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

Abstract

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.

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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.

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Acknowledgements

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.

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Contributions

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.

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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.

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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

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