apoE Treatment Research Articles

Apoliprotein E-mediated ferroptosis controls cellular proliferation in chronic lymphocytic leukemia.

Unraveling vulnerabilities in chronic lymphocytic leukemia (CLL) represents a key approach to understand molecular basis for its indolence and a path toward developing tailored therapeutic approaches. In this study, we found that CLL cells are particularly sensitive to the inhibitory action of abundant serum protein, apolipoprotein E (ApoE). Physiological concentrations of ApoE affect CLL cell viability and inhibit CD40-driven proliferation. Transcriptomics of ApoE-treated CLL cells revealed a signature of redox and metal disbalance which prompted us to explore the underlying mechanism of cell death. We discover, on one hand, that ApoE treatment of CLL cells induces lipid peroxidation and ferroptosis. On the other hand, we find that ApoE is a copper-binding protein and that intracellular copper regulates ApoE toxicity. ApoE regulation tends to be lost in aggressive CLL. CLL cells from patients with high leukocyte counts are less sensitive to ApoE inhibition, while resistance to ApoE is possible in transformed CLL cells from patients with Richter syndrome (RS). Nevertheless, both aggressive CLL and RS cells maintain sensitivity to drug-induced ferroptosis. Our findings suggest a natural suppression axis that mediates ferroptotic disruption of CLL cell proliferation, building up the rationale for choosing ferroptosis as a therapeutic target in CLL and RS.

Downregulating functional hyperactivity in APOE ε4 carriers: an important avenue for early‐life risk reduction

AbstractBackgroundHyperactivity in the brain regions supporting episodic memory (the hippocampus and default mode network) drives the accumulation of Alzheimer’s Disease (AD) neuropathology and the resulting cascade of brain network disruption. Pharmacologically reducing hippocampal hyperactivity using the anti‐seizure medication Levetiracetam improves memory in individuals with prodromal AD. Carriers of an APOE e4 genetic risk variant show patterns of brain hyperactivity from youth. This study tests if Levetiracetam can prophylactically manage hippocampal hyperactivity in mid‐age APOE e4 carriers, and if such a reduction is associated with improved cognitive performance.MethodFifty cognitively healthy adults (aged 45‐65 years), differentiated by APOE genotype (25 e4 carriers, 25 e33 carriers), are participating in a double‐blind, placebo‐controlled trial of Levetiracetam (125mg bidaily for two‐weeks). Analyses will primarily focus on change in task‐related and resting functional magnetic resonance imaging (fMRI) brain blood‐oxygen‐level‐dependent response (BOLD) following Levetiracetam. In addition, the everyday cognitive effects of this manipulation will be evaluated, including on daily smartphone‐based measures of memory and attention.ResultTo date, 43 participants are enrolled in the 8‐week intervention (23 of whom have completed). Adverse events relating to the study drug have been minimal; only one participant has withdrawn in response to experiencing side‐effects. APOE genotype and treatment order (Levetiracetam, placebo) will be unblinded at the end of data collection (anticipated April 2023).ConclusionConclusions from this work will help expose routes to AD risk reduction, and the potential for repurposing an existing treatment for mid‐age individuals at elevated genetic risk for future dementia. This research will motivate longer‐term clinical trials for risk reduction strategies across the lifespan.

Associations between pituitary-ovarian hormones and cognition in recently menopausal women independent of type of hormone therapy

