High-density lipoproteins (HDL), inflammation pathways, and associated proteins likelipopolysaccharide binding protein (LBP) play complex and interrelated roles in major age- related diseases including cardiovascular disease and Alzheimer’s disease (AD). This dissertation integrates findings across multiple key studies to elucidate novel insights of the “lipoprotein-gastrointestinal-inflammation axis” underlying these conditions. By reviewing evidence on HDL glycoprotein alterations in disease, identifying elevated LBP in Alzheimer’s patients and analyzing impacts of fiber supplementation on inflammation in preliminary trials, new insights emerge. These discoveries pave the way for improved risk prediction, prevention, and treatment strategies that leverage knowledge of HDL, LBP, and glycosylation aberrations. This body of work catalyzes future research to enable transformative advances against society’s most pressing health burdens.

1. Chapter one reviews research on glycosylation of HDL-associated proteins, showing it isaltered in diseases like cardiovascular disease (CVD) and has potential as a diagnostic biomarker. It discusses analytical methods for profiling HDL glycoproteins and evidence linking glycosylation to HDL function. The chapter emphasizes the need for large cohort studies on HDL glycan profiles and health outcomes.

2. Chapter two presents findings from an Alzheimer's study identifying significantly higherLBP concentrations in plasma fractions of apolipoprotein E (APOE)3E3 AD patients compared to APOE3E4 controls. LBP was associated with dementia severity and verbal memory scores. This implicates LBP-driven inflammation in AD pathogenesis in genetically low-risk individuals.

3. Chapter three analyzes data from a prebiotic fiber supplementation trial, finding limitedoverall impacts of the fiber supplement on plasma concentrations of LBP and HDL function, but reductions in LBP selectively in those individuals with higher baseline LBP. Our findings suggest that some individuals who consume low-fiber diets have increased gut permeability, and for these individuals, even a low-dose daily fiber supplement can improve gut barrier function.

Together, these discoveries reveal new aspects of lipoproteins, LBP, and glycosylation in majorage-related disease. This dissertation promotes future research leveraging this knowledge for enhanced prediction, prevention, and treatment of conditions like AD and CVD. Key priorities include large glycoprofiling studies, elucidating LBP's role in AD, and assessing early-life precision nutrition anti-inflammatory interventions in at-risk groups.

The purpose of this dissertation is to update the current methodology in understanding the function and structure of high-density lipoprotein (HDL), to describe improved methods for HDL isolation and deep-learning-assisted particle size measurement, and to show how HDL size and function alter in in vitro treatments with antioxidant carotenoids and environmental insults, as well as in clinical samples from study participants with Alzheimer’s disease (AD), mild cognitive impairment (MCI), or normal cognition. The dissertation is dived into four chapters with the corresponding objectives: 1. The first chapter is a literature review on recent updates and advancement of methods in understanding HDL characteristics, including physical properties, biological functions, and metabolism. 2. The second chapter reports an improved method for HDL isolation using ultracentrifugation followed by size exclusion chromatography. The isolated HDL fractions were evaluated on yield and purity, showing high HDL recovery and low levels of protein markers that are not HDL-associated. HDL fractions isolated using this method were further analyzed for HDL-associated protein markers, and we identified specific HDL-associated proteins that have preference for HDL subpopulations of specific size ranges. 3. The third chapter reports experiments to evaluate HDL’s size and functional alterations under direct treatment with lutein, zeaxanthin, oxidants (CuSO4, H2O2, HOCl), glycation, fructosylation, glycosylation removal (N-glycan, sialic acid), or acidic pH, as well as HDL pre-treated with antioxidant carotenoids (lutein or zeaxanthin) and then treated with H2O2 oxidant. We showed that HDL functions, including cholesterol efflux capacity (CEC), lecithin-cholesterol acyltransferase (LCAT) activity, and paraoxonase-1 (PON1) activity were altered by the treatment. Pre-incubating HDL with lutein attenuated the negative effects on HDL function caused by H2O2 treatment. 4. The fourth chapter reports an innovative method to use transmission electron microscopy followed by deep-learning-assisted particle detection for HDL particle size measurement of isolated HDL particles. The method showed improved precision and repeatability compared to a common image analysis software ImageJ on the specific task of HDL particle size measurement. We further use the method to characterize HDL particle size distribution from a large-scale data set from 183 study participants with AD, MCI, or normal cognition (age matched) and discovered a specific shift of HDL size from large to small in participants with dementia, with APOE genotype-dependent effects.

