ApoE4 Astrocytes Research Articles

APOE traffics to astrocyte lipid droplets and modulates triglyceride saturation and droplet size.

The E4 variant of APOE strongly predisposes individuals to late-onset Alzheimer's disease. We demonstrate that in response to lipogenesis, apolipoprotein E (APOE) in astrocytes can avoid translocation into the endoplasmic reticulum (ER) lumen and traffic to lipid droplets (LDs) via membrane bridges at ER-LD contacts. APOE knockdown promotes fewer, larger LDs after a fatty acid pulse, which contain more unsaturated triglyceride after fatty acid pulse-chase. This LD size phenotype was rescued by chimeric APOE that targets only LDs. Like APOE depletion, APOE4-expressing astrocytes form a small number of large LDs enriched in unsaturated triglyceride. Additionally, the LDs in APOE4 cells exhibit impaired turnover and increased sensitivity to lipid peroxidation. Our data indicate that APOE plays a previously unrecognized role as an LD surface protein that regulates LD size and composition. APOE4 causes aberrant LD composition and morphology. Our study contributes to accumulating evidence that APOE4 astrocytes with large, unsaturated LDs are sensitized to lipid peroxidation, which could contribute to Alzheimer's disease risk.

Multiomics of iPSC‐derived ApoE3‐ and ApoE4 astrocytes and microglia uncovers cell type specific dysregulation of immunometabolism

AbstractBackgroundApoE4 is the major genetic risk factor for Alzheimer’s disease and the main lipid transporter in brain. How ApoE4 drives AD pathogenesis, and how this is related to changes in lipid metabolism, is not well understood. This lack of mechanistic insight has hampered the development of interventions that can reverse the pathological effects of ApoE4.MethodsHere we perform integrated multiomic analysis (lipidomics, metabolomics, transcriptomics and proteomics) on isogenic iPSC‐derived glia carrying an ApoE4/4, ApoE3/3 or ApoE‐knockout genotype. We employ pathway enrichment to identify candidate biological processes that contribute to AD pathogenesis and perform a number of biochemical‐ and intervention experiments to gain insight into the biological mechanism. We feed iPSC‐derived glia with different lipid species to better understand the interaction between lipids and immune dysfunction. Lastly, we generate a new open‐access resource (the Neurolipid atlas), to allow others in the field to interact with our lipid data.ResultsOur lipidomic analysis shows that ApoE4 has a major effect on lipid metabolism in human iPSC‐derived astrocytes and microglia. In both astrocytes and microglia, ApoE4 causes accumulation of cholesteryl esters (CE), triglycerides (TAG) as well changes in ceramide metabolism. Interestingly, transcriptomic and proteomic analysis in both cell types shows that ApoE4 glia have strongly upregulated cholesterol synthesis despite having already high levels of stored cholesterol (CE). Astrocytes and microglia show very distinct adaptations to ApoE4 in terms of energy production. Whereas ApoE4 astrocytes have higher levels of mitochondrial proteins, ApoE4 microglia downregulate mitochondrial proteins but have increased levels of key glycolytic proteins. Immune profiles, including DAM signatures in microglia, are changed in a similar direction in both ApoE4 astrocytes and microglia. Lipid feeding also directly affect these immune pathways.ConclusionOur data indicate that astrocytes and microglia display both common and distinct immunometabolic adaptations to the ApoE4 genotype. Interestingly, both ApoE4 microglia and astrocytes have increased stored cholesterol yet keep upregulating cholesterol synthesis, indicating a possibly pathogenic broken feedback loop. We also show that this altered lipid metabolism is tightly coupled to immune dysfunction and propose novel mechanisms and interventions that may normalize ApoE4 dysfunction in human glia in AD.