ObjectivesTo examine associations of pituitary-ovarian hormone levels with cognition before and after different formulations of hormone therapy (HT) or placebo independent of treatment group. MethodsRecently menopausal, healthy women were randomized to 0.45 mg/day oral conjugated equine estrogens (o-CEE, n = 109), 50 μg/day transdermal 17β (tE2, n = 107) or placebo pills and patches (n = 146); women on active treatment received oral 200 mg/day micronized progesterone for 12 days per month. Levels of estrone, 17β-estradiol, follicle stimulating hormone, luteinizing hormone, androstenedione, and testosterone were determined prior to and after 48 months of study participation. Neuropsychological testing was administered at baseline, and months 18, 36 and 48. Latent growth curve models controlling for education level, age, APOE allele status, waist circumference, and treatment examined the trajectories of each cognitive domain after accounting for the effect of hormone levels at baseline and months 18, 36 and 48. A linear multivariate mixed model examined the effect of changes in hormone levels on changes in trajectories of complex attention tasks with varying degrees of difficulty. ResultsAll women were adherent to treatment at month 48. Higher baseline estrone levels were associated with poorer global cognition, auditory attention and working memory, visual attention, and executive function, but not working memory. Higher levels of baseline 17β-E2 were associated with poorer cognitive performance, with marginal significance at baseline in speeded language and mental flexibility (p = 0.013). Other hormone levels were not associated with cognition. Controlling for all treatments, hormone levels at baseline and at month 48 did not have any significant correlation with cognitive trajectories over time. SummaryIn healthy, recently menopausal women, baseline estrone levels were inversely associated with selected cognitive factors independent of two types of HT or placebo during 4 years of follow-up. Baseline levels of the other pituitary-ovarian hormones studied were not associated with baseline cognition, nor were changes in any hormones associated with changes in cognition during the study. The marginal association between estradiol levels and cognitive factors warrants further investigation. ClinicalTrials.gov numbersNCT00154180, NCT00623311.

Apolipoprotein E impairs aged bone fracture healing

Bone fracture healing and osteoblast differentiation are impaired with advanced age. Using a combination of parabiosis and proteomic models, we identified apolipoprotein E (ApoE) to be an aging factor in bone regeneration. Circulating levels of ApoE increased with age in patients and in mice. ApoE impaired bone fracture healing by decreasing bone deposition in the fracture callus which subsequently decreased the mechanical strength of healed tissue. Osteoblasts serve as the sole bone forming cells within the body. In tissue culture models, ApoE treatment decreased osteoblast differentiation and activity which led to decreased matrix formation and mineralization. This inhibition of osteoblast differentiation relied on down-regulation of the Wnt/β-catenin pathway. In mouse models, aged bone repair was rejuvenated when we lowered circulating ApoE levels using a hepatotropic AAV-siRNA model – serving as a proof of concept that ApoE can be targeted to improve bone repair in an older population. While promising, knockdown of circulating ApoE in such a fashion is likely not translatable to patient care. Thus, current work in our laboratory is focused on developing treatment strategies that temporally decrease circulating ApoE levels and consequently improve bone healing after acute injury and/or surgical orthopedic procedure in the geriatric population.

CXCR4-Binding Positron Emission Tomography Tracers Link Monocyte Recruitment and Endothelial Injury in Murine Atherosclerosis.

vMIP-II (viral macrophage inflammatory protein 2)/vCCL2 (viral chemotactic cytokine ligand 2) binds to multiple chemokine receptors, and vMIP-II-based positron emission tomography tracer (64Cu-DOTA-vMIP-II: vMIP-II tracer) accumulates at atherosclerotic lesions in mice. Given that it would be expected to react with multiple chemokine receptors on monocytes and macrophages, we wondered if its accumulation in atherosclerosis lesion-bearing mice might correlate with overall macrophage burden or, alternatively, the pace of monocyte recruitment. Approach and Results: We employed a mouse model of atherosclerosis regression involving adenoassociated virus 8 vector encoding murine Apoe (AAV-mApoE) treatment of Apoe-/- mice where the pace of monocyte recruitment slows before macrophage burden subsequently declines. Accumulation of 64Cu-DOTA-vMIP-II at Apoe-/- plaque sites was strong but declined with AAV-mApoE-induced decline in monocyte recruitment, before macrophage burden reduced. Monocyte depletion indicated that monocytes and macrophages themselves were not the only target of the 64Cu-DOTA-vMIP-II tracer. Using fluorescence-tagged vMIP-II tracer, competitive receptor blocking with CXCR4 antagonists, endothelial-specific Cre-mediated deletion of CXCR4, CXCR4-specific tracer 64Cu-DOTA-FC131, and CXCR4 staining during disease progression and regression, we show endothelial cell expression of CXCR4 is a key target of 64Cu-DOTA-vMIP-II imaging. Expression of CXCR4 was low in nonplaque areas but strongly detected on endothelium of progressing plaques, especially on proliferating endothelium, where vascular permeability was increased and monocyte recruitment was the strongest. Endothelial injury status of plaques is marked by CXCR4 expression and this injury correlates with the tendency of such plaques to recruit monocytes. Furthermore, our findings suggest positron emission tomography tracers that mark CXCR4 can be used translationally to monitor the state of plaque injury and monocyte recruitment.

