Complement C3aR depletion reverses HIF-1α–induced metabolic impairment and enhances microglial response to Aβ pathology
Manasee Gedam, … , Meng C. Wang, Hui Zheng
Manasee Gedam, … , Meng C. Wang, Hui Zheng
Published June 15, 2023
Citation Information: J Clin Invest. 2023;133(12):e167501. https://doi.org/10.1172/JCI167501.
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Research Article Neuroscience

Complement C3aR depletion reverses HIF-1α–induced metabolic impairment and enhances microglial response to Aβ pathology

Abstract

Microglia are the major cell type expressing complement C3a receptor (C3aR) in the brain. Using a knockin mouse line in which a Td-tomato reporter is incorporated into the endogenous C3ar1 locus, we identified 2 major subpopulations of microglia with differential C3aR expression. Expressing the Td-tomato reporter on the APPNL-G-F–knockin (APP-KI) background revealed a significant shift of microglia to a high-C3aR-expressing subpopulation and they were enriched around amyloid β (Aβ) plaques. Transcriptomic analysis of C3aR-positive microglia documented dysfunctional metabolic signatures, including upregulation of hypoxia-inducible factor 1 (HIF-1) signaling and abnormal lipid metabolism in APP-KI mice compared with wild-type controls. Using primary microglial cultures, we found that C3ar1-null microglia had lower HIF-1α expression and were resistant to hypoxia mimetic–induced metabolic changes and lipid droplet accumulation. These were associated with improved receptor recycling and Aβ phagocytosis. Crossing C3ar1-knockout mice with the APP-KI mice showed that C3aR ablation rescued the dysregulated lipid profiles and improved microglial phagocytic and clustering abilities. These were associated with ameliorated Aβ pathology and restored synaptic and cognitive function. Our studies identify a heightened C3aR/HIF-1α signaling axis that influences microglial metabolic and lipid homeostasis in Alzheimer disease, suggesting that targeting this pathway may offer therapeutic benefit.

Authors

Manasee Gedam, Michele M. Comerota, Nicholas E. Propson, Tao Chen, Feng Jin, Meng C. Wang, Hui Zheng

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Figure 2

Absence of C3aR dampens HIF-1α–induced metabolic changes.

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Absence of C3aR dampens HIF-1α–induced metabolic changes. (A) Representa...
(A) Representative images of HIF-1α immunofluorescence (green) and DAPI (gray) in WT and C3aR-KO primary microglial cultures. Scale bar: 15 μm. (B) Quantification of percentage area of HIF-1α fluorescence per cell (n = 180–198 cells from 6 replicates/group). (C) ATP concentration using luminescence assay in WT and C3aR-KO cells (n = 6 replicates/group). (D) Representative images of HIF-1α (green) and DAPI (gray) in BV-2 cells pretreated with 10 μM C3aR antagonist (C3aRA) prior to treatment with 100 μM CoCl2. Scale bar: 100 μm. (E) Quantification of HIF-1α expression per cell in BV-2 cells treated with vehicle, 10 μM C3aRA, 100 μM CoCl2, or C3aRA + CoCl2 (n = 250–270 cells from 6 replicates/group). (F) Representative images showing HIF-1α (green) immunofluorescence in WT and C3aR-KO primary microglia cells placed in hypoxia chamber (1% O2) for 24 hours compared with primary microglial cells placed in normoxia (21% O2). Scale bar: 15 μm. (G) Quantification of percentage area of HIF-1α fluorescence in WT and C3aR-KO cells exposed to hypoxic or normoxic conditions. (H) qPCR analysis of Hif1a in WT and C3aR-KO cells exposed to hypoxic or normoxic conditions. (I–K) qPCR analysis of HIF-1α targets Pdk1 (I), Pfkl (J), and Pfk2 (K) in response to 100 μM CoCl2 treatment in primary microglial cells. (L) Oxygen consumption rate (OCR) plot from Seahorse Mito Stress assay performed on WT and C3aR-KO cells with and without 24-hour CoCl2 treatment. Violin plots showing the quantification of basal OCR (M), maximal OCR (N), and ATP production OCR (O) across the 4 treatment groups. For all panels, data are presented as mean ± SEM (B–E and G–L) or median and quartile range (M–O). *P < 0.05; **P < 0.01; ***P < 0.001 by 2-sided Student’s t test (B and C) or 1-way ANOVA with Tukey’s correction (E, G–K, and M–O). Each experiment was repeated 2–3 times.