ApoE4 dysregulation incites depressive symptoms and mitochondrial impairments in mice.

Apolipoprotein E4 (ApoE4) is involved in the stress-response processes and is hypothesized to be a risk factor for depression by means of mitochondrial dysfunction. However, their exact roles and underlying mechanisms are largely unknown. ApoE4 transgenic mice (B6. Cg-ApoEtm1Unc Cdh18Tg( GFAP-APOE i4)1Hol /J) were subjected to stress (lipopolysaccharides, LPS) to elucidate the aetiology of ApoE4-induced depression. LPS treatment significantly aggravated depression-like behaviours, concurrent with neuroinflammation and impaired mitochondrial changes, and melatonin/Urolithin A (UA) + 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) reversed these effects in ApoE4 mice. Concurrently, ApoE4 mice exhibited mitophagy deficits, which could be further exacerbated by LPS stimulation, as demonstrated by reduced Atg5, Beclin-1 and Parkin levels, while PINK1 levels were increased. However, these changes were reversed by melatonin treatment. Additionally, proteomic profiling suggested mitochondria-related signalling and network changes in ApoE4 mice, which may underlie the exaggerated response to LPS. Furthermore, HEK 293T cells transfected with ApoE4 showed mitochondria-associated protein and mitophagy defects, including PGC-1α, TFAM, p-AMPKα, PINK1 and LC3B impairments. Additionally, it aggravates mitochondrial impairment (particularly mitophagy), which can be attenuated by triggering autophagy. Collectively, ApoE4 dysregulation enhanced depressive behaviour upon LPS stimulation.

A new andrographolide derivative ADA targeting SIRT3-FOXO3a signaling mitigates cognitive impairment by activating mitophagy and inhibiting neuroinflammation in Apoe4 mice.

BACKGROUND: Alzheimer's disease (AD) is one of the most common neurodegenerative diseases and mitophagy deficit was identified as the typical abnormality in early stage of AD. The neuroprotective effect of andrographolide (AGA) has been confirmed, anda acetylated derivative of AGA (3,14,19-triacetylandrographolide, ADA) was considered to have stronger efficacy. PURPOSE: The current study aims to investigate the impact of ADA on cognitive ability in a sporadic AD model and explore its potential mechanism. STUDY DESIGN/ METHODS: Apoe4 mouse was adopted for evaluating the impact of AGA on cognitive impairment through a serious of behavioral tests. The molecular mechanism of ADA involved in mitophagy and neuroinflammation was investigated in detailby Western blot, ELISA, immunofluorescence and transmission electron microscopy in Apoe4 mice, as well as Apoe4-transfected BV2 cells and HT22 cells. RESULTS: ADA application significantly improved cognitive impairment of Apoe4 mice, and lessened Aß load and neuronal damage, which has stronger activity than its prototype AGA. Accumulated mitophagy markers LC3II, P62, TOM20, PINK1 and Parkin, and decreased mitophagy receptor BNIP3 in hippocampus of Apoe4 mice were greatly reversed after ADA treatment. Meanwhile, ADA promoted the recruitment of BNIP3 to mitochondria, and the transport of damaged mitochondria to lysosome, indicating that disturbed mitophagy in AD mice was restored by ADA. Inhibited SIRT3 and FOXO3a in Apoe4 mice brains were elevated after ADA treatment. ADA also lightened the neuroinflammation caused by NLRP3 inflammasome activation. Additionally, damaged mitophagy and/or activated NLRP3 inflammasome were also observed in BV2 cells and HT22 cells transfected with Apoe4, all of which were rescued by ADA incubation. Noteworthily, SIRT3 inhibitor 3-TYP could abolish the impact of ADA on mitophagy and NLRP3 inflammasome in vitro. CONCLUSION: ADA exerted stronger cognition-enhancing ability in relative to AGA, and ADA could repaire mitophagy deficiency via SIRT3-FOXO3a pathway, and subsequently inhibite NLRP3 inflammasome to mitigate AD pathology.

ApoE4 associated with severe COVID-19 outcomes via downregulation of ACE2 and imbalanced RAS pathway.

