Defining the role of rare genetic variants that drive risk and pathogenesis of Alzheimer’s disease

Alzheimer’s disease (AD) is the leading cause of dementia and is pathologically defined by the aggregation of extracellular amyloid plaques and intracellular neurofibrillary tangles. Rare heritable mutations within the genes for amyloid precursor protein (APP) and presenilin 1 (PSEN1), and presenilin 2 (PSEN2) cause early onset AD and account for approximately 1% of AD cases. While the majority of AD cases are late-onset (LOAD), which is defined by a markedly more complex genetic architecture that is comprised of many genetic risk factors that influence AD through multiple cellular pathways. The advent of deep sequencing analyses have allowed for the identification of novel risk factors and genetic variants. By identifying these genetic risk factors, characterizing their cellular and molecular function, and then identifying the roles they play influencing AD risk, this knowledge may be leveraged for the development of more effective therapeutics. In this dissertation, I look more deeply into three risk factors: LMNA, RAB10, and PLD3. Through a combination of computational analysis and functional/biochemical assays, I attain a deeper understanding of the roles of these genes as they affect AD pathogenesis.The first of these genes, LMNA, encodes a nuclear lamin protein that is associated with the accelerated aging condition Hutchinson-Guilford Progeria Syndrome (HGPS). In this condition, a mutation in LMNA leads to the production of progerin, which is unable to be processed by ZMPSTE24 and constitutively incorporates into the nuclear membrane, causing nucleoskeletal dysfunction. In AD, some of these same nucleoskeletal deficiencies have also been observed. Through mRNA expression analysis of laser-captured microdissected neurons, we observe a significant elevation of LMNA expression coupled with a reduction of ZMPSTE24 expression in diseased brains, resulting in more immature, unprocessed prelamin A. When we induced progerin expression in iPSC-derived cortical neurons, we observed disruption in pathways associated with the oxidative stress response as well as the lysosome, which has been associated with AD pathology in the literature. Further genomic analyses in increasingly larger datasets has led to the identification of genetic variants that protect against AD. One study by our group identified a rare SNP in the 3’-untranslated region (3’-UTR) of RAB10 (rs142787485). As a Rab GTPase, RAB10¬ plays a role in the endo-lysosomal network (ELN), a central pathway to the processing of APP and the production of Aβ. In AD brains, we observe increased levels of RAB10 mRNA and RAB10 protein expression. Overexpressing RAB10 in N2A695 cells increases the ratio of extracellular Aβ42/40, which is associated with increased pathogenicity. Furthermore, when investigating the rs142787485 SNP, the location of this SNP is predicted to interfere with the binding of multiple miRNAs. However, these predictions still need to be validated through functional and biochemical assays. In our efforts to better understand AD genetics, there remain challenges posed by genes that remain uncharacterized in terms of their function. One such gene is PLD3, a member of the phospholipase D family, but lacks any confirmed phospholipase activity or substrate. Whole exome sequencing of families densely affected by LOAD uncovered the rare variant in PLD3 p.V232M. Further deep sequencing uncovered additional variants associated with increased disease risk. In our work we find that one particular synonymous variant, p.A442A, activates a cryptic splice site, resulting in the reduced expression of exon 11. Furthermore, studies of AD patients show that PLD3 is reduced in the neurons of AD brains. When we look at loss of Pld3 in amyloid model mice, we observe significantly decreased Aβ turnover and altered plaque pathology which we further attribute to reduced microglial plaques, mirroring what has been observed in Trem2-deficient amyloid mice. Together, these studies illustrate the critical role of nuclear and ELN function in AD risk and resilience

Systematic validation of variants of unknown significance in APP, PSEN1 and PSEN2

Alzheimer\u27s disease (AD) is a neurodegenerative disease that is clinically characterized by progressive cognitive decline. More than 200 pathogenic mutations have been identified in amyloid-β precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2). Additionally, common and rare variants occur within APP, PSEN1, and PSEN2 that may be risk factors, protective factors, or benign, non-pathogenic polymorphisms. Yet, to date, no single study has carefully examined the effect of all of the variants of unknown significance reported in APP, PSEN1 and PSEN2 on Aβ isoform levels in vitro. In this study, we analyzed Aβ isoform levels by ELISA in a cell-based system in which each reported pathogenic and risk variant in APP, PSEN1, and PSEN2 was expressed individually. In order to classify variants for which limited family history data is available, we have implemented an algorithm for determining pathogenicity using available information from multiple domains, including genetic, bioinformatic, and in vitro analyses. We identified 90 variants of unknown significance and classified 19 as likely pathogenic mutations. We also propose that five variants are possibly protective. In defining a subset of these variants as pathogenic, individuals from these families may eligible to enroll in observational studies and clinical trials

Hippocampal neuroinflammation, functional connectivity, and depressive symptoms in multiple sclerosis

Depression, a condition commonly comorbid with multiple sclerosis (MS), is associated more generally with elevated inflammatory markers and hippocampal pathology. We hypothesized that neuroinflammation in the hippocampus is responsible for depression associated with MS. We characterized the relationship between depressive symptoms and hippocampal microglial activation in patients with MS using the 18-kDa translocator protein radioligand [18F]PBR111. To evaluate pathophysiologic mechanisms, we explored the relationships between hippocampal neuroinflammation, depressive symptoms, and hippocampal functional connectivities defined by resting-state functional magnetic resonance imaging. Methods The Beck Depression Inventory (BDI) was administered to 11 patients with MS and 22 healthy control subjects before scanning with positron emission tomography and functional magnetic resonance imaging. We tested for higher [18F]PBR111 uptake in the hippocampus of patients with MS relative to healthy control subjects and examined the correlations between [18F]PBR111 uptake, BDI scores, and hippocampal functional connectivities in the patients with MS. Results Patients with MS had an increased hippocampal [18F]PBR111 distribution volume ratio relative to healthy control subjects (p = .024), and the hippocampal distribution volume ratio was strongly correlated with the BDI score in patients with MS (r = .86, p = .006). Hippocampal functional connectivities to the subgenual cingulate and prefrontal and parietal regions correlated with BDI scores and [18F]PBR111 distribution volume ratio. Conclusions Our results provide evidence that hippocampal microglial activation in MS impairs the brain functional connectivities in regions contributing to maintenance of a normal affective state. Our results suggest a rationale for the responsiveness of depression in some patients with MS to effective control of brain neuroinflammation. Our findings also lend support to further investigation of the role of inflammatory processes in the pathogenesis of depression more generally