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Overexpression of schizophrenia susceptibility factor human complement C4A promotes excessive synaptic loss and behavioral changes in mice

Abstract

The complement component 4 (C4) gene is linked to schizophrenia and synaptic refinement. In humans, greater expression of C4A in the brain is associated with an increased risk of schizophrenia. To investigate this genetic finding and address how C4A shapes brain circuits in vivo, here, we generated a mouse model with primate-lineage-specific isoforms of C4, human C4A and/or C4B. Human C4A bound synapses more efficiently than C4B. C4A (but not C4B) rescued the visual system synaptic refinement deficits of C4 knockout mice. Intriguingly, mice without C4 had normal numbers of cortical synapses, which suggests that complement is not required for normal developmental synaptic pruning. However, overexpressing C4A in mice reduced cortical synapse density, increased microglial engulfment of synapses and altered mouse behavior. These results suggest that increased C4A-mediated synaptic elimination results in abnormal brain circuits and behavior. Understanding pathological overpruning mechanisms has important therapeutic implications in disease conditions such as schizophrenia.

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Fig. 1: Human C4A and C4B are functionally active in a mouse model.
Fig. 2: Human C4A is more efficient than C4B in synaptic pruning.
Fig. 3: Increased C4A copy number induces excessive synaptic pruning via microglia engulfment.
Fig. 4: hC4A-overexpressing mice have increased synaptic material uptake by microglia in the mPFC.
Fig. 5: Adult hC4A-overexpressing mice have decreased synapse density in the mPFC.
Fig. 6: Human C4A overexpression alters mouse behavior.

Data availability

The bulk RNA-sequencing data have been uploaded to the NCBI GEO under accession code GSE161557. Source data for Extended Data Figs. 1a and 9c are provided in Supplementary Fig. 1. The datasets generated and analyzed during the current study, along with the detailed protocols and codes for the experiments, are available upon request from the corresponding author.

Code availability

The study utilized simple codes commonly used for bulk RNA sequencing. All scripts and codes are available upon request from the corresponding author.

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Acknowledgements

We thank members of the Carroll, Stevens and McCarroll laboratories for helpful comments on experimental design and feedback on the manuscript; E. M. Carroll for invaluable assistance; G. R. Andersen for providing the purified mouse C4 protein; X. Chen, D. Ghitza and L. Prince-Wright for technical support; B. Caldarone at the Mouse Behavior Core of Harvard Medical School and Brigham and Women’s Hospital; H. Leung at the Boston Children’s Hospital/PCMM microscopy core; the Boston Children’s Hospital/PCMM flow cytometry core; K. Holton at the HMS research computing core; and C. Usher and D. E. Potts for help with editing the manuscript. Parts of Figs. 2a and Fig. 3 were drawn by using pictures from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). Parts of Figs. 1a,b, 3a, 4a, 5a and 6c were created with BioRender (https://biorender.com/). This research was supported by funding from NIH P50MH112491–01 (to B.S., S.A.M. and M.C.C.) and AR072965 (to M.C.C.).

Author information

Affiliations

Authors

Contributions

M.Y., E.Y., J.P. and M.C.C. designed all the experiments. J.P. performed the BAC microinjections and created the human transgenic mice. M.Y. and J.P. conducted the splenic complement deposition, eye-specific segregation, microglia engulfment and behavior experiments and analyzed the corresponding data. M.Y. performed and analyzed the dendritic spine density analysis and the RNA sequencing. E.Y. performed and analyzed the synapse density analyses, the microglia reconstruction/engulfment assays and the C1q, C3 and C4 ELISAs for serum and tissue. E.A. assisted with the Cell Profiler analysis, the RNA-sequencing analysis and editing the manuscript. M.M. assisted in the generation of the human transgenic mice and the serum and tissue ELISAs. C.W.W. performed the whole-genome sequencing analysis. M.Y., E.Y. and J.P. wrote the manuscript. B.S. and S.A.M. provided expertise and comments on the experiments and the manuscript. M.C.C. conceived the development of the human C4A and C4B mice and advised on the design of experiments, participated in the analysis of the results and in writing the manuscript.

Corresponding author

Correspondence to Michael C. Carroll.

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Competing interests

B.S. serves on the scientific advisory board of Annexon LLC and is a minor shareholder of Annexon LLC; however, this is unrelated to the submitted work. All other authors declare no competing interests.