Activation of CREB Protein With Tabersonine Attenuates STAT3 During Atherosclerosis in Apolipoprotein E-Deficient Mice

Objective:Atherosclerosis is a pathological condition of fat deposition in the arteries, which causes cardiovascular disorders. Management of atherosclerosis remains a challenge and conventional drugs used for its management have several limitations. This study evaluated the protective effect of tabersonine against atherosclerosis and assessed its molecular mechanism of action.Methods:Atherosclerosis was induced by feeding apolipoprotein E (ApoE)-deficient mice a high-fat diet. Mice were treated with 20 or 40 mg/kg of tabersonine intraperitoneally for the 12-week duration of the study. Atherosclerosis markers and nitric oxide were measured in the sera of ApoE-deficient mice. Mediators of inflammation and markers of oxidative stress were assessed using enzyme-linked immunosorbent assays. Western blotting, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry analyses were conducted to determine the protein expression in aortic tissue.Results:The tabersonine-treatment groups had an improved lipid profile and enhanced liver function, compared to the ApoE treatment group. Tabersonine treatment resulted in reduced levels of nitric oxide, cytokines, and oxidative stress, compared to the ApoE group. The altered expression levels of protein inhibitor activated STAT-3 (PIAS3), signal transducer and activator of transcription-3 (STAT-3), and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα) in ApoE-deficient mice were ameliorated by tabersonine treatment. Moreover, cAMP-response-element-binding (CREB) expression was elevated in aortic tissue of tabersonine treatment groups, compared to the ApoE group.Conclusion:These results suggested that tabersonine ameliorates the expression of STAT-3 by activating CREB protein in atherosclerotic ApoE-deficient mice.

Lowering circulating apolipoprotein E levels improves aged bone fracture healing.

Age is a well-established risk factor for impaired bone fracture healing. Here, we identify a role for apolipoprotein E (ApoE) in age-associated impairment of bone fracture healing and osteoblast differentiation, and we investigate the mechanism by which ApoE alters these processes. We identified that, in both humans and mice, circulating ApoE levels increase with age. We assessed bone healing in WT and ApoE-/- mice after performing tibial fracture surgery: bone deposition was higher within fracture calluses from ApoE-/- mice. In vitro recombinant ApoE (rApoE) treatment of differentiating osteoblasts decreased cellular differentiation and matrix mineralization. Moreover, this rApoE treatment decreased osteoblast glycolytic activity while increasing lipid uptake and fatty acid oxidation. Using parabiosis models, we determined that circulating ApoE plays a strong inhibitory role in bone repair. Using an adeno-associated virus-based siRNA system, we decreased circulating ApoE levels in 24-month-old mice and demonstrated that, as a result, fracture calluses from these aged mice displayed enhanced bone deposition and mechanical strength. Our results demonstrate that circulating ApoE as an aging factor inhibits bone fracture healing by altering osteoblast metabolism, thereby identifying ApoE as a new therapeutic target for improving bone repair in the elderly.

Abstract TP103: Cholesterol Transporters Abca1 and Apoe Mediate Grey/White Matter Remodeling and Neurogenesis/Oligodendrogenesis After Stroke

