Abstract
Immune molecules, including complement proteins C1q and C3, have emerged as critical mediators of synaptic refinement and plasticity. Complement localizes to synapses and refines the developing visual system through C3-dependent microglial phagocytosis of synapses. Retinal ganglion cells (RGCs) express C1q, the initiating protein of the classical complement cascade, during retinogeniculate refinement; however, the signals controlling C1q expression and function remain elusive. Previous work implicated an astrocyte-derived factor in regulating neuronal C1q expression. Here we identify retinal transforming growth factor (TGF)-β as a key regulator of neuronal C1q expression and synaptic pruning in the developing visual system. Mice lacking TGF-β receptor II (TGFβRII) in retinal neurons had reduced C1q expression in RGCs and reduced synaptic localization of complement, and phenocopied refinement defects observed in complement-deficient mice, including reduced eye-specific segregation and microglial engulfment of RGC inputs. These data implicate TGF-β in regulating neuronal C1q expression to initiate complement- and microglia-mediated synaptic pruning.
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Change history
13 January 2022
A Correction to this paper has been published: https://doi.org/10.1038/s41593-021-00877-7
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Acknowledgements
We thank C. Chen, L. Benowitz, D.P. Schafer and M. Buckwalter for comments on the manuscript and critical discussion. In addition we thank A. Stephan, B. Barres and A. Tenner for assistance with anti-C1q antibody production and characterization. We also thank M. Rasband (Baylor College of Medicine) for the βIV spectrin antibody. Thank you to D.P. Schafer and E.K. Lehrman for technical expertise on the microglial engulfment assay and Imaris image analysis, T. Nelson for technical assistance, and the imaging core at Boston Children's Hospital, including T. Hill, for technical support. This work was supported by grants from the Smith Family Foundation (B.S.), Dana Foundation (B.S.), Ellison Foundation (B.S.), John Merck Scholars Program (B.S.), NINDS (RO1-NS-07100801; B.S.), NIDA (RO1-DA-15043; B.A. Barres) and NIH (P30-HD-18655; MRDDRC Imaging Core).
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A.R.B. conducted all experiments, performed data analysis and wrote the manuscript. B.S. advised on and supervised the project and co-wrote the manuscript.
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This article has been retracted. Please see the retraction notice for more detail:https://doi.org/10.1038/nn.3560
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Supplementary Figure 1 In vitro characterization of cytokine or ACM-induced C1q upregulation
(A) C1r and c1s, the two genes that associate with C1q to form the C1 complex, are also significantly upregulated at 15 min by qPCR (one-way ANOVA, n=3 experiments, **p<0.01, ***p<0.001, F(1,8)=73.29). (B) A transcriptional inhibitor, actinomycin, blocks C1q upregulation, supporting that C1q upregulation is a transcriptional event (two-way ANOVA, n= 3 experiments, ***p<0.001, F(1,8)=4949.46). (C) Boiled ACM does not upregulate C1q, suggesting that a protein in ACM upregulates C1q (two-way ANOVA, n=3 experiments, ***p<0.001, F(1,8)=18.53). (D) IL-12 concentration response curve showed that RGCs did not upregulate C1q significantly (after 15 min. treatment) at any concentration tested (two-way ANOVA, n=3 samples per treatment, **p<0.01, *p<0.05, F(5,12)=14.75). (E) CXCL1 failed to upregulate C1q in RGCs (after 15 min. treatment) at any concentration tested (two-way ANOVA, n=3 samples per treatment, ***p<0.001, F(5,12)=11.06). (F) RT-PCR analysis of c1qa and c1qb expression shows a robust upregulation of C1q by insert (I) versus control (C), while IL-6 or TNFα treatment induces a modest increase in C1q. (G) TGF-β3 mRNA is enriched in retinal astrocytes compared to other astrocytes (McCarthy and de Vellis (MD) preparation and purified cortical astrocytes (cortical), RGCs, and microglia (Mg) (two-way ANOVA, n=3 experiments, ***p<0.001, F(8,45)=15.88).
Supplementary Figure 2 Validation of retinal Tgfbr2−/− mouse
(A) C1q immunostaining is developmentally regulated in the IPL and RGC retinal layers. Scale bar = 20μm. (B) Co-localization of TGFβRII and calretinin by immunohistochemistry indicates that TGFβRII is expressed on RGCs in vivo. Scale bar = 20μm. (C) RT-PCR using RNA from P5 whole brain lysates from retinal TGFβRII-/- and WT littermates shows no difference in tgfbr2 expression. (D) Validation that a known TGF-β-dependent gene, TIEG, is downregulated in the retinal TGFβRII−/− mouse and when anti-TGF-β is injected into the eye (one-way ANOVA, n=3 samples/group, **p<0.01, ***p<0.001, F(3,8)=27.93). (E) Whole mount retina immunostaining for Tuj1 shows no difference in the number of RGCs in TGFβRII retinal KO mice vs. WT littermates. (t test, n=4 mice/group, p=0.4040 no significance, t(6)=0.897539). Scale bar =30μm. (F) Immunohistochemistry with anti-βIV spectrin to mark axon initial segments showed similar numbers in WT and retinal TGFβRII−/− retinas. Scale bar = 30μm. Bottom panel: Axon density was measured by counting the number of fascicles (labeled by intraocular injection of CTB conjugated to Alexa 488) in the nerve/area of the optic nerve cross section. (G) Axon initial segment density was determined by measuring the area of axons/area of the field. Density was measures for 10 fields of view per mouse and normalized to WT (t test, n=3 mice, p=0.2993 (ns), t(4)=1.192). (H) Quantification of axon density showed no difference between WT and retinal TGFβRII−/− mice (t test, n=3 mice, p=0.5575 (ns), t(4)=0.6391).
