Complement C3aR Inactivation Attenuates Tau Pathology and Reverses an Immune Network Deregulated in Tauopathy Models and Alzheimer's Disease

Abstract

Strong evidence implicates the complement pathway as an important contributor to amyloid pathology in Alzheimer's disease (AD); however, the role of complement in tau modulation remains unclear. Here we show that the expression of C3 and C3a receptor (C3aR1) are positively correlated with cognitive decline and Braak staging in human AD brains. Deletion of C3ar1 in PS19 mice results in the rescue of tau pathology and attenuation of neuroinflammation, synaptic deficits, and neurodegeneration. Through RNA sequencing and cell-type-specific transcriptomic analysis, we identify a C3aR-dependent transcription factor network that regulates a reactive glial switch whose inactivation ameliorates disease-associated microglia and neurotoxic astrocyte signatures. Strikingly, this C3aR network includes multiple genes linked to late-onset AD. Mechanistically, we identify STAT3 as a direct target of C3-C3aR signaling that functionally mediates tau pathogenesis. All together our findings demonstrate a crucial role for activation of the C3-C3aR network in mediating neuroinflammation and tau pathology.

Keywords: Alzheimer’s disease; C3aR; STAT3; complement; microglia; neuroinflammation; tau.

Figure 1.. Upregulation of C3 and C3aR in AD and tauopathy conditions.

(A, B) C3 (A) and C3aR1 (B) mRNA levels in the parahippocampal gyrus of NCI (n=32), MCI (n=34), or AD (Dementia, n=160). Spearman’s correlation, ρ (C3)=0.36, p<2.6E-08; ρ (C3aR)=0.33, p<3.4E-07. (C, D) Positive correlation of C3 (C) and C3aR1 (D) mRNA levels with Braak scores (from I to VI: n(I)=24, n(II)=34, n(III)=37, n(IV)=22, n(V)=25, n(VI)=62). Spearman’s correlation, ρ (C3)=0.34, p<4.4E-07; ρ (C3aR)=0.36, p<1.6E-07. Data used for A-D are from the Mount Sinai datasets available at the AMP-AD portal (Syn8484987). (E, F) qPCR analysis of C3 (E) and C3aR1 (F) mRNA levels in MFC of human tauopathy brain samples (mean ± s.e.m). NCI (n=6), CBD (n=5), Pick｀s disease (n=5), PSP (n=5). One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; *ρ<0.05; **ρ<0.01; ***ρ<0.001. (G, H) qPCR analysis of C3 (G) and C3ar1 (H) mRNA levels in 4, 6 and 9 month-old wild-type (WT) and PS19 mice (mean ± s.e.m). n=4/genotype/age. Two-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; ***ρ<0.001. (I) ELISA analysis of C3 protein levels in brain samples of 4, 6 and 9 month-old WT and PS19 mice (mean ± s.e.m). n=3/genotype/age. Two-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; *ρ<0.05; ***ρ<0.001. (J) Representative co-immunostaining of C3aR1 and Iba1 with DAPI in the hippocampus of 9 month-old WT and PS19 mice. C3aR KO is used as a negative control. Scale bar: 50 μM. (K) Quantification of (J) (mean ± s.e.m). n=6/genotype. Student｀s t-test. ***ρ<0.001. See also Figure S1.

Figure 2.. Genetic deletion of C3ar1 attenuates reactive gliosis and neuroinflammation in PS19 mice.

(A)Representative GFAP and Iba1 immunostainings with DAPI in the hippocampus of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. Scale bar: 50 μM. (B, C) Quantification of GFAP (B) and Iba1 (C) immunoreactivities (mean ± s.e.m). n=6 mice/genotype. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; ***p<0.001. (D)C3 and GFAP co-immunostaining in the hippocampus of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. Scale bar: 25 μM. (E)Quantification of (D) (mean ± s.e.m). n=6 mice/genotype. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; ***p<0.001. (F) CD68 and Iba1 co-immunostaining in the hippocampus of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. Scale bar: 25 μM. (G) Quantification of (E) (mean ± s.e.m). n=6 mice/genotype. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; ***p<0.001. (H-K) C3 (H), TNFα (I), IL6 (J) and IL 1β (K) protein levels in 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mouse brains measured by ELISA (mean ± s.e.m). n=6/genotype for all experiments. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; *p<0.05; ***p<0.001. See also Figure S2.

