Background: Dementia is a disease known to cause chronic deterioration of intellectual functions severe enough to interfere with the ability to perform activities of daily living. Alzheimer’s disease (AD) is the most frequent cause of dementia and is expected to have a substantial impact on the health care system as the Canadian population ages. Current therapies are ineffective at halting disease progression; thus, investigations examining risk factors for AD have become a popular avenue of research. A relationship between cerebrovascular disease and the risk of AD has been established, but the underlying mechanisms on how these morbidities are related remain unclear. The apolipoprotein E gene (ApoE) influences the development of AD with the apolipoprotein E-e4 allele (ApoE-e4) conferring increased risk. The underlying mechanism by which the ApoE-e4 allele influences AD is unclear. Since the ApoE-e4 allele is related to both AD and stroke, the impact of cerebral infarcts on AD may vary by ApoE-e4 allele status. Objective: The objective of this study was to assess if ApoE-e4 allele status modified the relationship between cerebral infarcts and AD. Methods: Secondary data from the Nun Study, a longitudinal clinico-pathologic study of aging representing 678 female participants 75+ years were used for this investigation. AD was diagnosed using criteria for clinical dementia and AD pathology. Dementia was diagnosed using standard criteria, including the Consortium to Establish a Registry for Alzheimer’s Disease battery of neuropsychological tests and performances on activities of daily living. AD pathology was diagnosed using a modified version of the National Institute on Aging and Reagan Institute criteria. Infarcts were identified during gross neuropathologic assessment at autopsy. Logistic regression was used to assess the relationship between AD and the presence, location, and size of cerebral infarcts. Regression models were then stratified by ApoE-e4 allele status to determine if this variable was a significant effect modifier. The relationship of ApoE-e4 allele status with AD, as well as presence of cerebral infarcts, was also explored. All regression models were adjusted for age at death, educational level, and, when appropriate, ApoE-e4 allele status. A sensitivity analysis using different definitions for the outcome AD was performed and showed that varying criteria for AD pathology did not change study results; however, the use of clinical dementia (regardless of pathology) as an outcome did produce significantly different results. Thus, the research questions were repeated using clinical dementia as an outcome. Results: The presence of cerebral infarcts was not significantly associated with AD; this relationship did not change when location and size of infarcts were examined. ApoE-e4 was significantly associated with an increased risk of AD. ApoE-e4 was not associated with presence of cerebral infarcts. ApoE-e4 did not modify the relationship between presence of cerebral infarcts and AD. When the outcome clinical dementia was investigated, presence of cerebral infarcts significantly increased the risk of dementia. This relationship remained when location and size of infarcts were analyzed. ApoE-e4 allele status slightly modified the relationship between presence of cerebral infarcts and dementia. Conclusions: The findings from this study suggest that individuals with severe AD pathology are unlikely to be affected by cerebral infarcts. Future studies should focus on examining levels of severity of AD pathology in relation to cerebral infarcts. Cerebral infarcts appear to have an impact on dementia and this relationship was found to slightly vary by ApoE-e4 status. Future studies are recommended to examine how ApoE interacts with a variety of age-related risk factors to increase the risk of AD.

Besides age, inheritance of the apoE-E4 allele is the main risk factor for late-onset AD. To determine the role of apoE in amyloid deposition, we studied mice expressing both mutant human amyloid [beta]-protein precursor (APP) and presenilin 1 (PS1) that were either normal or knocked-out for apoE. By 7 months, amorphous A[beta] deposition developed equally in both lines, indicating that A[beta] alone is sufficient for deposition to occur. In contrast, filamentous amyloid deposition was catalyzed at least 3000 fold by apoE. Electron micrographs further illustrate the filamentous nature of these plaques. These results and other, behavioral, data indicate that the primary function of apoE in AD is to promote the polymerization of A[beta] into mature, neurotoxic, amyloid. ApoE is also synthesized in the liver and is crucial in cholesterol metabolism, for mice lacking apoE exhibit hypercholesterolemia. We investigated neuropathology in mice using an uncommon technique, parabiosis, to determine whether apoE in the peripheral circulation influences brain amyloid formation. This surgical procedure allows exchange of proteins via peripheral circulation. We show that plasma apoE is found in parabiosed PS/APP/apoE-KO mice, rescuing their hypercholesterolemia. Unexpectedly, amyloid deposition is reduced in parabiosed PS/APP/apoE-KO mice compared to PS/APP controls. ApoE in the periphery seems to slightly reduce amyloid burden, by likely promoting efflux of A[beta];from the brain. These findings reinforce that the mechanisms whereby apoE affects A[beta] metabolism are complex, and the modulation of peripheral apoE metabolism is not likely to impact AD neuropathology. Since cognitive stimulation is associated with lower risk of AD, we sought to investigate the preventative potential of environmental enrichment (EE) using our mouse model.

