Abstract
Microglial cells consume adenosine triphosphate (ATP) during phagocytosis to clear neurotoxic β-amyloid in Alzheimer’s disease (AD). However, the contribution of energy metabolism to microglial function in AD remains unclear. Here, we demonstrate that hexokinase 2 (HK2) is elevated in microglia from an AD mouse model (5xFAD) and AD patients. Genetic deletion or pharmacological inhibition of HK2 significantly promotes microglial phagocytosis, lowers the amyloid plaque burden and attenuates cognitive impairment in male AD mice. Notably, the ATP level is dramatically increased in HK2-deficient or inactive microglia, which can be attributed to a marked upregulation in lipoprotein lipase (LPL) expression and subsequent increase in lipid metabolism. We further show that two downstream metabolites of HK2, glucose-6-phosphate and fructose-6-phosphate, can reverse HK2-deficiency-induced upregulation of LPL, thus supporting ATP production and microglial phagocytosis. Our findings uncover a crucial role for HK2 in phagocytosis through regulation of microglial energy metabolism, suggesting a potential therapeutic strategy for AD by targeting HK2.
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Data availability
The SRA accession number for RNA-seq data reported in this paper is: PRJNA843185 (SAMN28745444–SAMN28745459); PRJNA613212 (SRR11347559–SRR11347568). Transcription levels of HK1, HK2 and HK3 in the prefrontal cortex of human brains at different ages are from public data in the GSE44772 dataset. Single-cell characteristics of microglia were analysed using the public data in the GSE127893 database. Source data are provided with this paper.
Change history
14 October 2022
A Correction to this paper has been published: https://doi.org/10.1038/s42255-022-00682-x
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Acknowledgements
This work was supported by the National Nature Science Foundation of China (grant nos. 91849205, 81925010 and U1905207 to J.Z.; 81801337 and 82071520 to L.L.; 92149303 to H.X. and J.Z.; 92049202 to H.X.), the National Key Research and Development Program of China (grant no. 2021YFA1101402 to J.Z.), the Fundamental Research Funds for the Central Universities (grant nos. 20720190118 and 20720180049 to J.Z.; 20720190075 to L.L.), Fujian Province Nature Science Foundation (grant no. 2019J05006 to L.L.) and Xiamen Youth Innovation Fund (grant no. 3502Z20206031 to L.L.).
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Contributions
L.L. and J.Z. conceptualized the study. L.L., Z.Y., H.Lin, H.Li. and W.X. prepared and maintained AD mice. L.L., Z.Y., K.Z. and Z.C. designed and performed morphological analysis and biochemical assays. L.L., Z.Y and Z.C. performed behaviour tests. K.R., W.W., X.S. and J.X. performed electrophysiology experiments. H.W. prepared the primary microglia, astrocyte and neuron cultures. X.Z. and Z.-B.J. performed human microglia differentiation. J.G. and X.W. performed and analysed staining on AD patients’ samples. R.P. repeated the drug-treated AD experiments. L.L. and J.Z. wrote the manuscript. H.X., Z.Y., H.-M.C., R.P., Z.C., S.W. and X.W. discussed and edited the manuscript. J.Z. supervised the project. All authors reviewed and gave final approval to the manuscript.
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Nature Metabolism thanks Inhee Mook-Jung and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the Nature Metabolism team.
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Extended data
Extended Data Fig. 1 The expressions of HK1/2/3 in various mouse brain regions and various kinds of neuronal cells.
(a)Western blot analysis of HK1, HK2 and HK3 protein levels in various brain regions of mice. Immunoblots were probed with antibodies against the indicated proteins. Actin served as a loading control. (b) Western blot analysis of HK2 protein levels in skeletal muscle, liver, cortex and hippocampus of wildtype mice. Immunoblots were probed with antibodies against the indicated proteins. Actin served as a loading control. (c, d) Western blot analysis of HK1 and HK3 protein levels in primary microglia treated with Aβ. Immunoblots were probed with antibodies against the indicated proteins. Quantification of proteins levels are showed in (d), n = 4 independent experiments. Actin served as a loading control. (e) Schematic of the major enzymes and corresponding genes of glucose metabolism in primary microglia. (f) Heatmap showing the change in mRNA levels of the indicated glycolysis genes in microglia treated with oligo-Aβ(100 nM, 24 h), as measured via qPCR analysis (n = 4 per group). (g) PLX3397 or control vehicle was given 6-month-old 5xFAD mice at 290 mg/kg for 21 days, the brains were sectioned and stained with HK2 (red) and Iba1 (green). Scale bar: 5 μm. Representative confocal images are shown. (h) Immunofluorescence of HK2 (green) and Iba1 (red) in cortex from 4, 6, 8, 10-month-old 5×FAD mice. Scale bar: 20 μm. Representative confocal images are shown. (i-k) Representative immunofluorescent staining of HK2 (red) and Aβ (green) in the cortex, the hippocampal DG and CA1 areas from 6-month-old 5×FAD mice (i), and CA1 area from indicated aged 5×FAD mice (j). Quantifications of percentage of HK2+ Aβ+/ HK2+ are shown in (k), n = 4 slices from 3 mice. (l) 3D reconstruction after double immunofluorescent staining of HK2 (red) with Aβ (green) in the indicated aged 5×FAD mice. (m) Immunofluorescence of HK2 (green) and CD68 (red) in hippocampus from 4, 6, 8, 10-month-old 5×FAD mice. Scale bar: 20 μm. Representative confocal images are shown in m. n = 3 slices from 3 mice. Data represent mean ± SEM, n.s.: not significant. When compared with control group, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s post hoc analysis.
