Proteomic and metabolic profiling reveals APOE4-dependent shifts in whole brain, neuronal, and astrocytic mitochondrial function and glycolysis

Abstract

Apolipoprotein E (APOE) genetic variation is the strongest genetic risk factor for late onset Alzheimer's disease (LOAD). Studies on APOE genotype dependent changes have largely focused on amyloid beta (Aβ) aggregation, disease pathology, and lipid metabolism. Recently, there has been increased interest in the relationship between metabolic function and APOE genetic variation. In this study, we examined how APOE genotype can alter metabolism in the brains of young male and female APOE3 and APOE4 targeted replacement (TR) mice. In combination with this, we also examined cell type-specific differences using induced pluripotent stem cell (iPSC) derived astrocytes and neurons. We found sex and genotype dependent changes to metabolism in the brains of young APOE TR mice. Specifically, APOE4 mice show signs of metabolic stress and compensatory mechanisms in the brain. Using proteomics and stable isotope tracing metabolomics, we found that APOE4 iAstrocytes and iNeurons exhibit signs of inflammation, mitochondrial dysfunction, altered TCA cycle and malate-aspartate shuttle activity, and a metabolic shift toward glycolysis. Taken together, this data indicates APOE4 causes early changes to metabolism within the central nervous system. While this study establishes a relationship between APOE genotype and alterations in bioenergetics, additional studies are needed to investigate underlying mechanisms.

Figure 1.. Whole brain proteomics in male and female APOE3 and APOE4 TR mice.

A. Study schematic showing male and female APOE3 and APOE4 targeted replacement (TR) mice. Whole brain lysate or isolated brain mitochondria were used for proteomic (n=3 per group). B. Violin plot of APOE protein expression from whole brain in male and female APOE3 and APOE4 mice. C. The number of differentially expressed (DE) proteins in male and female APOE4 vs. APOE3 mice. D. Volcano plot showing up and down regulated proteins in female APOE4 vs. APOE3 mice. E. IPA showing up and down regulated pathways in female APOE4 mice. F. Volcano plot showing up and down regulated proteins in male APOE4 vs APOE3 mice. G. IPA showing up and down regulated pathways in male APOE4 mice. H. Venn diagram of upregulated protein overlap in male and female APOE4 vs. APOE3 mice. I. Venn diagram of down regulated protein overlap in male and female APOE4 vs. APOE3 mice. G=main effect of genotype.

Figure 2.. Mitochondrial respiration and isolated brain mitochondria proteomics in male and female APOE3 and APOE4 mice.

Carbohydrate (PM) driven respiration for A. state 2 B. state 3 C. state 3G D. state 3S and E. uncoupled respiration states. n=7 per genotype and sex. All data are shown as mean ±SD. *p<0.05, ** p<0.01. G=main effect of genotype, I=interaction between genotype and sex. F. Volcano plot showing up and down regulated mitochondrial proteins between female APOE4 and APOE3 mice. G. Volcano plot showing up and down regulated proteins between male APOE4 and APOE3 mice. H. IPA showing up and down regulated pathways between female APOE4 and APOE3 mice. I. IPA showing up and down regulated pathways between male APOE4 and APOE3 mice. J. Heatmap of proteins involved in mitochondrial translation between APOE4 and APOE3 mice.

Figure 3.. Proteomic analysis of isogenic APOE iAstrocytes and iNeurons.

A. Study schematic. Two pairs of isogenic iPSCs with either APOE3 or APOE4 genotype were used to generate iAstrocytes and iNeurons and examine proteomic and bioenergetic outcomes. B. S100B (red) and Hoechst (blue) staining in iAstrocytes, MAP2 (red) and Hoechst (blue) staining in iNeurons. C. APOE protein expression in iAstrocytes and D. APOE protein expression in iNeurons. E. Volcano plot showing up and down regulated proteins in iAstrocytes between APOE4 and APOE3. F. Volcano plot showing up and down regulated proteins in iNeurons between APOE4 and APOE3. G. IPA showing up and down regulated pathways in iAstrocytes between APOE4 and APOE3. H. IPA showing up and down regulated pathways in iNeurons between APOE4 and APOE3. I. Heatmap of the proteins involved in oxidative phosphorylation comparing APOE genotype mediated effects between iAstrocytes and iNeurons. n=5 per group. J. Violin plots showing disease associed markers S100A10, S100A6, STAT3, ALDOC, and CD9 in iAstrocytes along with BDNF levels in iNeurons. ns=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4.. Mitochondrial function in iAstrocytes.