Additional information

Peer review information Nature Neuroscience thanks Marie-Eve Tremblay, Michisuke Yuzaki, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Extended data

Extended Data Fig. 1 Characteristics of C4 BAC DNA integration.

a-d, Insertion sites and associated rearrangements are shown for each mouse strain. a, c, The BAC DNA insertion site is shown for each mouse strain. Normalized read depth in 2-kb genomic windows at the insertion sites of the BACs. Dashed vertical lines indicate the insertion site. The hC4A insertion was associated with a duplication, and the hC4B insertion was associated with a small deletion. b, d, The BAC DNA-associated rearrangements are shown for each mouse strain. Normalized read depth in 1-kb genomic windows shows the copy number of the inserted constructs. Gene models are shown beneath the read depth plots. Human C4A was likely inserted into hC4A transgenic mice at a normalized read depth between 5 and 9. Human C4B was likely inserted into hC4B transgenic mice at a copy number between 2 and 5. The inserted hC4B BAC constructs contain additional internal copy number variants. e, Pulse-field gel showing linearized hC4B and hC4A BAC DNA used for microinjection into zygotes (representative of 3 independent experiments). Unprocessed versions of the gels can be found in Supplementary Figure 1a. f, Sema6D mRNA expression level in cortex was not changed by the region duplication caused by hC4A BAC DNA insertion. Gapdh was used as a control housekeeping gene (n = 4 mice per group; ns = P > 0.05, Kruskal-Wallis test with Dunn’s multiple comparisons test). Bar graph shows mean ± s.e.m.

Extended Data Fig. 2 Peripheral expression and function of C4 in hC4 transgenic mice.

a, C4A- and C4B-specific mRNA level was measured by ddPCR in the spleen (left) and liver (right) of hC4A/- and hC4B/- mice. eiF4H was used as the control housekeeping gene (n = 4 mice per group; * PSpleen = 0.0286, * PLiver = 0.0286; two-tailed Mann-Whitney test). b, C4 hemolytic activity was measured with equal amounts of hC4A or hC4B protein using sensitized sheep red blood cells. Data were normalized to hC4A samples (n = 8 hC4A and n = 15 hC4B combined from 3 independent experiments; **** P < 0.0001, Unpaired, two-tailed t test). Bar graphs show mean ± s.e.m.

Extended Data Fig. 3 Complement activation and binding to synaptosomes.

a, Representative dot plots showing the FSC-A / SSC-A of 1-µm beads, 6-µm beads, and synaptosomes. Further analysis will be gated on 1-µm synaptosomes. b, Synaptosomes were permeabilized and stained with anti-SV2 antibody (+ SV2 Ab) or no antibody (FMO CT). More than 85% of the particles analyzed contain SV2 protein. c, Representative histogram plot of C4 staining on synaptosomes isolated from C4-/- mice and incubated with serum from C4-/- (red), hC4A (orange), and hC4B (blue) mice. d, C1q deposition is shown and quantified using serum from hC4A mice (orange; n = 2), hC4B mice (blue; n = 2), or no serum CT (red). e, C4 (left) and C1q (right) deposition was detected on synaptosomes using fresh (red) or heat-inactivated (blue) serum. Bar graph shows mean ± s.e.m.

Extended Data Fig. 4 Human C4 in the retinogeniculate system.

a, hC4AB/- mice: gene copy numbers for C4A, C4B, C4A-L (C4L), and C4B-S (C4S) were determined by ddPCR using Rpp30 as a reference gene. It showed an insertion of two C4A-L genes and one C4B-L gene (n = 5 representative mice). Whole-genome sequencing revealed the hC4AB BAC DNA was inserted in chromosome 3 and that one C4A copy was truncated resulting in only one C4A coding copy (data not shown). b-c, Absolute quantification by ddPCR was used to measure C4A- and C4B-specific mRNA level in the retina and LGN from P5 hC4A/- (n = 3), hC4B/- (n = 3), and hC4AB/- mice (n = 2). Bar graphs show mean ± s.e.m.

