Explore the vast range of titles available.

Background Cerebral glucose hypometabolism is consistently observed in individuals with Alzheimer’s disease (AD), as well as in young cognitively normal carriers of the Ε4 allele of Apolipoprotein E (APOE), the strongest genetic predictor of late-onset AD. While this clinical feature has been described for over two decades, the mechanism underlying these changes in cerebral glucose metabolism remains a critical knowledge gap in the field. Methods Here, we undertook a multi-omic approach by combining single-cell RNA sequencing (scRNAseq) and stable isotope resolved metabolomics (SIRM) to define a metabolic rewiring across astrocytes, brain tissue, mice, and human subjects expressing APOE4. Results Single-cell analysis of brain tissue from mice expressing human APOE revealed E4-associated decreases in genes related to oxidative phosphorylation, particularly in astrocytes. This shift was confirmed on a metabolic level with isotopic tracing of 13 C-glucose in E4 mice and astrocytes, which showed decreased pyruvate entry into the TCA cycle and increased lactate synthesis. Metabolic phenotyping of E4 astrocytes showed elevated glycolytic activity, decreased oxygen consumption, blunted oxidative flexibility, and a lower rate of glucose oxidation in the presence of lactate. Together, these cellular findings suggest an E4-associated increase in aerobic glycolysis (i.e. the Warburg effect). To test whether this phenomenon translated to APOE4 humans, we analyzed the plasma metabolome of young and middle-aged human participants with and without the Ε4 allele, and used indirect calorimetry to measure whole body oxygen consumption and energy expenditure. In line with data from E4-expressing female mice, a subgroup analysis revealed that young female E4 carriers showed a striking decrease in energy expenditure compared to non-carriers. This decrease in energy expenditure was primarily driven by a lower rate of oxygen consumption, and was exaggerated following a dietary glucose challenge. Further, the stunted oxygen consumption was accompanied by markedly increased lactate in the plasma of E4 carriers, and a pathway analysis of the plasma metabolome suggested an increase in aerobic glycolysis. Conclusions Together, these results suggest astrocyte, brain and system-level metabolic reprogramming in the presence of APOE4, a ‘Warburg like’ endophenotype that is observable in young females decades prior to clinically manifest AD.

The ε4 allele of apolipoprotein E (APOE) is the strongest genetic risk factor for late-onset Alzheimer's disease (LOAD). Impaired mitochondrial function disrupts metabolic homeostasis in the brains of ε4 carriers decades prior to disease onset. Microglia and astrocytes expressing ε4 display a 'broken' TCA cycle, including elevated levels of the metabolite succinate. However, it is unknown whether this accumulation of succinate in ε4 may lead to increased succinylation, a recently discovered post-translational modification which dramatically alters protein function by introducing four carbons and a -2 charge. Succinylated peptides were enriched from hippocampi of 12-month old male 5xFAD mice and wild-type controls using pan-succinyllysine magnetic beads (PTMScan) and detected by LC-MS/MS on the Orbitrap Eclipse Tribid mass spectrometer platform (Thermo Fisher Scientific) operating in data-independent acquisition mode. Data was processed using directDIA (Spectronaut). 588 succinylated peptides were quantified in the hippocampus of 5xFAD and wild-type mice. Of these, 3 succinylation sites were detected for apoE including lysine K252, located within the lipid-binding domain in a region known to be critical for apoE self-aggregation and amyloid plaque seeding. Succinylation at K252 increased >8-fold in 5xFAD hippocampi relative to wild-type controls (p =0.02). Studies on the posttranslational modification of apoE have so far focused primarily on its glycosylation. Little is known about other PTMs that may confer disease risk in ε4 carriers. Our discovery that succinylation of apoE is increased in the 5xFAD hippocampus suggests that this modification occurs in response to disease pathology and opens the door to a new mechanism by which apoE function is regulated across the disease trajectory. We hypothesize that succinylation of apoE impacts functions critical for neuroprotection against AD in an allelic-dependent manner, and are currently investigating whether modulation of succinylation represents a novel therapeutic approach for AD.