Extended Data Fig. 2 Lonidamine and 3-BP significantly attenuate amyloid plaque load in AD mice, and increase the protein levels in autophagy pathway without inducing obviously apoptosis, related to Figure 3.
(a) HK activity assay from hippocampus of 6-month-old 5×FAD mice treated with HK2 inhibitors (Lonidamine and 3-BP) and control by intraperitoneal (IP) injection for 7 days. (b) Large scale immunohistochemistry staining of Ab from 6-month-old 5×FAD mice treated with HK2 inhibitors (Lonidamine and 3-BP) and control by intraventricular (ICV) injection. Representative images of brain section (Bregma position) are shown on panel (b), Scale bar: 1mm, n=3 slices from 3 mice. (c) Immunofluorescence of Thioflavine T (blue), Ab (green) and Iba1(red) in cortex from 6-month-old 5×FAD mice and 5×FAD-CcKO mice. Representative images are shown on panel (c), Scale bar: 20μm. n=3 slices from 3 mice. (d) Immunofluorescence of Thioflavine T (blue), Ab (green) and Iba1(red) in cortex from 6-month-old 5×FAD mice, 5×FAD+Lonidamine mice and 5×FAD+3-BP mice. Representative images are shown on panel (d), Scale bar: 20μm. n=3 slices from 3 mice. (e) Double immunofluorescent staining of Ab (Red) and ATG9A (Green) in cortical and hippocampal regions from 6-month-old 5×FAD mice, 5×FAD+Lonidamine mice and 5×FAD+3-BP mice. Representative images are shown on panel (e). Scale bar: 100μm. n=3 slices from 3 mice. (f) Immunofluorescent staining of Ab (green) and C-caspase-3 (red) in hippocampus from the above mice, representative images are presented, n=3 slices from 3 mice. Scale bar:100μm. (g, h) 6-month-old 5xFAD mice and the aged-control littermate WT mice were treated with 3-BP or control by intraperitoneal (IP) injection. The protein levels of Caspase 3, ATG7 and LC3II/LCI in hippocampus were measured by western blotting. Immunoblots were probed with antibodies against the indicated proteins. Quantification of proteins levels are showed in (h), n=3 mice. Actin served as a loading control. Data represent mean±SEM, n.s.: not significant. When compared with control group, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s post hoc analysis.
Extended Data Fig. 3 Lonidamine and 3-BP attenuate neuroinflammation in AD mice and cultured microglia treated with Aβ.
(a) Six-month-old 5xFAD mice and the aged-control littermate WT mice were treated with 3-BP or control by intraperitoneal (IP) injection. The protein level of IL-1β in hippocampus and cortex were measured by western blotting. Immunoblots were probed with antibodies against the indicated proteins. Quantification of proteins levels are showed in (b), n = 3-4 mice. Actin served as a loading control. (d) Immunofluorescence staining of Iba1 in the above mice brain. Quantitation of microglia body diameter and microglia processes total length are showed in (d), n = 8 slices from 3 mice. (e) Real-time PCR in the cortex and hippocampus of 9-month-old 5×FAD mice after giving HK2 inhibition, n = 4 mice. (f) Real-time PCR in the primary microglia after giving HK2 inhibition, n = 4 mice. Data represent mean ± SEM. When compared with control group, *p < 0.05, **p < 0.01, ***p < 0.001. When compared with 5×FAD group, #p < 0.05, ##p < 0.01, ###p < 0.001, one-way ANOVA with Tukey’s post hoc analysis.
Extended Data Fig. 4 Lonidamine and 3-BP stimulate the migration of microglia and BV2 cells.