Extended Data Fig. 5 Complement profile in hC4 transgenic mice during adolescence (P40).

a-c, Level of classical complement cascade proteins were measured in serum by ELISA in adolescent (P40) WT, hC4A/-, and hC4A/A mice. a, C4 serum level was measured in WT (n = 8), hC4A/- (n = 10), and hC4A/A mice (n = 6). b, C3 serum level was measured in WT (n = 5), hC4A/- (n = 10), and hC4A/A mice (n = 6). c, C1q serum level was measured in WT (n = 5), hC4A/- (n = 9), and hC4A/A mice (n = 8). d-f, RNA expression of classical complement cascade components in the FC were measured by ddPCR in adolescent (P40) WT, hC4A/-, and hC4A/A mice. All RNA measurements were normalized to Hs2st1 expression level. d, C4 mRNA expression level in FC was measured in WT (n = 4), hC4A/- (n = 4), and hC4A/A mice (n = 2). e, C3 mRNA expression level in FC was measured in WT (n = 4), hC4A/- (n = 3), and hC4A/A mice (n = 2). f, C1qb mRNA expression level in FC was measured in WT (n = 4), hC4A/- (n = 4), and hC4A/A mice (n = 2). Bar graphs show mean ± s.e.m.

Extended Data Fig. 6 C4A overexpression doesn’t change LGN cellularity or morphology.

a, Representative images of contralateral (top), ipsilateral (middle), and overlapping regions (bottom; scale = 20 µm). b-d, dLGN size (b), contralateral area (c), and ipsilateral area (d) were measured in P10 hC4A/- and hC4A/A mice (n = 6 hC4A/- and n = 6 hC4A/A littermates from 3 independent cohorts; two-tailed Mann-Whitney test; ns = P > 0.05, * P = 0.0411; scale = 20 µm). e, Iba1 staining was used to count the total number of microglia in the dLGN FOV (n = 4 mice per group; two-tailed Mann-Whitney test; ns = P > 0.05). f, Mice were injected with fluorescent-conjugated cholera toxin subunit-B (CTB) in each eye at P4 and brains were harvested at P5. Representative pseudo-colored images of P5 dLGN from C4-/-, hC4A/-, and hC4A/A littermates of contralateral (red), ipsilateral territories (green), and overlap (yellow) between the two territories (scale = 20 µm). Percent of overlap in ipsilateral region was compared in C4-/- (n = 3), hC4A/- (n = 4), and hC4A/A mice (n = 2; littermates). g, DAPI was used to count the total number of cells in the dLGN field of view (FOV) (n = 6 hC4A/- and n = 5 hC4A/A littermates from 3 independent cohorts; two-tailed Mann-Whitney test; ns = P > 0.05). h, The number of Brn3a+ RGCs was compared between C4-/-, hC4A/-, and hC4A/A mice (n = 5 mice per group; Kruskal-Wallis test with Dunn’s multiple comparisons test; ns = P > 0.05; scale = 20 μm). Bar graphs show mean ± s.e.m.

Extended Data Fig. 7 Cell Profiler microglia morphological analysis.

a, Cell Profiler software was used to analyze microglia morphology and lysosomal activity in the frontal cortex of P40 hC4A/- and hC4A/A mice. Microglial soma and processes are identified by Iba1 signature by using the image-based watershed method (top row). Microglia are used as a mask to select and quantify intracellular CD68 puncta (bottom row; scale = 50 μm). b-h, Morphological parameters for mPFC microglia (b-f) and their soma (g-h) from hC4A/- (n = 4) and hC4A/A (n = 5) mice were calculated by Cell Profiler software at P40 timepoint (ns = P > 0.05, two-tailed Mann-Whitney test). Bar graphs show mean ± s.e.m.

Extended Data Fig. 8 Microglial RNA sequencing analysis reveals no transcriptomic alterations in hC4 transgenic mice.

a, Bulk RNA sequencing analysis of microglia isolated from the frontal cortex of adolescent (P40) mice from WT (n = 4), C4-/- (n = 5), and hC4A/- (n = 5), hC4B/- (n = 4), and hC4A/A (n = 2) groups. Heatmap representation of differentially expressed genes between all experimental groups shows no significant transcriptional profile difference between any two groups. b-c, Normalized gene counts for the TREM2/DAP12 signaling pathway (b) and TAM receptor genes (c) were calculated for WT (n = 4), C4-/- (n = 5), hC4A/- (n = 5), hC4B/- (n = 4), and hC4A/A (n = 2; ns = P > 0.05, Kruskal-Wallis test with Dunn’s test). Bar graphs show mean ± s.e.m.