The ATP-binding cassette transporter-A1 (ABCA1) and apolipoprotein-E (ApoE) are major cholesterol transporters in the brain. ABCA1 effluxes cholesterol and phospholipids to extra-cellular, and ApoE transport cholesterol and lipid back into cells. Brain-specific ABCA1-deficient (ABCA1 -B/-B ) mice exhibit reduced ApoE level in brain and cerebrospinal-fluid (CSF), and increased white matter damage and neurological deficits after stroke. Hypothesis: ApoE-treatment increases grey/white matter remodeling and neurogenesis/oligodendrogenesis in the ischemic brain in ABCA1 -B/-B -stroke mice. Methods: Adult ABCA1 -B/-B mice were subjected to distal middle cerebral artery occlusion (dMCAo) and were intraventricularly-infused with recombinant human ApoE2 (rhApoE2, 25μg in artificial-CSF) or artificial-CSF as vehicle-control by transplanting a micro-osmotic pump into the right lateral-ventricle starting 24h after dMCAo until sacrifice 14 days after dMCAo. Primary cultured cortical neurons (PCNs) and oligodendrocyte progenitor cells (OPCs) derived from ABCA1 -B/-B and ABCA1-floxed control (ABCA1 fl/fl ) mice were employed, and the PCN-axonal outgrowth and OPC proliferation/death were investigated. Results: No changes in the lesion volume between ABCA1 -B/-B +CSF and ABCA1 -B/-B +rhApoE2 mice were found. Compared with ABCA1 -B/-B +CSF mice, the ABCA1 -B/-B +rhApoE2 mice exhibit: 1) increased CSF ApoE level and improved functional outcome 7 and 14 days after dMCAo; 2) increased grey matter and white matter density in the ischemic boundary zone; 3) increased neurogenesis in the subventricular zone and oligodendrogenesis in the corpus collosum in the ischemic brain (p<0.05, n=11/group). In vitro data show that: 1) ABCA1 -B/-B decreased axonal-outgrowth and ApoE-treatment (2, 4 and 8 μg/ml) significantly attenuated ABCA1 -B/-B -induced reduction in axonal-outgrowth; 2) ABCA1 -B/-B decreased OPC proliferation and survival compared with ABCA1 fl/fl -OPCs and ApoE treatment increased OPC proliferation and OPC survival in primary cultured ABCA1 -B/-B -OPCs. Conclusion: ABCA1/ApoE contributes to grey and white matter remodeling, increases neurogenesis and oligodendrogenesis as well as improves functional recovery after stroke.

CX3CR1 deficiency promotes muscle repair and regeneration by enhancing macrophage ApoE production.

Muscle injury triggers inflammation in which infiltrating mononuclear phagocytes are crucial for tissue regeneration. The interaction of the CCL2/CCR2 and CX3CL1/CX3CR1 chemokine axis that guides phagocyte infiltration is incompletely understood. Here, we show that CX3CR1 deficiency promotes muscle repair and rescues Ccl2−/− mice from impaired muscle regeneration as a result of altered macrophage function, not infiltration. Transcriptomic analysis of muscle mononuclear phagocytes reveals that Apolipoprotein E (ApoE) is upregulated in mice with efficient regeneration. ApoE treatment enhances phagocytosis by mononuclear phagocytes in vitro, and restores phagocytic activity and muscle regeneration in Ccl2−/− mice. Because CX3CR1 deficiency may compensate for defective CCL2-dependant monocyte recruitment by modulating ApoE-dependent macrophage phagocytic activity, targeting CX3CR1 expressed by macrophages might be a powerful therapeutic approach to improve muscle regeneration.

Apolipoprotein E genotype‐specific short‐term cognitive benefits of treatment with the antihypertensive nilvadipine in Alzheimer's patients—an open‐label trial

Evidence suggests that dihydropyridine calcium channel blockers may be useful in preventing and treating Alzheimer's disease (AD). In an open-label trial of safety and tolerability of nilvadipine in patients with AD, we examined cognition and executive function over a short time period to determine an influence of nilvadipine on these outcomes. We investigated change in cognition using the Mini mental state examination and in executive function using the EXIT25 in 55 patients with AD who received nilvadipine 8 mg daily for 6 weeks compared with 30 non-treated subjects with AD. Apolipoprotein E genotyping was performed, and the study team and caregivers were kept blinded to APOE ε4 status during the trial. Aside from differences in gender and education, both the treatment and the control groups were similar in general demographics and on baseline cognition status. After correction for potential confounders, APOE ε4 status, and use of other antihypertensive medications, a significant impact of study intervention was observed on MMSE (F = 8.67, p < 0.01) and EXIT (F = 8.77, p < 0.03) scores. An interaction between APOE ε4 carrier status and treatment (p ≤ 0.05) was observed for both outcome measures. In this open-label trial, among APOE ε4 non-carriers, we observed stabilization of cognition and improvement in executive function among treated individuals compared with non-treated individuals. Among APOE ε4 carriers, cognitive stabilization was evident for treated individuals whereas a cognitive decline was observed in non-treated individuals. These findings provide additional evidence for potential therapeutic efficacy of nilvadipine in treating AD and warrant further investigation.

