The Alzheimer’s Genome Project™ (AGP) is aimed at analyzing our large Alzheimer’s disease (AD) genetics database consisting of our own, collaborative and all publicly available AD genome-wide association study (GWAS), whole genome sequence (WGS) and whole exome sequence (WES) data. In the AGP, we use these datasets along with a series of unique algorithms to identify genes harboring common and rare genomic variants associated with AD. We currently analyze WGS data from 2,247 subjects from 605 multiplex AD families, and then follow up with replication analyses in WGS from an independent case-control cohort composed of more than 1,650 individuals. To our knowledge, we are analyzing the largest amount of AD WGS data in the world today. For AD-associated genomic variants predicted to have functional consequences, we will analyze them in our various 3D cell culture models (Alzheimer’s in a Dish™). The most promising AD-associated functional variants also are shared with the Cure Alzheimer’s Fund Genes to Therapies™) (G2T) Committee, and any resulting mouse models then are made available to all Cure Alzheimer’s Fund investigators and the greater AD research community. As in the past, we also will continue to share our genetic data regarding AD-associated functional variants with a growing number of Cure Alzheimer’s Fund investigators.
The overarching goal of the Alzheimer’s Genome Project™ is two-pronged; first, to analyze an extensive Alzheimer’s disease genetics database consisting of approximately 1.5 petabytes of whole genome sequence (WGS) and whole exome (WES) data from family-based samples and other currently available AD samples to identify and functionally validate novel AD genes and AD-associated functional mutations and single nucleotide variants that are either common or rare; and second, to functionally validate and characterize their effects on multiple aspects of AD pathology in our 3D stem cell-derived neural glial culture models as well as AD mouse models, either in the Tanzi lab or other Cure Alzheimer’s Fund (CureAlz)-funded labs participating in the Genes to Therapies™ program. Induced pluripotent stem cell progenitors of either neurons or glia are generated, using CRISPR-Cas9 gene editing, that carry the identified potentially detrimental and protective mutations and SNV in AD-associated genes, which allows the observation and analysis of any differences in amyloid beta production, oligomerization and deposition, tau and neurofibrillary tangle formation, neuroinflammation (microglial activation and astrogliosis), and blood-brain barrier integrity from control iPSC-derived neurons or glia. These experiments employ the original 3D neural culture model (Choi et al., 2014), 3D neural-glial tri-culture system (Park et al., 2018) and new combined 3D neural-glial/blood-brain barrier model (Shin et al., 2019) developed in past years of this grant and in the CureAlz 3D Drug Screening Consortium’s efforts. We also test for effects on neural/synaptic activity using calcium imaging with GCaMP6 (courtesy of Dr. Clifford Wolfe, Harvard University). High priority will be given to gene variants identified through prior sex-specific genome-wide association studies investigation.
We now will focus on a major new effort of the AGP, testing and functionally validating potentially detrimental or protective mutations/SNVs in the 30 known AD-associated GWAS genes. The extensive collection of AD WGS and WES datasets, together with the 3D human tri-culture AD model, will be used to evaluate the pathogenic effects of functional variants in AD-associated innate immune genes linked to neuroinflammation. Notably, the Alzheimer’s in a Dish model and each of its more sophisticated iterations now are used in iPSC-derived neurons and glia from both male and female control and AD donors; this is always important, but particularly so given observed and as-yet unexplained sex-based differences in microglial behavior. The overarching goal is to comprehensively assess the pathogenic effects of functional variants in innate immune AD-risk genes on AD pathogenesis and explore underlying molecular networks in order to identify novel therapeutic targets. The Tanzi lab thus will test its hypothesis that microglia-related AD functional variants lead to reduced levels of microglial recruitment to AD pathology, that other AD functional variants reduce amyloid beta clearance and amyloid beta-reactive microgliosis that should cause microglial clustering around plaques and amyloid beta removal, and that microglial triggering of astrocyte transformation into “A1” states lead to chemical signaling that then leads to neuronal death. Pursuing these hypotheses will involve collaborations with other CureAlz-funded labs, particularly members of the 3DDS, CIRCUITS and Gifford Neuroinflammation Consortia.
The goal of this project is to evaluate our new Alzheimer’s disease gene candidates for effects on Alzheimer’s pathology and related biological pathways, including amyloid precursor protein processing, amyloid beta protein generation, tangle formation and cell death. These studies are being carried out as part of Phase II of the Alzheimer’s Genome Project™ and entail functional analyses of the Alzheimer’s gene candidates identified in phase I of the AGP. We have focused the Phase II studies on the novel Alzheimer’s genes known as ADAM10, ATXN1, and CD33, all identified in 2008 as part of Phase I of the AGP.
The functional studies, aimed at how these genes influence risk for Alzheimer’s, are carried out in both cell-based and animal models. We also have performed genetic follow-up and functional studies for AD-associated aberrations in the human genome, known as copy number variants (CNV). This has led to the identification of several CNVs in novel Alzheimer’s genes underlying the inheritance of cases of familial early-onset Alzheimer’s that were not explained by the known early-onset Alzheimer’s genes co-discovered by our lab in the 1980s and ’90s (amyloid precursor protein, presenilin 1 and presenilin 2).
The knowledge gained from how the newly identified Alzheimer’s genes (from Phase I) biologically increase or decrease risk for Alzheimer’s disease is being implemented to design new drug discovery efforts, also as part of Phase II of the AGP. Phase III of the AGP is being carried out parallel to Phase II and includes Whole Genome Sequencing of the human genomes of subjects from both early-onset and late-onset Alzheimer’s families. The goal of Phase III of the AGP is to identify all of the biologically relevant functional gene variants that influence risk for Alzheimer’s disease. Once identified, these gene variants will be analyzed using similar methods to those described here in Phase II of the AGP.