On Thursday, December 8, 2022 Cassi Friday successfully defended her dissertation and earned her doctoral degree. Congratulations Dr. Friday!
The Influence of APOE Genotype on Lipid Droplet Dynamics
Excess lipid droplet (LD) accumulation is associated with several pathological states, including multiple neurodegenerative disorders such as Alzheimer’s disease (AD). However, the mechanism(s) by which changes in LD composition and dynamics contribute to the pathophysiology of AD remains unclear. Apolipoprotein E (ApoE) is a droplet-related protein with a common variant (ApoE4) that confers the largest increase in genetic risk for late-onset AD. Interestingly, ApoE4 is associated with both increased neuroinflammation and excess LD accumulation. This dissertation work seeks to quantitatively profile the lipid and protein composition of LDs between the ‘neutral’ ApoE3 and ‘risk’ ApoE4 isoforms, in order to gain insight into potential LD-driven contributions to AD pathogenesis.
Targeted replacement mice expressing human ApoE3 or ApoE4 were injected with saline (control) or LPS (inflammatory stimulus) and after 24 hours, hepatic lipid droplets were isolated and droplet proteomes and lipidomes were analyzed. Quantitative proteomics showed that LD fractions from E4 mice are enriched for proteins involved in innate immunity, while E3 LDs are enriched for proteins involved in lipid ß-oxidation. Lipidomics revealed a shift in the distribution of glycerophospholipids in E4 LDs with an increase in multiple phosphatidylcholine species. To translate these findings to the brain, primary microglia from the same strain of mice were exposed to exogenous lipid, inflammatory stimulation, necroptotic N2A cells (nN2A), or a combination of treatments to evaluate lipid droplet accumulation and impact on cell function. Microglia from ApoE4 mice accumulated more LDs at baseline, with exogenous OA, LPS stimulation, and nN2As as a percentage of E3 control across multiple experiments. E4 microglia also secreted significantly more cytokines (TNF, IL-1β, IL-10) than E3 microglia in the control, oleic acid, and nN2A treatment conditions. Interestingly, droplet inhibitors for ACAT and DGAT both decreased droplet accumulation in cells, but did not ameliorate the cytokine response. Finally, we have established a biobank of APOE genotyped peripheral blood mononuclear cells (PBMCs) from research participants. These easily accessible immune cells will serve as a highly translational model to understand LD dynamics as it relates to ApoE and AD risk.
In summary, E4 cells accumulate more LDs compared to E3 under all conditions tested, while the proteomic profile of E4 LDs support the hypothesis that E4 expression increases inflammation under basal conditions. This increased LD formation in non-aged, non-diseased E4 cells may suggest preclinical dysfunction associated with the highest risk APOE genotype, and a better understanding of LD dynamics within these cells and their functional implications may provide novel targets to improve E4-related outcomes.
Acknowledgements
I would like to give my biggest ‘thank you’ to Dr. Lance Johnson - my mentor not only for this dissertation research, but for all general life endeavors. You took a chance on me as a returning PhD student, you challenged and encouraged me along the way, and you gave me the flexibility I needed as a parent. Thank you so much for rebuilding my confidence as a scientist and sharing your contagious passion for research and science communication with me. Thank you for sharing the same weakness as an over-committer to all opportunities and things and for being my check-point before I obligated myself to anything new – it was great to have you as a decision filter the last couple of years. Also, thank you for entertaining my anxious opposition to your chill procrastination. Finally, thank you for encouraging me to pursue a job that isn’t related to your work. I think this speaks volumes about you as a mentor and I will always appreciate you for being excited for me above all else. You are brilliant and I can’t wait to follow your journey through the illusive and exclusive ranks of the illuminati.
Thank you so much to my committee members. Your expertise, feedback, questions, and advice have been invaluable. Dr. Josh Morganti, thank you for the support you’ve shown since we first met – doing blood draws, PBMC prep, and talking through techniques with you was so helpful and I have loved collaborating with your lab. Dr. Jamie Sturgill, thank you for your mentorship in careers, parenting, and life. I’m amazed by the work you do and the passion and energy you devote to it – I loved shadowing you and learning about it all. Dr. Sarah D’Orazio, thank you for your insight and for the tough questions. You are impressively smart and I have learned so much from your guidance. Also, thank you for the opportunity to get out of the lab for science fair judging - it could not have come at a better time to give me a boost of excitement through a middle school student’s eyes. Dr. Ryan Temel, thank you for contributing knowledge about lipids and metabolism and your great advice on study design. You are practical and I have learned a lot by listening to your feedback about how not to overcomplicate things. Collectively, thank you all for believing in me and getting me to this point – I’ll always be grateful.