Apolipoprotein E reduces food intake via PI3K/Akt signaling pathway in the hypothalamus

Apolipoprotein E (apoE) is a satiation factor. While central apoE administration reduces food intake, the specific intracellular signaling mechanisms activated by apoE remain largely unknown. Using primary cultured hypothalamic neurons, we demonstrated that apoE treatment (50 nM) elicited rapid activation of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling cascade. Specifically, apoE induced the phosphorylation of Akt, peaking at 30 min, and the increased phosphorylation of Akt was significantly attenuated after pretreatment with LY294002 (50 μM), an inhibitor of the PI3K signaling pathway. To determine whether the activation of PI3K by apoE is required for the ability of apoE to reduce food intake, LY294002 (1 nmol) was infused into the 3rd-cerebral ventricle before injection of an anorectic dose of apoE. Consistent with our previous report, apoE (4 μg) exerted significant reduction of food intake in the 4-h fasted rats, compared with saline. Pretreatment with LY294002 significantly attenuated the potency of exogenous apoE to induce satiation, while the same dose of PI3K inhibitor by itself caused only a slight non-significant decrease of food intake. These results indicate that the activation of the PI3K/Akt pathway is necessary for the acute effects of apoE on food intake.

Apolipoprotein E-Mediated Immune Regulation in Sepsis

Lipids and lipoproteins have emerged as key constituents of the immune response to microbial infection. We, therefore, sought to understand the complex interaction between lipoprotein metabolism and sepsis. Apolipoprotein E (apoE), a component of plasma lipoproteins, has been suggested to bind and traffic Ags for NKT cell activation. However, apoE's role in sepsis has not been demonstrated. In this study, we examined the effect of exogenous apoE in a rat model of septic peritonitis, induced by cecal ligation and puncture. We demonstrate that 48 h after serial injections of apoE, septic mortality increased in a dose-dependent manner. While sepsis resulted in increased splenic and decreased hepatic and circulating NKT cell populations, serial injections of apoE for 24 h after cecal ligation and puncture increased the frequency, cell number, and BrdU uptake in splenic and hepatic NKT cell populations, while concomitantly depleting these populations in the circulation. These changes were correlated with elevated alanine amino transferase levels, an indicator of liver injury. Interestingly, while sepsis increased hepatic T cell apoptosis and necrosis, apoE reversed these changes. apoE also promoted increases in predominantly Th1 cytokine levels in sera and a decrease in IL-4, the main NKT cell-derived Th2 cytokine. Consequently, apoE treatment is associated with increased sepsis-induced mortality, and increased NKT cell frequency and proliferation in the liver and spleen, with concomitant decreases in these NKT cell parameters in the peripheral circulation. apoE treatment also promoted a Th1 cytokine response, increased the degree of liver injury, and decreased apoptosis in hepatic lymphocytes.

APOE ε3 Gene Transfer Attenuates Brain Damage after Experimental Stroke

Apolipoprotein E (apoE, protein; APOE, gene) is the major lipid-transport protein in the brain and plays an important role in modulating the outcome and regenerative processes after acute brain injury. The aim of the present study was to determine if gene transfer of the epsilon3 form of APOE improves outcome in a murine model of transient focal cerebral ischaemia. Mice received an intrastriatal injection of vehicle, a second-generation adenoviral vector containing the green fluorescent protein gene (Ad-GFP) or a vector containing the APOE epsilon3 gene (Ad-APOE) 3 days before 60 mins focal ischaemia. Green fluorescent protein expression was observed in cells throughout the striatum and subcortical white matter indicating successful gene transfer and expression. ApoE levels in the brain were significantly increased after Ad-APOE compared with Ad-GFP or vehicle treatment. Ad-APOE treatment reduced the volume of ischaemic damage by 50% compared with Ad-GFP or vehicle treatment (13+/-3 versus 29+/-4 versus 27+/-5 mm(3)). The extent of postischaemic apoE immunoreactivity was enhanced in Ad-APOE compared with Ad-GFP or vehicle treated mice. These results show the ability of APOE gene transfer to markedly improve outcome after cerebral ischaemia and suggest that modulating apoE levels may be a potential strategy in human stroke therapy.

