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RETRACTED ARTICLE: TGF-β signaling regulates neuronal C1q expression and developmental synaptic refinement

A Retraction to this article was published on 13 January 2022

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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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Figure 1: C1q is rapidly upregulated in neurons in response to astrocyte-secreted factors.
Figure 2: TGF-β is necessary and sufficient for neuronal C1q upregulation in vitro.
Figure 3: TGF-β expression corresponds to synaptic refinement period in the retinogeniculate system.
Figure 4: TGF-β signaling is required for neuronal C1q expression in vivo.
Figure 5: Retinal TGF-β signaling is required for complement localization in the dLGN.
Figure 6: TGF-β signaling and C1q are required for eye-specific segregation and microglia-mediated pruning in the retinogeniculate system.
Figure 7: Mice deficient in C1q or retinal TGF-β signaling show increased overlap of contralateral and ipsilateral areas in the dLGN.

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References

  1. 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 

  2. 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 

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

    CAS  PubMed  Google Scholar 

  4. Campbell, G. & Shatz, C.J. Synapses formed by identified retinogeniculate axons during the segregation of eye input. J. Neurosci. 12, 1847–1858 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Sretavan, D. & Shatz, C.J. Prenatal development of individual retinogeniculate axons during the period of segregation. Nature 308, 845–848 (1984).

    CAS  PubMed  Google Scholar 

  6. Hooks, B.M. & Chen, C. Distinct roles for spontaneous and visual activity in remodeling of the retinogeniculate synapse. Neuron 52, 281–291 (2006).

    CAS  PubMed  Google Scholar 

  7. 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 

  8. Boulanger, L.M. Immune proteins in brain development and synaptic plasticity. Neuron 64, 93–109 (2009).

    CAS  PubMed  Google Scholar 

  9. Ullian, E.M., Sapperstein, S.K., Christopherson, K.S. & Barres, B.A. Control of synapse number by glia. Science 291, 657–661 (2001).

    CAS  PubMed  Google Scholar 

  10. Christopherson, K.S. et al. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell 120, 421–433 (2005).

    CAS  PubMed  Google Scholar 

  11. Allen, N.J. et al. Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors. Nature 486, 410–414 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Foo, L.C. et al. Development of a method for the purification and culture of rodent astrocytes. Neuron 71, 799–811 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Ullian, E.M., Christopherson, K.S. & Barres, B.A. Role for glia in synaptogenesis. Glia 47, 209–216 (2004).

    PubMed  Google Scholar 

  14. Stephan, A.H. et al. A dramatic increase of C1q protein in the CNS during normal aging. J. Neurosci. 33, 13460–13474 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Massagué, J. How cells read TGF-β signals. Nat. Rev. Mol. Cell Biol. 1, 169–178 (2000).

    PubMed  Google Scholar 

  16. Inman, G.J. et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62, 65–74 (2002).

    CAS  PubMed  Google Scholar 

  17. de Melo, J., Qiu, X., Du, G., Cristante, L. & Eisenstat, D.D. Dlx1, Dlx2, Pax6, Brn3b, and Chx10 homeobox gene expression defines the retinal ganglion and inner nuclear layers of the developing and adult mouse retina. J. Comp. Neurol. 461, 187–204 (2003).

    CAS  PubMed  Google Scholar 

  18. Rowan, S. & Cepko, C.L. Genetic analysis of the homeodomain transcription factor Chx10 in the retina using a novel multifunctional BAC transgenic mouse reporter. Dev. Biol. 271, 388–402 (2004).

    CAS  PubMed  Google Scholar 

  19. Barres, B.A., Silverstein, B.E., Corey, D.R. & Chun, L.L.Y. Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning. Neuron 1, 791–803 (1988).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  21. Schafer, D.P., Lehrman, E.K. & Stevens, B. The “quad-partite” synapse: Microglia-synapse interactions in the developing and mature CNS. Glia 61, 24–36 (2012).

    PubMed  PubMed Central  Google Scholar 

  22. Tremblay, M.E. et al. The role of microglia in the healthy brain. J. Neurosci. 31, 16064–16069 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 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 

  24. Tsui, C.C. et al. Narp, a novel member of the pentraxin family, promotes neurite outgrowth and is dynamically regulated by neuronal activity. J. Neurosci. 16, 2463–2478 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Yuzaki, M. Synapse formation and maintenance by C1q family proteins: a new class of secreted synapse organizers. Eur. J. Neurosci. 32, 191–197 (2010).

    PubMed  Google Scholar 

  26. Beier, M., Franke, A., Paunel-Görgülü, A.N., Scheerer, N. & Dünker, N. Transforming growth factor beta mediates apoptosis in the ganglion cell layer during all programmed cell death periods of the developing murine retina. Neurosci. Res. 56, 193–203 (2006).

