Estradiol improves behavior in FAD transgenic mice that express APOE3 but not APOE4 after ovariectomy.

Increasing evidence suggests that female individuals have a higher Alzheimer's disease (AD) risk associated with post-menopausal loss of circulating estradiol (E2). However, clinical data are conflicting on whether E2 lowers AD risk. One potential contributing factor is APOE. The greatest genetic risk factor for AD is APOE4, a factor that is pronounced in female individuals post-menopause. Clinical data suggests that APOE impacts the response of AD patients to E2 replacement therapy. However, whether APOE4 prevents, is neutral, or promotes any positive effects of E2 is unclear. Therefore, our goal was to determine whether APOE modulates the impact of E2 on behavior and AD pathology in vivo. To that end, mice that express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce Aß42 were ovariectomized at either 4 months (early) or 8 months (late) and treated with vehicle or E2 for 4 months. In E3FAD mice, we found that E2 mitigated the detrimental effect of ovariectomy on memory, with no effect on Aß in the early paradigm and only improved learning in the late paradigm. Although E2 lowered Aß in E4FAD mice in the early paradigm, there was no impact on learning or memory, possibly due to higher Aß pathology compared to E3FAD mice. In the late paradigm, there was no effect on learning/memory and Aß pathology in E4FAD mice. Collectively, these data support the idea that, in the presence of Aß pathology, APOE impacts the response to E2 supplementation post-menopause.

Role of estrogen in women's Alzheimer's disease risk as modified by APOE.

Alzheimer's disease (AD) is characterized by numerous sexual dimorphisms that impact the development, progression, and probably the strategies to prevent and treat the most common form of dementia. In this review, we consider this topic from a female perspective with a specific focus on how women's vulnerability to the disease is affected by the individual and interactive effects of estrogens and apolipoprotein E (APOE) genotype. Importantly, APOE appears to modulate systemic and neural outcomes of both menopause and estrogen-based hormone therapy. In the brain, dementia risk is greater in APOE4 carriers, and the impacts of hormone therapy on cognitive decline and dementia risk vary according to both outcome measure and APOE genotype. Beyond the CNS, estrogen and APOE genotype affect vulnerability to menopause-associated bone loss, dyslipidemia and cardiovascular disease risk. An emerging concept that may link these relationships is the possibility that the effects of APOE in women interact with estrogen status by mechanisms that may include modulation of estrogen responsiveness. This review highlights the need to consider the key AD risk factors of advancing age in a sex-specific manner to optimize development of therapeutic approaches for AD, a view aligned with the principle of personalized medicine.

APOE4 homozygote females are resistant to the beneficial effects of 17ß-estradiol on memory and CA1 dendritic spine density in the EFAD mouse model of Alzheimer's disease.

Female APOE4 carriers are at greatest risk of Alzheimer's disease (AD). The potent estrogen 17ß-estradiol (E2) may mediate AD risk, as the onset of memory decline coincides with the menopausal transition. Whether APOE genotype mediates E2's effects on memory and neuronal morphology is poorly understood. We used the APOE+/+/5xFAD+/- (EFAD) mouse model to examine how APOE3 homozygote (E3FAD), APOE3/4 heterozygote (E3/4FAD), and APOE4 homozygote (E4FAD) genotypes modulate effects of E2 on object and spatial memory consolidation, dendritic spine density, and dorsal hippocampal estrogen receptor expression in 6-month-old ovariectomized EFAD mice. Dorsal hippocampal E2 infusion enhanced memory consolidation and increased CA1 apical spine density in E3FAD and E3/4FAD, but not E4FAD, mice. CA1 basal mushroom spines were also increased by E2 in E3FADs. E4FAD mice exhibited reduced CA1 and mPFC basal spine density, and increased dorsal hippocampal ERα protein, independent of E2. Overall, E2 benefitted hippocampal memory and structural plasticity in females bearing one or no APOE4 allele, whereas two APOE4 alleles impeded the memory-enhancing and spinogenic effects of E2.

The detrimental effects of APOE4 on risk for Alzheimer's disease may result from altered dendritic spine density, synaptic proteins, and estrogen receptor alpha.