(a) Primary microglial cells were plated onto transwell chamber inserts. Following 24 h incubation with solvent, cells were treated with Lonidamine (48 h, 300 μM) or 3-BP (48 h, 30 μM) in the present or absent of Aβ (24 h, 100 nM) as indicated in the figure. The cells were then stained with hematoxylin and eosin, and counted under Nikon inverted microscope. Scale bar, 20 µm. (b) Quantitation of the number of migrated cells, n = 3 independent experiments. (c) BV2 were plated onto transwell chamber inserts. Following 24 h incubation with solvent, cells were treated with Lonidamine (48 h, 300 μM) or 3-BP (48 h, 30 μM) in the present or absent of Aβ (24 h, 100 nM) as indicated in the figure. The cells were then stained with hematoxylin and eosin, and counted under Nikon inverted microscope. Scale bar, 20 µm. (d) Quantitation of the number of migrated BV2 cells, n = 3 independent experiments. (e) f/f or cKO primary microglia in the present or absent of Aβ (24 h, 100 nM) were plated onto transwell chamber inserts. Following 24 h incubation, the cells were then stained with hematoxylin and eosin, and counted under Nikon inverted microscope. Scale bar, 20 µm. (f) Quantitation of the number of migrated primary microglia, n = 3 independent experiments. Data represent mean ± SEM, n.s.: not significant. When compared with control group, *p < 0.05, **p < 0.01, ***p < 0.001. When compared with Aβ group, #p < 0.05, ##p < 0.01, ###p < 0.001, one-way ANOVA with Tukey’s post hoc analysis.
Extended Data Fig. 5 The volcano plots of DEGs of microglia treated with Aβ or 3-BP.
(a) RNA-Seq based transcriptome analysis of microglia treated with Aβ or lonidamine. Heatmap of differentially expressed genes (DEGs) are shown. (b-d) The DEGs were sub-grouped into three comparations, and the analyses of gene ontology of biological functions for these DEGs were presented: (b) Aβ vs control; (c) lonidamine vs control; (d) Aβ+ Lonidamine vs Aβ. The lipid metabolism pathway was significantly enriched (red). The volcano plots of DEGs are shown in e-j. The analysis was sub-grouped to three comparations: (e) Control vs Aβ; (f) Control vs 3-BP; (g) Aβ vs Aβ + 3-BP; (h) Control vs Aβ; (i) Control vs Lonidamine; (j) Aβ vs Ab+ Lonidamine. Red plots represent upregulated genes (p < 0.05). Green plots represent downregulated genes (p < 0.05). Blue points represent no significant differentially expressed genes (p > 0.05), one-way ANOVA with Tukey’s post hoc analysis.
Extended Data Fig. 6 Targeted metabolomics analysis of primary microglia treated with Aβ(100 nM)±3-BP(30 μM).
(a) Differentially expressed metabolites were identified from primary microglia treated with Aβ(100 nM)±3-BP(30 μM) and related metabolic networks were draw. (b) To allow for inverse modelling of biochemical regulation from metabolomics covariance data, a metabolic reconstruction and pathway reduction from available genome sequences is performed (RECON). The biological variance of the independent biological replicates per cell type is visible, which is further exploited first for the calculation of the Covariance matrix COV and subsequently for the Jacobian matrix JAC using the stochastic Lyapunov matrix Equation (1). (c) The levels of lactate were measured in microglia treated with 3-BP for 48 h in the present of Aβ, n = 4 independent experiments. Data represent mean ± SEM, not significant: fold change (Aβ + 3-BP/Aβ) at.0.8~1.2; significant increase: fold change (Aβ + 3-BP /Aβ) >1.2; significant decrease: fold change (Aβ + 3-BP /Aβ) <0.8, one-way ANOVA with Tukey’s post hoc analysis. Data represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s post hoc analysis.
Extended Data Fig. 7 Single cell characteristics of microglia with high or low expression of hk2.
(a) UMAP projections of the GSE127893 database coloured according to the expression of HK2. (b)Violin plots for the expression of HK2 of the GSE127893 database. (c-g) Heatmap showing clustering analysis of microglia cells, featuring the gene expression in lipid metabolism classified according to the expression of HK2: lipid metabolism (c); homoeostatic genes (d); lysosomal function (e); stage 1 DAM genes (f) and stage 2 DAM genes (g).
Extended Data Fig. 8 HK2 inhibition increases LPL expression in microglia of AD mice brain, and LPL inhibitor blocks the beneficial effects of 3-BP/Lonidamine on microglia.