Apolipoprotein E decreases tau kinases and phospho-tau levels in primary neurons

Apolipoprotein E (apoE) receptors act as signaling molecules in neurons, altering phosphorylation of numerous proteins after extracellular ligand binding and affecting neurite outgrowth, synapse formation, and neuronal migration. Since apoE is important in the pathogenesis of Alzheimer's disease (AD), we tested whether apoE treatment of neurons affected molecules important to phosphorylation of tau, such as GSK 3β, P35, and CDK5, and the phosphorylation of tau itself. Treatment of primary neurons with 2 uM apoE (or an apoE-derived peptide) decreased levels of phospho-GSK 3β, P35 and CDK5, and decreased levels of phosphorylated forms of tau. A lower concentration of apoE (100 nM) had no effect on these molecules. The alteration of tau phosphorylation by apoE was blocked by an inhibitor of the low-density lipoprotein receptor family, demonstrating the effects were due to receptor interactions. These results demonstrate that apoE affects several downstream signaling cascades in neurons: decreased tau kinases phosphorylation and inhibition of tau phosphorylation at Thr171 and Ser202/Thr205 epitopes. We conclude that apoE can alter levels of tau kinases and phospho-tau epitopes, potentially affecting tau neuropathological changes seen in AD brains.

Apolipoprotein E Receptor 2 Interactions with the N-Methyl-D-aspartate Receptor

In our previous studies we showed that apoE treatment of neurons activated ERK 1/2 signaling, and activation was blocked by treatment with inhibitors of the low density lipoprotein receptor family, the N-methyl-d-aspartate (NMDA) receptor antagonist MK 801, and calcium channel blockers. We hypothesized an interaction between the low density lipoprotein receptor family members and the NMDA receptor. In the present study, we confirmed through co-immunoprecipitation experiments an interaction between the apoE receptor, ApoEr2, and NMDAR1 through their extracellular domains. We also found that the PDZ1 domain of PSD95, a postsynaptic scaffolding protein, interacted with the C terminus of ApoEr2 via an alternatively spliced, intracellular exon. This interaction between ApoEr2 and PSD95 in neurons was modulated by NMDA receptor activation and an ApoEr2 ligand. We also found that the PDZ2 domain of PSD95 interacted with the NR2A and NR2B subunits of NMDA receptors. Full-length PSD95 increased cell surface levels of ApoEr2 and its cleavage, resulting in increases in secreted ApoEr2 and C-terminal fragments of ApoEr2. These studies suggest that ApoEr2 can form a multiprotein complex with NMDA receptor subunits and PSD95.

Multiple pathways of apolipoprotein E signaling in primary neurons

Apolipoprotein E is a genetic risk factor for Alzheimer's disease, and the apoE protein is associated with beta-amyloid deposits in Alzheimer's disease brain. We examined signaling pathways stimulated by apoE in primary neurons in culture. ApoE and an apoE-derived peptide activated several intracellular kinases, including prominently extracellular signal-regulated kinase 1/2 (ERK1/2). ERK1/2 activation by apoE was blocked by an inhibitor of the low-density lipoprotein receptor family, the specific NMDA glutamate receptor antagonist MK 801 and other calcium channel blockers. Activation of apoE receptors also induced tyrosine phosphorylation of Dab1, an adaptor protein of apoE receptors, but experiments in Dab1 knockout neurons demonstrated that Dab1 was not necessary for ERK activation. In contrast, apoE treatment of primary neurons decreased activation of c-Jun N-terminal kinase, a kinase that interacts with another apoE receptor adaptor protein, c-Jun N-terminal kinase-interacting protein. This change also depended on interactions with the low-density lipoprotein receptor family but was independent of calcium channels. c-Jun N-terminal kinase deactivation by apoE was blocked by gamma-secretase inhibitors and pertussis toxin. These results demonstrate that apoE affects several signaling cascades in neurons: increased disabled phosphorylation, activation of the ERK1/2 pathway (dependent on calcium influx via the NMDA receptor) and inhibition of the c-Jun N-terminal kinase 1/2 pathway (dependent on gamma-secretase and G proteins).

