Complement and microglia mediate early synapse loss in Alzheimer mouse models

Abstract

Synapse loss in Alzheimer's disease (AD) correlates with cognitive decline. Involvement of microglia and complement in AD has been attributed to neuroinflammation, prominent late in disease. Here we show in mouse models that complement and microglia mediate synaptic loss early in AD. C1q, the initiating protein of the classical complement cascade, is increased and associated with synapses before overt plaque deposition. Inhibition of C1q, C3, or the microglial complement receptor CR3 reduces the number of phagocytic microglia, as well as the extent of early synapse loss. C1q is necessary for the toxic effects of soluble β-amyloid (Aβ) oligomers on synapses and hippocampal long-term potentiation. Finally, microglia in adult brains engulf synaptic material in a CR3-dependent process when exposed to soluble Aβ oligomers. Together, these findings suggest that the complement-dependent pathway and microglia that prune excess synapses in development are inappropriately activated and mediate synapse loss in AD.

Fig. 1. C1q upregulation and deposition onto synapses precede pre-plaque synapse loss in J20 mice

(A) Super-resolution SIM images of synaptophysin (green)- and PSD95 (red)-immunoreactive puncta in stratum radiatum of 3 mo J20 or WT hippocampus (CA1). Quantification of synaptic puncta or their apposition using Imaris indicates selective loss of PSD95 in J20 hippocampus as compared to their WT littermate controls. See Fig. S1. (B) Region-specific upregulation of C1q (green) in 1 mo J20; DG=dentate gyrus, FC=frontal cortex, STR=striatum, CRB=cerebellum. See Fig. S2. (C) Orthogonal view of SIM image showing colocalization of C1q (green) and PSD95 (red). (D) Higher % PSD95 colocalized with C1q in 1 mo J20 dentate gyrus vs. WT. (E) Compound E reduces deposited soluble Aβ (red) and C1q (green) in 1 mo J20 dentate gyrus, with minimal effect on C1q levels in WT mice. Scale bar = 2 (A, C and D) or 10 (B and E) μm. Means ± SEM; n = 3-4 mice per genotype or per treatment group per genotype. *P < 0.05 or ***P < 0.001 using two-way ANOVA followed by Bonferroni posttest (A and B), two-tailed one-sample t-test (D) or two-tailed unpaired t-test (E).

Fig. 2. Oligomeric Aβ increases C1q and microglial phagocytic activity

(A and B) Soluble Aβ oligomers in WT mice led to elevation of C1q (green) (A) and a higher % PSD95 (red) colocalization with C1q vs. monomers (B). (C and D) oAβ induced high levels of CD68 (green) immunoreactivity in Iba1-positive (red) microglia in WT mice (C), but not in those of C1qa KO mice (D). Both had negligible changes in morphology. See Fig. S10. Scale bar = 10 (A), 5 (B) or 20 (C) μm. Means ± SEM; n = 3-5 mice per treatment group per genotype. *P < 0.05 using two-tailed t-test (B) or *P < 0.05, **P < 0.01 vs. control-treated or ^##P < 0.01 vs. Aβ monomer-treated using two-way ANOVA followed by Bonferroni posttest (C).

Fig. 3. Complement is necessary for synapse loss and dysfunction in AD models

(A) Aβ oligomers induced loss of colocalized synapsin- and PSD95-immunoreactive puncta in contralateral hippocampus of 3 mo WT mice (left panel); however, they failed to do so in C1qa KO mice (right panel). (B) Co-injection of Aβ oligomers with the function-blocking antibody against C1q, ANX-M1, but not with its IgG isotype control, prevented synapse loss in WT mice. (C) Pre-treatment of hippocampal slices with the C1q antibody, ANX-M1, prevented Aβ-mediated LTP inhibition (green) vs. IgG (red). IgG alone had a minimal effect (blue) vs. aCSF vehicle (black). n = 6-11 slices per group. (D) % PSD95 colocalized with C3 is increased in APP/PS1 hippocampus vs. that of WT mice. (E and F) Genetic deletion of C3 prevents synapse loss in 4 mo APP/PS1 mice. Quantification of colocalized immunoreactive puncta for synaptotagmin and homer in dentate gyrus (E) or synaptophysin and PSD95 in CA1 stratum radiatum (F) of WT, APP/PS1, APP/PS1xC3 KO and C3 KO hippocampi. Means ± SEM; n = 3-5 mice per genotype or per treatment group per genotype. *P < 0.05, **P < 0.01 or ***P < 0.001 using two-tailed one-sample t-test (D), one-way (A, C, E, F) or two-way (B) ANOVA followed by Bonferroni posttest.

Fig. 4. Microglia engulf synapses via CR3 upon oligomeric Aβ challenge

(A) Orthogonal view of high-resolution confocal image shows colocalization of Homer-GFP and Iba1 (red). (B) 3D reconstruction and surface rendering using Imaris demonstrate bigger volumes of Homer-GFP puncta inside microglia of oAβ-injected contralateral hippocampus vs. those of monomer-injected. (C) Microglia of Homer-GFPxCR3 KO mice (right panel) show less engulfment of Homer-GFP when challenged with oAβ vs. those of Homer-GFP mice (left panel). (D) Aβ oligomers failed to induce synapse loss in contralateral hippocampus of CR3 KO mice (right panel) as they did in WT mice (left panel). Scale bar = 5 μm (A and B). Means ± SEM; n = 3 mice per treatment group per genotype (n = 6-17 microglia analyzed per mouse). *P < 0.05 or **P < 0.01 using two-tailed t-test (B) or two-tailed one-sample t-test (C and D).