PROJECT SUMMARY Alzheimer’s disease (AD), a progressive neurodegenerative disease causing memory loss, is the primary cause of dementia. Recent FDA approval of an amyloid beta-targeting medicine represents a significant advance in AD treatment, yet effective therapies for tau pathology remain crucial. Tau accumulation and neurofibrillary tangles are central to AD pathology and strongly correlate with cognitive decline and neuronal loss1. Therefore, strategies that seek to interrupt tau deposition and propagation may be effective treatments. Regions associated with tau accumulation2 in postmortem AD brains show reduced proteasome activity, suggesting impaired protein degradation may contribute to tau pathology. Thus, increasing proteasome activity to degrade tau may be an efficient therapy to reduce tau burden and neurodegeneration. The ubiquitin-proteasome system is negatively regulated, in part, by enzymes that remove the ubiquitin side chains from proteins targeted to the proteasome. Among these enzymes, the ubiquitin-specific protease 14 (Usp14) selectively acts on substrates, including tau, to reduce or prevent their degradation. This proposal tests an antisense oligonucleotide (ASO)-mediated approach to reduce Usp14 in the central nervous system, increasing proteasomal degradation of tau. ASOs are short, single-stranded DNA that bind to target mRNA to decrease or modify the protein expression through the recruitment of RNAse H. In recent years, ASOs have become valuable tools for understanding and treating neurodegenerative diseases, and improved chemistries and delivery methods have made ASOs safe and effective in multiple clinical trials3,4. Preliminary data show that Usp14-lowering ASO treatment activates the proteasome system and decreases total tau in neurons and tau transgenic mice. Using this effective tool, this proposal will assess the effects of proteasome activation on tau pathology and tau seeding in a mouse model of tau-mediated neurodegeneration. Aim 1 will test the effects of proteasomal activation via Usp14 lowering on tau pathology and gliosis in mutant P301S tau transgenic mice (PS19). I will intraventricularly administer Usp14- lowering ASOs to PS19 mice at early (3 months), mid (6 months), and late (9 months) stage pathology to evaluate neurofibrillary tangle formation, neuronal loss, gliosis, and potential off-target effects using immunohistochemistry, mass spectrometry, and ELISA assays. Aim 2 will investigate proteasomal activation's role in preventing tau seeding in vitro and its propagation in vivo. In vitro, primary neurons will be treated with GFP-labeled bioactive tau seeds, followed by Usp14-lowering ASO to activate the proteasome system. I will assess amount of GFP-labeled tau seeds and total aggregated tau to evaluate seeding efficiency. In vivo, wildtype mice will be injected with tau seeds and subsequently treated with Usp14-lowering ASO. The extent of tau propagation will be quantified by assessing the sprea...