The endogenous neuronal complement inhibitor SRPX2 protects against complement-mediated synapse elimination during development


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: SRPX2 binds to C1q.
Fig. 2: SRPX2 is expressed by neurons and colocalizes with C1q.
Fig. 3: SRPX2 inhibits the classical complement pathway.
Fig. 4: SRPX2 knockout reduces the number of functional inputs to dLGN neurons.
Fig. 5: SRPX2 regulates complement-mediated RGC axon segregation in the dLGN.
Fig. 6: SRPX2 regulates complement-mediated microglial engulfment of synapses in the dLGN.
Fig. 7: SRPX2 inhibits complement activation in L4 of the SS cortex but not L2/3.
Fig. 8: SRPX2 regulates complement-mediated synapse elimination in the SS cortex.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.


  1. 1.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Stevens, B. et al. The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178 (2007).

    CAS  PubMed  Google Scholar 

  3. 3.

    Schafer, D. P. et al. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron 74, 691–705 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Chu, Y. et al. Enhanced synaptic connectivity and epilepsy in C1q knockout mice. Proc. Natl Acad. Sci. USA 107, 7975–7980 (2010).

    CAS  PubMed  Google Scholar 

  5. 5.

    Hong, S. et al. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712–716 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Shi, Q. et al. Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice. Sci. Transl Med. 9, eaaf6295 (2017).

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Lui, H. et al. Progranulin deficiency promotes circuit-specific synaptic pruning by microglia via complement activation. Cell 165, 921–935 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Simonetti, M. et al. Nuclear Calcium Signaling in Spinal Neurons Drives a Genomic Program Required for Persistent Inflammatory Pain. Neuron 77, 43–57 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Vasek, M. J. et al. A complement–microglial axis drives synapse loss during virus-induced memory impairment. Nature 534, 538–543 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Sekar, A. et al. Schizophrenia risk from complex variation of complement component 4. Nature 530, 177–183 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Ricklin, D., Hajishengallis, G., Yang, K. & Lambris, J. D. Complement—a key system for immune surveillance and homeostasis. Nat. Immunol. 11, 785–797 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    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).

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Roll, P. et al. SRPX2 mutations in disorders of language cortex and cognition. Hum. Mol. Genet. 15, 1195–1207 (2006).

    CAS  PubMed  Google Scholar 

  15. 15.

    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).

  16. 16.

    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).

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Kirkitadze, M. D. & Barlow, P. N. Structure and flexibility of the multiple domain proteins that regulate complement activation. Immunol. Rev. 180, 146–161 (2001).

    CAS  PubMed  Google Scholar 

  18. 18.

    Håvik, B. et al. The complement control-related genes CSMD1 and CSMD2 associate to schizophrenia. Biol. Psychiatry 70, 35–42 (2011).

    PubMed  Google Scholar 

  19. 19.

    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).

    CAS  PubMed  Google Scholar 

  20. 20.

    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).

    CAS  PubMed  Google Scholar 

  21. 21.

    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).

    CAS  PubMed  Google Scholar 

  22. 22.

    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).

    CAS  PubMed  Google Scholar 

  23. 23.

    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).

    CAS  PubMed  Google Scholar 

  24. 24.

    Kölm, R. et al. Von Willebrand factor interacts with surface-bound C1q and induces platelet rolling. J. Immunol. 197, 3669–3679 (2016).

    PubMed  Google Scholar 

  25. 25.

    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).

    CAS  PubMed  Google Scholar 

  26. 26.

    Krasemann, S. et al. The TREM2–APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity 47, 566–581.e9 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27.

    Chen, C. & Regehr, W. G. Developmental remodeling of the retinogeniculate synapse. Neuron 28, 955–966 (2000).

    CAS  PubMed  Google Scholar 

  28. 28.

    Lehrman, E. K. et al. CD47 protects synapses from excess microglia-mediated pruning during development. Neuron 100, 120–134.e6 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Bialas, A. R. & Stevens, B. TGF-β signaling regulates neuronal C1q expression and developmental synaptic refinement. Nat. Neurosci. 16, 1773–1782 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Petersen, C. C. H. The functional organization of the barrel cortex. Neuron 56, 339–355 (2007).

    CAS  PubMed  Google Scholar 

  31. 31.

    Petreanu, L., Mao, T., Sternson, S. M. & Svoboda, K. The subcellular organization of neocortical excitatory connections. Nature 457, 1142–1145 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    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).

    PubMed  Google Scholar 

  33. 33.

