PROJECT SUMMARY In the aged brain, reduced brain size and altered synaptic function are observed in conjunction with motor and cognitive decline. Despite the importance of identifying what underlies these changes with aging, the molecular drivers of brain aging and synaptic decline are not well characterized. Recently a role for non-neuronal glial cells, i.e., astrocytes, microglia, and oligodendrocytes, has been proposed in typical aging. Astrocytes are 15% of brain cells and provide metabolic support for neurons, perform neurotransmitter recycling, and directly modulate neuronal synapses in the developing and adult brain. Studies show mouse astrocytes undergo regionally-specific molecular alterations with aging, with unique transcriptional alterations in astrocytes of the cortex, hippocampus, striatum, hypothalamus, and cerebellum. In particular, astrocytes of the cerebellum show greater transcriptional dysregulation than those in cortex, including upregulation of immune system genes in the interferon pathway. Prior work identified loss of cerebellar neurons in aging, raising the question of whether regionally specialized changes to cerebellar astrocytes that occur with aging contribute to this neuronal decline. To address this question RNA sequencing of astrocytes from mouse and human, adult and aged, cerebellum was performed, identifying Stat1 as a candidate transcriptional regulator of the heightened interferon state of cerebellar astrocytes in the aged brain. Upregulation of Stat1 occurs concomitant with reduced numbers of excitatory synapses in cerebellum, and mouse behavioral alterations that are linked to cerebellar function. In Aim 1 experiments are performed to ask if Stat1 is responsible for driving the aged astrocyte signature in the cerebellum by: decreasing expression of Stat1 in astrocytes in aged mice using viral delivery of the RNA degrading enzyme CasRx to ask if this is sufficient to revert cerebellar astrocyte aging signatures, and rescue synaptic and behavioral deficits that are present; knocking-out Stat1 from adult astrocytes to ask if this is sufficient to prevent the induction of aging-related alterations in cerebellar astrocytes; over-expressing Stat1 in cerebellar astrocytes in juvenile mice to ask if this is sufficient to drive aging-related transcriptional signatures and motor decline. In Aim 2 experiments address the cues that drive upregulation of Stat1 and the genes it regulates in aged cerebellar astrocytes, focusing on signaling through the interferon receptor in astrocytes, and asking if the source of interferon originates within the brain or in the periphery. These experiments will provide mechanistic insight into the transcriptional regulation of aging-associated astrocyte states and reveal their contribution to synaptic and behavioral decline in the aging brain.