A. Mitochondrial stress test (MST) tracing from isogenic Pair A. B. Quantification of basal respiration, maximal respiration, proton leak, and ATP-production from isogenic Pair A. C. MST tracing from isogenic Pair B. D. Quantification of basal respiration, maximal respiration, proton leak, and ATP-production from isogenic Pair B. E. Electron transport chain (ETC) flux/driven respiration through complex I, II, III, and IV in isogenic Pair A. F. ETC flux/driven respiration through complex I, II, III, and IV in isogenic Pair B. G. TMRE, MitoSOX, Amplex Red, Rhod-2, AM, and Fura-2, AM fluorescence intensity from isogenic Pair A. H. TMRE, MitoSOX, Amplex Red, Rhod-2, AM, and Fura-2, AM fluorescence intensity from isogenic Pair B. n=16. All data are shown as mean ±SD. ns=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5.. Mitochondrial function in iNeurons.

A. Mitochondrial stress test (MST) tracing from isogenic Pair A. B. Quantification of basal respiration, maximal respiration, proton leak, and ATP-production from isogenic Pair A. C. MST tracing from isogenic Pair B. D. Quantification of basal respiration, maximal respiration, proton leak, and ATP-production from isogenic Pair B. E. Electron transport chain (ETC) flux/driven respiration through complex I, II, III, and IV in isogenic Pair A. F. ETC flux/driven respiration through complex I, II, III, and IV in isogenic Pair B. G. TMRE, MitoSOX, Amplex Red, Rhod-2, AM, and Fura-2, AM fluorescence intensity from isogenic Pair A. H. TMRE, MitoSOX, Amplex Red, Rhod-2, AM, and Fura-2, AM fluorescence intensity from isogenic Pair B. n=16. All data are shown as mean ±SD. ns=not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 6.. Glycolytic function in iAstrocytes and iNeurons.

A. Glycolytic stress test (GST) tracing from Pair A iAstrocytes. B. Quantification of basal glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification from iAstrocytes. C. GST tracing from Pair A iNeurons. D. Quantification of basal glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification from iNeurons. E. Glucose, glutamine, and fatty acid fuel dependency in Pair A and B iAstrocytes. F. Glucose, glutamine, and fatty acid fuel capacity in Pair A and B iAstrocytes. G. Glucose, glutamine, and fatty acid fuel dependency in Pair A and B iNeurons. H. Glucose, glutamine, and fatty acid fuel capacity in Pair A and B iNeurons. I. Heatmap of proteins involved in glycolysis I network comparing iAstrocyte and iNeuron outcomes between APOE4 and APOE3. All data are shown as mean ±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 7.. ¹³C-Glucose stable isotope labeling of TCA cycle metabolites and intermediates in iAstrocytes and iNeurons.

A. Schematic of stable isotope labeling experiment using ¹³C-glucose. iAstrocyte mass isotopologue labeling of B. aspartic acid, C. cis-aconitic acid, D. glutamic Acid, E. succinate, F. malic acid. G. Overview of TCA cycle metabolite labeling outcomes in iAstrocytes. iNeuron mass isotopologue labeling of H. aspartic acid, I. cis-aconitic acid, J. glutamic acid, K. succinate, L. malic acid. M. Overview of TCA cycle metabolite labeling outcomes in iNeurons. n=3 per group. All data are shown as mean ±SD. *p<0.05, **p<0.01, ***p<0.001. Data were analyzed using a multiple un-paired t-test with Holm-Sidak correction.

Figure 8.. Proteins involved in the TCA cycle and malate-aspartate shuttle in isogenic APOE iAstrocytes.

A. Heatmap of TCA cycle proteins in iAstrocytes and iNeurons. Violin plots showing levels of TCA cycle and related proteins in iAstrocytes including B. PDHA1, C. PDHB, D. FH, E. MDH1, F. MDH2, G. PC, H. GOT1, I. GOT2. J. Correlation analysis of MDH1 and GOT1 protein expression levels. K. Correlation analysis of MDH2 and GOT2 protein expression levels. Violin plots showing levels of L. SLC25A11 and M. SLC25A13. *p<0.05, ***p<0.001, ****p<0.0001.

Figure 9.. Stable isotope labeling of nucleotide/energy transfer/redox pair molecules, and evaluation of hexosamine/GlcNAc pathway in iAstrocytes and iNeurons.

iAstrocyte and iNeuron labeled fraction of total pool for A. ATP, B. ADP, C. GTP, D. GDP, E. NADP, F. NAD, G. NADH, H. UTP, I. UDP, J. UDP-GlcNAc. K. Heatmap of proteins involved in the Hexosamine and GlcNAc synthesis pathway in APOE4 vs. APOE3 iAstrocytes and iNeurons. L. O-GlcNAc western blot on APOE3 and APOE4 iAstrocytes and iNeurons with densitometry quantification. M. Schematic overview of hexosamine and GlcNAc synthesis pathway. n=3 per group. All data are shown as mean ±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.