Supplementary Figure 3 Blocking TGF-β signaling with anti-TGF-β reduces C1q expression levels
(A) In situ for c1qa shows expression of C1q in the RGC layer is significantly reduced in the retinal TGFβRII-/- mouse and shows a patchy reduction in anti-TGF-β injected mice. Scale bar =100μm. (B) RGCs acutely isolated from P5 WT saline injected (white bar) and anti-TGF-β injected (grey bar) mouse retinas using immunopanning showed a significant reduction in C1q expression (two-way ANOVA, n= 4 mice/group, *p<0.05, F(2,18)=7.108). Microglia acutely isolated using CD45 immunopanning show no difference in C1q levels. (C) Acutely isolated RGCs and microglia were checked for the expression of neuron specific and microglia specific genes, nse and iba1, respectively. RGCs were significantly enriched for nse compared to microglia and microglia were significantly enriched for iba1 compared to RGCs (two-way ANOVA, n=5 samples/group, **p<0.01,***p<0.001, F(1,16)=224.5). (D) Quantification of the relative fluorescence intensity in the IPL of anti-TGF-β injected and WT vehicle injected littermates shows a significant reduction in C1q localization to the IPL when TGF-β signaling is blocked (one-way ANOVA, n= 4 mice/group, *p<0.05, F(3,12)=6.306). Scale bar = 50μm. (E) Immunohistochemistry validation of C1q localization to RGCs and microglia as shown by C1q co-localization with markers for microglia (CD68) and RGCs (Calretinin). Co-localized cells indicated by arrows. Scale bar = 20μm. (F) Immunohistochemistry for C3 (Cappel, goat anti-C3) shows a reduction in C3 levels in the C1q-/- relative to WT littermate controls. Images shown are representative of 4 mice. Scale bar = 20μm.
Supplementary Figure 4 Microglia numbers and localization are unaffected in retinal Tgfbr2−/− mice
(A) Representative images stained for Iba1 show that microglia morphology and distribution are similar in WT, retinal TGFβRII-/-, and C1q-/- mice. Scale bar= 100μm. (B) Quantification of microglia density within the dLGN shows similar densities in WT, retinal TGFβRII-/-, and C1q-/- mice (one-way ANOVA, n= 4 animals/group, p=0.7849 (ns), F(2,9)=0.2489). Density was calculated as the number of microglia divided by the dLGN area, excluding optic tract. Numbers were normalized to WT. (C) Representative microglia from P5 dLGN showed similar activation states in WT, retinal TGFβRII-/-, and C1q-/- mice based on established activation state markers7. (D) WT, retinal TGFβRII-/-, and C1q-/- mice showed a similar distribution of microglia activation state in the P5 dLGN. Microglia from two dLGN sections were sampled and immunostained for Iba1 (Dako) and CD68. (two-way ANOVA, n=4 mice/condition, p=0.6463 (ns), F(4,20)=0.63).
Supplementary Figure 5 Model of complement-dependent synapse elimination
Our data and previous work support a model in which RGCs express C1q in response to TGF-β signaling and secrete C1q from their axon terminals in the dLGN. Once secreted, C1q localizes to inappropriate synapses where it triggers the classical complement cascade. C3 becomes activated and binds to inappropriate synapses, recruiting phagocytic microglia, expressing CR3, to remove inappropriate synapses by engulfment7. Microglia preferentially engulf less active retinogeniculate inputs (red axon)7; however it is not yet known whether or how complement targets weak inputs for elimination or if other mechanisms protect the specific (ie. stronger) synaptic inputs from elimination.
Supplementary Figure 6 Full-length pictures of the blots presented in the main figures
(A) Fig. 1d (B) Fig.3a (C) Fig. 3b (D) Fig. 2i (E) Fig. 3e
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Bialas, A., Stevens, B. RETRACTED ARTICLE: TGF-β signaling regulates neuronal C1q expression and developmental synaptic refinement. Nat Neurosci 16, 1773–1782 (2013). https://doi.org/10.1038/nn.3560
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DOI: https://doi.org/10.1038/nn.3560
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