Women carriers of APOE4, the greatest genetic risk factor for late-onset Alzheimer's disease (AD), are at highest risk of developing AD, yet factors underlying interactions between APOE4 and sex are not well characterized. Here, we examined how sex and APOE3 or APOE4 genotypes modulate object and spatial memory, dendritic spine density and branching, and protein expression in 6-month-old male and female E3FAD and E4FAD mice (APOE+/+/5xFAD+/-). APOE4 negatively impacted object recognition and spatial memory, with male E3FADs exhibiting the best memory across 2 object-based tasks. In both sexes, APOE4 reduced basal dendritic spine density in the medial prefrontal cortex and dorsal hippocampus. APOE4 reduced dorsal hippocampal levels of PDS-95, synaptophysin, and phospho-CREB, yet increased levels of ERα. E4FAD females exhibited strikingly increased GFAP levels, in addition to the lowest levels of PSD-95 and pCREB. Overall, our results suggest that APOE4 negatively impacts object memory, dendritic spine density, and levels of hippocampal synaptic proteins and ERα. However, the general lack of sex differences or sex by genotype interactions suggests that the sex-specific effects of APOE4 on AD risk may be related to factors unexplored in the present study.

Discovery of Nonlipogenic ABCA1 Inducing Compounds with Potential in Alzheimer's Disease and Type 2 Diabetes.

Selective liver X receptor (LXR) agonists have been extensively pursued as therapeutics for Alzheimer's disease and related dementia (ADRD) and, for comorbidities such as type 2 diabetes (T2D) and cerebrovascular disease (CVD), disorders with underlying impaired insulin signaling, glucose metabolism, and cholesterol mobilization. The failure of the LXR-focused approach led us to pursue a novel strategy to discover nonlipogenic ATP-binding cassette transporter A1 (ABCA1) inducers (NLAIs): screening for ABCA1-luciferase activation in astrocytoma cells and counterscreening against lipogenic gene upregulation in hepatocarcinoma cells. Beneficial effects of LXRß agonists mediated by ABCA1 include the following: control of cholesterol and phospholipid efflux to lipid-poor apolipoproteins forming beneficial peripheral HDL and HDL-like particles in the brain and attenuation of inflammation. While rare, ABCA1 variants reduce plasma HDL and correlate with an increased risk of ADRD and CVD. In secondary assays, NLAI hits enhanced cholesterol mobilization and positively impacted in vitro biomarkers associated with insulin signaling, inflammatory response, and biogenic properties. In vivo target engagement was demonstrated after oral administration of NLAIs in (i) mice fed a high-fat diet, a model for obesity-linked T2D, (ii) mice administered LPS, and (iii) mice with accelerated oxidative stress. The lack of adverse effects on lipogenesis and positive effects on multiple biomarkers associated with T2D and ADRD supports this novel phenotypic approach to NLAIs as a platform for T2D and ADRD drug discovery.

Effects of Docosahexaenoic Acid and Its Peroxidation Product on Amyloid-ß Peptide-Stimulated Microglia.

Growing evidence suggests that docosahexaenoic acid (DHA) exerts neuroprotective effects, although the mechanism(s) underlying these beneficial effects are not fully understood. Here we demonstrate that DHA, but not arachidonic acid (ARA), suppressed oligomeric amyloid-ß peptide (oAß)-induced reactive oxygen species (ROS) production in primary mouse microglia and immortalized mouse microglia (BV2). Similarly, DHA but not ARA suppressed oAß-induced increases in phosphorylated cytosolic phospholipase A2 (p-cPLA2), inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNF-α) in BV2 cells. LC-MS/MS assay indicated the ability for DHA to cause an increase in 4-hydroxyhexenal (4-HHE) and suppress oAß-induced increase in 4-hydroxynonenal (4-HNE). Although oAß did not alter the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, exogenous DHA, ARA as well as low concentrations of 4-HHE and 4-HNE upregulated this pathway and increased production of heme oxygenase-1 (HO-1) in microglial cells. These results suggest that DHA modulates ARA metabolism in oAß-stimulated microglia through suppressing oxidative and inflammatory pathways and upregulating the antioxidative stress pathway involving Nrf2/HO-1. Understanding the mechanism(s) underlying the beneficial effects of DHA on microglia should shed light into nutraceutical therapy for the prevention and treatment of Alzheimer's disease (AD).