Multi‐omic profiling of isogenic ApoE3/3 and ApoE4/4 iPSC‐derived astrocytes uncovers altered lipid metabolism and mitochondrial function of ApoE4 astrocytes

AbstractBackgroundAs the main genetic risk factor for AD, glial ApoE4 has been implicated in a number of early disease relevant processes, such as lipid dyshomeostasis, mitochondrial dysfunction and altered immune function. This ApoE4 glial phenotype is not yet well characterized and this hampers the rationale design of ApoE4‐targeting interventions.MethodWe characterize the phenotypes of ApoE3/3 and ApoE4/4 iPSC‐derived astrocytes by multi‐omic profiling. In addition, we use lipidomic and metabolic profiling of interventions in ApoE4 astrocytes to define compounds that normalize lipid counts and metabolic function.ResultIn the ApoE4 astrocytes we observe strong accumulation of cholesterol esters (storage product of cholesterol) and triglycerides (storage products of fatty acids), in comparison to the ApoE3 astrocytes. Interestingly, our proteomic and transcriptomic profiling of these cells reveal an increase in cholesterol synthesis, despite the already high levels of cholesterol esters in the cell. Moreover, our proteomic and transcriptomic results display an increase in mitochondrial pathways, such as beta‐oxidation, TCA cycle and oxidative phosphorylation and a decrease in glycolysis. Not only mitochondrial function is altered but also mitochondrial organization, as we see an upregulation of the most relevant proteins for mitochondrial fusion, MFN1, MFN2 and OPA1. Our lipidomic and metabolic profiling of ApoE4 astrocytes after treatment with a number of different interventions reveals new insights into these altered lipid metabolic pathways.ConclusionOur multi‐omic profiling of iPSC‐derived ApoE3/3 and ApoE4/4 astrocytes uncovers that ApoE4 astrocytes have altered lipid metabolism, mitochondrial function and mitochondrial organization. Treatment with interventions reveals new insights into these altered pathways.

Statin therapy reduces the risk of AD: Clinic to bench translation for protective mechanism

AbstractBackgroundCholesterol dysregulation is a well‐documented factor involved in the pathophysiology of Alzheimer’s Disease (AD). APOE4, which is involved in cholesterol transport, is recognized as the strongest genetic risk factor for late‐onset AD. In a previous study using clinical data, we showed that exposure to statins, a first‐line cholesterol lowering therapeutic, was associated with a reduced risk of developing AD. Another study showed the risk reduction associated with statins was greater in APOE4 carriers. Although the effects of statins in the periphery are well documented, the mechanisms by which statins act in the brain remain poorly understood.MethodThis study aimed to identify potential mechanisms by which statins impact AD pathology at a cellular level and the different response by APOE genotype. Humanized APP/APOE3 and APOE4 mouse neurons and astrocytes were co‐cultured and treated with vehicle or atorvastatin at clinically relevant concentrations. Metabolic flux analysis was used to determine the impact of statins on mitochondrial bioenergetics and dynamics. Multi‐electrode array (MEA) was used to conduct electrophysiological recordings on hippocampal neurons. Lipid droplet burden in astrocytic cells was determined using LipidTOX staining and confocal microscopy. qPCR and Western Blot were used to determine the expression of key proteins involved in cholesterol transport.ResultIn this study, hippocampal neurons treated with atorvastatin in the presence of astrocytic cells induced a significant increase in the number of spikes and spike frequency, as well as the number of bursts and number of spikes within a burst. In contrast, in the absence of astrocytes atorvastatin exposure in neurons did not exhibit a significant difference compared to vehicle. Electrophysiological properties were increased in a dose‐dependent manner and the effect was greater 24‐hours following treatment. Atorvastatin also increased astrocyte mitochondrial respiration whereas it did not in neurons or in neurons co‐cultured with astrocytes.ConclusionOutcomes of these analyses indicated that atorvastatin acts primarily on astrocytes. Specifically, atorvastatin increased mitochondrial respiration in astrocytes and increased synaptic activity on neurons which was astrocyte dependent. These results provide evidence of a neuroprotective role of atorvastatin by regulating lipid metabolism deficits in a cell‐specific manner.Acknowledgments: NIA P01AG026572 and R01AG057931 to RDB.

