Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, with genetic factors playing critical roles in its pathogenesis. Mutations in APP, PSEN1 and PSEN2 genes are confirmed to be causative risk factors for early-onset Alzheimer’s disease (EOAD). For late-onset Alzheimer’s disease (LOAD), growing evidence suggests it is caused by multiple genetic risk factors in corporation with the environmental exposures. Although, so far, APOE is the most well recognized common genetic risk factor for LOAD, other susceptible candidate genes, such as CR1, CLU and PICALM, have recently been identified in Caucasians using genome-wide association approach. In order to have a better understanding on the genetic components of LOAD in Chinese as well as identify other potential genetic risk factors for Chinese ethnic population, we conducted a case-control study using candidate gene association approach. In view of increasing evidence on the neural protective effects of sex steroid hormones both in vivo and in vitro, we hypothesized variations on sex steroid metabolic pathway genes were associated with LOAD. Four candidate genes (ESR1, ESR2, CYP19A1, CYP11A1) were evaluated based on 462 cases and 350 non-demented controls. Apart from consistent result for APOE, polymorphisms in ESR2 and CYP11A1 were found to be significantly associated with the disease. When stratification according to gender, marginally significant associations were detected for ESR1 and ESR2 variants in men while CYP11A1 variants relevant to LOAD risk were detected exclusively in women. Additionally, genotypic and phenotypic correlation analysis revealed CYP19A1 was significantly relevant to serum 17-estradiol (E2) levels in 689 subgroup participants, especially in 400 LOAD patients of subgroup. Further gene-level analyses based on whole sample confirmed above disease association for ESR2 and CYP11A1 and pathway-level analyses highlighted the impact of sex steroid metabolic pathway on disease predisposition. The independent follow-up study for CR1, CLU and PICALM previously reported by genome-wide association study (GWAS) in Caucasians was conducted in the same Chinese cohort. Similar to the Caucasian cohort, polymorphisms in CR1 and CLU were found to be significantly different between cases and non-demented controls. However, significant disease association for PICAML was detected only in the APOE ε4 (-) subgroup of our Chinese cohort. In conclusions, genetic abnormalities were founded in Chinese LOAD patients. In addition to confirmation disease susceptibility for APOE, CR1, CLU and PICALM, we were first to report the associations between several sex steroid metabolic pathway genes and LOAD. This valuable genetic information obtained from Chinese patients may lead to the development of novel diagnostic strategies and therapeutic interventions in LOAD. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Alzheimer’s disease (AD) is one of the common diseases of older people. Although several genes have been identified for Familial Alzheimer’s Disease (FAD), a reliable diagnostic, especially for those patients in their early or intermediate phases of AD, is still not available. There is neither effective treatment nor drugs that can stop or reverse AD progression. Breakthroughs in diagnosis or treatment development likely require understanding of the molecular mechanisms of AD. Studies in FAD have shown that APP, PS1, PS2 and some other genes are related to FAD. Mutations of APP and PS1 lead to amyloid plaque accumulation which is also prominent in Sporadic AD. Transgenic animals closely mimic human AD pathologies in many aspects. A mouse model carrying both APP Swedish mutation and PS1 deltaE9 mutation is used in this study. This mouse model accumulates amyloid plaque rapidly, and the plaque shows up as early as 6 months of age. Using microarrays, we have isolated 176 genes with significant expression changes and 14 turned on/off genes from AD mouse cortex. From this cDNA microarray measurement of global gene expression, several functional groups were regulated significantly in our mouse model of AD pathology. Mt2 and Atp7a were identified and may be candidates for further studies of AD pathology, as well as potential drug targets. Five significant microRNAs were found from AD mouse cortex, providing evidence that microRNAs could play a role in AD. cDNA arrays were also used to identify potential biomarkers from whole blood samples that distinguish AD mice from their non-transgenic littermates. / text

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Presenilin-1 (PS1) mutations caused by the PSEN1 gene mutations are the major cause of early onset familial Alzheimer’s disease (EOFAD). Two Chinesespecific EOFAD related PS1 mutations, V97L and A136G, have been found. Studies suggested that V97L mutation lead to the overexpression of Aβ42 and tau hyperphosphorylation, which are the major hallmarks of Alzheimer’s disease (AD), while properties of A136G were unclear. Since calcium dysregulation was suggested to play an important role in AD, the research project investigated if V97L and A136G mutations also lead to altered endoplasmic reticulum (ER) 〖Ca〗^(2+) regulation. SH-SY5Y cells transduced with retrovirus carrying V97L mutant or A136 mutant PSEN1 were used as the experiment models. In Western blotting, while the PS1 expression level was unaffected in V97L mutant, the expression level was significantly lower in A136G mutant. In carbachol (CCh) perfusion experiment, V97L mutant was found to exaggerate ER 〖Ca〗^(2+) release when stimulated by higher concentration (30, 100 and 300 μM) CCh, while A136G mutant exaggerated ER Ca2+ release when stimulated by 30 μM and 300 μM CCh, but not 100 μM CCh. In 5% fetal bovine serum (FBS) perfusion experiment, both V97L and A136G mutants were found to sensitize 〖Ca〗^(2+) oscillation, which the sensitization effect of V97L was 3 folds of A136G. The results suggested that V97L mutation exaggerates ER 〖Ca〗^(2+) release, possibly via interaction with IP3R. However the results of A136G were inconclusive and contradicting, therefore further investigation is needed. / published_or_final_version / Physiology / Master / Master of Medical Sciences