A small molecule ApoE4-targeted therapeutic candidate that normalizes sirtuin 1 levels and improves cognition in an Alzheimer's disease mouse model.

We describe here the results from the testing of a small molecule first-in-class apolipoprotein E4 (ApoE4)-targeted sirtuin1 (SirT1) enhancer, A03, that increases the levels of the neuroprotective enzyme SirT1 while not affecting levels of neurotoxic sirtuin 2 (SirT2) in vitro in ApoE4-transfected cells. A03 was identified by high-throughput screening (HTS) and found to be orally bioavailable and brain penetrant. In vivo, A03 treatment increased SirT1 levels in the hippocampus of 5XFAD-ApoE4 (E4FAD) Alzheimer's disease (AD) model mice and elicited cognitive improvement while inducing no observed toxicity. We were able to resolve the enantiomers of A03 and show using in vitro models that the L-enantiomer was more potent than the corresponding D-enantiomer in increasing SirT1 levels. ApoE4 expression has been shown to decrease the level of the NAD-dependent deacetylase and major longevity determinant SirT1 in brain tissue and serum of AD patients as compared to normal controls. A deficiency in SirT1 level has been recently implicated in increased tau acetylation, a dominant post-translational modification and key pathological event in AD and tauopathies. Therefore, as a novel approach to therapeutic development for AD, we targeted identification of compounds that enhance and normalize brain SirT1 levels.

Rexinoids as Therapeutics for Alzheimer's Disease: Role of APOE.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by amyloid plaques, composed of amyloid-beta peptide (Aß) and neurofibrillary tangles, composed of aberrantly phosphorylated tau. APOE4 is the greatest genetic risk factor for AD, increasing risk up to 12- fold with a double allele compared to APOE3. In contrast, APOE2 reduces AD risk ~2-fold per allele. Accumulating evidence demonstrates that apolipoprotein E4 (apoE4) plays a multifactorial role in AD pathogenesis, although the exact mechanisms remain unclear. Further data support roles for apoE4 as a toxic gain of function or loss of positive function in AD, a discrepancy that has significant implications for the future of apoE-directed therapeutics. However, recent evidence repurposing retinoid X receptor (RXR) agonists, or rexinoids, for the treatment of AD demonstrates conflicting, though potentially beneficial effects in familial AD-transgenic (FAD-Tg) mouse models. Of particular note is bexarotene (Targretin®), a selective rexinoid previously utilized in cancer treatment emerging as a viable candidate for AD clinical trials. However, the mechanism of action of bexarotene and similar rexinoids remains controversial, particularly in the context of human APOE. In addition, rexinoids demonstrate distinct adverse event profiles in humans that may have greater detrimental effects in an elderly AD population. Therefore, this special issue review discusses the implications for rexinoiddirected therapeutic strategies in AD, the potential mechanistic targets, and future directions for the improvement of rexinoid-based therapies in AD.

Arabidopsis thaliana extracts optimized for polyphenols production as potential therapeutics for the APOE-modulated neuroinflammation characteristic of Alzheimer's disease in vitro.

Although the cause of Alzheimer's disease (AD) is unknown, glial-induced neuroinflammation is an early symptom. Familial AD is caused by increases in amyloid-beta (Aß) peptide, particularly soluble oligomeric (oAß), considered a proximal neurotoxin and neuroinflammatory stimuli. APOE4, a naturally occurring genotype of APOE, is the greatest genetic risk factor for AD; increasing risk up to 12-fold compared to APOE3 and APOE2. oAß-induced neuroinflammation is greater with APOE4 compared to APOE3 and APOE2. As sinapates and flavonoids have anti-inflammatory properties, a protocol was developed for optimizing polyphenol production in seedlings of Arabidopsis thaliana (A. thaliana). Three mutants (cop1, prn1, xpf3) were identified, and the extracts treated with liver microsomes to mimic physiological metabolism, with HPLC and MS performed on the resulting metabolites for peak identification. These extracts were used to treat primary glial cells isolated from human APOE-targeted-replacement (APOE-TR) and APOE-knock-out (KO) mice, with neuroinflammation induced by lipopolysaccharide (LPS) or oAß. The dose-response data for TNFα secretion demonstrate the followed the order: APOE-KO > APOE4 > APOE3 > APOE2, with xpf3 the most effective anti-neuroinflammatory across APOE genotypes. Thus, the plant-based approach described herein may be particularly valuable in treating the APOE4-induced neuroinflammatory component of AD risk.