Background The ε4 allele of apolipoprotein E (APOE) is the strongest genetic risk factor for late‐onset Alzheimer’s disease (LOAD). Impaired mitochondrial function disrupts metabolic homeostasis in the brains of ε4 carriers decades prior to disease onset. Microglia and astrocytes expressing ε4 display a ‘broken’ TCA cycle, including elevated levels of the metabolite succinate. However, it is unknown whether this accumulation of succinate in ε4 may lead to increased succinylation, a recently discovered post‐translational modification which dramatically alters protein function by introducing four carbons and a ‐2 charge. Method Succinylated peptides were enriched from hippocampi of 12‐month old male 5xFAD mice and wild‐type controls using pan‐succinyllysine magnetic beads (PTMScan) and detected by LC‐MS/MS on the Orbitrap Eclipse Tribid mass spectrometer platform (Thermo Fisher Scientific) operating in data‐independent acquisition mode. Data was processed using directDIA (Spectronaut). Result 588 succinylated peptides were quantified in the hippocampus of 5xFAD and wild‐type mice. Of these, 3 succinylation sites were detected for apoE including lysine K252, located within the lipid‐binding domain in a region known to be critical for apoE self‐aggregation and amyloid plaque seeding. Succinylation at K252 increased >8‐fold in 5xFAD hippocampi relative to wild‐type controls (p =0.02). Conclusion Studies on the posttranslational modification of apoE have so far focused primarily on its glycosylation. Little is known about other PTMs that may confer disease risk in ε4 carriers. Our discovery that succinylation of apoE is increased in the 5xFAD hippocampus suggests that this modification occurs in response to disease pathology and opens the door to a new mechanism by which apoE function is regulated across the disease trajectory. We hypothesize that succinylation of apoE impacts functions critical for neuroprotection against AD in an allelic‐dependent manner, and are currently investigating whether modulation of succinylation represents a novel therapeutic approach for AD.

Cerebral glucose hypometabolism is consistently observed in individuals with Alzheimer's disease (AD), as well as in young cognitively normal carriers of the Î4 allele of Apolipoprotein E (APOE), the strongest genetic predictor of late-onset AD. While this clinical feature has been described for over two decades, the mechanism underlying these changes in cerebral glucose metabolism remains a critical knowledge gap in the field. Here, we undertook a multi-omic approach by combining single-cell RNA sequencing (scRNAseq) and stable isotope resolved metabolomics (SIRM) to define a metabolic rewiring across astrocytes, brain tissue, mice, and human subjects expressing APOE4. Single-cell analysis of brain tissue from mice expressing human APOE revealed E4-associated decreases in genes related to oxidative phosphorylation, particularly in astrocytes. This shift was confirmed on a metabolic level with isotopic tracing of .sup.13C-glucose in E4 mice and astrocytes, which showed decreased pyruvate entry into the TCA cycle and increased lactate synthesis. Metabolic phenotyping of E4 astrocytes showed elevated glycolytic activity, decreased oxygen consumption, blunted oxidative flexibility, and a lower rate of glucose oxidation in the presence of lactate. Together, these cellular findings suggest an E4-associated increase in aerobic glycolysis (i.e. the Warburg effect). To test whether this phenomenon translated to APOE4 humans, we analyzed the plasma metabolome of young and middle-aged human participants with and without the Î4 allele, and used indirect calorimetry to measure whole body oxygen consumption and energy expenditure. In line with data from E4-expressing female mice, a subgroup analysis revealed that young female E4 carriers showed a striking decrease in energy expenditure compared to non-carriers. This decrease in energy expenditure was primarily driven by a lower rate of oxygen consumption, and was exaggerated following a dietary glucose challenge. Further, the stunted oxygen consumption was accompanied by markedly increased lactate in the plasma of E4 carriers, and a pathway analysis of the plasma metabolome suggested an increase in aerobic glycolysis. Together, these results suggest astrocyte, brain and system-level metabolic reprogramming in the presence of APOE4, a 'Warburg like' endophenotype that is observable in young females decades prior to clinically manifest AD.

