ABSTRACT The rhesus monkey shows age-related cognitive decline similar to humans. Surprisingly both microscopic and MRI studies show that neurons and gray matter are not lost in the aging monkey brain. Instead, there is a progressive loss of white matter and accumulation of myelin damage that both predict age-related cognitive decline. Yet it is unknown whether myelin damage results from intrinsic changes in myelin-producing oligodendroglia or from interactions with microglia that normally clear myelin debris and facilitate remyelination. Data published by us and others show that microglia in the white matter become more inflammatory and phagocytically active with age, but lose phagocytic efficacy. These data correlate with myelin damage and cognitive decline. Here we propose experiments to test the hypothesis that functional changes in microglia impair oligodendroglial function and underlie age-related myelin pathology and cognitive impairment. The experiments will use tissue from the prefrontal cortex of 25 cognitively tested monkeys of both sexes that cover the adult life span from 5 years old to nearly 30 years old (human equivalent of 15 ? 90). This will be supplemented with archived and cryopreserved tissue from 50 monkeys behaviorally tested over the previous decade, providing unprecedented statistical power. In all cases, after cognitive testing, fresh brain tissue is harvested for use in novel in vitro and ex vivo assays while and the remainder of the brain is fixed for histopathological analyses. Studies proposed here will evaluate mechanisms underlying age-related changes in the role of oligodendroglia and microglia in the development of age-related myelin pathology according to 3 aims. In Aim 1, we will use primary cultures to assess OPC differentiation, immunohistochemistry (IHC) to quantify the ratio of OPCs to mature myelinating oligodendrocytes (OL) and spectral confocal reflectance (SCoRe) microscopy to quantify percent myelinated fibers and internode length. Functional effects of myelin pathology on the action potential will be assessed with in vitro slice electrophysiology followed by electron microscopy to validate the SCoRe measures. This will test the hypothesis that there is age-related impairment in the capacity of OPCs to differentiate into OLs leading to impaired myelin sheath maintenance. In Aim 2, we will use an ex vivo slice culture to assess phagocytic capacity by challenging microglia with myelin debris from cognitively impaired aged subjects and we will use IHC to assess microglial phagocytosis using markers for lysosomes and phagocytosed myelin. This will test the hypothesis that phagocytic functionality is impaired in aging as myelin debris is less efficiently cleared, which correlates with impaired OPC/OL differentiation and myelin sheath maintenance. In Aim 3, we will examine gene expression changes in the frontal white matter to identify transcriptional changes associated with myelin pathology and follow this up with cell-specific PCR to quantify transcriptional changes in microglia and oligodendroglia. Impact: Together, these aims will provide unique and novel mechanistic insights into of the role of oligodendroglia and microglia in age-related myelin pathology and cognitive impairment.