Apolipoprotein E isotype-dependent modulation of microRNA-146a in plasma and brain.

The Apolipoprotein E (ApoE) isotype ApoE4 is a prevalent genetic risk factor for Alzheimer's disease (AD) that can modulate systemic and central inflammation, independent of amyloid accumulation. Although disruption of innate immune toll receptor signaling is modulated by ApoE and observed in AD, ApoE isotype-specific effects remain poorly understood. Therefore, we examined the effect of the ApoE isotype on the brain levels of major regulators of TLR signaling including miR146a, a microRNA enriched in the brain. We used 6-month-old ApoE3 or ApoE4 targeted replacement mice with and without mutant familial AD transgenes. ApoE4 reduced the levels of miR146a compared with ApoE3, both in the brain (29%; P<0.0001) and in plasma (47%; P<0.05), which correlated with each other (r=0.74; P<0.05). The presence of 5xFAD transgenes increased brain miR146a in both ApoE3 (E3FAD) and ApoE4 (E4FAD) mice; however, miR146a levels in E4FAD mice remained lower than those in E3FAD mice (62%; P<0.05), despite increased amyloid and inflammation. Supporting these observations, ApoE4 brains showed increased expression of interleukin receptor-associated kinase-1 (160%; P<0.05) (normally downregulated by miR146) that correlated inversely with miR146a levels (r=0.637; P<0.0001). Reduced negative feedback of toll-like receptor signaling (by miRNA146a) can explain early-life hypersensitivity to innate immune stimuli (including Aß) in ApoE4 carriers. Thus, ApoE4 causes early dysregulation of a central controller of the innate immune system both centrally and systemically. This defect persists with familial AD pathology and may be relevant to ApoE4 AD risk.

APOE modulates the effect of estrogen therapy on Aß accumulation EFAD-Tg mice.

The post-menopausal loss of estrogen is key in the increased incidence of Alzheimer's disease (AD) in women. However, estrogen therapy (ET) clinical trials have produced conflicting results. The APOE gene of apolipoprotein E (apoE) likely modulates the effects of ET in AD. APOE4 is the greatest genetic risk factor for AD, increasing risk up to 15-fold compared with APOE3, and the negative effect of APOE4 on AD risk and neuropathology is greater in women than men. The interactive effects of APOE and ET may converge on modulation of amyloid-beta (Aß) levels, as independently both the loss of estrogen and APOE4 increases Aß accumulation. Thus, in this study, 3-month old female EFAD mice (5XFAD mice crossed with apoE-targeted replacement mice), which express increased levels of Aß42 and human APOE were ovariectomized and treated for 3 months with either 17-ß estradiol (OVX(ET+), 0.25mg total) or vehicle control (OVX(ET-)) and the effects on Aß accumulation were determined. Compared to the OVX(ET-) cohort, in the OVX(ET+) cohort, extracellular amyloid and Aß deposition in the hippocampus and cortex were decreased with APOE2 and APOE3, but were increased with APOE4 by IHC. Biochemical analysis demonstrated increased total and insoluble Aß levels with APOE4, and decreased soluble Aß42 levels with both APOE3 and APOE4, after ET. These data suggest that ET administered at menopause may benefit APOE4 negative women by decreasing extracellular and soluble Aß42. However, for APOE4 carriers, the efficacy of ET will be dependent on the relative impact of extracellular and soluble Aß on AD-induced neurodegeneration.

Differential regulation of amyloid-ß endocytic trafficking and lysosomal degradation by apolipoprotein E isoforms.