Transcriptomic Profiling Across Age, APOE and Sex for Models of Alzheimer’s Disease at a Single Cell Resolution

AbstractBackgroundAdvanced age, apolipoprotein E4(APOE4), and female sex are well‐established risk factors for Alzheimer’s Disease(AD). Almost two‐thirds of people with AD are women. The APOE4 genotype, the strongest genetic risk factor for AD, is associated with higher AD risk in women than in men between ages 55 to 70. We propose to study the interplay between these risk factors by analyzing single‐nucleus RNA‐sequencing(snRNA‐seq) data of the hippocampus of human APOE4 and APOE3 knock‐in(KI) female and male mice across age.MethodsA sex‐balanced cohort of APOE3‐KI and APOE4‐KI homozygous mice were sacrificed at 6 (young), 12 (adult), and 18 (old) months of age(Figure1A). Single nuclei were prepared from the hippocampus from each mouse and sequenced. Demultiplexed fastq files were aligned to a custom reference genome mm10‐1.2.0, using Cellranger v2.0.1. Quality assurance, count normalization, clustering, cell type identification, and differentially expressed gene(DEG) analysis were performed using Seurat4.0.ResultsOur dataset consists of a total of 527,395 cells covering 27,153 genes. A total of 36 cell clusters were determined with six major brain cell types including astrocytes, microglia, oligodendrocytes, oligodendrocyte precursor cells, and excitatory and inhibitory neurons (Figure1B). We investigated the abundance of each cell type and found that cellular composition is consistent across ages, APOE genotypes, and sexes for all cell types except astrocytes. Astrocyte abundance is significantly higher in old females compared to old males(padj = 0.025, Tukey’s multiple comparison test) and in APOE4 young males compared to APOE3 young males (padj = 0.042)(Figure1C).Sex and age stratified differential gene expression analysis comparing APOE4 versus APOE3 astrocytes reveals many shared DEGs between young and adult males but not with old males, while females have few consistent DEGs across the three age groups(Figure1D). We identified several DEGs associated with plasma membrane assembling and post‐synaptic transmission and signaling that are upregulated in APOE4 old females versus APOE3 old females while downregulated in APOE4 young females, young and adult male compared to their APOE3 counterparts.ConclusionWe identify clear interplay between age, APOE genotype, and sex in mouse models of AD and investigate the interaction and convergence of these risk factors at a single cell resolution.

Isoform‐ and cellular metabolism‐specific apolipoprotein E (ApoE) lipidation in astrocytes

AbstractBackgroundApolipoprotein E (ApoE) is the major lipoprotein in the brain and connects the metabolic interactions between astrocytes and neurons. The E4 variant (APOE4) affects this function and represents a genetic predisposition for Alzheimer’s disease (AD), but the molecular mechanisms remain unclear.MethodWe have integrated cell‐free in vitro assays, a new cell system based on isogenic iPSC‐derived astrocytes and lipidomics to study ApoE lipidation, and how this is affected by genetic polymorphism.ResultWe show that ApoE produces different types of lipoprotein particles via distinct lipidation pathways. ApoE forms high‐density lipoprotein (HDL)‐like, cholesterol‐rich particles via the ATP‐binding cassette transporter 1 (ABCA1), a mechanism largely unaffected by ApoE polymorphism. Alternatively, ectopic accumulation of fat in astrocytes, a stress‐associated condition, redirects ApoE toward the assembly and secretion of triacylglycerol‐rich lipoprotein particles, a process boosted by the APOE4 variant. We demonstrate in vitro that ApoE can detect triacylglycerol in membranes and spontaneously assemble lipoprotein particles (10‐20 nm) rich in unsaturated triacylglycerol, and that APOE4 has remarkable properties behaving as a strong triacylglycerol binder. Fatty APOE4 astrocytes have reduced ability to clear toxic fatty acids from the extracellular milieu, because APOE4 reroutes them back to secretion.ConclusionThe molecular mechanisms discussed here provide a new model explaining the interaction between genetic polymorphism and environmental factors, such as dyslipidaemia or aging, in the aetiology of late‐onset AD, as well as inspire possible new strategies for therapeutic intervention.Graphical abstract from https://doi.org/10.1016/j.celrep.2022.110435.

