ma, Y., Ramachandran, A., Ford, N., Parada, I. & Prince, D. A. Remodeling of dendrites and spines in the C1q knockout model of genetic epilepsy. Epilepsia 54, 1232–1239 (2013).
Gunner, G. et al. Sensory lesioning induces microglial synapse elimination via ADAM10 and fractalkine signaling. Nat. Neurosci. 22, 1075–1088 (2019).
Welsh, C. A., Stephany, C.-É., Sapp, R. W. & Stevens, B. Ocular dominance plasticity in binocular primary visual cortex does not require C1q. J. Neurosci. 40, 769–783 (2020).
Harris, C. L., Heurich, M., Cordoba, S. Rde & Morgan, B. P. The complotype: dictating risk for inflammation and infection. Trends Immunol. 33, 513–521 (2012).
Turner, J. P. & Salt, T. E. Characterization of sensory and corticothalamic excitatory inputs to rat thalamocortical neurones in vitro. J. Physiol. 510, 829–843 (1998).
Torborg, C. L. & Feller, M. B. Unbiased analysis of bulk axonal segregation patterns. J. Neurosci. Methods 135, 17–26 (2004).
Torborg, C. L., Hansen, K. A. & Feller, M. B. High frequency, synchronized bursting drives eye-specific segregation of retinogeniculate projections. Nat. Neurosci. 8, 72–78 (2005).
Datwani, A. et al. Classical MHCI molecules regulate retinogeniculate refinement and limit ocular dominance plasticity. Neuron 64, 463–470 (2009).
van Steensel, B. et al. Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons. J. Cell. Sci. 109, 787–792 (1996).
Bolte, S. & Cordelières, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).
Guido, W. Development, form, and function of the mouse visual thalamus. J. Neurophysiol. 120, 211–225 (2018).
Schafer, D. P., Lehrman, E. K., Heller, C. T. & Stevens, B. An engulfment assay: a protocol to assess interactions between CNS phagocytes and neurons. J. Vis. Exp. 88, 51482 (2014).
We thank A. Tenner (UC Irvine) for complement-related reagents. We thank D. Lodge (UTHSCA) for use of his microscope system, and D. Morilak (UTHSCSA) for use of histology equipment. We also thank M. Baum (Beth Steven’s Lab, Harvard) for technical advice on eye injections. CRISPR–Cas9 pronuclear injections were performed at the Johns Hopkins Transgenic Core Laboratory, and screening of founders and subsequent work was performed at UTHSCSA. This work was funded by the NARSAD Young Investigator grant number 25248 (to G.-M.S.), the William and Ella Owens Medical Research Foundation (to G.-M.S.), the Rising STARs award from the University of Texas System (to G.-M.S.), NINDS-R01NS112389 (to G.-M.S.) and NIDCD-R01DC013157 (to J.H.K.). Images were generated at the Core Optical Imaging Facility, which is supported by UTHSCSA, NCI-P30CA54174 (CTRC at UTHSCSA) and NIA-P01AG19316.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Schematic of targeting site for SRPX2-FLAG KI mouse, depicting sgRNA sequences (magenta), PAM sequence (green), FLAG sequence (gold), restriction sites (blue), and stop codon (yellow). Arrow shows site of Cas9-induced double stranded break. b, Partial chromatograph of the genome sequence of the SRXP2-FLAG KI mouse, showing correct insertion of FLAG sequence. c, Western blot of SRPX2 from whole brain (WB), cortex (CX), and midbrain (MD) lysates from SRPX2+/Y and SRPX2FLAG/Y mouse brain, showing comparable amounts of endogenous SRPX2 in both genotypes. These experiments were repeated independently 3 times. Source data
a, Representative images of RNAscope data presented in Fig. 2a, showing SRPX2 mRNA (red) and cell-specific markers NeuN/IbaI/GFAP/Olig2 (green) in separate channels for clarity. Nuclei were stained with DAPI (blue). Scale bar 20 µm. b, Synaptosome preparation from P60 mouse was immunoblotted for C3b, PSD95, SRPX2, and C1q. Fractions were brain homogenate (H), supernatant 1 (S1), pellet 1 (P1), supernatant 2 (S2), pellet 2 (P2) and synaptosomal fraction (Syn). This experiment was repeated independently 3 times. Source data
a-c, Representative images of P10 dLGN and P60 L4 SS cortex from SRPX2+/Y and SRPX2-/Y mice stained for neurodegeneration markers APP (a), ATF3 (b), and cleaved caspase 3 (c). Scale bar 100 µm. No staining for any neurodegeneration markers was observed in all mice. d, e, Representative images of P10 dLGN (d) and P60 L4 SS cortex (e) from SRPX2+/Y and SRPX2-/Y mice stained for microglial homeostatic state-associated marker P2RY12 and neurodegeneration state-associated marker Clec7a. Scale bar 10 µm. f, g, Representative images of P10 dLGN (f) and P60 L4 SS cortex (g) from SRPX2+/Y and SRPX2-/Y mice stained for microglial homeostatic state-associated marker TMEM119 and neurodegeneration state-associated marker ApoE. Scale bar 10 µm. Comparable levels of all microglial markers were present in SRPX2+/Y and SRPX2-/Y mice.
Representative images of coronal brain sections of P60 mice stained with Nissl, myelin, and DAPI stains with the BrainStainTM kit (Invitrogen). Scale bar 2000 µm.
Representative images of microglial CTB engulfment in the dorsolateral geniculate nucleus at P4, P10 and P30. Inset shows 3D rendered engulfed CTB-labelled inputs (red) within CD68+ lysosomes (blue) in microglia (green). Scale bar 10 µm.
Representative fluorescence & 3D rendered images of microglia engulfing VGlut2 in L4 somatosensory cortex at P30, P60, P90. Inset shows engulfed VGlut2 (magenta) within microglial (green) CD68+ lysosomes (blue). Scale bar 10 µm.
Unprocessed western blots for Fig. 1.
Unprocessed western blots for Fig. 3.
Statistical source data for Fig. 3.
Statistical source data for Fig. 4.
Statistical source data for Fig. 5.
Statistical source data for Fig. 6.
Statistical source data for Fig. 7.
Statistical source data for Fig. 8.
Unprocessed western blots for Extended Fig. 1.
Unprocessed western blots for Extended Fig. 2.
About this article
Cite this article
Cong, Q., Soteros, B.M., Wollet, M. et al. The endogenous neuronal complement inhibitor SRPX2 protects against complement-mediated synapse elimination during development. Nat Neurosci (2020). https://doi.org/10.1038/s41593-020-0672-0