Background Aging microglia accumulate lipid droplets (LDs), secrete pro‐inflammatory cytokines, and are defective in phagocytosis. The E4 allele of Apolipoprotein E (APOE) is the strongest genetic risk factor for late‐onset Alzheimer’s disease (LOAD) and is associated with increased neuroinflammation and LD accumulation. Here, we aimed to determine if the effects of aging and the E4 allele are synergistic in causing the accumulation of LDs seen in LOAD. Specifically, we hypothesize that APOE genotype modulates the LD proteome and lipidome at baseline and with inflammation. Additionally, we show the effects of APOE genotype and age on LD accumulation in monocyte‐derived human macrophages treated with sex‐matched exogenous serum. Method Primary microglia were isolated from mice expressing human ApoE3 and ApoE4. Microglia were exposed to oleic acid (OA), LPS, OA+LPS, dead N2A cells, or dead N2As+LPS to stimulate an inflammatory response, then lipid droplet content was analyzed. To examine the impact of inflammation on LDs in vivo, ApoE3 and ApoE4 mice were injected with saline or LPS and liver tissue collected at 24h. Density gradient centrifugation was used for isolation of LD‐enriched fractions and these were analyzed using mass spectrometry proteomics. To determine the impact of age on circulating factors that may affect LD dynamics, human Peripheral Blood Mononuclear Cells (PBMCs) were treated with serum from different aged donors and the impact on LDs was analyzed. Result Primary microglia from ApoE4 mice accumulated significantly more LDs at baseline, with OA, LPS, and N2As as a percentage of E3 control. Proteomics revealed that LD fractions from E4 mice are enriched for proteins involved in innate immunity, while E3 LDs are enriched for lipid b‐oxidation proteins. Lipidomics showed an increase in phosphatidylcholine distribution in the LD membrane of E4‐control and LPS‐treated droplets. Finally, a significant positive correlation between the age of serum samples used to treat human macrophages and LD accumulation in the PBMCs was observed. Conclusion In agreement with prior studies, E4 microglia accumulate more LDs compared to E3 microglia under all conditions tested. The proteomic profile of E4 LDs support the hypothesis that E4 expression increases inflammation under basal and stimulated conditions, causing a more robust response.

Excess lipid droplet (LD) accumulation is associated with several pathological states, including Alzheimer's disease (AD). However, the mechanism(s) by which changes in LD composition and dynamics contribute to pathophysiology of these disorders remains unclear. Apolipoprotein E (ApoE) is a droplet associated protein with a common risk variant (E4) that confers the largest increase in genetic risk for late-onset AD. E4 is associated with both increased neuroinflammation and excess LD accumulation. In the current study, we sought to quantitatively profile the lipid and protein composition of LDs between the 'neutral' E3 and risk variant E4, to gain insight into potential LD-driven contributions to AD pathogenesis. Targeted replacement mice expressing human E3 or E4 were injected with saline or lipopolysaccharide (LPS), and after 24 hours, hepatic lipid droplets were isolated for proteomic and lipidomic analyses. Lipidomics revealed a shift in the distribution of glycerophospholipids in E4 LDs with a concomitant increase in phosphatidylcholine species, and overall, the baseline profile of E4 LDs resembled that of the LPS-treated groups. Quantitative proteomics showed that LDs from E4 mice are enriched for proteins involved in protein/vesicle transport but have decreased levels of proteins involved in fatty acid β-oxidation. Interestingly, proteins associated with LDs showed substantial overlap with previously published lists of AD postmortem tissue and microglia 'omics studies, suggesting a potential role for LDs in modulating AD risk or progression. Given this, we exposed primary microglia from the same E3 or E4 mice to exogenous lipid, inflammatory stimulation, necroptotic N2A cells (nN2A), or a combination of treatments to evaluate LD formation and its impact on the cells' immune state. Microglia from E4 mice accumulated more LDs in every condition tested - at baseline and following addition of fatty acids, LPS stimulation, or nN2As. E4 microglia also secreted significantly more cytokines (TNF, IL-1β, IL-10) than E3 microglia in the control, oleic acid, and nN2A treatment conditions, yet showed a blunted response to LPS. In sum, these results suggest that E4 microglia accumulate more LDs compared to E3 microglia and that E4 is associated with a basal LD composition that resembles a pro-inflammatory cell. Together with the high overlap of the LD proteome with established AD-associated datasets, these data further support the idea that alterations in LD dynamics, particularly within microglia, may contribute to the increased risk for AD associated with .

