Congratulations Holden Williams, PhD!

On October 18, 2021 Holden Williams successfully defended his dissertation and earned his doctoral degree. Congratulations, Dr. Williams!

APOE GENOTYPE AND CEREBRAL GLUCOSE METABOLISM: A MULTI-OMICS APPROACH

Doctoral Committee Members
Dr. Lance Johnson, Department of Physiology, MentorDr. Ming Cui Gong, Department of PhysiologyDr. Matthew Gentry, Department of Molecular & Cellular Biochemistry
Dr. Bret Smith, Department of Neuroscience
Dr. Greg Jicha, Department of Neurology
Dr. Christopher Norris, Department of Pharmacology & Nutritional Sciences, Outside Examiner

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 developed 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.

Acknowledgements
I’d like to thank a lot of very special people in my life, and I’ll start with the one who I truly owe the most thanks, my grandfather, John Reuben Hillin.

I owe a great deal to my loving parents who were always by my side, even despite some troubling teenage years. To my mom, my friend; and my dad, my shepherd: I owe this opportunity to you for the unwavering support, for the guidance and wisdom, for all that you provided, and especially for always loving me. I seek nothing less than to make you proud. You both have given me the strength to get through many difficult times and offer the guidance for those too difficult to surpass alone. To my oldest brother Austin, for showing me what it means to be a leader; and to my youngest brother Hunter, for being my first friend. To my grandmother, Gene Hillin, for your unconditional, tender love and pure spirit…and for never holding back if I needed a reality check. To my Aunt Suzanne and cousins Claire and Rhett (aka Dr. Rhettule), your intelligence and successes in academia were always inspiring to me and I thank you for wonderful insight in times when I thought I had no idea what in the hell I was doing.
Lance, oh how I owe to thee, Fearless Leader. You’ve always had my back, especially when times were grim, and I knew you would always go to battle for me. Although you probably feel like you took it too easy on me, believe me I learned what it meant for “hell hath no fury…” It’s an honor that I reminded you of yourself in graduate school, even though you never had the heart to tell me that yourself (probably because I did it better). You were exactly the kind of PI I didn’t know I needed. In the beginning I wanted an experienced PI with a hands-on approach in the lab, someone with a great scientific background and a little grey hair (sort of like Ramon). What I got was the youngest, most junior PI with a background in journalism who was probably better off not in the lab. Joking aside, I probably would not have made it in any other lab. I learned more than just science and technical skills during my time under your mentorship. You taught me about etiquette, compassion, and professionalism in the workplace but also how to work and how to have fun at the same time. When any prospective student asked me about you, my answer was always that you truly couldn’t ask for a better mentor in every aspect. I wholeheartedly believe that and there’s ample proof in your actions – if only you paid more, I’d work for you another four years.

I also want to acknowledge my committee members: Drs. Ming Gong, Bret Smith, Matt Gentry, and Greg Jicha. Thank you all for dedicating your time to my training. You all have been instrumental in shaping this project and especially in guiding my development as a student and as a scientist. I have enjoyed having each of you on my committee. Ming, you have been an excellent co-chair and someone I admire for your compassion and insight. Bret, our conversations arising from your knowledge of cutting-edge brain metabolism have been a source of great satisfaction. Matt, your breadth of experience and mentorship is truly inspiring. Dr. Jicha, I cherish the opportunity of shadowing in your clinic, of which I found very rewarding, and I appreciate always pressing the translational implications of my research.
Ramon, my adopted, behind the scenes mentor. Normally one might say “you taught me everything I know about metabolomics” but in my case your approach was more so “metabolism isn’t something I can teach you, you just have to learn it yourself…and by the way, good luck!” As I painstakingly found out later, that actually was the best approach because I truly learned everything I needed to. However, you spent a great deal of time always answering all of my questions and showing me the ropes of mass spec in the early days. For that you deserve much credit, and I sincerely thank you. As for the later days, Lyndsay, you became my go-to resource if anything went awry with those darned machines. Your technical support surpassed Agilent’s ten times out of ten. I am glad we became friends and still hope you’ll take me marlin fishing!
I’d like to thank everyone in the Johnson Lab who I am leaving behind and congratulate all of those lucky enough to escape before me. Being the great team we were, everyone has had at least some part in my project and I want to express my gratitude for all of your help whether it was technical, moral, or otherwise. Especially I’d like to acknowledge Maggie Piron and Elizabeth Allenger who are two wonderful ladies whose incredible work dissecting mice, grinding brains, and harvesting cells is almost as commendable as their ability to put up with my excruciatingly particular instruction. And Brandon, I mostly thank you for taking the bullet in going first, but also for the great joy it was having you in lab from conversations we had exchanging ideas, talking smack, and just for being a great friend.
Lastly, to the most important (former) graduate student, Dr. Gabrielle Keb. Though my chivalry may have wooed you from our very first bar crawl, it pales in comparison to your kindness and compassion that you demonstrate each day. You make me a better person and bring great joy to my life.