Aggregation of amyloid-ß (Aß) peptides leads to synaptic disruption and neurodegeneration in Alzheimer disease (AD). A major Aß clearance pathway in the brain is cellular uptake and degradation. However, how Aß traffics through the endocytic pathway and how AD risk factors regulate this event is unclear. Here we show that the majority of endocytosed Aß in neurons traffics through early and late endosomes to the lysosomes for degradation. Overexpression of Rab5 or Rab7, small GTPases that function in vesicle fusion for early and late endosomes, respectively, significantly accelerates Aß endocytic trafficking to the lysosomes. We also found that a portion of endocytosed Aß traffics through Rab11-positive recycling vesicles. A blockage of this Aß recycling pathway with a constitutively active Rab11 mutant significantly accelerates cellular Aß accumulation. Inhibition of lysosomal enzymes results in Aß accumulation and aggregation. Importantly, apolipoprotein E (apoE) accelerates neuronal Aß uptake, lysosomal trafficking, and degradation in an isoform-dependent manner with apoE3 more efficiently facilitating Aß trafficking and degradation than apoE4, a risk factor for AD. Taken together, our results demonstrate that Aß endocytic trafficking to lysosomes for degradation is a major Aß clearance pathway that is differentially regulated by apoE isoforms. A disturbance of this pathway can lead to accumulation and aggregation of cellular Aß capable of causing neurotoxicity and seeding amyloid.

APOE genotype alters glial activation and loss of synaptic markers in mice.

The ε4 allele of the Apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and affects clinical outcomes of chronic and acute brain damages. The mechanisms by which apoE affect diverse diseases and disorders may involve modulation of the glial response to various types of brain damage. We examined glial activation in a mouse model where each of the human APOE alleles are expressed under the endogenous mouse APOE promoter, as well as in APOE knock-out mice. APOE4 mice displayed increased glial activation in response to intracerebroventricular lipopolysaccharide (LPS) compared to APOE2 and APOE3 mice by several measures. There were higher levels of microglia/macrophage, astrocytes, and invading T-cells after LPS injection in APOE4 mice. APOE4 mice also displayed greater and more prolonged increases of cytokines (IL-1ß, IL-6, TNF-α) than APOE2 and APOE3 mice. We found that APOE4 mice had greater synaptic protein loss after LPS injection, as measured by three markers: PSD-95, drebin, and synaptophysin. In all assays, APOE knock-out mice responded similar to APOE4 mice, suggesting that the apoE4 protein may lack anti-inflammatory characteristics of apoE2 and apoE3. Together, these findings demonstrate that APOE4 predisposes to inflammation, which could contribute to its association with Alzheimer's disease and other disorders.

Preparing synthetic Aß in different aggregation states.

This chapter outlines protocols that produce homogenous preparations of oligomeric and fibrillar amyloid-ß peptide (Aß). While there are several isoforms of this peptide, the 42 amino acid form is the focus because of its genetic and pathological link to Alzheimer's disease (AD). Past decades of AD research highlight the dependence of Aß42 function on its structural assembly state. Biochemical, cellular and in vivo studies of Aß42 usually begin with purified peptide obtained by chemical synthesis or recombinant expression. The initial steps to solubilize and prepare these purified dry peptide stocks are critical to controlling the structural assembly of Aß. To develop homogenous Aß42 assemblies, we initially monomerize the peptide, erasing any "structural history" that could seed aggregation, by using a strong solvent. It is this starting material that has allowed us to define and optimize conditions that consistently produce homogenous solutions of soluble oligomeric and fibrillar Aß42 assemblies. These preparations have been developed and characterized by using atomic force microscopy (AFM) to identify the structurally discrete species formed by Aß42 under specific solution conditions. These preparations have been used extensively to demonstrate a variety of functional differences between oligomeric and fibrillar Aß42. We also present a protocol for fluorescently labeling oligomeric Aß42 that does not affect structure, as measured by AFM, or function, as measured by a cellular uptake assay. These reagents are critical experimental tools that allow for defining specific structure/function connections.

Overexpression of human apolipoprotein A-I preserves cognitive function and attenuates neuroinflammation and cerebral amyloid angiopathy in a mouse model of Alzheimer disease.

