ABSTRACT Changes in the sialic acid content of neural glycoproteins have been reported in neurodegenerative and neuroinflammatory diseases, including Alzheimer’s disease (AD). However, how these changes govern neuropathogenic processes remains poorly understood. The central hypothesis of these studies is that impaired de-sialylation affects the physiological and functional properties of specific sialo-glycoproteins in neurons and microglia, initiating a pathogenic cascade leading to neurodegeneration and neuroinflammation. We will test this hypothesis from the angle of the sialic acid-cleaving enzyme NEU1, a lysosomal sialidase, using a mouse model of the lysosomal storage disease, sialidosis, as an experimental tool. We have shown that Neu1–/– mice develop a hippocampal neuropathology with features of an amyloidosis, including accumulation of an oversialylated APP, a substrate of NEU1, and widespread neuroinflammation. Ablating NEU1 expression in a canonical model of AD (5xFAD) leads to exacerbation of the amyloidosis phenotype, while increasing NEU1 expression, via a gene therapy approach, reduces plaque formation. These findings implicate NEU1 as the potential underpinning cause, although the mechanisms linking the cause to the effects still need to be deciphered. The studies proposed in this application are directed to closing this knowledge gap, because of these previous observations and new compelling preliminary data. Besides APP, we have identified additional potential substrates of NEU1, including those that participate directly in the amyloidogenic process (BACE1 and nicastrin) and those that are responsible for neuroinflammatory processes, such as microglial phagocytosis (CD68 and TREM2). We will explore the idea that by controlling the levels of sialic acids on these specific AD-related glycoproteins in neurons (Aim 1) and microglia (Aim 2), NEU1 functions as a crucial regulator of key biological processes that maintain brain homeostasis. In Aim 1, we will examine the effects of altered sialylation on APP, and its sialylated proteolytic enzymes, BACE1 and nicastrin, in Neu1–/– mice and in established AD mouse models crossed into the Neu1–/– background. We will use a combination of subcellular fractionation methods, immunofluorescent microscopy, and high-resolution 3D imaging to determine alterations in intracellular distribution, processing and turnover rate of these glycoproteins. In Aim2, we will study how impaired de-sialylation of microglial receptors and signaling proteins affects their subcellular localization, turnover rate, and the cellular functions they are known to control. To focus our studies exclusively on microglia we will cross the above-mentioned mice with a mouse model that expresses GFP almost solely in microglia. Overall the experimental design of this grant application will enable us to elucidate mechanisms of pathogenesis leading to neuronal and microglial dysfunction with direct translational implications for pa...