OBJECTIVE: The brain reserve hypothesis was examined in a secondary analysis of cross-sectional data from a community-based sample of 468 older adults residing in Charlotte County, Florida. The objective of the analysis was to determine the association between head circumference and eight cognitive outcomes and to assess any potential effect modification of existing associations by Apolipoprotein E (APOE) genotype. METHODS: Cognitive outcomes include scores from the Modified Mini-Mental State Exam (3MS), the Hopkins Verbal Learning Test-Revised (HVLT-R), Stroop Color-Word Test, Trail-Making Test A and B, and a word-stem completion task measuring implicit memory. Descriptive statistics were calculated for each variable. Head circumference and dependent cognitive outcomes were modeled as dichotomous variables using logistic regression, adjusting for gender, age, education, income, height, and Spot The Word test score, a measure of pre-morbid IQ. For dichotomized test scores, poor outcomes (cases) were defined as having scores in the lowest quintile; the remaining top four quintiles were considered non-cases. RESULTS: small head circumference was significantly associated with low 3MS scores [OR(95%CI): 2.97 (1.12, 7.89), p=0.03], after adjustment for age, income and pre-morbid IQ. The association remained statistically significant after adjustment for gender and education as well. After adjustment, head circumference was not found to be statistically significantly associated with any other cognitive outcome. No effect modification was found by APOE genotype or years of education. CONCLUSION: This analysis confirms previous findings that exposure to low head circumference significantly impacts cognition in late life.

Apolipoprotein B (apoB) is the main protein of very-low-density lipoprotein (VLDL). As apoB is translated and moves through the secretory pathway, lipids from cytoplasmic lipid droplets (LDs) are added to form VLDL particles. Without adequate lipid availability, apoB is misfolded and undergoes proteasomal degradation; however, evidence now shows that apoB can be degraded through autophagy. Inhibiting autophagy decreased apoB localization to autophagosomes in HepG2 cells, but also decreased apoB recovered from cells and media. Inducing autophagy increased apoB localization to autophagosomes and decreased apoB recovery. LDs are also degraded through autophagy however LDs were not affected by autophagy modulation in HepG2 cells. In primary hamster hepatocytes, inhibiting autophagy reduced apoB-autophagosome co-localization and increased LD numbers. These data suggest that autophagy may play a complex role in VLDL assembly by regulating degradation of both apoB and LDs. This dual role is more evident in primary hepatocytes indicating a potential physiological role.

High plasma concentrations of Lipoprotein(a) (Lp(a)) have been identified as a risk factor for a variety of atherogenic disorders such as cerebrovascular disease, peripheral vascular disease, and coronary heart disease. Lp(a) consists of a lipoprotein moiety containing apolipoproteinB-100 (apoB-100), as well as apolipoprotein(a) (apo(a)), a unique glycoprotein to which the majority of Lp(a) functions are attributed. Variation in the number of identically repeated kringle IV type 2 (KIV2) motifs of apo(a) forms the molecular basis of Lp(a) isoform size heterogeneity, which is a hallmark of this lipoprotein. There is a general inverse correlation between apo(a) size and plasma Lp(a) concentrations, attributed in part to less efficient secretion of larger apo(a) isoforms from hepatic cells. The present study provides a preliminary investigation into processes involved in apo(a) secretion, with respect to isoform size, to understand this inverse correlation at a molecular level. Pulse-chase experiments were performed in human embryonic kidney (HEK 293) cells and human hepatoma (HepG2) cells, both stably expressing differently-sized recombinant apo(a) isoforms representing the range of apo(a) sizes observed in the population. The folding kinetics for the different apo(a) isoforms were determined by changes in the mobility of the non-reduced radiolabelled species on SDS-PAGE gels. In HEK 293 cells, the rate at which apo(a) is folded correlated well with isoform size. In HepG2 cells, however, folding times were comparable regardless of isoform size. Apo(a) secretion from both cell lines exhibited size-dependency. Preliminary experimentation on endoplasmic reticulum (ER)-resident protein modifications of apo(a) was performed, resulting in the identification of apo(a) interactions with PDI, Erp57, Calnexin, Grp78, Grp94, and EDEM. Preliminary experiments indicate a role for intracellular apo(a) degradation in the amount of apo(a) that is secreted from HepG2 cells, although an isoform size dependency of this degradation process cannot be established with current experimental data. Further experimentation is required to confirm enzyme interactions with differently-sized apo(a) isoforms, to identify other chaperones involved in apo(a) secretion, and to confirm the role of proteasomes in intracellular apo(a) degradation. This may, in turn, provide information regarding the mechanism of how apo(a) secretion from hepatic cells is regulated. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-20 19:10:09.497

