NFκB-activated astroglial release of complement C3 compromises neuronal morphology and function associated with Alzheimer's disease

Abstract

Abnormal NFκB activation has been implicated in Alzheimer's disease (AD). However, the signaling pathways governing NFκB regulation and function in the brain are poorly understood. We identify complement protein C3 as an astroglial target of NFκB and show that C3 release acts through neuronal C3aR to disrupt dendritic morphology and network function. Exposure to Aβ activates astroglial NFκB and C3 release, consistent with the high levels of C3 expression in brain tissue from AD patients and APP transgenic mice, where C3aR antagonist treatment rescues cognitive impairment. Therefore, dysregulation of neuron-glia interaction through NFκB/C3/C3aR signaling may contribute to synaptic dysfunction in AD, and C3aR antagonists may be therapeutically beneficial.

Figure 1. C3 is overexpressed in IκBα-deficient astroglia

(A) Quantitative RT-PCR measurement of C3 mRNA expression in hippocampal samples of 2-month-old Nestin-Cre; IκBα^fl/− (NcKO), CamKIIα-Cre; IκBα^fl/− (CcKO), and GFAP-Cre; IκBα^fl/− (GcKO) mice and their littermate controls (Ctrl). (B) C3 protein levels in GcKO and Ctrl hippocampi measured by ELISA. (C) C3 mRNA levels in wild-type (WT) and IκBα knockout (KO) primary neurons or astroglia. (D) ELISA quantification of C3 protein levels in conditioned media of WT or IκBα KO astroglial cultures. (E) C3 mRNA expression in WT or IκBα KO astroglial cultures treated with different combinations of TNFα (50 ng/ml) or NFκB inhibitor JSH23 (20 μM). N=3 per group per experiment except N=6 in (B). A, B, and D: Student’s t-test; C: Two-way ANOVA followed by pairwise comparison. E: Three-way ANOVA followed by pairwise comparison. *P < 0.05; **P < 0.01; ***P < 0.001; NS: non-significant. See also Figure S1.

Figure 2. Astroglial C3 alters synaptic and dendritic morphology

(A) Double-immunostaining of wild-type or IκBα KO astroglia (WTA or KOA) co-cultured neurons with anti-synaptophysin (Syn) and anti-MAP2 (MAP2) antibodies. (B) Same as (A) except that anti-VGluT1 (VGluT1) antibody was used instead of Syn. Images underneath each panel are enlarged view of the bracketed areas. (C) Quantification of number of Syn⁺MAP2⁺, VGluT1⁺MAP2⁺ or VGAT⁺MAP2⁺ synaptic puncta per 10 μm of dendrite in WTA and KOA co-cultured neurons. N_{WTA Syn}=31; N_{KOA Syn}=28; N_{WTA VGluT1}=57; N_{KOA VGluT1}=52; N_{WTA VGAT}=45; N_{KOA VGAT}=52. (D) Quantification of total MAP2-positive dendritic length in WTA and KOA co-cultured neurons. N_WTA=38; N_KOA=42. (E) Representative dendritic structure, (F) Quantification of dendritic complexity of WTA and KOA co-cultured neurons by Sholl analysis. N_WTA=39; N_KOA=44. (G) Double staining of Syn and MAP2 of WT neurons treated with vehicle (PBS) or 5 μg/ml C3. (H) Quantified synaptic density of neurons treated with PBS or C3 at 1, 2, or 5 μg/ml. N_PBS=36; N_1μg/ml=33; N_2μg/ml=37; N_5μg/ml=29. (I) Representative dendritic structures of WT neurons treated with PBS or 5 μg/ml C3. (J) Dendritic complexity quantification of WT neurons treated with PBS or C3 at 1, 2, and 5 μg/ml. N_PBS=116; N_1μg/ml=66; N_2μg/ml=53; N_5μg/ml=68. Scale bar: 10 μm in (A, B, G) and 20 μm in (E, I). C and D: Student’s t-test. F: Two-way ANOVA. H: One-way ANOVA followed by Bonferroni post-hoc analysis. J: Two-way ANOVA followed by Bonferroni post-hoc analysis. *P < 0.05; **P < 0.01; ***P < 0.001; NS: non-significant. See also Figure S2.