Apolipoprotein E protects against oxidative stress in mixed neuronal–glial cell cultures by reducing glutamate toxicity

Apolipoprotein E (ApoE) deficiency has been shown to adversely affect outcome after transient cerebral ischemia and head trauma. Since oxidative stress contributes to these injuries, the ability of ApoE to reduce irreversible oxidative damage was studied in primary mixed neuronal–glial cell cultures. Cells (13–16 days in vitro) were exposed to 50 μM hydrogen peroxide (H 2O 2) for 30 min, and toxicity was determined by the release of lactate dehydrogenase (LDH) 24 h after exposure. The presence of recombinant human ApoE2 (100, 300, or 1000 nM) in the culture media partially protected against oxidative injury. This protection was not reversed by pre-treatment with receptor associated protein. The NMDA receptor antagonist, MK-801, also provided partial protection against H 2O 2 toxicity. The degree of protection was similar to that conferred by ApoE treatment. The protective effects of ApoE and MK-801 were not additive; no ApoE protection was observed in cultures treated with MK-801 prior to H 2O 2 exposure. ApoE treatment had no effect on H 2O 2 stimulated glutamate release, but did increase the rate of glutamate uptake via the high affinity glutamate transporter in H 2O 2 treated cultures. Pre-treatment with ApoE also conferred partial protection against glutamate-induced LDH release. Taken together, these findings suggest that ApoE protects mixed neuronal–glial cell cultures against irreversible oxidative injury from H 2O 2 by reducing secondary glutamate excitotoxicity.

Modulation of Nitric Oxide Production in Human Macrophages by Apolipoprotein-E and Amyloid-Beta Peptide

Induction of oxidative stress has been implicated as a causative factor in chronic neurodegenerative diseases such as Alzheimer's disease. Apolipoprotein-E (apoE) and amyloid-βeta peptide (Aβ) have been reported to alter the redox state of the brain. Using human monocyte-derived macrophages as a model of brain microglia, physiological levels of apolipoprotein-E were found to stimulate nitric oxide (NO) production in polyinosinic:polycytidylic acid (poly I:C) primed cells. ApoE treatment released 68% more NO than cells treated with poly I:C alone and almost threefold more NO than unprimed cells. In contrast to mouse microglia, human cells failed to generate NO in response to Aβ peptides, with or without poly I:C treatments. Furthermore, the combination of Aβ plus apoE inhibited the increase in NO production induced by apoE. Since Alzheimer's is strongly associated with the presence of an APOE4allele, our study predicts a mechanism where apoE and Aβ regulate nitric oxide production in human brain.

Effect of Apolipoprotein E on Neurite Outgrowth and β‐Amyloid‐Induced Toxicity in Developing Rat Primary Hippocampal Cultures

The correlation between the epsilon 4 allele of apolipoprotein E (apoE) and Alzheimer's disease is well established. However, the role of apoE in normal as well as pathological brain processes remains unclear. We evaluated the effect of apoE treatment on development and beta-amyloid (A beta)-induced toxicity using primary cultures of developing rat hippocampal neurons. The source of apoE was conditioned media from HEK cells stably transfected with human apoE3 or apoE4 cDNA, a preparation where apoE is lipid-associated. Morphological and biochemical changes in the cultures were assessed at 1 and 3 days following low- and high-density plating with either apoE3 or E4 with or without A beta. Both apoE isoforms were neurotrophic, as measured by increased neurite length. Aged A beta(1-42), a peptide preparation exhibiting extensive fibril and aggregate formation, is toxic to these cultures. Addition of apoE3 and E4 significantly and comparably attenuated the A beta-induced reduction in both neurite length and cell viability. The level of protection against this toxicity was proportional to the neurotrophic actions of the two apoE isoforms. Thus, apoE acts as a potent growth factor in both the absence and the presence of A beta, supporting a potentially important role for apoE in neurobiology.