ApoE4-dependent lysosomal cholesterol accumulation impairs mitochondrial homeostasis and oxidative phosphorylation in human astrocytes

Recent developments in genome sequencing have expanded the knowledge of genetic factors associated with late-onset Alzheimer's disease (AD). Among them, genetic variant ε4 of the APOE gene (APOE4) confers the greatest disease risk. Dysregulated glucose metabolism is an early pathological feature of AD. Using isogenic ApoE3 and ApoE4 astrocytes derived from human induced pluripotent stem cells, we find that ApoE4 increases glycolytic activity but impairs mitochondrial respiration in astrocytes. Ultrastructural and autophagy flux analyses show that ApoE4-induced cholesterol accumulation impairs lysosome-dependent removal of damaged mitochondria. Acute treatment with cholesterol-depleting agents restores autophagic activity, mitochondrial dynamics, and associated proteomes, and extended treatment rescues mitochondrial respiration in ApoE4 astrocytes. Taken together, our study provides a direct link between ApoE4-induced lysosomal cholesterol accumulation and abnormal oxidative phosphorylation.

ApoE4 expression disrupts tau uptake, trafficking, and clearance in astrocytes.

Tauopathies are a collection of neurodegenerative diseases characterized by the accumulation of pathogenic aggregates of the microtubule-associated protein tau. Despite the prevalence and diversity of tau astrogliopathy in tauopathies, the interactions between astrocytes and tau in the brain, and the influence of neurodegenerative genetic risk factors like the apolipoprotein E4 (apoE4) isoform, are largely unknown. Here, we leveraged primary and immortalized astrocytes expressing humanized apoE isoforms to characterize the mechanisms by which astrocytes interact with and eliminate extracellular tau, and the influence of apoE genotype on these processes. Our work indicates that astrocytes rapidly internalize, process, and release tau via an exosomal secretory mechanism under physiological conditions. However, we found that apoE4 disrupted these processes in comparison to apoE3, resulting in an astrocytic phenotype prone to intracellular tau accumulation. Furthermore, exposure to repetitive mild traumatic brain injuries exacerbated the apoE4-induced impairments in tau processing and elimination by astrocytes in apoE4 targeted-replacement mice. The diminished ability of apoE4 astrocytes to eliminate extracellular tau can lead to an accumulation of pathogenic tau, which induces mitochondrial dysfunction, as demonstrated by our studies. In total, our findings suggest that the apoE4 isoform lowers the threshold of astrocytic resilience to pathogenic tau, rendering them susceptible to bioenergetic deficits in the early stages of neurodegenerative diseases such as traumatic brain injury, potentially contributing to neurological decline.

Astrocytic APOE4 genotype-mediated negative impacts on synaptic architecture in human pluripotent stem cell model.

The APOE4 genotype is the strongest risk factor for the pathogenesis of sporadic Alzheimer's disease (AD), but the detailed molecular mechanism of APOE4-mediated synaptic impairment remains to be determined. In this study, we generated a human astrocyte model carrying the APOE3 or APOE4 genotype using human induced pluripotent stem cells (iPSCs) in which isogenic APOE4 iPSCs were genome edited from healthy control APOE3 iPSCs. Next, we demonstrated that the astrocytic APOE4 genotype negatively affects dendritic spine dynamics in a co-culture system with primary neurons. Transcriptome analysis revealed an increase of EDIL3, an extracellular matrix glycoprotein, in human APOE4 astrocytes, which could underlie dendritic spine reduction in neuronal cultures. Accordingly, postmortem AD brains carrying the APOE4 allele have elevated levels of EDIL3 protein deposits within amyloid plaques. Together, these results demonstrate the novel deleterious effect of human APOE4 astrocytes on synaptic architecture and may help to elucidate the mechanism of APOE4-linked AD pathogenesis.

