Author ORCID Identifier

https://orcid.org/0000-0003-4041-5190

Date Available

7-7-2022

Year of Publication

2022

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Physiology

First Advisor

Dr. Lance A. Johnson

Second Advisor

Dr. Ming C. Gong

Abstract

Apolipoprotein E (APOE) is encoded by the APOE gene, present in humans as three main isoforms (E2, E3, and E4). E4 carriers face up to a 15-fold increased risk for developing late-onset Alzheimer’s disease (AD), while E2 carriers are protected. Understanding the risk conferred by E4 has been an extensive research focus for nearly three decades, but the exact mechanism has yet to be proven. Many studies have demonstrated attenuated roles of E4 in classical hallmarks of AD, notably amyloid processing and neurofibrillary formation, which normally present later in disease progression. How APOE influences hallmarks that present much earlier are not as well- characterized and may indicate how E4 confers greater risk with an earlier age of disease onset. One such hallmark of AD is cerebral glucose hypometabolism, which happens to also be present in young cognitively normal E4 carriers. The specific cell types responsible for this reduction are unknown; however, an important role of astrocytes, which are the main producers of APOE in the brain, is to facilitate glucose uptake from the vasculature to provide metabolic support for neurons. Therefore, I hypothesize astrocytes are primarily responsible for the reduction in cerebral glucose uptake associated with E4, specifically due to impairments in glucose metabolism. In vivo brain metabolism in human APOE mice (E2, E3, or E4) was examined using an oral gavage of [U-13C] glucose. Glucose metabolism in immortalized astrocytes expressing human APOE E2, E3, or E4 was measured using stable isotope resolved metabolomics via a [U-13C] glucose tracer supplemented growth media. Cell culture and brain metabolite profiles were analyzed using mass spectrometry to determine glucose utilization by tracing the enrichment of 13C atoms in central carbon metabolism pathways. Lastly, a combined multi-omics approach was utilized to determine the metabolic and transcriptomic changes associated with inflammation in astrocytes. Immortalized astrocytes (E3 and E4) were subjected to an acute inflammatory challenge consisting of pro-inflammatory mediators (TNFα, IL-1α, and C1q). Metabolic phenotyping of E3 and E4 astrocytes was conducted by Seahorse mitochondrial and glycolytic rate assays, steady state metabolomics, and further examined using stable isotope resolved metabolomics with a [U-13C] glucose tracer in primary mixed glial cultures. Transcriptional changes in E3 and E4 astrocytes after inflammatory challenge were determined using Nanostring neuroinflammatory gene expression arrays. E4 mice exhibited alterations in glucose flux through central carbon metabolism, specifically in glycolysis and the TCA cycle. In vitro findings revealed E4 astrocytes redirected glucose flux through glycolysis into the non-oxidative pentose phosphate pathway. Glutathione, phospholipid species, NADH, and nucleotide biosynthesis from

glucose was also increased in E4 astrocytes, suggesting an inflammatory or oxidative stress component. Following acute inflammatory stimulation, E4 astrocytes exhibited altered glycolytic function, disrupted metabolic responses, and blunted inflammatory gene expression compared to E3 astrocytes. These findings begin to shed light on the cell type specific effects of APOE by which immunometabolism, especially in E4 carriers, may lead to progression of AD and may direct future development of therapeutics.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2022.017

Funding Information

This study was supported by the National Institutes of Health in 2018 (no.:1T32GM118292-01A1) and in 2019-21 (no.:1T32GM118292-01A1).

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