BACKGROUND: Recent numerous epidemiology and clinical association studies reported that ApoE polymorphism might be associated with the risk and severity of coronavirus disease 2019 (COVID-19), and yielded inconsistent results. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection relies on its spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptor expressed on host cell membranes. METHODS: A meta-analysis was conducted to clarify the association between ApoE polymorphism and the risk and severity of COVID-19. Multiple protein interaction assays were utilized to investigate the potential molecular link between ApoE and the SARS-CoV-2 primary receptor ACE2, ApoE and spike protein. Immunoblotting and immunofluorescence staining methods were used to access the regulatory effect of different ApoE isoform on ACE2 protein expression. RESULTS: ApoE gene polymorphism (ε4 carrier genotypes VS non-ε4 carrier genotypes) is associated with the increased risk (P = 0.0003, OR = 1.44, 95% CI 1.18-1.76) and progression (P < 0.00001, OR = 1.85, 95% CI 1.50-2.28) of COVID-19. ApoE interacts with both ACE2 and the spike protein but did not show isoform-dependent binding effects. ApoE4 significantly downregulates ACE2 protein expression in vitro and in vivo and subsequently decreases the conversion of Ang II to Ang 1-7. CONCLUSIONS: ApoE4 increases SARS-CoV-2 infectivity in a manner that may not depend on differential interactions with the spike protein or ACE2. Instead, ApoE4 downregulates ACE2 protein expression and subsequently the dysregulation of renin-angiotensin system (RAS) may provide explanation by which ApoE4 exacerbates COVID-19 disease.

Exercise modulates APOE expression in brain cortex of female APOE3 and APOE4 targeted replacement mice.

Apolipoprotein E (ApoE) is the main cholesterol carrier of the brain and the ε4 gene variant (APOE4) is the most prevalent genetic risk factor for Alzheimer's disease (AD), increasing risk up to 15-fold. Several studies indicate that APOE4 modulates critical factors for neuronal function, including brain-derived neurotrophic factor (BDNF) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Both proteins show exercise-induced upregulation, which is presumed to mediate many of the beneficial effects of physical activity including improved cognition; however, there is variability in results between individuals potentially in-part due to genetic variations including APOE isoform. This study aimed to determine if the two most prevalent human APOE isoforms influence adaptive responses to exercise-training. Targeted replacement mice, homozygous for either APOE3 or APOE4 were randomized into exercised and sedentary groups. Baseline locomotor function and voluntary wheel-running behavior was reduced in APOE4 mice. Exercised groups were subjected to daily treadmill running for 8 weeks. ApoE protein in brain cortex was significantly increased by exercise in both genotypes. PGC-1α mRNA levels in brain cortex were significantly lower in APOE4 mice, and only tended to increase with exercise in both genotypes. Hippocampal BDNF protein were similar between genotypes and was not significantly modulated by treadmill running. Behavioral and biochemical variations between APOE3 and APOE4 mice likely contribute to the differential risk for neurological and vascular diseases and the exercise-induced increase in ApoE levels suggests an added feature of the potential efficacy of physical activity as a preventative and therapeutic strategy for neurogenerative processes in both genotypes.

Effect of Chronic Cadmium Exposure on Brain and Liver Transporters and Drug-Metabolizing Enzymes in Male and Female Mice Genetically Predisposed to Alzheimer's Disease.