To date there is no effective therapy for Alzheimer disease (AD). High levels of circulating high density lipoprotein (HDL) and its main protein, apolipoprotein A-I (apoA-I), reduce the risk of cardiovascular disease. Clinical studies show that plasma HDL cholesterol and apoA-I levels are low in patients with AD. To investigate if increasing plasma apoA-I/HDL levels ameliorates AD-like memory deficits and amyloid-ß (Aß) deposition, we generated a line of triple transgenic (Tg) mice overexpressing mutant forms of amyloid-ß precursor protein (APP) and presenilin 1 (PS1) as well as human apoA-I (AI). Here we show that APP/PS1/AI triple Tg mice have a 2-fold increase of plasma HDL cholesterol levels. When tested in the Morris water maze for spatial orientation abilities, whereas APP/PS1 mice develop age-related learning and memory deficits, APP/PS1/AI mice continue to perform normally during aging. Interestingly, no significant differences were found in the total level and deposition of Aß in the brains of APP/PS1 and APP/PS1/AI mice, but cerebral amyloid angiopathy was reduced in APP/PS1/AI mice. Also, consistent with the anti-inflammatory properties of apoA-I/HDL, glial activation was reduced in the brain of APP/PS1/AI mice. In addition, Aß-induced production of proinflammatory chemokines/cytokines was decreased in mouse organotypic hippocampal slice cultures expressing human apoA-I. Therefore, we conclude that overexpression of human apoA-I in the circulation prevents learning and memory deficits in APP/PS1 mice, partly by attenuating neuroinflammation and cerebral amyloid angiopathy. These findings suggest that elevating plasma apoA-I/HDL levels may be an effective approach to preserve cognitive function in patients with AD.

Low-density lipoprotein receptor-related protein 1 (LRP1) mediates neuronal Abeta42 uptake and lysosomal trafficking.

BACKGROUND: Alzheimer's disease (AD) is characterized by the presence of early intraneuronal deposits of amyloid-beta 42 (Abeta42) that precede extracellular amyloid deposition in vulnerable brain regions. It has been hypothesized that endosomal/lysosomal dysfunction might be associated with the pathological accumulation of intracellular Abeta42 in the brain. Our previous findings suggest that the LDL receptor-related protein 1 (LRP1), a major receptor for apolipoprotein E, facilitates intraneuronal Abeta42 accumulation in mouse brain. However, direct evidence of neuronal endocytosis of Abeta42 through LRP1 is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that LRP1 endocytic function is required for neuronal Abeta42 uptake. Overexpression of a functional LRP1 minireceptor, mLRP4, increases Abeta42 uptake and accumulation in neuronal lysosomes. Conversely, knockdown of LRP1 expression significantly decreases neuronal Abeta42 uptake. Disruptions of LRP1 endocytic function by either clathrin knockdown or by removal of its cytoplasmic tail decreased both uptake and accumulation of Abeta42 in neurons. Finally, we show that LRP1-mediated neuronal accumulation of Abeta42 is associated with increased cellular toxicity. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that LRP1 endocytic function plays an important role in the uptake and accumulation of Abeta42 in neuronal lysosomes. These findings emphasize the central function of LRP1 in neuronal Abeta metabolism.

Abeta42 neurotoxicity in primary co-cultures: effect of apoE isoform and Abeta conformation.

Autosomal dominant mutations that increase amyloid-beta(1-42) (Abeta42) cause familial Alzheimer's disease (AD), and the most common genetic risk factor for AD is the presence of the epsilon4 allele of apolipoprotein E (apoE). Previously, we characterized stable preparations of Abeta42 oligomers and fibrils and reported that oligomers induced a 10-fold greater increase in neurotoxicity than fibrils in Neuro-2A cells. To determine the effects of apoE genotype on Abeta42 oligomer- and fibril-induced neurotoxicity in vitro, we co-cultured wild type (WT) neurons with glia from WT, apoE-knockout (apoE-KO), and human apoE2-, E3-, and E4-targeted replacement (TR) mice. Dose-dependent neurotoxicity was induced by oligomeric Abeta42 with a ranking order of apoE4-TR>KO=apoE2-TR=apoE3-TR>WT. Neurotoxicity induced by staurosporine or glutamate were not affected by apoE genotype, indicating specificity for oligomeric Abeta42-induced neurotoxicity. These in vitro data demonstrate a gain of negative function for apoE4, synergistic with oligomeric Abeta42, in mediating neurotoxicity.

Apolipoprotein E modulates gamma-secretase cleavage of the amyloid precursor protein.