Figure 3. Impaired dendritic morphology in astroglial IκBα-deficient (GcKO) mice

(A) Schematic representation of the steps taken for dendritic spine analysis in vivo. Four-month-old littermate control (Ctrl) and GcKO mouse brains were stereotaxically injected with AAV-GFP virus followed by measurement of spine density 3 weeks later. IMARIS was used to reconstruct the 3D renderings of the spines on dendrites based on confocal images of GFP-positive neurons and classify the four spine types (mushroom, stubby, long-thin and filopodia). (B) Total spine density in Ctrl and GcKO mouse brains. N_Ctrl=25; N_GcKO=22. (C) Density of each of the four spine types in Ctrl and GcKO mouse brains. N_Ctrl=39; N_GcKO=31. (D) Frequency of each spine types. Scale bar: 5 μm. Dendritic segments were derived from 3 animals/group. Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. See also Video S1 and Figure S3.

Figure 4. Neuronal C3aR mediates dendritic morphology and network function

(A) Representative images of MAP2-positive dendritic structures in wild-type or IκBα KO astroglia (WTA or KOA) co-cultured neurons treated with DMSO or 1 μM C3aRA for 4 days. (B) Sholl analysis of dendritic complexity of WTA or KOA co-cultured neurons treated with DMSO or C3aRA. N_{WTA DMSO}=47; N_{KOA DMSO}=55; N_{WTA C3aRA}=63; N_{KOA C3aRA}=57. (C) Representative MAP2-positive dendrites of C3aR wild-type (C3aRWTN) or knockout (C3aRKON) neurons co-cultured with IκBα WT or KO astroglia (WTA or KOA). (D) Sholl analysis of dendritic complexity of WTA or KOA co-cultured C3aRWTN or C3aRKON neurons. N_{C3aRWTN WTA}=46; N_{C3aRKOA WTA}=51; N_{C3aRWTN KOA}=44; N_{C3aRKOA KOA}=46. (E) Representative dendritic spines in 10 month-old Ctrl or GcKO mouse brains treated with DMSO or C3aRA (1 mg/kg i.p.) for 3 weeks. (F) Quantification of spine density in Ctrl or GcKO mouse brains treated with DMSO or C3aRA. N_{Ctrl DMSO}=69; N_{GcKO DMSO}=80; N_{Ctrl C3aRA}=41; N_{GcKO C3aRA}=43. Dendritic segments from 3 animals/group were selected for spine density quantification. (G) Reduced contextual freezing in GcKO mice N_Ctrl=14; N_GcKO=18. (H) Rescue of contextual memory defects of GcKO mice by C3aRA treatment. DMSO: vehicle treated controls. N_{Ctrl DMSO}=6; N_{GcKO DMSO}=7; N_{Ctrl C3aRA}=8; N_{GcKO C3aRA}=6. (I) Slope of field excitatory postsynaptic potential (fEPSP) in response to theta burst stimulation delivered to the Schaffer collateral pathway from Ctrl or GcKO mice treated with DMSO or C3aRA. (J) Quantification of average fEPSP slope in the last 10 minutes. N_{Ctrl DMSO}=6; N_{GcKO DMSO}=5; N_{Ctrl C3aRA}=8; N_{GcKO C3aRA}=6. Scale bar: 20 μm in (A, C) and 10 μm in (E). B and D: Three-way ANOVA followed by pairwise comparison; F, H, and J: Two-way ANOVA followed by pairwise comparison. G: Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; NS: non-significant. See also Figure S4.

Figure 5. Astroglial NFκB activation leads to aberrant intraneuronal calcium

(A) Representative images of GCaMP fluorescence in AAV-GCaMP6s-infected neurons co-cultured with wild-type or IκBα KO astroglia (WTA or KOA). (B) Mean GFP fluorescence in co-cultured neurons. N_WTA=221; N_KOA=234. (C) Representative GFP fluorescence in Ctrl or GcKO derived organotypic hippocampal slice cultures infected with AAV-GCaMP6s. (D) Quantification of basal GFP fluorescence intensity. N=65/ group. (E) Mean GCaMP fluorescence of Ctrl and GcKO slice cultures treated with DMSO, 10 μM C3aRA or 50 μM BAPTA/AM. N_{Ctrl DMSO}=103; N_{GcKO DMSO}=104; N_{Ctrl BAPTA/AM}=60; N_{GcKO BAPTA/AM=}60; N_{Ctrl C3aRA}=180; N_{GcKO C3aRA}=104. (F) Representative images of MAP2-positive dendritic morphologies in WTA or KOA co-cultured neurons treated with DMSO or 1 μM BAPTA/AM for 4 days. (G) Sholl analysis of dendritic complexity of WTA or KOA co-cultured neurons treated with DMSO or BAPTA/AM. N_{WTA DMSO}=52; N_{KOA DMSO}=62; N_{KOA BAPTA/AM}=70. Scale bar: 50 μm in (A), 100 μm in (C), 20 μm in (F). B and D: Student’s t-test; E: Two-way ANOVA followed by Bonferroni post-hoc analysis; G: Three-way ANOVA followed by pairwise comparison. *P < 0.05; **P < 0.01; ***P < 0.001. See also Video S2.