Alzheimer’s Disease (AD) is the most common cause of dementia, characterized by the extracellular deposition of β-amyloid plaques and intracellular neurofibrillary tangles resulting from hyperphosphorylated tau protein. AD has emerged as a looming concern in the U.S., with an estimated 6.7 million Americans projected to live with AD by 2023, and about 1 in 9 people aged 65 and older affected by Alzheimer’s dementia. Unfortunately, there is still no effective treatment to reverse or stop AD progression. Glycosylation is a post-translational modification where glycans are attached to peptide chains or lipids. Emerging studies have demonstrated a close link between aberrant protein glycosylation and AD pathology. Given its complexity and diversity, glycosylation holds promise to understand the precise pathogenesis of AD from a novel perspective. Meanwhile, nutritional interventions hold potential to modify glycosylation patterns, as evidenced by the widespread use of oral monosaccharide administration in managing certain congenital disorders of glycosylation. Hence, it is pivotal to investigate the involvement of glycosylation in AD pathology and explore the possibility of nutritional management in modulating glycosylation patterns. This dissertation includes four chapters that highlight work focusing on glycosylation alterations in AD and dietary impacts on glycosylation.The first chapter delved into profiling glycosylation alterations in the postmortem brains of AD individuals, focusing on alterations in glycosyltransferases and N-linked glycans. Leveraging publicly available RNA-seq datasets encompassing seven brain regions and incorporating 1724 samples, we identified differentially expressed glycosyltransferases, including MGAT1, B4GALT1, GALNTs, subsequently confirmed by qPCR in an independent set of human medial temporal cortex (MTC) samples. N-glycan-related changes predicted by differential expression of these glycosyltransferases were confirmed by liquid chromatography-mass spectrometry (LC-MS)-based N-glycan analysis in the MTC (n=9 AD vs. 6 controls). Our findings indicated that MGAT1 and B4GALT1, which are involved in the formation of complex N-linked glycans and galactosylation, were upregulated in multiple brain regions. This upregulation was reflected in an increased abundance of corresponding N-glycans. Additionally, we identified transcription factors (TFs), including nutrient-stimulated TFs, regulating glycosyltransferases, indicating the potential of dietary components to impact glycosylation. Furthermore, heterogeneous differential expression of glycosylation isoenzymes underscored the necessity to investigate the cell-specific functions of these isoenzymes. The second chapter focused on glycosylation alterations in microglia, innate immune cells in the central nervous system. AD pathology has highlighted the importance of microglia due to their role in orchestrating neuroinflammation and phagocytosis. We characterized glycosylation changes in human microglia following (Amyloid-β Oligomer) AβO and (lipopolysaccharide) LPS stimulation. Both treatments had distinct impacts on the N-glycan profile of human microglia, as evidenced by distinct clustering of AβO- and LPS-activated human microglia glycan signatures on the PCA plot. AβO treatment increased sialylated N-glycan abundance, coupled with the upregulation of genes encoding for sialyltransferases, ST3GAL2, ST3GAL4, and ST3GAL6 in AβO-activated microglia. In contrast, LPS stimulation increased fucosylation in N-glycans, alongside the upregulation of genes encoding for fucosyltransferases, including FUT4, and the downregulation of fucosidase, FUCA1. Additionally, LPS stimulation decreased the abundance of complex-type N-glycans, aligned with downregulation of mannosidase genes (MAN1A1, MAN2A2, MAN1C1). However, the glycosphingolipid (GSL) profile did not exhibit significant changes in either AβO- or LPS-activated microglia. While protein glycosylation alterations observed in the brain and in microglia reveal potential mechanisms by which glycosylation is involved in AD, brain tissue samples are not useful for diagnosis. In the third chapter we explored the potential utility of serum glycoproteins as biomarkers for AD diagnosis. Utilizing serum samples from around 100 AD patients and 100 age- and gender-matched controls, we quantified glycopeptides of the most abundant serum proteins. The results indicate that among the differentially expressed glycopeptides, the majority belonged to proteins involved in immune function, including immunoglobulins and acute-phase proteins such as alpha-1-antitrypsin (A1AT), alpha-2-macroglobulin (A2MG), and complement C3 (CO3). Non-fucosylated and mono-fucosylated immunoglobulins G1 and G2 were key discriminators between control and AD individuals. Partial Least Square-Discriminant Analysis demonstrated the potential of these glycopeptides in distinguishing AD from normal controls, albeit with some overlap. Confounding factors, such as age, sex, BMI, and ethnicity, had impacts on glycopeptide abundances. Given the pivotal role of glycosylation in AD, the final chapter investigated the impacts of dietary supplements on glycosylation patterns. We developed a targeted glycoproteomic method to monitor site-specific glycoprofiles and quantities of the most abundant HDL-associated proteins using Orbitrap LC-MS for (glyco)peptide target discovery and QqQ LC-MS for quantitative analysis. Using this glycopeptide quantification workflow, we conducted a pilot study to determine whether HDL protein glycoprofiles are altered in healthy human participants in response to dietary glycan supplementation. Twenty-two healthy adult men/women (age 18-45) with a Body Mass Index (BMI) range of 18.5-25 were randomized to four treatment groups: placebo (n=4), N-acetylglucosamine (n=6), Spirulina (n=6), and galactose (n=6). Blood samples were collected at baseline and after four weeks of supplementation. Peptides and glycopeptides on HDL were quantified using the new workflow. The results demonstrated the potential of dietary intervention in modifying the HDL glycoprofile even within a short-term period. Further studies with larger sample sizes are required to validate diet-induced HDL glycoprofile alterations.