The aim of this thesis was to investigate the possibility of using buccal cells derived from a multi layered epithelial tissue from the oral mucosa as a model to identify potential biomarkers of genomic instability in relation to normal ageing and premature ageing syndromes such as AD and DS. A buccal micronucleus cytome assay was developed and used to investigate biomarkers for DNA damage, cell proliferation and cell death in healthy young, healthy old and young Down’s syndrome cohorts. Cells with micronuclei, karyorrhectic cells, condensed chromatin cells and basal cells increased significantly with normal ageing (P<0.0001). Cells with micronuclei and binucleated cells increased (P<0.0001) and condensed chromatin, karyorrhectic, karyolytic and pyknotic cells decreased (P<0.002) significantly in Down’s syndrome relative to young controls. The buccal micronucleus cytome assay was used to measure ratios of buccal cell populations and micronuclei in clinically diagnosed Alzheimer’s patients compared to age and gender matched controls. Frequencies of basal cells (P<0.0001), condensed chromatin cells (P<0.0001) and karyorrhectic cells (P<0.0001) were found to be significantly lower in Alzheimer’s patients, possibly reflecting changes in the cellular kinetics or structural profile of the buccal mucosa. Changes in telomere length were investigated using a quantitative RTm-PCR method to measure absolute telomere length (in Kb per diploid genome) and show agerelated changes in white blood cells and buccal cell telomere length (in kb per diploid genome) in normal healthy individuals and Alzheimer’s patients. We observed a significantly lower telomere length in white blood cells (P<0.0001) and buccal cells (P<0.01) in Alzheimer’s patients relative to healthy age-matched controls (31.4% and 32.3% respectively). However, there was a significantly greater telomere length in hippocampus cells of Alzheimer’s brains (P=0.01) compared to control samples (49.0). Buccal cells were also used to investigate chromosome 17 and 21 aneuploidy. A 1.5 fold increase in trisomy 21 (P<0.001) and a 1.2 fold increase in trisomy 17 (P<0.001) was observed in buccal cells of Alzheimer’s patients compared to age and gender matched controls. Chromosome 17 and chromosome 21 monosomy and trisomy increase significantly with age (P<0.001). Down’s syndrome, which exhibits similar neuropathological features to those observed in Alzheimer’s disease also showed a strong increase in chromosome 17 monosomy and trisomy compared to matched controls (P<0.001). However, aneuploidy rate for chromosome 17 and 21 in the nuclei of hippocampus cells of brains from Alzheimer’s patients and controls were not significantly different. Observations that AD individuals have altered plasma folate, B12 and Hcy levels compared to age-matched controls who have not been clinically diagnosed with AD were investigated. Genotyping studies were undertaken to determine whether polymorphisms within particular genes of the folate methionine pathway contributed to AD pathogenesis. Correlations between folate, B12 and Hcy status with previously determined buccal micronucleus assay cytome biomarkers for DNA damage, cell proliferation and cell death markers was investigated. Lastly, the potential protective effects of phytonutrient polyphenols on genomic instability events in a transgenic mouse model for AD were investigated. We determined the effects of curcumin and GSE polyphenols on DNA damage by testing the mice over a 9 month period utilizing a buccal micronucleus cytome assay, an erythrocyte micronucleus assay and measuring telomere length in both buccal cells and olfactory lobe brain tissue. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1313395 / Thesis (Ph.D.) -- The University of Adelaide, School of Molecular and Biomedical Science, 2008

The aim of this thesis was to investigate the possibility of using buccal cells derived from a multi layered epithelial tissue from the oral mucosa as a model to identify potential biomarkers of genomic instability in relation to normal ageing and premature ageing syndromes such as AD and DS. A buccal micronucleus cytome assay was developed and used to investigate biomarkers for DNA damage, cell proliferation and cell death in healthy young, healthy old and young Down’s syndrome cohorts. Cells with micronuclei, karyorrhectic cells, condensed chromatin cells and basal cells increased significantly with normal ageing (P<0.0001). Cells with micronuclei and binucleated cells increased (P<0.0001) and condensed chromatin, karyorrhectic, karyolytic and pyknotic cells decreased (P<0.002) significantly in Down’s syndrome relative to young controls. The buccal micronucleus cytome assay was used to measure ratios of buccal cell populations and micronuclei in clinically diagnosed Alzheimer’s patients compared to age and gender matched controls. Frequencies of basal cells (P<0.0001), condensed chromatin cells (P<0.0001) and