    CAS  PubMed  Google Scholar 

  27. Peterziel, H., Unsicker, K. & Krieglstein, K. TGFβ induces GDNF responsiveness in neurons by recruitment of GFRα1 to the plasma membrane. J. Cell Biol. 159, 157–167 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Yi, J.J., Barnes, A.P., Hand, R., Polleux, F. & Ehlers, M.D. TGF-[beta] Signaling Specifies Axons during Brain Development. Cell 142, 144–157 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Diniz, L.P. et al. Astrocyte-induced synaptogenesis is mediated by transforming growth factor β signaling through modulation of d-serine levels in cerebral cortex neurons. J. Biol. Chem. 287, 41432–41445 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Feng, Z. & Ko, C.-P. Schwann cells promote synaptogenesis at the neuromuscular junction via transforming growth factor-β1. J. Neurosci. 28, 9599–9609 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Fuentes-Medel, Y. et al. Integration of a retrograde signal during synapse formation by glia-secreted TGF-β ligand. Curr. Biol. 22, 1831–1838 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Ball, R.W. et al. Retrograde BMP signaling controls synaptic growth at the NMJ by regulating trio expression in motor neurons. Neuron 66, 536–549 (2010).

    CAS  PubMed  Google Scholar 

  33. Awasaki, T., Huang, Y., O'Connor, M.B. & Lee, T. Glia instruct developmental neuronal remodeling through TGF-β signaling. Nat. Neurosci. 14, 821–823 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Yu, X.M. et al. Plum, an immunoglobulin superfamily protein, regulates axon pruning by facilitating TGF-β signaling. Neuron 78, 456–468 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Heupel, K. et al. Loss of transforming growth factor-beta 2 leads to impairment of central synapse function. Neural Dev. 3, 25 (2008).

    PubMed  PubMed Central  Google Scholar 

  36. Chin, J., Angers, A., Cleary, L.J., Eskin, A. & Byrne, J.H. Transforming growth factor beta 1 alters synapsin distribution and modulates synaptic depression in Aplysia. J. Neurosci. 22, RC220 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Lu, C.C., Appler, J.M., Houseman, E.A. & Goodrich, L.V. Developmental profiling of spiral ganglion neurons reveals insights into auditory circuit assembly. J. Neurosci. 31, 10903–10918 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Huang, L.-C., Thorne, P.R., Housley, G.D. & Montgomery, J.M. Spatiotemporal definition of neurite outgrowth, refinement and retraction in the developing mouse cochlea. Development 134, 2925–2933 (2007).

    CAS  PubMed  Google Scholar 

  39. Wyss-Coray, T. et al. Amyloidogenic role of cytokine TGF-β1 in transgenic mice and in Alzheimer's disease. Nature 389, 603–606 (1997).

    CAS  PubMed  Google Scholar 

  40. Town, T. et al. Blocking TGF-β–Smad2/3 innate immune signaling mitigates Alzheimer-like pathology. Nat. Med. 14, 681–687 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Afagh, A., Cummings, B.J., Cribbs, D.H., Cotman, C.W. & Tenner, A.J. Localization and cell association of C1q in Alzheimer's disease brain. Exp. Neurol. 138, 22–32 (1996).

    CAS  PubMed  Google Scholar 

  42. Fonseca, M.I., Zhou, J., Botto, M. & Tenner, A.J. Absence of C1q leads to less neuropathology in transgenic mouse models of Alzheimer's disease. J. Neurosci. 24, 6457–6465 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. McCarthy, K.D. & De Vellis, J. Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J. Cell Biol. 85, 890–902 (1980).

    CAS  PubMed  Google Scholar 

  44. Botto, M. et al. Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat. Genet. 19, 56–59 (1998).

    CAS  PubMed  Google Scholar 

  45. Meyer-Franke, A., Kaplan, M.R., Pfrieger, F.W. & Barres, B.A. Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron 15, 805–819 (1995).

    CAS  PubMed  Google Scholar 

  46. Doyle, K. et al. TGFβ signaling in the brain increases with aging and signals to astrocytes and innate immune cells in the weeks after stroke. J. Neuroinflam. 7, 62 (2010).

    Google Scholar 

  47. Dyer, M.A. & Cepko, C.L. p57 (Kip2) regulates progenitor cell proliferation and amacrine interneuron development in the mouse retina. Development 127, 3593–3605 (2000).

    CAS  PubMed  Google Scholar 

  48. Lázár-Molnár, E. et al. Programmed death-1 (PD-1)–deficient mice are extraordinarily sensitive to tuberculosis. Proc. Natl. Acad. Sci. USA 107, 13402–13407 (2010).

    PubMed  PubMed Central  Google Scholar 

  49. Truckenmiller, M.E. et al. AF5, a CNS cell line immortalized with an N-terminal fragment of SV40 large T: growth, differentiation, genetic stability, and gene expression. Exp. Neurol. 175, 318–337 (2002).

    CAS  PubMed  Google Scholar 

  50. Liu, G. et al. Requirement of Smad3 and CREB-1 in Mediating Transforming Growth Factor-β (TGFβ) Induction of TGFβ3 Secretion. J. Biol. Chem. 281, 29479–29490 (2006).

    CAS  PubMed  Google Scholar 

  51. Jaubert-Miazza, L. et al. Structural and functional composition of the developing retinogeniculate pathway in the mouse. Vis. Neurosci. 22, 661–676 (2005).

    PubMed  Google Scholar 

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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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Correspondence to Beth Stevens.

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This article has been retracted. Please see the retraction notice for more detail:https://doi.org/10.1038/nn.3560

Integrated supplementary information

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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