Mitochondrial control of microglial phagocytosis by the translocator protein and hexokinase 2 in Alzheimer's disease

Abstract

Microglial phagocytosis is an energetically demanding process that plays a critical role in the removal of toxic protein aggregates in Alzheimer's disease (AD). Recent evidence indicates that a switch in energy production from mitochondrial respiration to glycolysis disrupts this important protective microglial function and may provide therapeutic targets for AD. Here, we demonstrate that the translocator protein (TSPO) and a member of its mitochondrial complex, hexokinase-2 (HK), play critical roles in microglial respiratory-glycolytic metabolism and phagocytosis. Pharmacological and genetic loss-of-function experiments showed that TSPO is critical for microglial respiratory metabolism and energy supply for phagocytosis, and its expression is enriched in phagocytic microglia of AD mice. Meanwhile, HK controlled glycolytic metabolism and phagocytosis via mitochondrial binding or displacement. In cultured microglia, TSPO deletion impaired mitochondrial respiration and increased mitochondrial recruitment of HK, inducing a switch to glycolysis and reducing phagocytosis. To determine the functional significance of mitochondrial HK recruitment, we developed an optogenetic tool for reversible control of HK localization. Displacement of mitochondrial HK inhibited glycolysis and improved phagocytosis in TSPO-knockout microglia. Mitochondrial HK recruitment also coordinated the inflammatory switch to glycolysis that occurs in response to lipopolysaccharide in normal microglia. Interestingly, cytosolic HK increased phagocytosis independent of its metabolic activity, indicating an immune signaling function. Alzheimer's beta amyloid drastically stimulated mitochondrial HK recruitment in cultured microglia, which may contribute to microglial dysfunction in AD. Thus, targeting mitochondrial HK may offer an immunotherapeutic approach to promote phagocytic microglial function in AD.

Keywords: Alzheimer’s disease; hexokinase; immunometabolism; mitochondria; translocator protein.

Fig. 1.

TSPO function associated with mitochondrial bioenergetics, lipid metabolism and phagocytosis pathways in mouse models of AD-related neuroinflammation. (A) Volcano plot showing DEGs in hippocampus of WT vs. TSPO-KO mice (n = 4 mice/group). (B) Normalized enrichment score of top enriched Gene Ontology pathways related to immune and inflammatory responses, mitochondrial and lipid metabolism identified by fGSEA analysis of LPS treated WT vs. TSPO-KO mice. (C) Heat map of mean gene expression for selected enriched immune, mitochondrial and lipid metabolism pathways in LPS treated WT and TSPO-KO mice. (D) Schematic of proteomic TSPO interactome analysis in brain. TSPO complexes were isolated via immunoprecipitation from wildtype and APP-KI mouse brains, using TSPO-KO as a background control. Enriched proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the interactome network mapped using bioinformatics approaches. Created with BioRender.com. (E) Volcano plot showing proteins identified by TSPO IP-MS, with candidate TSPO interactors (orange data points) identified as greater than twofold significantly enriched compared to background (TSPO-KO). (F) Brain TSPO interactome protein-protein interaction network showing significantly enriched functional clusters in immune system and metabolic pathways. A, B, and F: Benjamini Hochberg (BH) corrected FDR <0.05; NS, non-significant. E: Limma differential enrichment analysis, BH corrected FDR <0.1.

Fig. 2.