karyorrhectic cells (P<0.0001) were found to be significantly lower in Alzheimer’s patients, possibly reflecting changes in the cellular kinetics or structural profile of the buccal mucosa. Changes in telomere length were investigated using a quantitative RTm-PCR method to measure absolute telomere length (in Kb per diploid genome) and show agerelated changes in white blood cells and buccal cell telomere length (in kb per diploid genome) in normal healthy individuals and Alzheimer’s patients. We observed a significantly lower telomere length in white blood cells (P<0.0001) and buccal cells (P<0.01) in Alzheimer’s patients relative to healthy age-matched controls (31.4% and 32.3% respectively). However, there was a significantly greater telomere length in hippocampus cells of Alzheimer’s brains (P=0.01) compared to control samples (49.0). Buccal cells were also used to investigate chromosome 17 and 21 aneuploidy. A 1.5 fold increase in trisomy 21 (P<0.001) and a 1.2 fold increase in trisomy 17 (P<0.001) was observed in buccal cells of Alzheimer’s patients compared to age and gender matched controls. Chromosome 17 and chromosome 21 monosomy and trisomy increase significantly with age (P<0.001). Down’s syndrome, which exhibits similar neuropathological features to those observed in Alzheimer’s disease also showed a strong increase in chromosome 17 monosomy and trisomy compared to matched controls (P<0.001). However, aneuploidy rate for chromosome 17 and 21 in the nuclei of hippocampus cells of brains from Alzheimer’s patients and controls were not significantly different. Observations that AD individuals have altered plasma folate, B12 and Hcy levels compared to age-matched controls who have not been clinically diagnosed with AD were investigated. Genotyping studies were undertaken to determine whether polymorphisms within particular genes of the folate methionine pathway contributed to AD pathogenesis. Correlations between folate, B12 and Hcy status with previously determined buccal micronucleus assay cytome biomarkers for DNA damage, cell proliferation and cell death markers was investigated. Lastly, the potential protective effects of phytonutrient polyphenols on genomic instability events in a transgenic mouse model for AD were investigated. We determined the effects of curcumin and GSE polyphenols on DNA damage by testing the mice over a 9 month period utilizing a buccal micronucleus cytome assay, an erythrocyte micronucleus assay and measuring telomere length in both buccal cells and olfactory lobe brain tissue. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1313395 / Thesis (Ph.D.) -- The University of Adelaide, School of Molecular and Biomedical Science, 2008

Understanding the effect of the environment on human health has benefited from progress made in measuring the exposome. High resolution mass spectrometry (HRMS) has made it possible to measure small molecules across a large dynamic range, allowing researchers to study the role of low abundance environmental toxicants in causing human disease, including examining their effects on biochemistry. Alzheimer’s disease is the most prevalent neurodegenerative disease in the world. While aging is the largest risk factor of the disease, evidence of risk factors for dementias show that lifestyle choices and the environment may modify disease onset and alter the projected prevalence. Observational epidemiological studies have linked exposure to the persistent pesticide dichlorodiphenytrichloroethane (DDT) with increased risk of Alzheimer’s disease (AD). In Chapter 2, using an aging cohort based in Washington Heights and Inwood in Northern Manhattan, I investigated systemic biochemical changes associated with Alzheimer’s disease (AD). Small molecules in plasma were measured in 59 AD cases and 60 healthy participants of African American, Caribbean Hispanic, and non-Hispanic white ancestry using untargeted liquid-chromatography–based ultra-high-resolution mass spectrometry. Metabolite differences between AD and healthy, the different ethnic groups and apolipoprotein E gene (APOE) ε allele status were analyzed. Untargeted network analysis identified pathways enriched by AD-associated metabolites. Then, in Chapter 3, using the genetically tractable nematode model Caenorhabditis elegans, I investigated whether DDT can exacerbate AD-related pathology. DDT is a persistent organic pollutant which, despite its ban in 1972, can be detected in the blood of most people in the U.S. I investigated whether DDT can exacerbate AD-related pathology using a transgenic C. elegans strain that expresses a mutant tau protein fragment that is prone to aggregation, as well as a mutant strain expressing a non-aggregating form of tau protein. DDT restricted the growth in all strains; however, the restriction was more severe in the aggregating tau transgenic strain. Further, I found that DDT exacerbates the inhibitory effects of aggregating tau protein on basal mitochondrial respiration, and increases the amount of time the worms spent curled/coiled. High-resolution metabolomics in the whole worm suggests that DDT reduces levels of several amino acids but increases levels of uric acid and adenosylselenohomocysteine. Surprisingly, developmental exposure to DDT blunts the lifespan reduction caused by aggregating tau protein suggesting a mitohormetic effect of the “double-hit” from DDT and aggregating tau protein or an antagonistic effect which could ultimately turn on lifespan extension pathways. Our data suggest that exposure to DDT likely exacerbates the mitochondrial