Apolipoprotein E ε4 modulates astrocyte neuronal support functions in the presence of amyloid-β.

Apolipoprotein E (APOE) is a lipid transporter produced predominantly by astrocytes in the brain. The ε4 variant of APOE (APOE4) is the strongest and most common genetic risk factor for Alzheimer's disease (AD). Although the molecular mechanisms of this increased risk are unclear, APOE4 is known to alter immune signaling and lipid and glucose metabolism. Astrocytes provide various forms of support to neurons, including regulating neuronal metabolism and immune responses through cytokine signaling. Changes in astrocyte function because of APOE4 may therefore decrease neuronal support, leaving neurons more vulnerable to stress and disease insults. To determine whether APOE4 alters astrocyte neuronal support functions, we measured glycolytic and oxidative metabolism of neurons treated with conditioned media from APOE4 or APOE3 (the common, risk-neutral variant) primary astrocyte cultures. We found that APOE4 neurons treated with conditioned media from resting APOE4 astrocytes had similar metabolism to APOE3 neurons treated with media from resting APOE3 astrocytes, but treatment with astrocytic conditioned media from astrocytes challenged with amyloid-β (Aβ), a key pathological protein in AD, caused APOE4 neurons to increase their basal mitochondrial and glycolytic metabolic rates more than APOE3 neurons. These changes were not because of differences in astrocytic lactate production or glucose utilization, but instead correlated with increased glycolytic ATP production and a lack of cytokine secretion in response to Aβ. Additionally, we identified that astrocytic cytokine signatures could predict basal metabolism of neurons treated with the astrocytic conditioned media. Together, these findings suggest that in the presence of Aβ, APOE4 astrocytes alter immune and metabolic functions that result in a compensatory increase in neuronal metabolic stress.

ApoE3 vs. ApoE4 Astrocytes: A Detailed Analysis Provides New Insights into Differences in Cholesterol Homeostasis.

The strongest genetic risk factor for sporadic Alzheimer's disease (AD) is the presence of the ε4 allele of the apolipoprotein E (ApoE) gene, the major apolipoprotein involved in brain cholesterol homeostasis. Being astrocytes the main producers of cholesterol and ApoE in the brain, we investigated the impact of the ApoE genotype on astrocyte cholesterol homeostasis. Two mouse astrocytic cell lines expressing the human ApoE3 or ApoE4 isoform were employed. Gas chromatography-mass spectrometry (GC-MS) analysis pointed out that the levels of total cholesterol, cholesterol precursors, and various oxysterols are altered in ApoE4 astrocytes. Moreover, the gene expression analysis of more than 40 lipid-related genes by qRT-PCR showed that certain genes are up-regulated (e.g., CYP27A1) and others down-regulated (e.g., PPARγ, LXRα) in ApoE4, compared to ApoE3 astrocytes. Beyond confirming the significant reduction in the levels of PPARγ, a key transcription factor involved in the maintenance of lipid homeostasis, Western blotting showed that both intracellular and secreted ApoE levels are altered in ApoE4 astrocytes, as well as the levels of receptors and transporters involved in lipid uptake/efflux (ABCA1, LDLR, LRP1, and ApoER2). Data showed that the ApoE genotype clearly affects astrocytic cholesterol homeostasis; however, further investigation is needed to clarify the mechanisms underlying these differences and the consequences on neighboring cells. Indeed, drug development aimed at restoring cholesterol homeostasis could be a potential strategy to counteract AD.

