Eugene Hone

Apolipoprotein E, cholesterol metabolism, diabetes, and the convergence of risk factors for Alzheimer's disease and cardiovascular disease

High fat diets and sedentary lifestyles are becoming major concerns for Western countries. They have led to a growing incidence of obesity, dyslipidemia, high blood pressure, and a condition known as the insulin-resistance syndrome or metabolic syndrome. These health conditions are well known to develop along with, or be precursors to atherosclerosis, cardiovascular disease, and diabetes. Recent studies have found that most of these disorders can also be linked to an increased risk of Alzheimers disease (AD). To complicate matters, possession of one or more apolipoprotein E ɛ4 (APOE ɛ4) alleles further increases the risk or severity of many of these conditions, including AD. ApoE has roles in cholesterol metabolism and Aβ clearance, both of which are thought to be significant in AD pathogenesis. The apparent inadequacies of ApoE ɛ4 in these roles may explain the increased risk of AD in subjects carrying one or more APOE ɛ4 alleles. This review describes some of the physiological and biochemical changes that the above conditions cause, and how they are related to the risk of AD. A diversity of topics is covered, including cholesterol metabolism, glucose regulation, diabetes, insulin, ApoE function, amyloid precursor protein metabolism, and in particular their relevance to AD. It can be seen that abnormal lipid, cholesterol and glucose metabolism are consistently indicated as central in the pathophysiology, and possibly the pathogenesis of AD. As diagnosis of mild cognitive impairment and early AD are becoming more reliable, and as evidence is accumulating that health conditions such as diabetes, obesity, and coronary artery disease are risk factors for AD, appropriate changes to diets and lifestyles will likely reduce AD risk, and also improve the prognosis for people already suffering from such conditions.

Expanding the association between the APOE gene and the risk of Alzheimer's disease: possible roles for APOE promoter polymorphisms and alterations in APOE transcription

Alzheimers disease (AD) is the most commonly diagnosed form of dementia in the elderly. Predominantly this disease is sporadic in nature with only a small percentage of patients exhibiting a familial trait. Early‐onset AD may be explained by single gene defects; however, most AD cases are late onset (> 65 years) and, although there is no known definite cause for this form of the disease, there are several known risk factors. Of these, the ε4 allele of the apolipoprotein E (apoE) gene (APOE) is a major risk factor. The ε4 allele of APOE is one of three (ɛ2 ɛ3 and ɛ4) common alleles generated by cysteine/arginine substitutions at two polymorphic sites. The possession of the ɛ4 allele is recognized as the most common identifiable genetic risk factor for late‐onset AD across most populations. Unlike the pathogenic mutations in the amyloid precursor or those in the presenilins, APOEɛ4 alleles increase the risk for AD but do not guarantee disease, even when present in homozygosity. In addition to the cysteine/arginine polymorphisms at the ɛ2/ɛ3/ɛ4 locus, polymorphisms within the proximal promoter of the APOE gene may lead to increased apoE levels by altering transcription of the APOE gene. Here we review the genetic and biochemical evidence supporting the hypothesis that regulation of apoE protein levels may contribute to the risk of AD, distinct from the well known polymorphisms at the ɛ2/ɛ3/ɛ4 locus.

Apolipoprotein E influences amyloid-beta clearance from the murine periphery

The relationship between amyloid-beta protein (Abeta) metabolism and Alzheimers disease is currently poorly understood. While it is well known that the generation of Abeta results from enzymatic cleavage of its parent molecule, the amyloid beta protein precursor (AbetaPP), there is little information available regarding its in vivo clearance. The E4 isoform of apolipoprotein E (apoE) has been associated with poor clearance of Abeta under in vitro conditions. This is thought to be due to its poor ability to bind Abeta compared with the other common isoforms, apoE2 and apoE3. Although cell culture studies support the notion that Abeta clearance depends upon apoE isoform, validation of these findings requires Abeta clearance studies in vivo. In this study, we examined the clearance of Abeta in vivo from the periphery in mice that expressed apoE (C57BL/6J) or lacked apoE (APOE knockout). We measured the clearance of peripherally injected Abeta over time and additionally, the quantities sequestered by peripheral organs. Western blot analysis of the murine plasma indicated that the half-life of Abeta in the periphery was approximately 15 minutes. The livers of the C57BL/6J mice were found to have sequestered approximately 40% of the total injected Abeta at 90 minutes post-injection, whilst their kidneys contained 5% of the total injected Abeta. In contrast, the livers and kidneys of the APOE knockout animals were found to contain no detectable Abeta. These findings indicate that Abeta is rapidly removed from the plasma by murine peripheral tissues and the rate of its clearance is affected by apoE.