ABSTRACT

Establishing mitochondrial cholesterol enrichment as a fundamental causal mechanism underlying the dysfunctional clearance of neurotoxic substances by microglia would help pave the way toward the discovery of potential new therapeutics for Alzheimer’s disease (AD). The dissertation is divided into four chapters with the corresponding objectives:1. The first chapter is a literature review describing the mechanisms by which cholesterol has been implicated in influencing microglia phenotype and function, and the implications for a role in contributing to the pathophysiology of AD. We describe both what is known and the research gaps that exist and should be explored for the discovery of novel therapies for AD 2. The second chapter is an investigation on the effects of a 36h fast on HDL particle size distribution by transmission electron microscopy (TEM), and ability to efflux cholesterol from activated microglia. Importantly, HDL from 36h fasted individuals effluxed cholesteryl esters from immortalized human microglia (HMC3) loaded with cholesterol + amyloid beta oligomers (AβO) 10-fold more effectively than HDL from postprandial individuals, and microglia treated with cholesterol+AβO had reduced apolipoprotein E (ApoE) secretion, which was attenuated more effectively by HDL from 36h fasted individuals than HDL from postprandial individuals. 3. In the third chapter we use a multi-stimulant approach to test the usefulness of the HMC3 microglia cell line in duplicating critical aspects of the dysfunctional microglia phenotype and to better mimic the multi-factorial nature of AD, which is associated with multiple comorbidities, including diabetes and cardiovascular disease. HMC3 microglia were treated with cholesterol (Chol), AβO, lipopolysaccharide (LPS), and fructose individually and in combination. HMC3 microglia demonstrated changes in morphology consistent with activation when treated with the combination of Chol+AβO+fructose+LPS. Multiple treatments increased the cellular content of Chol and cholesteryl esters (CE), but only the combination treatment of Chol+AβO+ fructose+LPS increased mitochondrial Chol content. Microglia treated with combinations containing cholesterol+AβO had lower apolipoprotein E (ApoE) secretion, with the combination of Chol+AβO+fructose+LPS having the strongest effect. Combination treatment with Chol+AβO+fructose+LPS also induced APOE and TNF-alpha expression, reduced ATP production, increased reactive oxygen species (ROS) concentration, and reduced phagocytosis events.

The purpose of this dissertation is to highlight the role of high-density lipoprotein (HDL) function, structure, and composition across different life stages and how HDL influences health and disease outcomes, from early childhood, to pregnancy, to old age and Alzheimer’s disease (AD) dementia. The dissertation is divided into four chapters with the corresponding objectives:1. The first chapter is a literature review on the role of HDL across the lifespan, particularly in children, during pregnancy, and in the elderly. These populations are often less studied in the context of HDL and health outcomes. 2. In the second chapter we determine whether small-quantity lipid-based nutrient supplements (SQ-LNS) provided to mothers during the second trimester (≤ 20 weeks), postpartum (0 – 6 months), and their children (6-18 months) improved HDL cholesterol efflux capacity (CEC) compared to children of mothers supplemented with the standard iron and folic acid (IFA) supplement. We further explored whether SQ-LNS altered HDL lipidomic and glycoproteomic composition. We found that children in the SQ-LNS group had improved HDL CEC and altered HDL protein glycosylation. CEC was also found to be associated with site-specific glycans on HDL. 3. The third chapter evaluates whether SQ-LNS altered HDL CEC and plasma enzyme activities involved in HDL metabolism of pregnant mothers at 36 weeks gestation and whether these functional metrics are associated with pregnancy outcomes. SQ-LNS did not alter HDL CEC or plasma enzyme activities in mothers, but these metrics were heavily influenced by factors mediated by season at the time of blood draw. 4. In the fourth chapter we determine whether the function and structure of HDL are altered in AD patients compared to non-demented controls and whether these alterations are influenced by apolipoprotein E (APOE) genotype. We found for the first time that changes in HDL function and size were APOE genotype-specific, and that there is a link between HDL function, size, and cognitive function.