Calcium-dependent cytosolic phospholipase A2 activation is implicated in neuroinflammation and oxidative stress associated with ApoE4

BackgroundApolipoprotein E4 (APOE4) is associated with a greater response to neuroinflammation and the risk of developing late-onset Alzheimer’s disease (AD), but the mechanisms for this association are not clear. The activation of calcium-dependent cytosolic phospholipase A2 (cPLA2) is involved in inflammatory signaling and is elevated within the plaques of AD brains. The relation between APOE4 genotype and cPLA2 activity is not known.MethodsMouse primary astrocytes, mouse and human brain samples differing by APOE genotypes were collected for measuring cPLA2 expression, phosphorylation, and activity in relation to measures of inflammation and oxidative stress.ResultsGreater cPLA2 phosphorylation, cPLA2 activity and leukotriene B4 (LTB4) levels were identified in ApoE4 compared to ApoE3 in primary astrocytes, brains of ApoE-targeted replacement (ApoE-TR) mice, and in human brain homogenates from the inferior frontal cortex of persons with AD dementia carrying APOE3/4 compared to APOE3/3. Higher phosphorylated p38 MAPK but not ERK1/2 was found in ApoE4 primary astrocytes and mouse brains than that in ApoE3. Greater cPLA2 translocation to cytosol was observed in human postmortem frontal cortical synaptosomes with recombinant ApoE4 than ApoE3 ex vivo. In ApoE4 astrocytes, the greater levels of LTB4, reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) were reduced after cPLA2 inhibition.ConclusionsOur findings implicate greater activation of cPLA2 signaling system with APOE4, which could represent a potential drug target for mitigating the increased neuroinflammation with APOE4 and AD.

Isoform- and cell-state-specific lipidation of ApoE in astrocytes.

Apolipoprotein E transports lipids and couples metabolism between astrocytes and neurons. The E4 variant (APOE4) affects these functions and represents a genetic predisposition for Alzheimer's disease, but the molecular mechanisms remain elusive. We show that ApoE produces different types of lipoproteins via distinct lipidation pathways. ApoE forms high-density lipoprotein (HDL)-like, cholesterol-rich particles via the ATP-binding cassette transporter 1 (ABCA1), a mechanism largely unaffected by ApoE polymorphism. Alternatively, ectopic accumulation of fat in astrocytes, a stress-associated condition, redirects ApoE toward the assembly and secretion of triacylglycerol-rich lipoproteins, a process boosted by the APOE4 variant. We demonstrate invitro that ApoE can detect triacylglycerol in membranes and spontaneously assemble lipoprotein particles (10-20nm) rich in unsaturated triacylglycerol, and that APOE4 has remarkable properties behaving as a strong triacylglycerol binder. We propose that fatty APOE4 astrocytes have reduced ability to clear toxic fatty acids from the extracellular milieu, because APOE4 reroutes them back to secretion.

APOE4 expression is associated with impaired autophagy and mitophagy in astrocytes.

APOE4 expression is associated with impaired autophagy and mitophagy in astrocytes.

APOE4-carrying human astrocytes oversupply cholesterol to promote neuronal lipid raft expansion and Aβ generation.

SummaryThe ε4 allele of APOE-encoding apolipoprotein (ApoE) is one of the strongest genetic risk factors for Alzheimer’s disease (AD). One of the overarching questions is whether and how this astrocyte-enriched risk factor initiates AD-associated pathology in neurons such as amyloid-β (Aβ) accumulation. Here, we generate neurons and astrocytes from isogenic human induced pluripotent stem cells (hiPSCs) carrying either APOE ε3 or APOE ε4 allele and investigate the effect of astrocytic ApoE4 on neuronal Aβ production. Secretory factors in conditioned media from ApoE4 astrocytes significantly increased amyloid precursor protein (APP) levels and Aβ secretion in neurons. We further found that increased cholesterol secretion from ApoE4 astrocytes was necessary and sufficient to induce the formation of lipid rafts that potentially provide a physical platform for APP localization and facilitate its processing. Our study reveals the contribution of ApoE4 astrocytes to amyloidosis in neurons by expanding lipid rafts and facilitating Aβ production through an oversupply of cholesterol.