Cadmium (Cd) exposure is associated with increased Alzheimer's disease (AD) risks. The human Apolipoprotein E (ApoE) gene encodes a lipid-transporting protein that is critical for brain functions. Compared with ApoE2 and E3, ApoE4 is associated with increased AD risk. Xenobiotic biotransformation-related genes have been implicated in the pathogenesis of AD. However, little is known about the effects of Cd, ApoE, and sex on drug-processing genes. We investigated the Cd-ApoE interaction on the transcriptomic changes in the brains and livers of ApoE3/ApoE4 transgenic mice. Cd disrupts the transcriptomes of transporter and drug-processing genes in brain and liver in a sex- and ApoE-genotype-specific manner. Proinflammation related genes were enriched in livers of Cd-exposed ApoE4 males, whereas circadian rhythm and lipid metabolism related genes were enriched in livers of Cd-exposed ApoE3 females. In brains, Cd up-regulated the arachidonic acid-metabolizing Cyp2j isoforms only in the brains of ApoE3 mice, whereas the dysregulation of cation transporters was male-specific. In livers, several direct target genes of the major xenobiotic-sensing nuclear receptor pregnane X receptor were uniquely upregulated in Cd-exposed ApoE4 males. There was a female-specific hepatic upregulation of the steroid hormone-metabolizing Cyp2 isoforms and the bile acid synthetic enzyme Cyp7a1 by Cd exposure. The dysregulated liver transporters were mostly involved in intermediary metabolism, with the most significant response observed in ApoE3 females. In conclusion, Cd dysregulated the brain and liver drug-processing genes in a sex- and ApoE-genotype specific manner, and this may serve as a contributing factor for the variance in the susceptibility to Cd neurotoxicity. SIGNIFICANCE STATEMENT: Xenobiotic biotransformation plays an important role in modulating the toxicity of environmental pollutants. The human ApoE4 allele is the strongest genetic risk factor for AD, and cadmium (Cd) is increasingly recognized as an environmental factor of AD. Very little is known regarding the interactions between Cd exposure, sex, and the genes involved in xenobiotic biotransformation in brain and liver. The present study has addressed this critical knowledge gap.

Chimeric cerebral organoids reveal the essentials of neuronal and astrocytic APOE4 for Alzheimer's tau pathology.

The apolipoprotein E4 (APOE4) genotype is one of the strongest genetic risk factors for Alzheimer's disease (AD), and is generally believed to cause widespread pathological alterations in various types of brain cells. Here, we developed a novel engineering method of creating the chimeric human cerebral organoids (chCOs) to assess the differential roles of APOE4 in neurons and astrocytes. First, the astrogenic factors NFIB and SOX9 were introduced into induced pluripotent stem cells (iPSCs) to accelerate the induction of astrocytes. Then the above induced iPSCs were mixed and cocultured with noninfected iPSCs under the standard culturing condition of cerebral organoids. As anticipated, the functional astrocytes were detected as early as 45 days, and it helped more neurons matured in chCOs in comparation of the control human cerebral organoids (hCOs). More interestingly, this method enabled us to generate chCOs containing neurons and astrocytes with different genotypes, namely APOE3 or APOE4. Then, it was found in chCOs that astrocytic APOE4 already significantly promoted lipid droplet formation and cholesterol accumulation in neurons while both astrocytic and neuronal APOE4 contributed to the maximum effect. Most notably, we observed that the co-occurrence of astrocytic and neuronal APOE4 were required to elevate neuronal phosphorylated tau levels in chCOs while Aß levels were increased in chCOs with neuronal APOE4. Altogether, our results not only revealed the essence of both neuronal and astrocytic APOE4 for tau pathology, but also suggested chCOs as a valuable pathological model for AD research and drug discovery.

A Time-to-Event Exposure-Response Model for Amyloid-Related Imaging Abnormalities Following Administration of Aducanumab to Subjects With Early Alzheimer Disease.

Amyloid-related imaging abnormalities with edema (ARIA-E) have been reported in patients with early Alzheimer disease treated with aducanumab. ARIA-E incidence has been observed to be dependent on both dose and apolipoprotein E4 carrier status. A time-to-event (TTE) approach applying data from 2 phase 3 studies (studies 301 and 302) was used to describe the effect of aducanumab serum exposure on the instantaneous risk of 2 end points: the first incidence of ARIA-E and time to ARIA-E resolution. A total of 3251 subjects with 826 events supported the TTE model to characterize the first ARIA-E event. The TTE resolution model was supported by data from 768 of 826 subjects who had ARIA-E resolved. Relationships between drug concentrations and ARIA-E events were modeled with a hazard function dependent on time, aducanumab serum concentrations, attenuation of aducanumab exposure effects with time (ie, potential for tolerance to aducanumab exposure), study, and apolipoprotein E4 carrier status. The TTE model showed that ARIA-E incidence rates were higher during the first 200 days, followed by a reduction in rates. The change in event rate reflects the attenuation of drug effect, thereby providing support for the current proposed titration regimen. Time to ARIA-E resolution was characterized by a constant baseline hazard with a probability to resolution affected by baseline ARIA-E severity and aducanumab concentration. ARIA-E resolution was found to be driven primarily by baseline hazard and time and suggested that aducanumab concentration effect is a minor contributor to the time to resolution of ARIA-E.