Figure 3.. Genetic deletion of C3ar1 reduces tau pathology and rescues behavioral deficits in PS19 mice.

(A) Representative Western blots of PHF1- and CP13-positive phospho-tau and total tau in PS19 and PS19/C3aR KO mouse brains at 9 months. γ-tubulin is used as a loading control. (B) Quantification (A) (mean ± s.e.m). n=6/genotype. Student｀s t-test. n.s.: non-significant; ***p<0.001. (C) Representative PHF1 immunohistochemical staining in hippocampus (HPC) and cortex (CTX) of 9 month-old PS19 and PS19/C3aR KO mice. Scale bar: 0.5 mm. (D) Quantification of (C) (mean ± s.e.m). n=6/genotype. Student｀s t-test. ***p<0.001. (E) Contextual (left) and cued (right) fear conditioning test performed in 8 month-old wild-type (WT, n=14), C3aR KO (KO, n=17), PS19 (PS, n=14), and PS19/C3aR KO (PS/KO, n=15) mice. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; *p<0.05; **p<0.01. Data shown is mean ± s.e.m. (F) Slope of field excitatory postsynaptic potential (fEPSP) in response to theta burst stimulation delivered to the Schaffer collateral pathway from WT, C3aR KO, PS19, and PS19/C3aR KO mice. Top: example fEPSP traces taken before (blue) or after (red) stimulation. Calibration: 2 mV, 5 msec. n=number of slices per genotype. Two-way repeated measures ANOVA (between groups) followed by Tukey｀s HSD test. ***p<0.001. Data shown is mean ± s.e.m. See also Figure S3.

Figure 4.. Genetic deletion of C3ar1 rescues synaptic deficits and neurodegeneration in PS19 mice.

(A) Representative synaptophysin (Syp) and Syp/NeuN co-immunostaining in area CA3 of hippocampus of WT, C3aR KO, PS19, and PS19/C3aR KO mice at 9 months. Scale bar: 50 μM. (B) Quantification of (A) (mean ± s.e.m). n=6/genotype. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; ***p<0.001. (C) Representative high magnification confocal images of Syp and PSD-95 co-immunostaining in area CA3 of hippocampus of WT, C3aR KO, PS19, and PS19/C3aR KO mice at 9 months. Colocolized punctas are marked by circles. Scale bar: 5 μM. (D) Quantification of (C) (mean ± s.e.m). n=6/genotype with 5-7 planes per mouse. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; **p<0.01; ***p<0.001. (E) Representative 3D reconstruction and rendering of Syp signals inside Iba1-positive microglia (Iba1/Syp) from WT, C3aR KO, PS19, and PS19/C3aR KO mice at 9 months using the Imaris software. Scale bar: 5 μM. (F) Quantification of (E) (mean ± s.e.m). n=6/genotype; 10-15 cells/mouse were quantified. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; ***p<0.001. (G) Representative NeuN immunohistochemical staining in hippocampus of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. Rectangles mark CA1 (upper) and CA3 (lower) areas selected for quantification. Scale bar: 0.5 mm. (H) Estimate of neuronal numbers in CA1 (left) and CA3 (right) using unbiased stereology. n=6/genotype; three equidistant planes separated by 150 μM per animal. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; **p<0.01; ***p<0.001. See also Figure S3.

Figure 5.. C3aR regulates the expression of immune pathway genes in PS19 mice conserved in human AD.

