PROJECT SUMMARY Alzheimer's Disease (AD) is the primary cause of progressive dementia associated with cognitive decline, neurodegeneration, and neurocircuit dysfunction. Results from recent attempts to address classical amyloid- beta (Aβ) deposition and hyperphosphorylated tau (pTau) deposits have been limited in providing significant clinical benefits. As a result, researchers are now exploring alternative disease mechanisms that contribute to the pathogenesis of AD. One such mechanism is the loss of perineuronal nets (PNNs), specialized extracellular matrix (ECM) structures that regulate the activity of key neurons involved in memory and cognition. PNNs are significantly reduced in the brains of AD patients and thus represent a novel target for therapeutic investigation. PNN matrices consist of chondroitin and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs) that are uniquely modified with sulfate attachments linked to specific biological functions within the brain. Within the mono-sulfated class, the 6S-CS isomer has been shown to directly destabilize PNN matrices and induce neurocircuit re- organization. Moreover, we recently showed increased cortical 6S-CS expression occurs prior to AD clinicopathology, which is then worsened in association with advanced stages of AD and pTau accumulation. These results suggest that favoring the 6S-CS isomer in PNN CS/DS-GAG composition may destabilize PNN formation in AD brain tissue, leading to reorganization of the underlying circuitry and causing deficits in learning, memory, and cognition while potentially worsening the neurodegenerative process. To investigate the functional impact of increased 6S-CS expression in PNNs surrounding GABAergic neurons, we first induced GABAergic neuronal expression of the 6S-CS isomer using an AAV1-mChst3 (6S- sulfotransferase) in vgat-Cre mice. Our Preliminary results show that GABAergic overexpression of the 6S-CS isomer reduced stable PNN labeling but also unexpectedly increased neuroinflammatory markers, including those for astrogliosis (GFAP) and microgliosis (Iba1). These preliminary results indicate a potential novel role for 6S-CS in driving neuroinflammation associated with the AD pathogenesis. Specific Aim 1 highlighted within this proposal focuses on determining the cell-specific effects of 6S-CS overexpression on neuroinflammation and pTau accumulation using different neuronal, glial, and tauopathy Cre-mouse models. Meanwhile, Specific Aim 2 proposes to utilize MALDI imaging mass spectrometry to spatially resolve CS/DS isomer patterns in human AD brain tissue in association with cellular and pathogenic neuropathology in AD and non-AD human brain tissue. Overall, this fellowship proposal seeks to understand the role of 6S-CS overexpression in neuroinflammation and AD neuropathology accumulation using innovative viral and imaging mass spectrometry techniques, the results of which will contribute to a better understanding of how CS/DS-GAGs drive the AD pathogene...