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

BACKGROUND: Apolipoprotein 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. METHODS: Mouse 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. RESULTS: Greater 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. CONCLUSIONS: Our 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.

ApoE4 inhibition of VMAT2 in the locus coeruleus exacerbates Tau pathology in Alzheimer's disease.

ApoE4 enhances Tau neurotoxicity and promotes the early onset of AD. Pretangle Tau in the noradrenergic locus coeruleus (LC) is the earliest detectable AD-like pathology in the human brain. However, a direct relationship between ApoE4 and Tau in the LC has not been identified. Here we show that ApoE4 selectively binds to the vesicular monoamine transporter 2 (VMAT2) and inhibits neurotransmitter uptake. The exclusion of norepinephrine (NE) from synaptic vesicles leads to its oxidation into the toxic metabolite 3,4-dihydroxyphenyl glycolaldehyde (DOPEGAL), which subsequently activates cleavage of Tau at N368 by asparagine endopeptidase (AEP) and triggers LC neurodegeneration. Our data reveal that ApoE4 boosts Tau neurotoxicity via VMAT2 inhibition, reduces hippocampal volume, and induces cognitive dysfunction in an AEP- and Tau N368-dependent manner, while conversely ApoE3 binds Tau and protects it from cleavage. Thus, ApoE4 exacerbates Tau neurotoxicity by increasing VMAT2 vesicle leakage and facilitating AEP-mediated Tau proteolytic cleavage in the LC via DOPEGAL.

ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes.

The APOE ε4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). ApoE protein aggregation plays a central role in AD pathology, including the accumulation of ß-amyloid (Aß). Lipid-poor ApoE4 protein is prone to aggregate and lipidating ApoE4 protects it from aggregation. The mechanisms regulating ApoE4 aggregation in vivo are surprisingly not known. ApoE lipidation is controlled by the activity of the ATP binding cassette A1 (ABCA1). ABCA1 recycling and degradation is regulated by ADP-ribosylation factor 6 (ARF6). We found that ApoE4 promoted greater expression of ARF6 compared with ApoE3, trapping ABCA1 in late-endosomes and impairing its recycling to the cell membrane. This was associated with lower ABCA1-mediated cholesterol efflux activity, a greater percentage of lipid-free ApoE particles, and lower Aß degradation capacity. Human CSF from APOE ε4/ε4 carriers showed a lower ability to induce ABCA1-mediated cholesterol efflux activity and greater percentage of aggregated ApoE protein compared with CSF from APOE ε3/ε3 carriers. Enhancing ABCA1 activity rescued impaired Aß degradation in ApoE4-treated cells and reduced both ApoE and ABCA1 aggregation in the hippocampus of male ApoE4-targeted replacement mice. Together, our data demonstrate that aggregated and lipid-poor ApoE4 increases ABCA1 aggregation and decreases ABCA1 cell membrane recycling. Enhancing ABCA1 activity to reduce ApoE and ABCA1 aggregation is a potential therapeutic strategy for the prevention of ApoE4 aggregation-driven pathology.SIGNIFICANCE STATEMENT ApoE protein plays a key role in the formation of amyloid plaques, a hallmark of Alzheimer's disease (AD). ApoE4 is more aggregated and hypolipidated compared with ApoE3, but whether enhancing ApoE lipidation in vivo can reverse ApoE aggregation is not known. ApoE lipidation is controlled by the activity of the ATP binding cassette A1 (ABCA1). In this study, we demonstrated that the greater propensity of lipid-poor ApoE4 to aggregate decreased ABCA1 membrane recycling and its ability to lipidate ApoE. Importantly, enhancing ABCA1 activity to lipidate ApoE reduced ApoE and ABCA1 aggregation. This work provides critical insights into the interactions among ABCA1, ApoE lipidation and aggregation, and underscores the promise of stabilizing ABCA1 activity to prevent ApoE-driven aggregation pathology.

Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation.

BACKGROUND: Apolipoprotein E (ApoE) is the major carrier protein that mediates the transport and delivery of cholesterol and other lipids in the brain. Three isoforms of ApoE (ApoE2, ApoE3, ApoE4) exist in humans, and their relative expression levels impact HIV-1 infection, HIV-1/AIDS disease progression, and cognitive decline associated with HIV-1-associated neurocognitive disorder. Because HIV-1 Tat, a viral protein essential for HIV-1 replication, can bind to low-density lipoprotein receptor-related protein 1 (LRP1) that controls ApoE uptake in the brain, we determined the extent to which different isoforms of ApoE affected Tat-mediated HIV-1 LTR transactivation. METHODS: Using U87MG glioblastoma cells expressing LTR-driven luciferase, we determined the extent to which LRP1 as well as ApoE2, ApoE3, and ApoE4 affected Tat-mediated HIV-1 LTR transactivation. RESULTS: A specific LRP1 antagonist and siRNA knockdown of LRP1 both restricted significantly Tat-mediated LTR transactivation. Of the three ApoEs, ApoE4 was the least potent and effective at preventing HIV-1 Tat internalization and at decreasing Tat-mediated HIV-1 LTR transactivation. Further, Tat-mediated LTR transactivation was attenuated by an ApoE mimetic peptide, and ApoE4-induced restriction of Tat-mediated LTR transactivation was potentiated by an ApoE4 structure modulator that changes ApoE4 into an ApoE3-like phenotype. CONCLUSIONS: These findings help explain observed differential effects of ApoEs on HIV-1 infectivity and the prevalence of HAND in people living with HIV-1 infection and suggest that ApoE mimetic peptides and ApoE4 structure modulator might be used as a therapeutic strategy against HIV-1 infection and associated neurocognitive disorders.

ApoE4 Accelerates Early Seeding of Amyloid Pathology.

Accumulation and aggregation of amyloid-ß (Aß) in the brain is an initiating step in the pathogenesis of Alzheimer's disease (AD). The ε4 allele of apolipoprotein E (apoE) gene is the strongest genetic risk factor for late-onset AD. Although there is strong evidence showing that apoE4 enhances amyloid pathology, it is not clear what the critical stage(s) is during amyloid development in which apoE4 has the strongest impact. Using apoE inducible mouse models, we show that increased expression of astrocytic apoE4, but not apoE3, during the seeding stage of amyloid development enhanced amyloid deposition and neuritic dystrophy in amyloid model mice. ApoE4, but not apoE3, significantly increased brain Aß half-life measured by in vivo microdialysis. Furthermore, apoE4 expression increased whereas apoE3 reduced amyloid-related gliosis in the mouse brains. Together, our results demonstrate that apoE4 has the greatest impact on amyloid during the seeding stage, likely by perturbing Aß clearance and enhancing Aß aggregation.

Effects of ApoE on intracellular calcium levels and apoptosis of neurons after mechanical injury.

OBJECTIVE: The current study aimed to explore the effects of apolipoprotein e (ApoE) on intracellular calcium ([Ca(2+)]i) and apoptosis of neurons after mechanical injury in vitro. METHODS: A neuron mechanical injury model was established after primary neurons obtained from APOE knockout and wild-type (WT) mice, and four experimental groups were generated: Group-ApoE4, Group-ApoE3, Group-ApoE(-) and Group-WT. Recombinant ApoE4 and ApoE3 were added to Group-ApoE4 and Group-ApoE3 respectively, and Group-ApoE(-) and Group-WT were control groups. Intracellular calcium was labeled by fluo-3/AM and examined using laser scanning confocal microscope and flow cytometry, and the apoptosis of neurons was also evaluated. RESULTS: The intracellular calcium levels and apoptosis rates of mice neurons were significantly higher in Group-ApoE4 than in Group-ApoE3 and Group-WT after mechanical injury. However, without mechanical injury on neurons, no significant differences in intracellular calcium levels and apoptosis rates were found among all four experimental groups. The effects of ApoE4 on intracellular calcium levels and apoptosis rates of injured neurons were partly decreased by EGTA treatment. CONCLUSION: Compared with ApoE3-treatment and WT neurons, ApoE4 caused higher intracellular calcium levels and apoptosis rates of neurons after mechanical injury. This suggested APOE polymorphisms may affect neuron apoptosis after mechanical injury through different influences on intracellular calcium levels.