TSPO deletion induces mtHK binding and impairs mitochondrial bioenergetics in mouse microglia. (A) Seahorse Mitostress test of WT and TSPO-KO cultured primary mouse microglia showing mean ± SEM OCR normalized to WT. Basal respiration (basal), ATP production (ATP) and maximal respiration (max.) indicated on WT curve. ATP synthase inhibitor, oligomycin was injected to inhibit ATP-linked mitochondrial respiration as a measure of mitochondrial ATP production. To determine maximal respiration, the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) was injected. To determine the contribution of non-mitochondrial respiration to the OCR, a mixture of rotenone and antimycin was injected, inhibiting mitochondrial respiration. (n = 7 to 8 wells/group, average of two independent experiments). (B) Quantification of basal respiration, maximal respiration, and ATP production calculated from Mitostress assay. Dots represent individual wells. (C) Representative image of MitoTracker (green) and intracellular ATP signals (red) in cultured WT and TSPO-KO primary mouse microglia. Reduced ATP signals in the mitochondrial compartment evident in the merged panel (colocalization indicated in yellow). Histogram indicates ATP-Red 1 and MitoTracker intensity in line indicated on merged image. (D) HK activity measured in primary mouse microglia cell lysates. Dots represent individual wells (n = 9 to 10/group, average of two independent experiments). (E) mtHK binding measured by mean HK immunoreactive intensity within mitochondrial volumes determined using ATP synthase subunit beta (ATPB) staining in WT and TSPO-KO mouse microglial cultures. Dots represent individual cells (n = 40/group, average of two independent experiments). (F) Representative confocal images of HK immunoreactivity (Left, HK, green), mitochondria (Middle, ATPB, red), and colocalization index of HK relative to ATPB immunoreactivity (Right). AU: arbitrary units. (G) Glycolysis test curve (Left, mean ± SEM) and quantification of glycolytic rate (Right) in cultured WT and TSPO-KO primary mouse microglia. Data shown as mean ± SEM ECAR normalized to WT. Glucose was injected as a substrate for glycolysis. ATP synthase inhibitor, oligomycin was injected to stimulate maximal dependence on glycolysis. 2DG was injected to competitively inhibit glucose uptake to determine non-glycolytic ECAR. (n = 15 wells/group, average of two independent experiments). (H) Lactate concentrations measured in culture media of WT and TSPO-KO primary mouse microglia. Dots represent individual wells. (n = 4/group). WT vs. TSPO-KO groups in all figures analyzed by unpaired two-sided permutation t test. *P < 0.05. **P < 0.002. ***P < 0.0001.

Fig. 3.

TSPO deletion promotes lipid droplet accumulation and impairs phagocytosis in microglia. (A) Flow cytometry quantification (Left) and representative histogram (Right) of BODIPY+ cells in TSPO-KO and WT primary mouse microglia stimulated with or without LPS. Dots represent individual wells (n = 8 to 9/group, average of three experiments). (B) Representative confocal images of BODIPY (green) with DAPI (blue) staining in WT and TSPO-KO mouse microglial cultures. (C) Flow cytometry quantification (Left) and representative histogram (Right) of BODIPY⁺ cells in BV2 immortalized mouse microglia treated with TSPO agonist, Ro5-4864 (Ro5), or Vehicle. Dots represent individual wells (n = 9/group, average of three experiments). (D and E) Flow cytometry quantification (D) and representative confocal images of Aβ uptake (E) in TSPO-KO and WT cultured primary mouse microglia. Dots represent individual wells (n = 9/group, average of three experiments). Representative 4 °C control showing negligible surface binding of Aβ in absence of phagocytosis indicated on histoplot. (F and G) Flow cytometry quantification (F) and representative confocal images of Aβ uptake (G) in cultured BV2 microglia treated with TSPO agonist, Ro5-4864 (Ro5, 10 nM), or Vehicle (1% Ethanol). Dots represent individual wells (n = 12/group, average of three experiments). Representative 4 °C control showing negligible surface binding of Aβ in absence of phagocytosis indicated on histoplot. A and C: Two-way ANOVA including LPS and genotype (A) or treatment (C) as factors with Bonferroni-corrected pairwise comparisons. D and F: Unpaired two-sided permutation t test. *P < 0.05, **P < 0.002, ***P < 0.0001.

Fig. 4.