(A) Unsupervised clustering gene expression heatmap from RNAseq analysis of hippocampal tissues of 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. 1726 DEGs were identified by comparing the PS19 vs WT group (1155 genes upregulated, red, and 571 downregulated, blue). n=4-5/genotype, adjusted p<0.01. (B) Venn diagram with number of DEGs identified in four comparisons: PS19 vs WT (red circle); PS19/C3aR KO vs PS19 (blue circle); PS19/C3aR KO vs WT (grey circle) and C3aR KO vs WT (unfilled circle). The 526 genes rescued by C3aR KO (significant between PS19/C3aR KO vs PS19 and insignificant between PS19/C3aR KO vs WT) are highlighted in bold. (C) Comparison of expression changes between PS19 vs WT (y-axis) and PS19/C3aR KO vs PS19 (x-axis) showing that the 526 genes rescued by C3aR KO (blue) are predominately clustered within the upregulated DEGs in PS19 vs WT (red, n=499) in the upper left quadrant. (D) Gene Ontology analysis of the 1155 upregulated genes in PS19 vs WT comparison (red) and the 499 upregulated genes rescued by C3aR KO (blue). (E) A C3AR1-centered network confirmed in both human AD brain studies (using consensus C3AR1-correlated gene signatures) and PS19/C3aR KO mice. Genes within this network significantly correlate with C3AR1 expression in at least five human AD datasets (MSSM BM10, 22, 36, 44; HBTRS PFC, VC, CR; ROS-MAP). The node size is proportional to the number of datasets in which a particular gene significantly correlates with C3AR1 expression. Red nodes represent genes that are positively correlated with C3AR1 in human AD studies and are downregulated in PS19/C3aR KO mice (n=301). Blue nodes represent the genes negatively correlated with C3AR1 in human AD datasets and upregulated in PS19/C3aR KO mice (n=7). Pink nodes represent the genes negatively correlated with C3AR1 in human AD datasets but not upregulated in PS19/C3aR KO mice (n=3). Human AD risk factors correlated with C3AR1 expression are as indicated. See also Figure S4.

Figure 6.. C3aR signaling activates the inflammatory and microglia proliferation transcription factors in PS19 mice.

(A) Gene-set enrichment analyses (GSEA) of the microglia sensome gene list in PS19 vs WT (left, NES=2.81, FDR<0.001) and in PS19/C3aR KO vs PS19 (right, NES= −3.0, FDR<0.001) comparisons. NES: normalized enrichment score; FDR: false discovery rate. (B) GSEA of the reactive astrocyte gene list in PS19 vs WT (NES=2.22, FDR<0.001) and in PS19/C3aR KO vs PS19 (NES = −2.36, FDR<0.001) comparisons. (C) GSEA of the complement pathway gene list in PS19 vs WT (NES=2.22, FDR<0.001) and in PS19/C3aR KO vs PS19 (ES= −1.95, FDR<0.001) comparisons. (D, E) qPCR analysis of mRNA expression in CoBrA-isolated microglia (D) and astrocytes (E) from WT, C3aR KO, PS19, and PS19/C3aR KO mice. The asterisks in PS19 column represent the comparison between PS19 and WT, and in PS19/C3aR KO represent the comparison between PS19/C3aR KO and PS19. n=6/genotype. Two-way ANOVA followed by Tukey’s HSD test. *p<0.05; **p<0.01; ***p<0.001. (F) Transcription factor network analysis of the 1726 DEGs in PS19 vs WT mice generated using the TRANSFAC database. Hub genes in the network denote the TFs that regulate the transcription of DEGs in PS19. Other network nodes represent the TFs’ downstream targets. The size of the node corresponds to the number of gene connections while the color intensity represents the degree of changes comparing PS19 vs WT (red: upregulated; green: downregulated). Genes rescued in PS19/C3aR KO are highlighted by a dark red circle around the node. (G, H) qPCR analysis of hub gene expression levels in CoBrA-isolated microglia (G) and astrocytes (H). The asterisks above PS19 represent the PS19 vs WT comparison and those above PS19/C3aR KO is for PS19 vs PS19/C3aR KO comparison. n=6/genotype. Student｀s t-test. *p<0.01; **p<0.01; ***p<0.001. All data is presented as mean ± s.e.m. See also Figures S5-S7.

Figure 7.. Direct regulation of STAT3 phosphorylation and signaling by C3-C3aR.