Diet is known to be a strong determinant of gut microbiome composition and function, and it is also known that gut microbiome dysbiosis is strongly linked to the risk of chronic disease. However, it is not clear how to intervene through diet or supplementation to beneficially alter the gut microbiome in individuals who have dysbiosis or who are at risk for chronic diseases such as cardiovascular disease and Alzheimer’s disease (AD). Hypothesis driven studies to determine how dietary changes affect the diet-gut microbiome-host interrelationship are critical for the discovery of solutions to improve health and mitigate disease risk. In this dissertation, three chapters are included to discuss important aspects of study design and prebiotic selection, and the outcomes of a study investigating the effects of a prebiotic supplement.

In the first chapter, critical factors in successful study design for diet-microbiome research are discussed. For example, the importance of detailed dietary data collection and analysis, tight control of background diet, and stabilizing diet in the days prior to sample collection is critical for reducing noise and amplifying the signal that can be detected of the effects of the intervention diet. Specific recommendations such as including multiple consecutive sampling per study timepoint and collection of multiple consecutive days of diet records, particularly the days prior to sample collection, are discussed. High interindividual variability in gut microbiome composition is a well-established phenomenon that has been known since the early results from the Human Microbiome Project. Because of this high interindividual variability, some study designs are not well suited to discovering the effects of a dietary intervention. Instead, we discuss the double-blinded, randomized order, placebocontrolled, cross-over study design is the optimal study design for gut microbiome research, enabling the detection of the effects of even a subtle diet intervention. We also discuss the importance of precision nutrition approaches for nutritional recommendations. As an example, we discuss nutritional recommendations for egg intake, taking into account the link between egg-derived components (i.e., choline and cholesterol) and their interaction with the gut microbiome and subsequent health outcome measures important in cardiovascular disease.

The second chapter emphasizes why we care about changes in gut microbiome in human health and how we can better utilize certain dietary interventions such as prebiotics to modulate human health and diseases focusing particularly on AD. Prebiotics are oligosaccharide and polysaccharide structures isolated from plants or synthesized from sugars that are shown to modulate the microbiome and are associated with health benefits. We summarized the importance of dietary fiber intake for maintaining a healthy gut microbiota, with a focus on short chain fatty acid production, mucus secretion, and protection from pathogens. The specific links between healthy gut physiology, gut immune function and the brain are also discussed. The importance of anti-inflammatory metabolites produced by gut microbes consuming specific substrates in modulating critical signaling pathways in the brain is discussed in the context of AD prevention. We conclude by discussing which specific bacterial genera are increased and decreased in AD patients and highlight the potential of increasing bifidobacteria abundance through the intake of specific prebiotic substrates as a promising approach for beneficially modulating the gut microbiome and improving health outcomes in AD patients.

The final chapter summarizes the findings from our human clinical trial in healthy subjects consuming a low fiber diet (< 15 g/day) to determine whether a low-dose, easy to use bifidogenic prebiotic supplement formulation increases bifidobacteria and produces measurable metabolomic and metagenomic changes over the course of 4 weeks. We used the double-blinded, placebo-controlled, randomized order crossover study design highlighted as the optimal study design for diet-microbiome studies in chapter 1, to study the effects of the prebiotic supplements in 20 otherwise healthy young individuals consuming diets low in fiber, typical of the average American diet. As hypothesized, the relative abundance of bifidobacteria significantly increased after the prebiotic supplement, as did the gene counts of genes associated with prebiotic utilization, acetate production, and choline oxidation. Importantly, these changes in gut microbiome composition and function were reflected by changes in the concentrations of microbially-derived metabolites in the plasma, and point to novel pathways by which increasing the abundance of bifidobacteria may confer beneficial health effects on the host.

This article focuses on the role of omega-3 fatty acids as precursors for lipid signaling molecules known as oxylipins. Although omega-3 fatty acids are beneficial in autoimmune disorders, inflammatory diseases and heart disease, they are generally underrepresented in the American diet. A literature review confirms that the consumption of omega-3 fatty acids — whether in food sources such as walnuts, flax seeds and fatty fish (including salmon and sardines), or in supplements — is associated with decreased morbidity and mortality. This growing body of evidence, including the results of a recent study of patients with kidney disease, highlights the need to measure omega-3 fatty acids and their oxylipin products as markers of metabolic health and biomarkers of disease. In addition, there is substantial evidence of the need to increase the omega-3 fatty acid content of American diets to optimize metabolic health.