A huge thank you to Tanya Graf. Had you not been the amazing human and friend you are, we would not have kept in touch when I left in 2015 and I would have likely not returned. Thank you for working with Dr. Ken Campbell to have me reinstated to the graduate school and the suggestion to talk with Lance (and thank you Dr. Campbell for your mentorship, support, and advice throughout my journey!). Tanya, thank you for always having an open door, for your prayers, and for cheering me on. Every time I stepped in your office and spilled my guts, I was always met with love and reassurance. You’re the best!
Thank you to the past and present members of the Johnson Lab for your help with projects that complete this dissertation and for your comradery (and often debauchery (Lesley)). You make being in lab so much fun and working with mice tolerable – something I never thought I would say! A special thanks to the students I have had the chance to mentor or act as ‘lab mom’ to – you are all so smart and I know you will do great things! I will be following you and cheering you on from wherever I go. My door is always open - please call, come visit, and reach out any time for any reason.
Because my PhD journey began in another lab over ten years ago, I would be remiss if I did not thank my previous committee and mentor for laying a foundation of knowledge and skills to build on. Thank you, Dr. Brandon Fornwalt, for growing my interest in clinical research, for introducing me to every person you knew, building my networking and presentation skills, and for a great first-half of my degree. Thank you to Dr. Jody Clasey and Dr. Karen Esser for the incredible outreach opportunities with kids in Eastern Kentucky and for your help with my project. Also, thank you Dr. Moriel Vandsburger and Dr. Dennis Bruemmer for being on my first committee, allowing me to rotate in your lab, and supporting me through tough decisions. Thank you also to Dr. Speck – I learned an incredible amount from you through the teaching certification, and you planted a seed for education and teaching physiology that grew into several fun opportunities down the line.
Thank you to the children who bravely volunteered for my first research study. It is not an easy task to ask kids to complete a 45 minute MRI, but every single one rocked it! Their enthusiasm and curiosity drove me forward and made a lasting impact on my motivation to do research. Also, thank you to the hundreds of volunteers for my current study. Without your altruism and interest in research, the science community would truly be at a loss.
Thank you to my friends and my family for your constant encouragement, prayers, Starbucks gift cards, and cheers. I am blessed beyond measure to have sisters and friends to check in on me, offer help, and send me goodies. I did not intend to finish a PhD during a pandemic with three kids at home and a husband serving overseas, but here we are and I couldn’t do it without your help and inspiration. To my parents – Mom and Dad – thank you for raising me on a farm, for making me work hard, for keeping me humble, and for giving me everything you could when you had little. You have never missed an opportunity to tell me you are proud of me and those words always keep me going. I love you so incredibly much and owe you big for shaping me into the person I am today.
To my forever boyfriend and our sweet wildlings. Anthony, I love you and I am so glad to have met you at the beginning of this journey over a decade ago. Who knew it would have unfolded like this? You have been my biggest advocate, my loudest encourager, and my most practical advisor. You mean the world to me and the sacrifices we have shared and endured for the betterment of one another and our family make me love you even more. Eleanor Lucille, you are by far my biggest cheerleader and you have been a constant source of light when I am overwhelmed with everything. Your optimism is infectious and you have carried me more than you know. Theodore Gregory Isaac, your terrible twos extended into your threes and fours and I thought we would never survive all of this together, but you keep me on my toes, give me endless reasons to be thankful for you, and you always make me laugh. Graham Radley, thank you for always being happy, chill, and hilarious – you bring me so much joy. I love you all so much!
A lengthier acknowledgements than I intended, but I have so many people to thank for believing in me. However, none of this would be possible without my belief in a gracious God that has kept me grounded and at peace during the chaos of life and the many ups and down of this ride.