inhibitory effects of aggregating tau protein in C. elegans. DDT may mimic some of the mitochondrial inhibitory effects induced by increased tau protein aggregation, suggesting that the genetic and environmental insult converge on a common mitochondrial inhibitory pathway, which has been associated with AD in several other studies. Finally, in Chapter 4, I determined changes in global metabolism associated with aggregating tau protein in both C. elegans and humans. We performed high-resolution metabolomic analysis on cerebrospinal fluid (CSF) and plasma obtained from patients of AD and mild cognitive impairment, and cognitively normal controls. Using a transgenic strain of C. elegans which expresses aggregating tau protein in all neurons, I studied the effect of aggregating tau protein on metabolism using high-resolution metabolomic analysis in the whole worm. In the population study, I found >300 features associated (p < 0.05) with phosphorylated tau levels in CSF. Metabolic pathway enrichment identified alterations in fatty acid and amino acid metabolism. Worms expressing aggregating tau showed >900 features altered. Pathway enrichment suggested alterations in glycerophospholipid, fatty acid and amino acid metabolism pathways. To determine which metabolic features are altered in both species, I analyzed annotated features for overlap. Five metabolites were concordant between human plasma and C. elegans, and four concordant between human CSF and C. elegans. Thus, in this analysis I provide evidence in support of using C. elegans to study changes in global metabolism associated with Alzheimer’s disease. In conclusion, using liquid and gas-based chromatography coupled with high-resolution mass spectrometry, we can measure levels of endogenous and exogenously derived small molecules in different biological matrices. By using the appropriate study design, we can identify candidate molecules and biochemical pathways associated with environmental exposures or disease in human populations. These candidates can be followed up by exposing an appropriate C. elegans strain: transgenic strains, mutant strains, or strains that are susceptible to RNAi based knockdown. Given their short life cycle and being amenable to high-throughput behavioral assays, they can readily provide functional and molecular readouts of the perturbation. The findings can provide leads for relevant policy around environmental exposures, understanding mechanisms of toxicity and disease, and identifying potential therapeutic targets.

Alzheimer’s Disease (AD) is a complex brain disorder that affects at least one in every ten persons aged 65 and above worldwide. The pathogenesis of this disorder remains elusive. In this work, we utilized a rich set of publicly available gene expression data to elucidate the genes and molecular processes that may underlie its pathogenesis. We developed a new ranking score to prioritize molecular pathways enriched in differentially expressed genes during AD. After applying our new ranking score, GO categories such as cotranslational protein targeting to membrane, SRP-dependent cotranslational protein targeting to membrane, and spliceosomal snRNP assembly were found to be significantly associated with AD. We also confirm the protein-protein interaction between APP, NPAS4 and ARNT2 and explain that this interaction could be implicated in AD. This interaction could serve as a theoretical framework for further analyses into the role of NPAS4 and other immediate-early genes in AD pathogenesis. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

Alzheimer’s disease (AD) affects tens of millions of people worldwide. Its cause is unknown, with no cure, and disease-modifying treatment options have only recently become available. Emerging research has made a strong case for the involvement of immune cells, such as microglia and T cells, in modulating AD pathology. Newer technologies in RNA-sequencing have detailed specific phenotypic changes to microglia and T cells over the course of neurodegenerative disease. Some researchers have also used whole-genome sequencing to correlate genetic variants with changes in gene expression. However, no studies thus far have conducted this type of genotype-phenotype correlation in immune cells from aged individuals or AD patients. We have collected gene expression data from four sorted T cell subtypes in peripheral blood samples from 96 subjects in ROSMAP, a cohort of AD patients and age-matched controls. 78 of these subjects also have whole-genome sequencing data, which we used to detect genetic variants associated with changes in T cell gene expression. These are known as expression quantitative trait loci (eQTL). We found genes related to T cell cytotoxicity and immunosenescence in gene co-expression modules, among the eQTL, and in correlation with AD neuropathological traits or risk variants for several disease traits. We extended our findings related to disease association by calculating polygenic risk scores (PRSs) in our cohort from whole-genome sequencing data for 19 traits related to immune function and disease, including AD. Genes associated with the PRS for one or more disease traits often were in biological pathways related to downstream cytokine signaling, regulation of T cell receptor signaling, and T cell migration and trafficking. Overall, our findings indicate that the use of aged and AD patients in T cell genotype-phenotype correlation studies highlights genetic variants and differentially expressed genes that are not seen in studies using young, healthy individuals.