the formation of mature neural circuits
The Classical Complement Cascade Mediates CNS Synapse Elimination Beth Stevens,1,* Nicola J. Allen,1 Luis E. Vazquez,1 Gareth R. Howell,3,4 Karen S. Christopherson,1 Navid Nouri,1
Kristina D. Micheva,2 Adrienne K. Mehalow,3,4 Andrew D. Huberman,1 Benjamin Stafford,5 Alexander Sher,5
Alan M. Litke,5 John D. Lambris,6 Stephen J. Smith,2 Simon W.M. John,3,4 and Ben A. Barres1 1Department of Neurobiology 2Department of Molecular and Cellular Physiology 3Stanford University School of Medicine, Stanford, CA 94305, USA Howard Hughes Medical Institute 4The Jackson Laboratory, Bar Harbor, ME 04609, USA 5Santa Cruz Institute for Particle Physics, University of California, Santa Cruz, Santa Cruz, CA 95064, USA 6Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical School, Pennsylvania, PA 19104, USA *Correspondence: beths@stanfordmedalumni.org DOI 10.1016/j.cell.2007.10.036
SUMMARY
During development, the formation of mature neural circuits requires the selective elimination of inappropriate synaptic connections. Here we show that C1q, the initiating protein in the clas- sical complement cascade, is expressed by postnatal neurons in response to immature as- trocytes and is localized to synapses through- out the postnatal CNS and retina. Mice deficient in complement protein C1q or the downstream complement protein C3 exhibit large sustained defects in CNS synapse elimination, as shown by the failure of anatomical refinement of retino- geniculate connections and the retention of ex- cess retinal innervation by lateral geniculate neurons. Neuronal C1q is normally downregu- lated in the adult CNS; however, in a mouse model of glaucoma, C1q becomes upregulated and synaptically relocalized in the adult retina early in the disease. These findings support a model in which unwanted synapses are tagged by complement for elimination and sug- gest that complement-mediated synapse elim- ination may become aberrantly reactivated in neurodegenerative disease.
INTRODUCTION
The formation of mature neural circuits requires the activ- ity-dependent pruning of inappropriate synapses (Katz and Shatz, 1996; Sanes and Lichtman, 1999; Hua and Smith, 2004), but the specific molecular mechanisms that drive synapse elimination are not known. The mouse retinogeniculate system has proven to be an excellent model system for studying developmental CNS synapse
elimination (Jaubert-Miazza et al., 2005; Hooks and Chen, 2006). Axons from retinal ganglion cells (RGCs) ter- minate in distinct nonoverlapping eye-specific domains in the dorsal lateral geniculate nucleus (dLGN). The majority of eye-specific segregation occurs postnatally before the onset of vision, but synaptic pruning continues in monoc- ular regions of the LGN during a 2 week period spanning eye opening (P8-P30 in mouse). Initially, dLGN neurons are multiply innervated by up to ten RGC axons, but by the third postnatal week, each dLGN neuron receives sta- ble inputs from only one or two RGC axons (Hooks and Chen, 2006). This developmental shift in synaptic conver- gence represents the elimination of inappropriate retino- geniculate synapses and the maintenance and strength- ening of appropriate synaptic connections. This dynamic period of synaptic refinement coincides with the appear- ance of astrocytes in the postnatal brain, and recent evi- dence indicates a role for astrocyte-derived signals in synapse development (Christopherson et al., 2005; Ullian et al., 2001).
Here we identify an unexpected role for astrocytes and the classical complement cascade in mediating CNS syn- apse elimination in the retinogeniculate pathway. By gene profiling, we found that all three chains of the complement protein C1q are strongly upregulated when purified RGCs are exposed to astrocytes. C1q is the initiating protein of the classical complement cascade, which is part of the innate immune system. When C1q binds to and coats (opsonizes) dead cells, pathogens, or debris, it triggers a protease cascade, leading to the deposition of the downstream complement protein C3 (Gasque, 2004). Opsonization with activated C3 fragments (C3b and iC3b) leads to cell or debris elimination in one of two ways. Deposited C3 can directly activate C3 receptors on macrophages or microglia, thereby triggering elimina- tion by phagocytosis, or activated C3 can trigger the ter- minal activation of the complement cascade, leading to cell lysis through the formation of a lytic membrane attack complex. We show that complement proteins opsonize or
1164 Cell 131, 1164–1178, December 14, 2007 ª2007 Elsevier Inc.
mailto:beths@stanfordmedalumni.org
‘‘tag’’ CNS synapses during a discrete window of postna- tal development and that the complement proteins C1q and C3 are required for synapse elimination in the devel- oping retinogeniculate pathway. We also show that C1q becomes aberrantly upregulated and relocalized to adult retinal synapses in a mouse model of glaucoma at an early stage of the disease prior to overt neurodegeneration, suggesting that the complement cascade also mediates synapse loss in glaucoma and other CNS neurodegenera- tive diseases.