(a-c) Western blot and Real-time PCR analysis of LPL expression in cortex samples from 9-month-old 5×FAD mice and aged control WT mice treated with HK2 inhibitors or control vehicle. Immunoblots were probed with antibodies against the indicated proteins. Quantification of protein levels are showed in (b), n = 3 mice. Quantification of Lpl mRNA levels were showed in (c), n = 6 mice. (d, e) Immunofluorescence staining of LPL (red) with Iba1(green) or HK2(green) in cortex from 9-month-old 5×FAD mice treated with HK2 inhibitors. Representative confocal images are shown on panel (e), Scale bar:20 μm and 5 μm. Quantitation of LPL fluorescence intensity are showed in (e), n = 6 slices from 3 mice. Data represent mean ± SEM. When compared with control group, *p < 0.05, **p < 0.01, ***p < 0.001. When compared with Aβ group, #p < 0.05, ##p < 0.01, ###p < 0.001, one-way ANOVA with Tukey’s post hoc analysis.
Extended Data Fig. 9 The inhibitions of PFKFB3, PKM2, and PDH did not promote phagocytosis and ATP production in microglia.
(a) Schematic diagram of inhibitors target enzymes in glycolysis pathway. (b) The ATP production of microglia treated with these inhibitors were measured, n = 3 independent experiments. (c) The ATP production of microglia treated with PFK158 (PFK inhibitor) in different dose and time quantum, n = 3 independent experiments. (d) RT-PCR was performed to test the effect of siRNA-induced mRNA Knockdown of PFKFB3 in primary microglia, n = 3 independent experiments. (e) The ATP production of microglia treated with PFK158 (PFK siRNA). n = 3 independent experiments. Data represent mean ± SEM. When compared with control group, *p < 0.05, **p < 0.01, ***p < 0.001. When compared with Aβ group, #p < 0.05, ##p < 0.01, ###p < 0.001, one-way ANOVA with Tukey’s post hoc analysis.
Extended Data Fig. 10 HK2 inhibitors decreased NAPDH production, and NAPDH blocked the phagocytosis, ATP production, and LPL expression induced by HK2 inhibition.
NADP+ (a), NADPH (b), and ratio of total NADP+/NADPH (c) in primary microglia after treated with HK2 inhibitors with or without Aβ were measured, n = 3 independent experiments. (d) The flow cytometry of FAM-microspheres uptake experiments was performed to test the phagocytosis of primary microglia treated by HK2 inhibitors (48 h) with or without NADPH (24 h). Values were normalized to the vehicle control, n = 3 independent experiments. (e) The ATP production of microglia treated by HK2 inhibitors (48 h) with or without NADPH (24 h), n = 3 independent experiments. (f) The protein level of LPL in primary microglia were measured by western blotting. Immunoblots were probed with antibodies against the indicated proteins. Quantification of proteins levels are showed in (g), n = 4 independent experiments. Actin served as a loading control. (h) Real-time PCR was performed to test the level of LPL in primary microglia after treating NAPDH, n = 3 independent experiments. (i) LPL-mediated transactivation in BV2 cells treated with NADPH, n = 3 independent experiments. Data represent mean ± SEM. When compared with control group or compared with the specific group indicated, *p < 0.05, **p < 0.01, ***p < 0.001. When compared with Aβ group, #p < 0.05, ##p < 0.01, ###p < 0.001, one-way ANOVA with Tukey’s post hoc analysis.
Supplementary information
Supplementary Information
Primer sequence list, Supplementary datasheet legends and Figs. 1–5.
Supplementary Data 1
Differential expressed gene (DEG) lists in microglia-treated groups: Aβ versus control, 3-BP versus control and Aβ + 3-BP versus Aβ.
Supplementary Data 2
Differential expressed gene (DEG) lists in microglia-treated groups: Aβ versus control, lonidamine versus control and Aβ + lonidamine versus Aβ.
Supplementary Data 3
Fraction contribution in microglia-treated groups in 13C metabolic flux.
Supplementary Data 4
Targeted metabolomics analysis of primary microglia treated with Aβ ± 3-BP.
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Unprocessed western blot gels.
Source Data Extended Data Fig. 8
Statistical source data.
Source Data Extended Data Fig. 9
Statistical source data.
Source Data Extended Data Fig. 10
Unprocessed western blot gels.
Source Data Extended Data Fig. 10
Statistical source data.
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Leng, L., Yuan, Z., Pan, R. et al. Microglial hexokinase 2 deficiency increases ATP generation through lipid metabolism leading to β-amyloid clearance. Nat Metab 4, 1287–1305 (2022). https://doi.org/10.1038/s42255-022-00643-4
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DOI: https://doi.org/10.1038/s42255-022-00643-4
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