TSPO deletion impairs Aβ phagocytosis in Alzheimer’s mice and exacerbates pathology. (A) Representative flow cytometry gating of Aβ+ phagocytes in WT (Left) and APP-KI mice (Middle). Quantification of Aβ+ phagocytes in APP-KI mice at 2, 4, and 9 mo of age (Right, n = 6 to 7/group). Age-matched WT mice shown for comparison (n = 4 to 5/group). Dots in quantification graph represent individual brains. (B) Representative flow cytometry scatter plot showing CD11b and microglial marker, TMEM199 in Aβ+ cells isolated from APP-KI mouse (>9 mo age), confirming the Aβ⁺ phagocytes are microglia. (C) Uniform manifold approximation and projection analysis showing 10,000 randomly sampled CD45⁺ cells from 9-mo-old APP-KI mice (n = 7) with distribution of Aβ, TSPO, MHCII, CD68 and CD45 enriched populations indicated as a heatmap. Red arrow indicates Aβ⁺TSPO^hi population. (D) Representative flow cytometry histograms showing gating of CD45^hiAβ⁺ and CD45^intAβ⁻ microglial populations isolated from 10-mo-old APP-KI mouse and relative expression of CD11c and microglial marker P2RY12. (E) Flow cytometry histograms showing CD45, MHCII and CD11c increase with age/disease progression in Aβ⁺ microglia (5 mo vs. 10 mo old APP-KI mice; concatenate of CD45+ cells, 5 mo group: n = 3 mice, 10 mo group: n = 5 mice). (F and G) Quantification and representative confocal images of TSPO immunoreactivity (TSPO-IR) in IBA-1+ microglia located proximal (10 μm) vs. distal (80 μm) to amyloid plaques in (F) APP-KI mice at 9 mo of age (n = 5/group) and (G) human AD brain (n = 7/group). (H) Flow cytometry quantification (Left) and representative histogram (Middle) of Aβ⁺ microglial cells gated on CD45⁺/CD11b⁺ in 9-mo-old APP-KI and APP-KI × TSPO-KO mice. No difference in total isolated CD45⁺/CD11b⁺ microglia detected by flow cytometry (Right). Dots represent individual mouse brains (n = 5/group). (I) Quantification and representative confocal images of Aβ and IBA-1 immunoreactivity in 11-mo-old APP-KI and APP-KI × TSPO-KO mice (n = 3 to 4/group). HIP: hippocampus; CTX: cortex. A: One-way ANOVA analysis with Bonferroni pairwise comparisons used on APP-KI groups. F, G, and H: Unpaired two-sided permutational t test. I: Two-way ANOVA with Bonferonni corrected pairwise comparisons. ***P < 0.001, ****P < 0.0001.

Fig. 5.

Mitochondrial HK displacement inhibits glycolysis and improves phagocytosis in cultured TSPO-KO microglia. (A) Schematic showing strategy to competitively displace endogenous HK-2 (HK) from mitochondria using a cell permeable, truncated N-terminal HK peptide lacking the enzymatic domain (HKp). Created with BioRender.com. (B) Quantification of mtHK binding in BV2 microglia treated with vehicle or HKp. mtHK measured by mean HK immunoreactive intensity within mitochondrial volumes determined using immunostaining of mitochondrial marker, ATPB. Dots represent individual cells (n = 5/group). (C) Representative confocal images showing mitochondria (Left, ATPB, red), HK immunoreactivity (Middle, HK, purple) and colocalization index of HK relative to ATPB immunoreactivity (Right) in BV2 microglia following treatment with vehicle or HKp. (D) Glycolysis test curve (Left) and quantification of glycolysis (Right) in WT vs. TSPO-KO cultured microglia treated with either scrambled peptide (control) or HKp. Data shown as mean ± SEM ECAR. Dots represent individual wells (n = 6 wells/group). (E) Schematic of light-activated control of HKp-sspb-RFP localization. With light activation (“ON”), HKp-sspb-RFP translocates to the plasma membrane (PM) to bind ssrA-iLID-CAAX-Venus, enabling endogenous HK binding to the mitochondria and increasing glycolysis. In dark start (‘OFF’) HKp-sspb-RFP binds to the mitochondria, displacing endogenous mitochondrial HK and inhibiting glycolysis. (F) Colocalization index (Pearson correlation coefficient, PCC) of HKp-sspb-RFP with outer mitochondrial membrane vs. PM marker in dark state showing HKp localizes to mitochondria in OFF condition. (G) Representative live-cell confocal images of a cell expressing GlycoSwitch: HKp-sspb-RFP (red) coexpressed with ssrA-iLID-CAAX-Venus (green). Time-lapse of cell prior to blue-light stimulation (dark state, 'OFF”), following 1 min light-induced dimerization (light state, ‘ON’), and following 2 min dark (dark state reversal, “OFF”). Arrow indicates region of co-localization after light stimulation. (H) Quantification of GlycoSwitch-HKp-RFP colocalization with the PM targeted dimer, GlycoSwitch-CAAX measured by PCC. Data normalized to prestimulation OFF condition. Individual dots represent single cells. (n = 5 cells). (I) Glycolysis test curve (Left) and quantification of glycolysis (Right) in BV2 cells expressing GlycoSwitch in either ON or OFF state. Data shown as mean ECAR ± SEM measured in the Seahorse Glycostress test. Data normalized to control group expressing optogenetic vector lacking the HKp domain. n = 12 wells/group, average of two experiments. (J) Lactate concentrations measured in media of BV2 microglia expressing GlycoSwitch in either ON or OFF state. Individual dots represent wells. n = 6/group. (K) Quantification of phagocytic uptake of latex beads measured by flow cytometry in cultured primary mouse TSPO-KO microglia expressing GlycoSwitch in either ON or OFF state. N = 4 well/group. Outlier indicated as hollow dot. (L) Representative confocal images of TSPO-KO microglia expressing GlycoSwitch activated in either OFF or ON state showing ingested beads (red), F-actin marker, phalloidin (green) and nuclear stain (DAPI, blue). B and H–K: Unpaired two-sided permutational t test. D: One-way ANOVA with FDR corrected pairwise comparisons. F: Paired two-sided t test. *P < 0.05, ****P < 0.0001.