    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).

    CAS  PubMed  Google Scholar 

  34. 34.

    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).

    CAS  PubMed  Google Scholar 

  35. 35.

    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).

    CAS  PubMed  Google Scholar 

  36. 36.

    Huberman, A. D., Feller, M. B. & Chapman, B. Mechanisms underlying development of visual maps and receptive fields. Annu. Rev. Neurosci. 31, 479–509 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Bjartmar, L. et al. Neuronal pentraxins mediate synaptic refinement in the developing visual system. J. Neurosci. 26, 6269–6281 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    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).

    CAS  PubMed  Google Scholar 

  39. 39.

    Huh, G. S. et al. Functional requirement for class I MHC in CNS development and plasticity. Science 290, 2155–2159 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40.

    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).

    CAS  PubMed  Google Scholar 

  41. 41.

    Salmi, M. et al. Tubacin prevents neuronal migration defects and epileptic activity caused by rat Srpx2 silencing in utero. Brain 136, 2457–2473 (2013).

    PubMed  Google Scholar 

  42. 42.

    Huttenlocher, P. R. Synaptic density in human frontal cortex—developmental changes and effects of aging. Brain Res. 163, 195–205 (1979).

    CAS  PubMed  Google Scholar 

  43. 43.

    Huttenlocher, P. R. & Dabholkar, A. S. Regional differences in synaptogenesis in human cerebral cortex. J. Comp. Neurol. 387, 167–178 (1997).

    CAS  PubMed  Google Scholar 

  44. 44.

    Petanjek, Z. et al. Extraordinary neoteny of synaptic spines in the human prefrontal cortex. Proc. Natl Acad. Sci. USA 108, 13281–13286 (2011).

    CAS  PubMed  Google Scholar 

  45. 45.

    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).

    CAS  PubMed  Google Scholar 

  46. 46.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    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).

    PubMed  PubMed Central  Google Scholar 

  48. 48.

    Gunner, G. et al. Sensory lesioning induces microglial synapse elimination via ADAM10 and fractalkine signaling. Nat. Neurosci. 22, 1075–1088 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49.

    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).

    CAS  PubMed  Google Scholar 

  50. 50.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    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).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Torborg, C. L. & Feller, M. B. Unbiased analysis of bulk axonal segregation patterns. J. Neurosci. Methods 135, 17–26 (2004).

    CAS  PubMed  Google Scholar 

  53. 53.

    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).

    CAS  PubMed  Google Scholar 

  54. 54.

    Datwani, A. et al. Classical MHCI molecules regulate retinogeniculate refinement and limit ocular dominance plasticity. Neuron 64, 463–470 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    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).

    PubMed  Google Scholar 

  56. 56.

    Bolte, S. & Cordelières, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Guido, W. Development, form, and function of the mouse visual thalamus. J. Neurophysiol. 120, 211–225 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    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).

    Google Scholar 

Download references


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.

Author information




Q.C. and B.M.S. conducted the experiments. G.-M.S. generated the SRPX2 transgenic mice, designed the experiments and wrote the manuscript. M.W. and J.H.K. performed the electrophysiology experiments.

Corresponding author

Correspondence to Gek-Ming Sia.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Generation of SRPX2-FLAG knock-in mice.

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

Extended Data Fig. 2 Neuronal expression of SRPX2.

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

Extended Data Fig. 3 Neurodegeneration and microglial state markers are unchanged in SRPX2-/Y mice.

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.

Extended Data Fig. 4 Major cell layers and axon tracts are intact in all mouse genotypes.

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.

Extended Data Fig. 5 Representative images of microglia engulfment assay in dLGN.

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.

Extended Data Fig. 6 Representative images of microglia engulfment assay in L4 SS cortex.

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.

Supplementary information

Source data

Source Data Fig. 1

Unprocessed western blots for Fig. 1.

Source Data Fig. 3

Unprocessed western blots for Fig. 3.

Source Data Fig. 3

Statistical source data for Fig. 3.

Source Data Fig. 4

Statistical source data for Fig. 4.

Source Data Fig. 5

Statistical source data for Fig. 5.

Source Data Fig. 6

Statistical source data for Fig. 6.

Source Data Fig. 7

Statistical source data for Fig. 7.

Source Data Fig. 8

Statistical source data for Fig. 8.

Source Data Extended Data Fig. 1

Unprocessed western blots for Extended Fig. 1.

Source Data Extended Data Fig. 2

Unprocessed western blots for Extended Fig. 2.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

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).

Download citation