Figure 6. C3aR and intraneuronal calcium mediates excitatory synaptic transmission and surface AMPAR expression

(A) Example mEPSC traces of wild-type or IκBα KO astroglia (WTA or KOA) co-cultured neurons. (B) Increased mEPSC amplitude in KOA neurons compared to WTA controls. (C) No differences of mEPSC frequency between WTA and KOA cultures. (D) mEPSC amplitude fractionation curve of WTA and KOA neurons. N_WTA=13; N_KOA=14. (E) Representative images of co-cultured WTA and KOA neurons stained against surface GluR1 (Surf. GluR1) and synaptophysin (Syn) and counter-stained with DAPI. Inset: Enlarged images of the bracketed areas. (F) Relative fluorescence intensity of Surf. GluR1 over whole cell surface (N_WTA=20; N_KOA=19) and (G) Relative fluorescence intensity of Surf. GluR1 in Syn⁺ puncta (N_WTA=60,000; N_KOA=45,000). (H) Blots of surface GluR1 in co-cultured neuronal lysates. Cell surface protein samples were prepared by surface biotinylation. Ctrl lanes are samples from neurons without biotin incubation. Loading was quantified by blotting with an anti-γ-tubulin antibody in cell lysates before neutravidin pulldown. (I) Quantification of the blot in (H). (J) Sample mEPSC traces of wild-type or IκBα KO astroglia (WTA or KOA) co-cultured neurons treated with DMSO or 1 μM C3aRA for 4 days. (K) Mean mEPSC amplitude of treated neurons. N_{WTA DMSO}=8; N_{KOA DMSO}=9; N_{WTA C3aRA}=12; N_{KOA C3aRA}=17. (L) Representative images of surface GluR1 staining with DAPI counter-staining in WTA or KOA neurons treated with DMSO or C3aRA. (M) Quantification of fluorescence intensity of (L). N_{WTA DMSO}=35; N_{KOA DMSO}=31; N_{WTA C3aRA}=33; N_{KOA C3aRA}=37. (N) Quantification of Surf. GluR1 fluorescence in co-cultured neurons treated with DMSO or 1 μM BAPTA/AM for 15 min. N_{WTA DMSO}=29; N_{KOA DMSO}=24; N_{WTA BAPTA/AM}=34; N_{KOA BAPTA/AM}=31. Scale bar: 10 μm. B, C, F, G, and I: Student’s t-test; K, M, and N: Two-way ANOVA followed by pairwise comparison. *P < 0.05; **P < 0.01; ***P < 0.001; NS: non-significant. See also Figure S5.

Figure 7. Activation of the NFκB/C3 in AD and beneficial effect of C3aR antagonist

(A) p65 and GFAP double-staining of primary wild-type astroglial cultures incubated with Aβ42 peptide showing p65 nuclear translation induced by Aβ42. Reverse Aβ42 peptide (rAβ42) was used as a negative control. Scale bar: 20 μm. (B) Quantification of nuclear/cytoplasmic p65 ratio. N=50 cells per group. (C) qPCR measurement of C3 mRNA expression in astroglial cultures treated with Aβ42 or rAβ42. N=3 per group. (D) C3 mRNA expression in the APP/TTA AD mouse model compared to the control TTA mice. N_TTA=6; N_APP/TTA=7. (E) Rescue of Morris water maze deficits in APP/TTA mice by C3aRA. N_{TTA DMSO}=7; N_{APP/TTA DMSO}=8; N_{TTA C3aRA}=6; N_{APP/TTA C3aRA}=7. (F) Rescue of radial arm water maze deficits in APP/TTA mice by C3aRA. N_{TTA DMSO}=7; N_{APP/TTA DMSO}=8; N_{TTA C3aRA}=5; N_{APP/TTA C3aRA}=7. (G) ELISA measurement of NFκB subunit p65 in nuclear fractions of human brain lysates from control subjects (Ctrl) and AD patients. N_Ctrl=7; N_AD=8. (H) Western blotting of IκBα levels in Ctrl and AD human total protein lysates. Neuron specific enolase (NSE) was used as a loading control. (I) Quantification of blots in (H). (J) C3 protein concentration in total protein lysates from control and AD brain samples. N_Ctrl=7; N_AD=8. B, C, D, G, I, and J: Student’s t-test; E and F: Two-way ANOVA followed by pairwise comparison. *P < 0.05; **P < 0.01; ***P < 0.001; NS: non-significant. See also Table S1.