RESULTS
Astrocytes Upregulate All Three C1q Subunit mRNAs in Retinal Ganglion Cells We used a gene profiling approach to screen candidate neuronal genes that are regulated by astrocytes. RNA was collected from purified postnatal RGCs that had been cultured for 1 week in the presence or absence of a feeding layer of cultured neonatally derived astrocytes, and the target RNA was hybridized to an Affymetrix gene chip. We found that mRNA for all three C1q chains was upregulated by astrocytes in purified RGCs by 10- to 30- fold, and we verified this upregulation using semiquantita- tive RT-PCR (Figure 1A). To confirm that C1q mRNA is normally expressed by developing RGCs in vivo, we per- formed RT-PCR analysis on mRNA collected from purified
RGCs that were acutely isolated (Figure 1B) as well as in situ hybridization on whole retina (Figure 1C). We found that C1q mRNA levels were highest in postnatal RGCs between P5 and P10 and declined significantly by P30 (Figures 1B and 1C). Thus, C1q mRNA is expressed by RGCs in vitro in response to astrocytes and is normally ex- pressed by postnatal, but not adult, RGCs in vivo during a window of development corresponding to the presence of immature astrocytes.
C1q Immunoreactivity Is Localized to Synapses throughout the Developing CNS We next investigated whether C1q protein could be de- tected in the developing mouse CNS by immunostaining cryosections. Using several different C1q antisera, we ob- served bright, punctate C1q immunoreactivity throughout the developing retina that was enriched in the synaptic inner plexiform layer (IPL) of postnatal mouse retinas and was also observed in developing RGCs (Figure 2A). Consistent with the expression pattern of C1q mRNA in postnatal RGCs in vivo (Figure 1), C1q protein expression and synaptic localization followed a similar developmental pattern, being higher in the IPL at P5 and decreasing in the mature retina (Figure 2A). In addition, many C1q-positive puncta in the IPL were associated with synaptic puncta identified by double immunostaining with synaptic markers such as PSD-95 (Figure 2B). Together, these
Figure 1. Astrocytes Upregulate C1q Expression by Neurons (A) RT-PCR validation of our gene chip analysis
confirms that astrocyte exposure upregulates
mRNAs for all three chains (A, B, and C) of
C1q in purified postnatal RGC neurons in
culture.
(B) C1q is highly expressed by RGCs in vivo.
RT-PCR analysis of mRNA isolated from
RGCs that were acutely isolated from P5 retina
(left lane) and PBS perfused whole postnatal
mouse retina from different developmental
time points (P5–P30).
(C) In situ hybridization confirmation that C1q is
expressed by developing, but not mature,
RGCs in vivo. Expression of C1qA was pre-
dominantly localized to retinal ganglion neu-
rons (arrows) in fresh frozen sections of post-
natal (P5) retina, but C1q gene expression
was largely absent in RGCs by P30. RGC, ret-
inal ganglion cell layer; IPL, inner plexiform
layer; INL, inner nuclear layer; OPL, outer plex-
iform layer; ONL, outer nuclear layer. Scale bar,
20 mm.
Cell 131, 1164–1178, December 14, 2007 ª2007 Elsevier Inc. 1165
findings indicate that C1q protein is expressed by RGCs in vivo and that C1q is synaptically localized to the postnatal, but not mature, IPL.
To find out if C1q protein is also present in the brain, we next immunostained cryosections from postnatal and ma- ture brain. We again observed punctate C1q staining in synaptic regions, particularly between P4 and P10, that
closely resembled the pattern of immunoreactivity ob- served with synaptic markers such as SV2 (Figure 2C). Importantly, a similar expression pattern was observed in the postnatal dLGN, a major target of RGC axons (Figure 2D). As in the retina, synaptic C1q immunoreactiv- ity in the cortex and LGN was developmentally downregu- lated (Figures 2C and 2D) and was largely absent when the
Figure 2. C1q Is Localized to Developing CNS Synapses In Vivo (A) Longitudinal cryosection of a P8 mouse retina stained with anti-C1q. Layers of the retina are labeled on the right. Punctate C1q immunoreactivity is
enriched in the synaptic inner plexiform layer (IPL). RGC, retinal ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plex-
iform layer; ONL, outer nuclear layer. Scale bar, 20 mm. Confocal imaging demonstrates the developmental enrichment of C1q in synaptic IPL of the
mouse retina (panels, right). Zoomed-in images of IPL are from a region comparable to the boxed area in the epifluorescence image in panel (left).
Punctate C1q immunoreactivity in the IPL was highest at P5 and largely decreased after P15. Scale bar, 20 mm.
(B) Double labeling of C1q (green) with the postsynaptic marker, PSD95 (red), demonstrates C1q-positive puncta in close proximity to PSD95 puncta
in postnatal P5 retina….