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] have been identified as an independent risk factor for vascular diseases including coronary heart disease and stroke. In the current study, we have examined the binding and degradation of recombinant forms of apolipoprotein(a) [r-apo(a)], the unique kringle-containing moiety of Lp(a), using a cultured cell model. We found that the incubation of human hepatoma (HepG2) cells with an iodinated 17 kringle-containing (17K) recombinant form of apo(a) resulted in a two-component binding system characterized by a high affinity (Kd = 12 nM), low capacity binding site, and a low affinity (Kd = 249 nM), high capacity binding site. We subsequently determined that the high affinity binding site on HepG2 cells corresponds to the LDL receptor. In the HepG2 cell model, association of apo(a) with the LDL receptor was shown to be dependent on the formation of Lp(a) particles from endogenous LDL. Using an apo(a) mutant incapable of binding to the high affinity site through its inability to form Lp(a) particles (17KΔLBS7,8), we further demonstrated that the LDL receptor does not participate in Lp(a) catabolism. The low affinity binding component observed on HepG2 cells, familial hypercholesterolemia (FH) fibroblasts and human embryonic kidney (HEK) 293 cells may correspond to a member(s) of the plasminogen receptor family, as binding to this site(s) was decreased by the addition of the lysine analogue epsilon-aminocaproic acid. The lysine-dependent nature of the low affinity binding site was further confirmed in HepG2 binding studies utilizing r-apo(a) species with impaired lysine binding ability. We observed a reduction maximum binding capacity for 17K r-apo(a) variants lacking the strong lysine binding site (LBS) in kringle IV type 10 (17KΔAsp) and the very weak LBS in kringle V (17KΔV). Degradation of Lp(a)/apo(a) was found to be mediated exclusively by the low affinity component on both HepG2 cells and FH fibroblasts. Fluorescence confocal microscopy, using the 17K r-apo(a) variant fused to green fluorescent protein, further confirmed that degradation by the low affinity component on HepG2 cells does not proceed by the activity of cellular lysosomes. Taken together, these data suggest a potentially significant route for Lp(a)/apo(a) clearance in vivo. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-26 02:15:50.754

No description available.

Apolipoprotein B (apoB) is the main protein of very-low-density lipoprotein (VLDL). As apoB is translated and moves through the secretory pathway, lipids from cytoplasmic lipid droplets (LDs) are added to form VLDL particles. Without adequate lipid availability, apoB is misfolded and undergoes proteasomal degradation; however, evidence now shows that apoB can be degraded through autophagy. Inhibiting autophagy decreased apoB localization to autophagosomes in HepG2 cells, but also decreased apoB recovered from cells and media. Inducing autophagy increased apoB localization to autophagosomes and decreased apoB recovery. LDs are also degraded through autophagy however LDs were not affected by autophagy modulation in HepG2 cells. In primary hamster hepatocytes, inhibiting autophagy reduced apoB-autophagosome co-localization and increased LD numbers. These data suggest that autophagy may play a complex role in VLDL assembly by regulating degradation of both apoB and LDs. This dual role is more evident in primary hepatocytes indicating a potential physiological role.