Variation at the APOE -491 promoter locus is associated with altered brain levels of apolipoprotein E

The apolipoprotein E (APOE, gene; apoE, protein) type 4 isoform is a well-established risk factor for late-onset Alzheimers disease (AD), and new data suggest that APOE promoter polymorphisms might also modulate AD risk, perhaps by altering transcription of the APOE gene. The current study was undertaken to determine whether the presence of the APOE promoter −491AA genotype (that appears to increase the risk for AD) is associated with an increase in the levels of apoE in brain tissue. Among 40 control and 20 autopsy-confirmed AD brain samples, levels of apoE were increased in the frontal cortex of AD cases (P < 0.001), consistent with the well-recognized up-regulation of APOE expression in reactive astrocytes. Among controls, the −491A allele appeared to impart a gene dose-dependent effect on the levels of apoE in frontal cortex. The levels of apoE in the brains of AD patients with the −491AA genotype were increased as compared to control subjects with the same genotype (P< 0.001). These data support the notion that the −491AA APOE promoter genotype is associated with elevated brain apolipoprotein E levels, suggesting that the risk for AD may be modulated by the apoE protein level as well as by the apoE protein isoform.

Reduction of inclusion body pathology in ApoE-deficient mice fed a combination of antioxidants

Clinical studies have shown that the antioxidant vitamin E can slow the progression of Alzheimers disease (AD). Other antioxidants reported to affect cognitive function include ginkgo biloba, vitamin C, and lipoic acid. To examine the effects of combination antioxidant therapy (CAT) on longevity and neuropathology in mice, we supplemented the diet of ApoE-deficient mice with vitamin E, ginkgo biloba, pycnogenol, and ascorbyl palmitate. ApoE-deficient mice normally exhibit increased numbers of PAS-positive inclusion bodies with aging. However, supplementation with CAT resulted in a significant increase in life span and a marked reduction of inclusion body histopathology in the hippocampus. In addition, while untreated apoE-deficient mice exhibited increased levels of TUNEL staining, a marker of DNA fragmentation, supplementation with CAT resulted in a significant reduction in the levels of TUNEL staining. These findings suggest that oxidative mechanisms, perhaps related to neuronal apoptosis, are integral to inclusion body formation in aging mice. The association between the reduced number of apoptotic cells and the reduction in inclusion bodies may explain in part the increased longevity of mice fed CAT, and supports the contention that the combined actions of selected antioxidants may be therapeutically effective against neurodegenerative diseases.

Increased levels of apolipoprotein E in the frontal cortex of subjects with schizophrenia.

BACKGROUND It is unclear whether altered expression of a specific isoform of apolipoprotein E (apoE) is associated with the pathology of schizophrenia. METHODS To address whether apoE may be involved in the pathology of schizophrenia, we measured the genotypic and allelic frequency of polymorphisms in its gene and transcriptional regulatory region in DNA from Brodmanns area (BA) 9 obtained postmortem from schizophrenic and control subjects as well as its levels in the same tissue using Western blot analysis. RESULTS The genotypic or allelic frequencies of any polymorphism studied did not vary between diagnostic cohorts. There was a significant increase in the levels of apoE protein in BA 9 from the schizophrenic subjects (Mean +/- SEM: 270 +/- 8.3 vs. 238 +/- 7.1 ng apoE/mg protein, p =.008) and a decrease in tissue from an analogous cortical region from rats treated with haloperidol compared with vehicle-treated animals (50 +/- 6.4 vs. 116 +/- 9.2 ng apoE/mg protein; p =.0002). CONCLUSIONS These data support the hypothesis that increased levels of apoE may be associated with the pathology of schizophrenia and that antipsychotic drugs decrease apoE levels as part of their therapeutic actions.