The effect of apolipoprotein E4 on synchronous neural interactions in brain cultures.

In a previous study, we assessed the synchronous neural interactions (SNI) in a developing neural network in brain cultures on multielectrode arrays (Christopoulos et al. in J Neural Eng 9:046008, 2012). Here, we report on the effects of apolipoprotein E4 (apoE4) on these neural interactions. We carried out six experiments (five using rodent brain cultures and one using neuroblastoma cultures) in which we recorded local field potentials (LFP) from 59 sites for several days in vitro under the following conditions. In one experiment, we added to the culture media triglyceride (TG)-rich lipoproteins from a human subject with the apoE4/4 genotype, whereas in the other experiments, we added recombinant human apoE4. We found that SNI in the apoE4-treated cultures had higher coefficient of SNI variation, as compared to control cultures. These findings further document the role of SNI as a fundamental aspect of the dynamic organization of neural networks (Langheim et al. in Proc Natl Acad Sci USA 103:455-459, 2006. doi: 10.1073/pnas.0509623102 ; Georgopoulos et al. in J Neural Eng 4:349-355, 2007) and extend the effect of apoE4 on SNI (Leuthold et al. in Exp Brain Res 226:525-536, 2013) across different brain species (human, rodents), apoE source (TG-rich lipoproteins, recombinant), neural signals (MEG, LFP), and brain network (intact brain, developing brain in vitro). To our knowledge, this is the first study of the effects of apoE4 on neural network function in vitro.

Apolipoprotein E isoform-specific effects on lipoprotein receptor processing.

Recent findings indicate an isoform-specific role for apolipoprotein E (apoE) in the elimination of beta-amyloid (Aß) from the brain. ApoE is closely associated with various lipoprotein receptors, which contribute to Aß brain removal via metabolic clearance or transit across the blood­brain barrier (BBB). These receptors are subject to ectodomain shedding at the cell surface, which alters endocytic transport and mitigates Aß elimination. To further understand the manner in which apoE influences Aß brain clearance, these studies investigated the effect of apoE on lipoprotein receptor shedding. Consistent with prior reports, we observed an increased shedding of the low-density lipoprotein receptor (LDLR) and the LDLR-related protein 1 (LRP1) following Aß exposure in human brain endothelial cells. When Aß was co-treated with each apoE isoform, there was a reduction in Aß-induced shedding with apoE2 and apoE3, while lipoprotein receptor shedding in the presence of apoE4 remained increased. Likewise, intracranial administration of Aß to apoE-targeted replacement mice (expressing the human apoE isoforms) resulted in an isoform-dependent effect on lipoprotein receptor shedding in the brain (apoE4 > apoE3 > apoE2). Moreover, these results show a strong inverse correlation with our prior work in apoE transgenic mice in which apoE4 animals showed reduced Aß clearance across the BBB compared to apoE3 animals. Based on these results, apoE4 appears less efficient than other apoE isoforms in regulating lipoprotein receptor shedding, which may explain the differential effects of these isoforms in removing Aß from the brain.

Apolipoprotein E4 impairs in vivo hippocampal long-term synaptic plasticity by reducing the phosphorylation of CaMKIIα and CREB.