Polymorphisms in the apolipoprotein E (APOE) gene affect the risk of Alzheimer disease and the amount of amyloid beta-protein (Abeta) deposited in the brain. The apoE protein reduces Abeta levels in conditioned media from cells in culture, possibly through Abeta clearance mechanisms. To explore this effect, we treated multiple neural and non-neural cell lines for 24 h with apoE at concentrations similar to those found in the cerebrospinal fluid (1-5 microg/mL). The apoE treatment reduced Abeta40 by 60-80% and Abeta42 to a lesser extent (20-30%) in the conditioned media. Surprisingly, apoE treatment resulted in an accumulation of amyloid precursor protein (APP)-C-terminal fragments in cell extracts and a marked reduction of APP intracellular domain-mediated signaling, consistent with diminished gamma-secretase processing of APP. All three isoforms of apoE, E2, E3 and E4, had similar effects on Abeta and APP-C-terminal fragments, and the effects were independent of the low-density lipoprotein receptor family. Apolipoprotein E had minimal effects on Notch cleavage and signaling in cell-based assays. These data suggest that apoE reduces gamma-secretase cleavage of APP, lowering secreted Abeta levels, with stronger effects on Abeta40. The apoE modulation of Abeta production and APP signaling is a potential mechanism affecting Alzheimer disease risk.

Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability.

Genetic evidence predicts a causative role for amyloid-beta (A beta) in Alzheimer's disease. Recent debate has focused on whether fibrils (amyloid) or soluble oligomers of A beta are the active species that contribute to neurodegeneration and dementia. We developed two aggregation protocols for the consistent production of stable oligomeric or fibrillar preparations of A beta-(1-42). Here we report that oligomers inhibit neuronal viability 10-fold more than fibrils and approximately 40-fold more than unaggregated peptide, with oligomeric A beta-(1-42)-induced inhibition significant at 10 nm. Under A beta-(1-42) oligomer- and fibril-forming conditions, A beta-(1-40) remains predominantly as unassembled monomer and had significantly less effect on neuronal viability than preparations of A beta-(1-42). We applied the aggregation protocols developed for wild type A beta-(1-42) to A beta-(1-42) with the Dutch (E22Q) or Arctic (E22G) mutations. Oligomeric preparations of the mutations exhibited extensive protofibril and fibril formation, respectively, but were not consistently different from wild type A beta-(1-42) in terms of inhibition of neuronal viability. However, fibrillar preparations of the mutants appeared larger and induced significantly more inhibition of neuronal viability than wild type A beta-(1-42) fibril preparations. These data demonstrate that protocols developed to produce oligomeric and fibrillar A beta-(1-42) are useful in distinguishing the structural and functional differences between A beta-(1-42) and A beta-(1-40) and genetic mutations of A beta-(1-42).

Apolipoprotein E structural requirements for the formation of SDS-stable complexes with beta-amyloid-(1-40): the role of salt bridges.

Of the three major isoforms of human apolipoprotein E (apoE), apoE4 is a risk factor for the development of Alzheimer's disease. Among possible neurologically relevant differences in the properties of apoE3 and apoE4 is the fact that apoE3 forms an SDS-stable complex with beta-amyloid-(1-40) (Abeta40) with greater avidity than does apoE4. This interaction may sequester potentially toxic species of Abeta or facilitate clearance. To understand more about this difference, we examined whether differences in salt bridges between apoE domains influence the capacity of apoE isoforms to form complexes with Abeta. In apoE3 there is a salt bridge between Arg-61 and Asp-65, while in apoE4 there are salt bridges between Arg-61 and Glu-255, and Arg-112 and Glu-109. Mutation of position 112, which is Cys in apoE3 and Arg in apoE4, to Ala or Lys abolished complex formation, while mutant apoE with Ser at this position retained the capacity to form complex. Substituting Ala for Glu-109 had no effect on the ability of either apoE4 or apoE3 to form complexes. On the other hand, substitution of Thr for Arg-61 in apoE3 abolished, and truncation of apoE3 at position 201 substantially lowered, but did not abolish, complex formation. Neither of these mutations within apoE4 had any affect on its complex formation with Abeta. These results suggest that the nature of the cysteine residue in apoE3 and interactions between the N-terminal and C-terminal domains of human apoE are important for the ability of apoE3 to form an SDS-stable complex with Abeta40.