Effects of apolipoprotein E on the human immunodeficiency virus protein Tat in neuronal cultures and synaptosomes

Human immunodeficiency virus type 1 (HIV‐1)‐associated dementia is observed in 20–30% of patients with acquired immunodeficiency syndrome (AIDS). The ϵ4 allele of the apolipoprotein E (APOE) gene currently is thought to play a role as a risk factor for the development of HIV dementia. The HIV protein Tat is neurotoxic and binds to the same receptor as apoE, the low‐density lipoprotein receptor‐related protein (LRP). In this study, we investigated the role apoE plays in Tat toxicity. Synaptosomes from wild‐type mice treated with Tat had increased reactive oxygen species (ROS), increased lipid and protein oxidation, and decreased mitochondrial membrane potential. Synaptosomes from APOE‐knockout mice also had increased ROS, increased protein oxidation, and decreased mitochondrial membrane potential, but to a significantly lesser degree. Treatment of synaptosomes with heparinase and Tat increased Tat‐induced oxidative stress, consistent with the notion of Tat requiring interaction with neuronal membranes to induce oxidative damage. Human lipidated apoE3 greatly protected neurons from Tat‐induced toxicity, whereas human lipidated apoE4 showed no protection. We demonstrated that human apoE3 has antioxidant properties against Tat‐induced toxicity. Taken together, the data suggest that murine apoE and human apoE4 act similarly and do not protect the cell from Tat‐induced toxicity. This would allow excess Tat to remain outside the cell and interact with synaptosomal membranes, leading to oxidative stress and neurotoxicity, which could contribute to dementia associated with HIV. We show that the antioxidant properties of apoE3 greatly outweigh the competition for clearance in deterring Tat‐induced oxidative stress.

APOE Genotype Results in Differential Effects on the Peripheral Clearance of Amyloid-β42 in APOE Knock-in and Knock-out Mice

The epsilon4 allele of apolipoprotein E (APOE) is currently the major genetic risk factor identified for Alzheimers disease (AD). Previous in vivo data from our laboratory has demonstrated that amyloid-beta (Abeta) is rapidly removed from the plasma by the liver and kidney and that the rate of its clearance is affected by ApoE in C57BL/6J and APOE-/- mice. To expand upon these findings, we assessed the peripheral clearance of human synthetic Abeta42 in APOE epsilon2, epsilon3, and epsilon4 knock-in and APOE knock-out mice injected with lipidated recombinant apoE2, E3, and E4 protein. Our results show that APOE does influence the rate at which the mice are able to clear Abeta42 from their bloodstream. Both APOE epsilon4 mice and APOE knock-out mice treated with lipidated recombinant apoE4 demonstrated increased retention of plasma Abeta42 over time compared to APOE epsilon2/APOE knock-out rE2 and APOE epsilon3/APOE knock-out rE3 mice. These findings suggest that the peripheral clearance of Abeta42 is significantly altered by APOE genotype. Given that APOE epsilon4 is a risk factor for AD, then these novel findings provide some insight into the role of ApoE isoforms on the peripheral clearance of Abeta which may impact on clearance from the brain.

Gonadotropins and cognition in older women.

Recent research studies associate elevated gonadotropin levels with dementia. Specifically, an age associated increase in levels of luteinizing hormone has been linked to an increased risk of Alzheimers disease. The objective of this study was to investigate the association between gonadotropin levels and cognition in older, healthy postmenopausal women. Cognitive functioning was compared with plasma levels of estradiol, luteinizing hormone, follicle stimulating hormone, Abeta40 and APOE genetic status in 649 community-dwelling, non-demented older women residing in Western Australia. High endogenous luteinizing hormone levels were associated with a lower cognitive score, especially in older women and in those women that were depressed. Unexpectedly, disproportionately well preserved cognitive functioning was found for the oldest women who had high endogenous levels of follicle stimulating hormone. The findings indicate that gonadotropins can impact upon cognitive functioning in older postmenopausal women, and that luteinizing hormone and follicle stimulating hormone may exert contrasting effects. Taken together, the findings have important implications for the development of possible preventive strategies for dementia.

Alzheimer's disease amyloid-beta peptide modulates apolipoprotein E isoform specific receptor binding.

The major protein component of the extracellular deposits in Alzheimers disease (AD) is a 4 kDa peptide termed amyloid-beta (Abeta). This peptide is known to bind apolipoprotein E (apoE), a key mediator of lipoprotein transport, in an isoform specific manner. Whilst these isoform specific effects on apoE are well recognized, the functional significance of this interaction is poorly understood. Here, we investigated the influence of Abeta on apoE-mediated lipoprotein binding to cells using fluorescently tagged lipoprotein-like emulsions. Using this approach, we demonstrate that Abeta enhanced the normally poor binding of apoE2 lipoprotein-like particles to fibroblasts in culture, whilst markedly reducing the binding of apoE3 and apoE4. This suggests that the action of apoE isoforms on cellular lipoprotein or cholesterol metabolism is differentially modulated by Abeta. This also suggests that Abeta may also compromise apoE function in the Alzheimer disease affected brain.