Abstract Glaucoma is a neurodegenerative disease characterized by loss of retinal ganglion cells (RGCs) and their axons. Damage from glaucoma is permanent and may cause irreversible blindness. Glaucoma is a major public health problem in the United States and worldwide, where it is the leading cause of irreversible blindness. Four classic risk factors have been identified for the most common type of glaucoma, primary open angle glaucoma (POAG): increasing age, race / ethnicity, family history, and increased intraocular pressure (IOP). Currently, all treatments for glaucoma slow or halt disease by targeting one risk factor, increased IOP. However, IOP-lowering therapies fail to prevent vision loss in many patients. Thus, glaucoma patients desperately need new, more effective therapies that target other aspects of glaucoma beyond elevated IOP, such as the mechanisms underlying family history of glaucoma. Unfortunately, most genetic risk associated with glaucoma remains poorly understood. Most cases of POAG have a complex genetic basis and involve many risk factors. To date, >127 risk factor loci have been identified with genome-wide association studies (GWAS's). In each of these loci, several single nucleotide polymorphisms (SNPs), have been associated with risk for POAG and the effect of these SNPs on the nearest gene has been presumed to be the source of risk. However, little progress has been made in precisely defining these loci, including the causative SNP(s), gene(s), or mechanism(s). Very few of the disease-associated SNPs the >127 glaucoma risk loci are in coding sequence. Consequently, we hypothesize that the functional SNPs in each locus confer glaucoma risk by increasing or decreasing transcript levels of effector genes in the locus. To test our hypothesis, we have prioritized three loci for detailed studies (chr 4p14, chr 21q21.3, and chr 17q21.3), which each containing an exemplar candidate for being the effector gene (APBB2, APP, MAPT), which are all expressed in RGCs and all share links with neurodegeneration associated with Alzheimer disease. To test whether these are indeed the effectors and study their mechanisms, we propose a complementary set of experiments using human, mouse, and molecular approaches. In Specific Aim 1 we will use genotyped human donor retinal tissue to identify how transcript and protein levels for the exemplar candidates are altered in association with the high-risk allele at each locus (using scRNAseq, IHC, and ELISA), as well as assess other transcriptomic changes across the locus (and genome). We will also use BiT-STARR-seq, to identify SNPs in each locus capable of changing transcription. In Specific Aim 2 we will use AAV2 constructs in mice to manipulate expression levels of the exemplar candidate genes and study how dysregulation of these genes influences RGC health. With completion of these experiments, we will advance understanding the basic biology of glaucoma risk factors identified by GWAS. We will...