RETRACTED ARTICLE: Calcium-dependent cytosolic phospholipase A2 activation is implicated in neuroinflammation and oxidative stress associated with ApoE4

BackgroundApolipoprotein E4 (APOE4) is associated with a greater response to neuroinflammation and the risk of developing late-onset Alzheimer’s disease (AD), but the mechanisms for this association are not clear. The activation of calcium-dependent cytosolic phospholipase A2 (cPLA2) is involved in inflammatory signaling and is elevated within the plaques of AD brains. The relation between APOE4 genotype and cPLA2 activity is not known.MethodsMouse primary astrocytes, mouse and human brain samples differing by APOE genotypes were collected for measuring cPLA2 expression, phosphorylation, and activity in relation to measures of inflammation and oxidative stress.ResultsGreater cPLA2 phosphorylation, cPLA2 activity and leukotriene B4 (LTB4) levels were identified in ApoE4 compared to ApoE3 in primary astrocytes, brains of ApoE-targeted replacement (ApoE-TR) mice, and in human brain homogenates from the inferior frontal cortex of patients with AD carrying APOE3/E4 compared to APOE3/E3. Greater cPLA2 phosphorylation was also observed in human postmortem frontal cortical synaptosomes and primary astrocytes after treatment with recombinant ApoE4 ex vivo. In ApoE4 astrocytes, the greater levels of LTB4, reactive oxygen species (ROS), and inducible nitric oxide synthase (iNOS) were reduced after cPLA2 inhibition.ConclusionsOur findings implicate greater activation of cPLA2 signaling system with APOE4, which could represent a potential drug target for mitigating the increased neuroinflammation with APOE4 and AD.

Expression and secretion of apoE isoforms in astrocytes and microglia during inflammation.

Neuroinflammation is a common feature in neurodegenerative diseases, modulated by the Alzheimer's disease risk factor, apolipoprotein E (APOE). In the brain, apoE protein is synthesized by astrocytes and microglia. We examined primary cultures of astrocytes and microglia from human APOE (E2, E3, and E4) targeted-replacement mice. Astrocytes secreted two species of apoE, whereas cellular apoE consisted of only one. Both forms of secreted astrocytic apoE were bound during glycoprotein isolation, and enzymatic removal of glycans produced a convergence of the two forms of apoE to a single form; thus, the two species of astrocyte-secreted apoE are differentially glycosylated. Microglia released only a single species of apoE, while cellular apoE consisted of two forms; the secreted apoE and one of the two forms of cellular apoE were glycosylated. We treated the primary glia with either endogenous (TNFα) or exogenous (LPS) pro-inflammatory stimuli. While LPS had no effect on astrocytic apoE, APOE2, and APOE3 microglia increased release of apoE; APOE4 microglia showed no effect. APOE4 microglia showed higher baseline secretion of TNFα compared to APOE2 and APOE3 microglia. TNFα treatment reduced the secretion and cellular expression of apoE only in APOE4 astrocytes. The patterns of apoE species produced by astrocytes and microglia were not affected by inflammation. No changes in APOE mRNA were observed in astrocytes after both treatments. Together, our data demonstrate that astrocytes and microglia differentially express and secrete glycosylated forms of apoE and that APOE4 astrocytes and microglia are deficient in immunomodulation compared to APOE2 and APOE3.

ApoE-Isoform-Dependent SARS-CoV-2 Neurotropism and Cellular Response.