Inheritance of the apolipoprotein E genotype ε4 (APOE4) is a powerful risk factor for most cases of late-onset Alzheimer's disease (AD). However, the effects of ApoE4 on the long-term synaptic plasticity and its underlying mechanism have not clearly investigated. In the present study, we examined the effects of ApoE4 on the hippocampal late-phase long-term potentiation (L-LTP) and investigated its probable molecular mechanisms by using in vivo field potential recording, immunohistochemistry, and western blotting. The results showed that: (1) intra-hippocampal injection of 0.2 µg ApoE4, but not ApoE2, before high frequency stimulations (HFSs) attenuated the induction of hippocampal L-LTP in the CA1 region, while injection of the same concentration of ApoE4 after HFSs, even at a higher concentration (2 µg), did not affect the long term synaptic plasticity; (2) ApoE4 injection did not affect the paired pulse facilitation in the hippocampal CA1 region; (3) ApoE4 injection before, not after, HFSs significantly decreased the levels of phosphorylated Ca2+/calmodulin-dependent protein kinase IIα (p-CaMKIIα) and phosphorylated cAMP response element-binding protein (p-CREB) in the hippocampus. These results demonstrated for the first time that ApoE4 could impair hippocampal L-LTP by reducing p-CaMKIIα and p-CREB, suggesting that the ApoE4-induced suppression of hippocampal long-term synaptic plasticity may contribute to the cognitive impairments in genetic AD; and both CaMKIIα and CREB are important intracellular targets of the neurotoxic ApoE4.

A multifaceted role for apoE in the clearance of beta-amyloid across the blood-brain barrier.

BACKGROUND: While apolipoprotein E4 (apoE4) is highly correlated with the development of Alzheimer's disease (AD), its role in AD pathology and, in particular, beta-amyloid (Aß) removal from the brain, is not clearly defined. OBJECTIVE: To elucidate the influence of apoE on the clearance of Aß across the blood-brain barrier (BBB). METHODS: Aß(1-42) was intracerebrally administered to transgenic mice expressing human apoE isoforms and examined in the periphery. RESULTS: apoE3 and apoE4 mice had 5 times and 2 times, respectively, more Aß(1-42) appearing in the plasma than wild-type or apoE knockout mice, indicating an enhanced clearance of Aß from the brain to the periphery. In vitro, unbound basolateral apoE3 (i.e., not bound to Aß), and to a lesser extent unbound apoE4, at concentrations ≤10 nM facilitated basolateral-to-apical fluorescein-Aß(1-42) transcytosis across a BBB model, while apoE isoforms bound to Aß significantly disrupted Aß transcytosis. Additionally, following apical exposure to the BBB model, we found that apoE4 bound to Aß is able to penetrate the BBB more readily than apoE3 bound to Aß and does so via the RAGE (receptor for advanced glycation end products) transporter. CONCLUSION: These studies indicate a multifaceted, isoform-dependent role for apoE in the exchange of Aß across the BBB and may partially explain the association of apoE4 and Aß brain accumulation in AD.

Postprandial apoE isoform and conformational changes associated with VLDL lipolysis products modulate monocyte inflammation.

OBJECTIVE: Postprandial hyperlipemia, characterized by increased circulating very low-density lipoproteins (VLDL) and circulating lipopolysaccharide (LPS), has been proposed as a mechanism of vascular injury. Our goal was to examine the interactions between postprandial lipoproteins, LPS, and apoE3 and apoE4 on monocyte activation. METHODS AND RESULTS: We showed that apoE3 complexed to phospholipid vesicles attenuates LPS-induced THP-1 monocyte cytokine expression, while apoE4 increases expression. ELISA revealed that apoE3 binds to LPS with higher affinity than apoE4. Electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels placed on specific amino acids of apoE3 showed that LPS interferes with conformational changes normally associated with lipid binding. Specifically, compared to apoE4, apoE bearing the E3-like R112→Ser mutation displays increased self association when exposed to LPS, consistent with a stronger apoE3-LPS interaction. Additionally, lipolysis of fasting VLDL from normal human donors attenuated LPS-induced TNFα secretion from monocytes to a greater extent than postprandial VLDL, an effect partially reversed by blocking apoE. This effect was reproduced using fasting VLDL lipolysis products from e3/e3 donors, but not from e4/e4 subjects, suggesting that apoE3 on fasting VLDL prevents LPS-induced inflammation more readily than apoE4. CONCLUSION: Postprandial apoE isoform and conformational changes associated with VLDL dramatically modulate vascular inflammation.

