- Cognitive impairment, or cognitive decline, a noticeable and measurable decline in cognitive abilities (e.g. memory and learning) that exceeds those attributed to normal aging, represents an early symptom of neurodegeneration and increased risk for progression to more severe dementias, such as Alzheimer's disease (AD). While the complex etiology of these conditions remains an area of active investigation, oxidative stress has been implicated as a primary factor in neurodegenerative disease pathogenesis. Zebrafish (Danio rerio) are a recognized model for studying the pathogenesis of cognitive deficits and the mechanisms underlying behavioral impairments, including the consequences of increased oxidative stress within the brain. The vertebrate brain is especially enriched in long-chain polyunsaturated lipids, such as ω-3 docosahexaenoic acid (DHA; 22:6 ω-3); therefore, lipid peroxidation is a likely contributor to neuropathology. Vitamin E (α-tocopherol; VitE), the body’s most potent lipophilic antioxidant, was first discovered in 1922 as an essential nutrient for preventing fetal resorption in rodents, and has since been linked with embryonic and neurological health in both numerous animal (including my own work with zebrafish) and human studies. VitE deficiency increases early miscarriage risk in humans, which poses public health concerns since estimates of inadequate dietary VitE intakes exceed 80% of the global adult population. However, nearly a century after its initial discovery, the underlying biological rationale explaining VitE’s essentiality for (neuro)development and brain function remains unknown.The purpose of this project was to provide for an evidence-based assessment of metabolic interactions between VitE and specific membrane lipids to elucidate the biochemical basis underlying VitE’s neurological function in vivo during neurodevelopmentand into adulthood. To accomplish this overall aim, I exploited a zebrafish model in which my lab group has pioneered the study of nutrition and dietary manipulation and the use of novel “omics” methodologies. I used both embryonic and adult conditions of VitE deficiency to publish compelling evidence that demonstrates the major role for VitE in the brain is to protect DHA and DHA-containing phospholipids (DHA-PLs) against oxidative stress, and without this antioxidant protection, ensuing secondary deficiencies in both DHA and choline coincide with increased morbidity, mortality, and or cognitive impairments. Further, my work shows that VitE’s antioxidant activity is vital for maintaining the cellular antioxidant network, and dysregulation of such aberrantly alters energy metabolism by severely compromising mitochondrial function. The studies included in this work, when considered together, provide insight as to how inadequate VitE perturbs DHA, phospholipid, and choline metabolism, resulting in dysregulation of other metabolic pathways as well as epigenetic methylation reactions, and how disruption of these processes compromises neurological and cognitive outcomes during neurodevelopment as well as in later life.My primary research goal was to help elucidate the mechanism(s) through which VitE contributes to lifetime brain health. To achieve this, I evaluated the consequences of inadequate VitE from the earliest stages of brain development through middle-age. My central hypothesis was that VitE protects DHA, a vital substrate for brain membrane phospholipid maintenance, and that dysregulation of DHA-PL status due to restricted dietary VitE severely perturbs critical events necessary for embryonic neurodevelopment that, ultimately, increase susceptibility for consequent, persistent cognitive impairments.First, I performed phenotypic assessments and lipidomics analyses, as well as developed a method to measure PL turnover in zebrafish embryos using H218O labeling, to gain mechanistic insight on the organism-level effects of developmental α-tocopherol deficiency. I hypothesized that VitE is required by the developing embryonic brain to prevent depletion of highly polyunsaturated fatty acids, especially DHA, the loss of which I predicted would underlie abnormal morphological and behavioral outcomes. Therefore, I fed adult 5D zebrafish defined diets without (E-) or with added VitE (E+, 500 mg RRR-α-tocopheryl acetate kg diet) for a minimum of 80 days, and then spawned them to obtain E- and E+ embryos. The E- compared with E+ embryos were behaviorally impaired at 96 hours post-fertilization (hpf), even in the absence of gross morphological defects. Evaluation of phospholipid (PL) and lysophospholipid (lyso-PL) composition using untargeted lipidomics in E- compared with E+ embryos at 24, 48, 72, and 120 hpf showedthat four PLs and three lyso-PLs containing DHA, including lysophosphatidylcholine (LPC 22:6, required for transport of DHA into the brain), were at lower concentrations in E- at all time-points. Additionally, H218O labeling experiments revealed enhanced turnover of LPC 22:6 and three other DHA-containing PLs in the E- compared with the E+ embryos, suggesting that increased membrane remodeling is a result of PL depletion. Overall, these data indicate that VitE deficiency in the zebrafish embryo causes the specific depletion and increased turnover of DHA-containing PL and lyso-PLs, which may compromise DHA delivery to the brain and thereby contribute to the functional impairments observed in E- embryos.Next, I investigated the underlying mechanisms causing developmental VitE deficiency-induced mortality in E- embryos using targeted metabolomics analyses embryos over five days of development, which coincided with their increased