Congratulations Ben Shaw, PhD
On Friday, February 25, 2022, Ben Shaw successfully defended his dissertation and earned his doctoral degree. Congratulations, Dr. Shaw!
IMMUNOREGULATORY RECEPTOR GENETICS, EXPRESSION, AND SPLICING STUDIES IN ALZHEIMER’S DISEASE
Doctoral Committee
Dr. Steve Estus, Department of Physiology, Mentor
Dr. Bret Smith, Department of Neuroscience
Dr. John Gensel, Department of Physiology
Dr. Chris Norris, Department of Pharmacology & Nutritional Sciences
Dr. Linda Van Eldik, Department of Neuroscience, Outside Examiner
Abstract
Microglia are the resident immune cells of the brain, undertaking many critical tissue maintenance functions such as immune surveillance and phagocytosis. Microglial dysfunction has recently been identified as a multi-stage signature of many neurodegenerative diseases, including late-onset Alzheimer’s Disease (LOAD). Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in over thirty genes that modulate risk of developing LOAD. In the central nervous system, roughly half of these LOAD-associated genes are primarily expressed in microglia. The proteins encoded by these genes include cell surface receptors that contain either immunomodulatory tyrosine-phosphorylated activating motifs (ITAMs) or inhibitory motifs (ITIMs), including TREM2, CD33, and SIGLEC14. Here, I studied the molecular genetics underlying these three genes and their respective contributions to LOAD risk.
First, I found that TREM2 undergoes extensive alternative splicing in multiple tissues, including brain. Total TREM2 expression is not different as a function of LOAD diagnosis (p = 0.1268), but TREM2 expression is increased by 34% in tissues with higher NIARI scores (p = 0.0033). I also found that a novel TREM2 isoform lacking exon 2, D2-TREM2, accounts for 11% of the total TREM2 mRNA in human brain, and that this splicing efficiency is not altered as a function of AD status (p = 0.4909) or brain pathology (p = 0.9502). I also found that the D2-TREM2 protein has similar subcellular localization to its parent TREM2 protein, as both are primarily retained in the Golgi apparatus.
Next, I studied the exon 2-lacking CD33 isoform, D2-CD33. I developed an in vitro model to study the function of the D2-CD33 using a CRISPR-Cas9 approach in the U937 human monocyte cell line. After validating this model with sequencing, qPCR, and flow cytometry, I found that a nearby pseudogene, SIGLEC22P, was used as a repair template in approximately 10% of edited cells. This finding also provided the highest resolution to date of the clinically relevant anti-CD33 P67.6 antibody clone, gemtuzumab.
Finally, I combined a recent LOAD GWAS with a protein quantitative trait loci (pQTL) study to uncover SIGLEC14 as a potentially overlooked LOAD risk factor. I found that a previously described SIGLEC14 genetic deletion occurs within a 692 bp crossover region. I also found additional copy number variation not previously described using both qPCR-based and in silico assays, with copy numbers identified ranging from zero to four. While SIGLEC14 deletion does correlate well with a proxy single nucleotide polymorphism (SNP), rs1106476, additional SIGLEC14 genomic copies do not correlate with this SNP. Further, the SIGLEC14 genomic deletions correlate stepwise with decreased SIGLEC14 expression (p = 0.0002), and also correlate significantly with decreased SIGLEC5 expression (p = 0.0389).
In conclusion, microglial cell surface receptors are heavily implicated in the risk of developing LOAD, and these studies advance the field by adding to the molecular mechanisms which underlie their risk contribution. Further studies will be needed to address whether these findings can be translated clinically to either potential druggable targets or biomarkers.
Acknowledgements
Graduate school is not without its challenges, and these challenges require the support of a broad range of individuals. Mentors, fellow students, friends, and family have each sustained me throughout this process, and I could not be more grateful.
Dr. Steve Estus picked me up in the middle of my training, providing me a home to finish my PhD when I was unable to move to Pittsburgh. Throughout this time, he has been profoundly supportive, mentoring me through four grant applications, four publications, and a behind-the-scenes view into how to run a lab from personnel to grant management. I’m sure I could have been an easier student to mentor, but he has been nothing but patient with me. The skills he has passed on to me will serve me well for decades to come. Dr. Brad Taylor also had an integral role in my training as my first mentor. While I was unable to continue with him after moving his laboratory to Pittsburgh, he provided formative training in experimental design, techniques, and grantsmanship. My time in his laboratory provided the foundation needed to successfully transfer to the Estus laboratory.
My committee members Dr. Bret Smith, Dr. John Gensel, and Dr. Chris Norris have also guided me along this journey. You have all written more letters on my behalf than you signed up for, sat through committee meetings that went far too long, and shifted to a completely different project with me. Bret, your counsel during the time I tried making a long-distance mentorship work, my transition between laboratories, and the way you’ve watched over me and acted as a second mentor is especially appreciated. Dr. Beth Garvy, while not acting as a mentor or committee member in any official capacity, has also been enormously supportive. From training me in experimental techniques to helping me navigate the postdoctoral market, I was lucky to find such a caring, knowledgeable, and candid guide.
None of this would have been possible without the seasoned experimentalists and bench scientists from whom I owe everything I’ve learned at the bench. Renee Donahue, Dr. Suzanne Doolen, Dr. Lilian Custodio-Patsey, Melissa Hollifield have helped me master techniques which I never thought possible. Jim Simpson, you have entertained every qPCR assay I’ve concocted since joining you and Steve, teaching me that as much experience as I have in a technique there’s always more to learn. You’ve been a great lab manager, and even better friend. I’ll certainly miss our frequent golf outings, constantly chasing a better score and being berated about that one shot that went “under the bridge.”
Nobody can relate to the unique struggles of this journey like those going through with you. Dr. Bethi Oates, Dr. Brooke Ahern, Dr. Brandon Farmer, and Dr. Ryan Cloyd have walked this path with me, and we have pulled each other up countless times. From office venting sessions to long lunches to our Bourbon Club, we’ve been there for each other for years. While we all seem to be scattering across the country as this phase ends, I know we will continue to support each other from afar.
Finally, I have to thank my family. My father, Craig Shaw, has been a shining example of discipline, integrity, and perseverance. Through every bump in the road these past six years, he has been a sounding board and reminded me how far I’ve come. Knowing I’ve made him proud is one of my greatest accomplishments. My in-laws, Susan & Walter Hirth and Rachel Mossop, have been a constant source of encouragement. My amazing wife, Heather Shaw, could not have been more supportive. I have worked 90-hour weeks, gone in early and come home late, left for weeks at a time to Pittsburgh, traveled for conferences, and far too often left my fair share of responsibilities at home on the back burner only for you to pick them up. You have shouldered that without complaint while maintaining your own career and keeping up with our wonderful daughter more than I should have let you on your own. Our beautiful daughter, Hayley, has grown up in the lab. I started this PhD when she was only two years old, and during this time she has been to the lab with me so many times she now looks forward to looking under microscopes and seeing what kind of new things I can show her. I can only hope that she maintains this level of curiosity in all that she does.