Extended Data Fig. 9 Synaptic protein expression is not affected by C4A overexpression in mice.

a-b, rt-PCR was used to measure mRNA expression of Sv2a (a) and Psd95 (b) in the FC in adult mice (C4-/- n = 2, hC4A/- n = 3, and hC4A/A littermates n = 7). RNA expression was normalized to Gapdh expression. c, SV2 and PSD95 protein level were analyzed by western blot. GAPDH was used as a loading control protein (C4-/- n = 4, hC4A/- n = 4, and hC4A/A n = 4 littermates from one experiment; Kruskal-Wallis test with Dunn’s multiple comparisons test; ns = P > 0.05). Unprocessed versions of the western blots can be found in Supplementary Figure 1b. d, Total SV2 area per FOV in the mPFC was calculated from immunofluorescence staining between WT (n = 3), C4-/- (n = 11), hC4A/- (n = 13), and hC4A/A (n = 6) groups (Kruskal-Wallis test with Dunn’s multiple comparisons test). e-f, Total Homer1 puncta and percentage of colocalized Homer1 puncta from the mPFC in adult mice were calculated for WT (n = 3), C4-/- (n = 11), hC4A/- (n = 13), and hC4A/A (n = 6) groups (Kruskal-Wallis test with Dunn’s multiple comparisons test; ns = P > 0.05, * PWT vs A/A = 0.0139, ** PWT vs A/A = 0.0139). g-h, Brain sections were stained with DAPI and NeuN, and cellularity was measured in frontal cortex at P180. Total cells (g) and neurons (h) per FOV in frontal cortex is represented for hC4A/- (n = 3) and hC4A/A mice (n = 4; two-tailed Mann-Whitney test; ns = P > 0.05). i, Method used for the manual counting of dendritic spines in mPFC of hC4 mice. j, Length of dendrites that have been used for spine density analysis for hC4A/- (80 dendrites) and hC4A/A (96 dendrites; two-tailed Mann-Whitney test; ns = P > 0.05). Bar graphs show mean ± s.e.m.

Extended Data Fig. 10 Human-C4A overexpression alters mouse behavior.

a-h, WT, C4-/-, hC4A/-, and hC4A/A mice were subjected to a battery of behavioral tests. a-b, Weight of male and female mice were compared between WT (n = 10), C4-/- (n = 4), hC4A/- (n = 9), and hC4A/A mice (n = 8; Kruskal-Wallis test with Dunn’s multiple comparisons test; ns = P > 0.05). c, Anxiety levels were measured in the light-dark box test by time spent in the light-zone between WT (n = 8), C4-/- (n = 22), hC4A/- (n = 20), and hC4A/A (n = 12) groups (two-tailed, unpaired t test; ** P = 0.0085; two-tailed Mann-Whitney test, *** P = 0.0009). Results were normalized to the C4-/- group to retain littermate controls. d, In the rotarod test, latency to fall was measured in seconds for WT (n = 10), C4-/- (n = 7), hC4A/- (n = 8), and hC4A/A (n = 7) groups (Kruskal-Wallis test with Dunn’s multiple comparisons test, ns = P > 0.05). e-f, Immobility time in seconds was compared in the tail suspension test (e) and the forced swim test (f) for WT (n = 10), C4-/- (n = 7), hC4A/- (n = 8), and hC4A/A (n = 7) groups (Kruskal-Wallis test with Dunn’s multiple comparisons test, ns = P > 0.05). g, Prepulse inhibition was measured between WT (n = 10), C4-/- (n = 7), hC4A/- (n = 8), and hC4A/A (n = 7) groups (two-way ANOVA with Tukey’s multiple comparisons test; ns = P > 0.05). h, Percent of correct decisions were recorded in the water t maze and the reversal water t maze for WT (n = 10), C4-/- (n = 7), hC4A/- (n = 8), and hC4A/A (n = 7) groups (two-way ANOVA with Tukey’s multiple comparisons test; ns = P > 0.05). Bar graphs show mean ± s.e.m. Box-and-whisker plots display the median (center line), 25th to 75th percentile (box), and minimum to maximum values (whiskers).

Supplementary information

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Supplementary Fig. 1 and titles for Supplementary Tables 1–5.

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Supplementary Tables 1–5.

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Yilmaz, M., Yalcin, E., Presumey, J. et al. Overexpression of schizophrenia susceptibility factor human complement C4A promotes excessive synaptic loss and behavioral changes in mice. Nat Neurosci 24, 214–224 (2021). https://doi.org/10.1038/s41593-020-00763-8

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