Pharmacodynamic and pharmacokinetic analysis of apoE4 [L261A, W264A, F265A, L268A, V269A], a recombinant apolipoprotein E variant with improved biological properties.

Physiological levels of wild-type (wt) apolipoprotein E (apoE) in plasma mediate the clearance of cholesterol-rich atherogenic lipoprotein remnants while higher than normal plasma apoE concentrations fail to do so and trigger hypertriglyceridemia. This property of wt apoE reduces significantly its therapeutic value as a lead biological for the treatment of dyslipidemia. Recently, we reported the generation of a recombinant apoE variant, apoE4 [L261A, W264A, F265A, L268A, V269A] (apoE4mut1) with improved biological functions. Specifically, in apoE-deficient (apoE(-/-)) mice this variant can normalize high plasma cholesterol levels without triggering hypertriglyceridemia, even at supraphysiological levels of expression. In the present study we performed pharmacodynamic and pharmacokinetic analysis of apoE4mut1 in experimental mice. Using adenovirus-mediated gene transfer in LDL receptor deficient (LDLr(-/-)) mice, we show that the cholesterol lowering potential of apoE4mut1 is dependent on the expression of a functional classical LDLr. Bolus infusion of apoE4mut1-containing proteoliposomes in apoE(-/-) mice fed western-type diet for 6 weeks indicated that exogenously synthesized apoE4mut1 maintains intact its ability to normalize the high cholesterol levels of these mice with a maximum pharmacological effect obtained at 10h post-treatment. Interestingly, plasma cholesterol levels remained significantly reduced up to 24h following intravenous administration of apoE4mut1 proteoliposomes. Measurements of plasma apoE levels indicated that apoE4mut1 in the form of proteoliposomes used in the study has a half-life of 15.8h. Our data suggest that purified apoE4mut1 may be an attractive new candidate for the acute correction of hypercholesterolemia in subjects expressing functional LDL receptor.

An apolipoprotein E4 fragment affects matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1 and cytokine levels in brain cell lines.

Apolipoprotein (apo) E4 isoform, a major risk factor for Alzheimer disease (AD), is more susceptible to proteolysis than apoE2 and apoE3 isoforms. ApoE4 fragments have been found in AD patients' brain. In the present study, we examined the effect of full-length apoE4 and apoE4 fragments apoE4[Δ(186-299)] and apoE4[Δ(166-299)] on inflammation in human neuroblastoma SK-N-SH and human astrocytoma SW-1783 cells. Western blot and zymography analysis showed that treatment of SK-N-SH cells with apoE4[Δ(186-299)], but not full-length apoE4 or the shorter apoE4[Δ(166-299)] fragment, leads to increased extracellular levels of matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1). Real-time PCR showed that interleukin (IL)-1ß gene expression is also increased in SK-N-SH cells treated with apoE4[Δ(186-299)]. Treatment of SK-N-SH cells with IL-1ß leads to increased MMP9 and TIMP1 extracellular levels, suggesting that the induction of IL-1ß may be the mechanism by which apoE4[Δ(186-299)] regulates MMP9 and TIMP1 levels in these cells. In contrast to SK-N-SH cells, treatment of SW-1783 cells with apoE4[Δ(186-299)], and to a lesser extent with apoE4, leads to increased TIMP1 extracellular levels without affecting MMP9 levels. Additionally, apoE4[Δ(186-299)] leads to decreased IL-10 gene expression in SK-N-SH cells, whereas both apoE4 and apoE4[Δ(186-299)] lead to decreased TNFα gene expression without affecting IL-1ß and IL-10 gene expression in SW-1783 cells. Overall, our findings indicate that a specific apoE4 fragment (apoE4[Δ(186-299)]), with molecular mass similar that of apoE4 fragments detected in AD patients' brain, can influence the level of inflammatory molecules in brain cell lines. It is possible that these phenomena contribute to AD pathogenesis.