ApoE4, a strong genetic risk factor for Alzheimer disease, has been associated with increased risk for severe COVID-19. However, it is unclear whether ApoE4 alters COVID-19 susceptibility or severity, and the role of direct viral infection in brain cells remains obscure. We tested the neurotropism of SARS-CoV2 in human-induced pluripotent stem cell (hiPSC) models and observed low-grade infection of neurons and astrocytes that is boosted in neuron-astrocyte co-cultures and organoids. We then generated isogenic ApoE3/3 and ApoE4/4 hiPSCs and found an increased rate of SARS-CoV-2 infection in ApoE4/4 neurons and astrocytes. ApoE4 astrocytes exhibited enlarged size and elevated nuclear fragmentation upon SARS-CoV-2 infection. Finally, we show that remdesivir treatment inhibits SARS-CoV2 infection of hiPSC neurons and astrocytes. These findings suggest that ApoE4 may play a causal role in COVID-19 severity. Understanding how risk factors impact COVID-19 susceptibility and severity will help us understand the potential long-term effects in different patient populations.

ApoE4 Impairs Neuron-Astrocyte Coupling of Fatty Acid Metabolism

SUMMARYAlzheimer’s disease (AD) risk gene ApoE4 perturbs brain lipid homeostasis and energy transduction. However, the cell-type-specific mechanism of ApoE4 in modulating brain lipid metabolism is unclear. Here, we describe a detrimental role of ApoE4 in regulating fatty acid (FA) metabolism across neuron and astrocyte in tandem with their distinctive mitochondrial phenotypes. ApoE4 disrupts neuronal function by decreasing FA sequestering in lipid droplets (LDs). FAs in neuronal LDs are exported and internalized by astrocytes, with ApoE4 diminishing the transport efficiency. Further, ApoE4 lowers FA oxidation and leads to lipid accumulation in both astrocyte and the hippocampus. Importantly, diminished capacity of ApoE4 astrocytes in eliminating neuronal lipids and degrading FAs accounts for their compromised metabolic and synaptic support to neurons. Collectively, our findings reveal a mechanism of ApoE4 disruption to brain FA and bioenergetic homeostasis that could underlie the accelerated lipid dysregulation and energy deficits and increased AD risk for ApoE4 carriers.

APOE4 impairs neuron‐astrocyte coupling of fatty acid metabolism

AbstractBackgroundAs the strongest genetic risk factor for late‐onset Alzheimer’s disease, the ε4 variant of apolipoprotein E (APOE4) is known to perturb both lipid homeostasis and energy metabolism in brain. However, the mechanistic role of APOE4 in modulating the bioenergetic aspects of lipid species, and its connection with cell‐type‐specific mitochondrial phenotypes are not well understood.MethodHere, in individual‐ and co‐culture models of primary neurons and astrocytes from humanized APOE3 or APOE4 knockin mice, we describe a loss‐of‐function role of APOE4 in regulating key mechanisms controlling neuron‐astrocyte coupling of lipid metabolism, from neuronal storage of fatty acids (FAs) in lipid droplets (LDs), to the mobilization of neuronal LDs and transport of FAs to astrocytes, and eventually to the energetic degradation of FAs in astrocytes.ResultCompared to APOE3, APOE4 decreases the sequestering of free fatty acids (FAs) in neuronal lipid droplets (LDs), elevates FA levels and leads to mitochondrial‐ and neuronal dysfunction. These neuronal LDs are eliminated upon astrocyte exposure and FAs are transported to astrocytes together with APOE, with APOE4 in either neurons or astrocytes diminishing the transport efficiency. In APOE4 astrocytes, increased mitochondrial fragmentation underlies a metabolic shift towards enhanced glucose metabolism and reduced FA β‐oxidation, which further elicits astrocytic LD accumulation. Moreover, the lesser capacity in eliminating neuronal lipids and performing β‐oxidation results in compromised ability of APOE4 astrocytes in providing metabolic‐ and synaptic support to neurons.ConclusionThese data support the coupling of neuron lipid homeostasis, astrocytic FA degradation with cell‐specific mitochondrial function, which is essential to protect neurons from bioenergetic deficits and lipotoxicity. Further, our findings reveal the mechanistic basis of APOE4 in disrupting inter‐cellular FA metabolism in brain that may underlie the exaggerated lipid dyshomeostasis and bioenergetic decline, and increased AD risk for APOE4 carriers.