Apolipoprotein E (ApoE) is a plasma protein that plays a prominent role in lipid metabolism and cholesterol transport. The gene is polymorphic: three alleles, ε2, ε3, and ε4 code for three different protein isoforms, E2, E3, and E4 respectively, each of which has different genetic implications. Carriers of ε2 and ε4 alleles have shown greater susceptibility to diseases such as lipid metabolism problems, cardiovascular disease, and Alzheimer’s disease. Although ε4 is the ancestral form of the gene, the most common allele in the human population currently is the ε3 allele. The three isoforms of ApoE generally occur in all populations at different frequencies. However, the frequencies of the various alleles have not been examined in the Midwest. The purpose of this project was to determine if a correlation existed between frequencies of ApoE mutation and heritage in Midwest individuals by using real-time PCR. It was hypothesized that this method could be an alternative and cost effective method to sequence an individual’s genome for personalized healthcare purposes. In 2000, Dr. Vann and her research assistants collected saliva samples from approximately 300 anonymous volunteers participating in the UniverCity fair hosted by Ball State University. The samples were catalogued in an Excel database with their corresponding information (gender, self- reported lineage). From this database, 50 samples were randomly selected for this study and the DNA was screened by real-time PCR for isoforms of ApoE. A handful of individuals with altered alleles were identified. The specific number of each isoform and the genotype of each individual were determined. Only one of the 50 individuals resulted in a non-wild type haplotype. A confirmatory melt curve analysis of this major heritage group A individual resulted in a homozygous E4 genotype. Unfortunately, a correlation between frequencies of ApoE mutation and heritage in Midwest individuals could not be inferred based on one differing individual. Thus, we did not have sufficient non-wild type haplotypes to permit us to amplify the variable regions of maternally inherited mitochondrial DNA for sequencing. Those sequences could have been aligned with the Cambridge Reference Sequence (Mitomap 2006) to determine maternal lineage or haplotype, which could then be correlated with self-reported lineage, and the presence of specific isoforms of ApoE. / Department of Biology

Background: Dementia is a disease known to cause chronic deterioration of intellectual functions severe enough to interfere with the ability to perform activities of daily living. Alzheimer’s disease (AD) is the most frequent cause of dementia and is expected to have a substantial impact on the health care system as the Canadian population ages. Current therapies are ineffective at halting disease progression; thus, investigations examining risk factors for AD have become a popular avenue of research. A relationship between cerebrovascular disease and the risk of AD has been established, but the underlying mechanisms on how these morbidities are related remain unclear. The apolipoprotein E gene (ApoE) influences the development of AD with the apolipoprotein E-e4 allele (ApoE-e4) conferring increased risk. The underlying mechanism by which the ApoE-e4 allele influences AD is unclear. Since the ApoE-e4 allele is related to both AD and stroke, the impact of cerebral infarcts on AD may vary by ApoE-e4 allele status. Objective: The objective of this study was to assess if ApoE-e4 allele status modified the relationship between cerebral infarcts and AD. Methods: Secondary data from the Nun Study, a longitudinal clinico-pathologic study of aging representing 678 female participants 75+ years were used for this investigation. AD was diagnosed using criteria for clinical dementia and AD pathology. Dementia was diagnosed using standard criteria, including the Consortium to Establish a Registry for Alzheimer’s Disease battery of neuropsychological tests and performances on activities of daily living. AD pathology was diagnosed using a modified version of the National Institute on Aging and Reagan Institute criteria. Infarcts were identified during gross neuropathologic assessment at autopsy. Logistic regression was used to assess the relationship between AD and the presence, location, and size of cerebral infarcts. Regression models were then stratified by ApoE-e4 allele status to determine if this variable was a significant effect modifier. The relationship of ApoE-e4 allele status with AD, as well as presence of cerebral infarcts, was also explored. All regression models were adjusted for age at death, educational level, and, when appropriate, ApoE-e4 allele status. A sensitivity analysis using different definitions for the outcome AD was performed and showed that varying criteria for AD pathology did not change study results; however, the use of clinical dementia (regardless of pathology) as an outcome did produce significantly different results. Thus, the research questions were repeated using clinical dementia as an outcome. Results: The presence of cerebral infarcts was not significantly associated with AD; this relationship did not change when location and size of infarcts were examined. ApoE-e4 was significantly associated with an increased risk of AD. ApoE-e4 was not associated with presence of cerebral infarcts. ApoE-e4 did not modify the relationship between presence of cerebral infarcts and AD. When the outcome clinical dementia was investigated, presence of cerebral infarcts significantly increased the risk of dementia. This relationship remained when location and size of infarcts were analyzed. ApoE-e4 allele status slightly modified the relationship between presence of cerebral infarcts and dementia. Conclusions: The findings from this study suggest that individuals with severe AD pathology are unlikely to be affected by cerebral infarcts. Future studies should focus on examining levels of severity of AD pathology in relation to cerebral infarcts. Cerebral infarcts appear to have an impact on dementia and this relationship was found to slightly vary by ApoE-e4 status. Future studies are recommended to examine how ApoE interacts with a variety of age-related risk factors to increase the risk of AD.