Fig. 6.

mtHK binding/displacement regulates microglial glycolysis and phagocytic function in inflammation. (A) Representative confocal images of BV2 microglia expressing full-length HK (FL-HK, green, Top panel) and truncated HK (tHK, green, Bottom panel) after 1 h treatment with 500 nM Aβ. Mitochondrial (ATPB) costaining indicated in red. (B) HK activity measured in lysates of microglial BV2 cells expressing full length HK-2 (FL-HK) or truncated HK (tHK). Dots represent individual wells, n = 4/group. (C) Glycolysis test curve (Left) and quantification of glycolysis (Right) in vehicle (Veh) or LPS treated BV2 cells expressing FL-HK, tHK or mutant HK (mHK) measured in the Seahorse Glycostress test. Control vehicle and LPS groups were transfected with an empty vector control. Data shown as mean ± SEM ECAR. n = 6 wells/group. (D) Lactate concentrations measured in culture media of BV2 cells expressing FL-HK (FL), tHK or mHK. Dots represent individual wells. n = 5/group. (E) Glycolysis test curve (Left) and quantification of glycolysis (Right) in primary WT mouse microglia treated with either vehicle or LPS ± HKp showing mean ECAR ± SEM. n = 5 to 6 wells/group. (F) Representative confocal images showing mitochondria (Left, ATPB, red), HK immunoreactivity (Middle, HK, green) and colocalization index of HK relative to ATPB immunoreactivity (Right) in primary mouse microglia treated with vehicle (Top), LPS (Middle) or Aβ (Bottom). (G) Quantification of mitochondrial HK (mt HK) enrichment in cultured mouse microglia treated with vehicle, LPS or Aβ. Dots represent individual cells (n = 20/group). (H) Phagocytosis calculated as % cells ingested latex beads measured via flow cytometry in BV2 microglia expressing FL-HK (FL), tHK or mHK. Data expressed relative to FL-HK group. Dots represent individual wells. n = 16/group, average of four independent experiments. (I) Effect of 2DG compared to vehicle (Veh) on phagocytosis of latex beads in cultured microglial BV2 cells measured by flow cytometry. n = 3/group. (J) Quantification of phagocytic uptake of latex beads measured by flow cytometry in cultured immortalized microglial BV2 cells expressing GlycoSwitch activated in either ON or OFF state. Data expressed relative to ON group. Dots represent individual wells. n = 14 well/group, average of two independent experiments. B, I, and J: Unpaired two-sided permutational t test. C–F and H: One-way ANOVA with FDR pairwise comparisons. *P < 0.05, **P < 0.003, ***P < 0.0002.