morbidity and death. VitE deficiency resulted in peroxidation of DHA, depleting DHA-PLs, especially phosphatidylcholine, which also caused choline depletion. This increased lipid peroxidation increased NADPH oxidation as well, which depleted glucose by shunting it to the pentose phosphate pathway. Using bioenergetic profiling analyses, I also found that VitE deficiency was associated with mitochondrial dysfunction with concomitant impairment of energy homeostasis. The observed morbidity and mortality outcomes could be attenuated, but not fully reversed, by glucose injection into VitE-deficient embryos at developmental day one. These studies together suggest that embryonic VitE deficiency in vertebrates leads to a metabolic reprogramming that adversely affects methyl donor status and cellular energy homeostasis, with ultimately lethal outcomes.I then shifted my focus to address outcomes of chronic VitE deficiency and to probe more thoroughly brain-specific consequences. I investigated behavioral perturbations due to isolated, chronic VitE deficiency in adult zebrafish fed diets that were either VitE-deficient (E- group) or sufficient (E+ group) for up to 18-months of age. I hypothesized that E- adult zebrafish would display significant cognitive impairments associated with elevated lipid peroxidation and additional metabolic disruptions in the brain. Using assays of both associative (avoidance conditioning) and non-associative (habituation) learning, I found E- adults were learning impaired compared with E+ fish, and that these functional deficits occurred concomitantly with the following observations in adult E- brains: decreased concentrations and increased peroxidation of polyunsaturated fatty acids (e.g. DHA), altered brain phospholipid and lysophospholipid composition, dysregulation of the cellular antioxidant network, and perturbed energy (glucose ketone), phosphatidylcholine, andcholine methyl-donor metabolism. Collectively, these data show that chronic VitE deficiency could lead to cognitive dysfunction through multiple potential mechanisms, including decreases in DHA, antioxidants, glucose, and choline, as well as corresponding dysfunction in related metabolic pathways (e.g. energy NAD(P)H and methyl-donor metabolism) within the brain.Finally, given the outcomes of my embryo studies demonstrating that increased lipid peroxidation in E– embryos perturbs their cellular antioxidant network, which ultimately disrupts aerobic energy metabolism, causing a significant decrease in whole-body (and, presumably, brain) glucose levels, and thus adversely impacts neurobehavioral outcomes, I investigated whether these consequences could be reversed via dietary remediation. Previous pilot studies showed that mortality and behavioral impairments are avoided with proactive VitE repletion, as an α-tocopherol emulsion administered into the yolk of 0 hpf E– embryos entirely prevented mortality and morbidity outcomes. However, remediation of VitE deficiency-induced (i.e. secondary) nutrient deficiencies only partially rescues E– embryos, as observed following glucose supplementation into the yolk at one day of age (24 hpf; after established VitE deficiency but prior to glucose depletion). Together, this data suggests the effects of developmental VitE deficiency may be prevented, but not necessarily reversed. I hypothesized, therefore, that deleterious outcomes of embryonic VitE deficiency cannot be ameliorated fully though later supplementation with VitE and other depleted nutrients (e.g. DHA and choline), and that long-term cognitive defects will persist in E– compared with E+ embryos despite dietary intervention.To test this hypothesis, I selected normal appearing E– or E+ embryos, then fed them a complete diet for 7 days and analyzed them for behavioral, biochemical, and morphological changes. I evaluated the embryo groups for up to 12 days post-fertilization (dpf). The E– group suffered significantly increased morbidity and mortality as well as altered DNA methylation status through 5 dpf when compared to E+ larvae, but upon feeding with a VitE-adequate diet from 5-12 dpf both the E– and E+ groups survived and grew normally; the DNA methylation profile also was similar between groups by 12 dpf. However, 12 dpf E– larvae still had behavioral defects. These observations coincided with sustained VitE deficiency in the E– vs. E+ larvae, despite adequate dietary supplementation. I also found continued DHA depletion and significantly increased lipid peroxidation in E– vs. E+ larvae. Further, targeted metabolomics analyses revealed persistent dysregulation of the cellular antioxidant network, the CDP-choline pathway, andglucose metabolism. While anaerobic processes were increased, aerobic metabolism was decreased in the E– vs. E+ larvae, potentially indicating mitochondrial damage and aberrant reliance on aerobic glycolysis (“Warburg effect”) in the E- group. Taken together, these outcomes indicate embryonic VitE deficiency causes lasting behavioral impairments due to persistent lipid peroxidation and metabolic perturbations that are not resolved via later dietary VitE supplementation.Collectively, the findings from these completed studies provide mechanistic evidence to explain VitE’s essentiality for human neurodevelopment and adult brain function, and yield new insights regarding the impact early-life VitE deficiency has on embryonic (neuro)development as well as on the inception of cognitive decline and ensuing neurological disorders. These outcomes may be used to support continued research investigating and promoting the importance of adequate VitE for optimal brain health throughout life.