(A, B) Total STAT3 (A) and Y705-phospho-STAT3 (B) levels in 9 month-old WT (n=10), C3aR KO (KO, n=10), PS19 (PS, n=7), and PS19/C3aR KO (PS/KO, n=7) mice measured by RPPA (mean ± s.e.m). The asterisks above PS19 are for PS19 vs WT comparison; those above PS19/C3aR KO are for PS19/C3aR KO vs PS19 comparison. Student｀s t-test. *p<0.05; **p<0.01. (C) Western blots of total- and phospho-STAT3 (pSTAT3) in 9 month-old WT, C3aR KO, PS19, and PS19/C3aR KO mice. γ-tubulin is used as a loading control. (D, E) Quantification of total STAT3/ γ-tubulin (D) and pSTAT3/STAT3 (E) levels. n=3-4/genotype. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; *p<0.05; **p<0.01; ***p<0.001. (F) Representative Western blots of pSTAT3 and STAT3 in BV2 cells treated with vehicle control (Ctrl), C3aR antagonist (C3aRA, 100 uM), IL6 (50 nM), C3 (100 nM) or C3 plus C3aRA (C3/C3aRA). γ-tubulin is used as a loading control.. (G) Representative pSTAT3 immunostaining with or without DAPI in BV2 cells with the same treatment as (F). Scale bar: 20 μM. (H) Quantification of nuclear/cytoplasmic pSTAT3. n=50 cells/treatment. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; ***p<0.001.. (I) Relative luciferase activity in BV2 cells infected with the luciferase reporter driven by the STAT3 response element and with the same treatment as (F). One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; ***p<0.001. (J) qPCR analysis of SOCS3 mRNA levels in BV2 cells with the same treatment as (F). Oneway ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; **p<0.01. (K) Representative Western blots of pSTAT3 and STAT3 in primary WT microglial cultures treated with vehicle (Ctrl), C3aRA, IL6, C3 or C3 plus C3aRA. γ-tubulin is used as a loading control. (L) Representative Western blot analysis of pSTAT3 and STAT3 in primary microglial cultures derived from C3aR KO mice treated with Ctrl, IL6, or C3. γ-tubulin is used as a loading control. (M, N) qPCR analysis of SOCS3 mRNA levels in primary WT (M) or C3aR KO (N) microglial cultures with treatments as indicated. One-way ANOVA followed by Sidak｀s HSD test. n.s.: nonsignificant; *p<0.05; **p<0.01; ***p<0.001. All data is presented as mean ± s.e.m. All cell culture experiments were repeated three to four times each in triplicates. See also Figure S8.

Figure 8.. STAT3 inhibitor treatment mitigates tau pathology and neuroinflammation in aged PS19 mice.

(A) Representative Western blots using PHF1 and CP13 phospho-tau antibodies and antibodies against total tau, pSTAT3 and total STAT3 in brain samples of WT and PS19 mice treated with vehicle control (VC) or SH-4-54 (SH, 10 mg/kg) starting at 7 months for 2 months. γ-tubulin is used as a loading control. (B) Quantification of (A) (mean ± s.e.m). n=8/genotype. Student｀s t-test. n.s.: non-significant; **p<0.01; ***p<0.001. (C) Representative PHF1 immunohistochemical staining in the hippocampus (HPC) and cortex (CTX) of vehicle (VC) or SH-treated PS19 mice. Scale bar: 0.5 mm. (D) Quantification of (C). n=8/genotype. Student｀s t-test. ***p<0.001. (E) Representative pSTAT3, GFAP and Iba1 co-immunostainings in HPC of WT or PS19 mice treated with VC or SH. Scale bar: 50 μM. (F, G) Quantification of GFAP (F) and Iba1 (F) immunoreactivities in above treated mice. n=8/genotype/treatment. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; **p<0.01; ***p<0.001. (H-J) TNFa (H), IL1 β (I), and IL6 (J) levels measured by ELISA in WT and PS19 mice treated with VC or SH. n=6/genotype/treatment. One-way ANOVA followed by Sidak｀s HSD test. n.s.: non-significant; **p<0.01; ***p<0.001. All data is shown as mean ± s.e.m. See also Figure S8.