Aging microglia accumulate lipid droplets (LDs), secrete pro-inflammatory cytokines, and are defective in phagocytosis. The E4 allele of Apolipoprotein E (APOE) is the strongest genetic risk factor for late-onset Alzheimer's disease (LOAD) and is associated with increased neuroinflammation and LD accumulation. Here, we aimed to determine if the effects of aging and the E4 allele are synergistic in causing the accumulation of LDs seen in LOAD. Specifically, we hypothesize that APOE genotype modulates the LD proteome and lipidome at baseline and with inflammation. Additionally, we show the effects of APOE genotype and age on LD accumulation in monocyte-derived human macrophages treated with sex-matched exogenous serum. Primary microglia were isolated from mice expressing human ApoE3 and ApoE4. Microglia were exposed to oleic acid (OA), LPS, OA+LPS, dead N2A cells, or dead N2As+LPS to stimulate an inflammatory response, then lipid droplet content was analyzed. To examine the impact of inflammation on LDs in vivo, ApoE3 and ApoE4 mice were injected with saline or LPS and liver tissue collected at 24h. Density gradient centrifugation was used for isolation of LD-enriched fractions and these were analyzed using mass spectrometry proteomics. To determine the impact of age on circulating factors that may affect LD dynamics, human Peripheral Blood Mononuclear Cells (PBMCs) were treated with serum from different aged donors and the impact on LDs was analyzed. Primary microglia from ApoE4 mice accumulated significantly more LDs at baseline, with OA, LPS, and N2As as a percentage of E3 control. Proteomics revealed that LD fractions from E4 mice are enriched for proteins involved in innate immunity, while E3 LDs are enriched for lipid b-oxidation proteins. Lipidomics showed an increase in phosphatidylcholine distribution in the LD membrane of E4-control and LPS-treated droplets. Finally, a significant positive correlation between the age of serum samples used to treat human macrophages and LD accumulation in the PBMCs was observed. In agreement with prior studies, E4 microglia accumulate more LDs compared to E3 microglia under all conditions tested. The proteomic profile of E4 LDs support the hypothesis that E4 expression increases inflammation under basal and stimulated conditions, causing a more robust response.

Apolipoprotein E4 (APOE4) is the strongest risk allele associated with the development of late onset Alzheimer's disease (AD). Across the CNS, astrocytes are the predominant expressor of while also being critical mediators of neuroinflammation and cerebral metabolism. APOE4 has been consistently linked with dysfunctional inflammation and metabolic processes, yet insights into the molecular constituents driving these responses remain unclear. Utilizing complementary approaches across humanized APOE mice and isogenic human iPSC astrocytes, we demonstrate that ApoE4 alters the astrocyte immunometabolic response to pro-inflammatory stimuli. Our findings show that ApoE4-expressing astrocytes acquire distinct transcriptional repertoires at single-cell and spatially-resolved domains, which are driven in-part by preferential utilization of the cRel transcription factor. Further, inhibiting cRel translocation in ApoE4 astrocytes abrogates inflammatory-induced glycolytic shifts and in tandem mitigates production of multiple pro-inflammatory cytokines. Altogether, our findings elucidate novel cellular underpinnings by which ApoE4 drives maladaptive immunometabolic responses of astrocytes.

The E4 allele of Apolipoprotein E (APOE) is associated with both metabolic dysfunction and a heightened pro-inflammatory response — two findings that may be intrinsically linked through the concept of immunometabolism. Here, we combined bulk, single-cell, and spatial transcriptomics with cell-specific and spatially resolved metabolic analyses to systematically address the role of APOE across age, neuroinflammation, and AD pathology. RNAseq highlighted immunometabolic changes across the APOE4 glial transcriptome, specifically in subsets of metabolically distinct microglia enriched in the E4 brain during aging or following an inflammatory challenge. E4 microglia display increased Hif1α expression, a disrupted TCA cycle, and are inherently pro-glycolytic, while spatial transcriptomics and MALDI mass spectrometry imaging highlight an E4-specific response to amyloid that is characterized by widespread alterations in lipid metabolism. Taken together, our findings emphasize a central role for APOE in regulating microglial immunometabolism. Competing Interest Statement The authors have declared no competing interest. Footnotes * http://www.ljohnsonlab.com/database.html