ABSTRACT Our long-term goal is to identify effective therapeutic targets for preventing neurodegeneration in Alzheimer's Disease (AD). In this proposal, we investigate a novel mechanism of AD pathogenesis following our discovery of HYPE as a central player in AD pathogenesis. HYPE is a newly discovered enzyme, which catalyzes both AMPylation and DeAMPylation. HYPE is highly expressed in the brain, specifically in areas susceptible to neurodegeneration in AD. HYPE and AMPylation have not yet been analyzed in AD. We uncovered a Jekyll and Hyde type scenario for HYPE in neuronal health and disease. HYPE is a neuroprotective enzyme that maintains neuronal homeostasis by upregulating several neuroprotective pathways. However, in AD, the balance between its AMPylase and DeAMPylase activities is perturbed, transforming HYPE into a highly neurotoxic enzyme. This switch in HYPE's function occurs due to its mislocalization from the ER to the cytoplasm under neurotoxic conditions, bringing it close to Cdk5, which drives HYPE's deregulation. Accordingly, HYPE's AMPylase activity exhibits strong correlation with disease progression in clinical specimens. Equally important is the finding that HYPE is the causative factor driving Cdk5 deregulation upon neurotoxic insults. Cdk5 deregulation is well established in AD and contributes extensively to AD pathogenesis. Thus, HYPE employs a two-pronged approach to promote neurotoxicity: indirectly by deregulating Cdk5; directly by aberrant AMPylation. Our central hypothesis is that neuroprotective HYPE turns neurotoxic in AD by engaging in a positive feedback loop with Cdk5 and thereby contributing heavily to disease pathogenesis. We will test this hypothesis using the following aims: 1: Dissect the regulatory mechanisms that transform neuroprotective HYPE into neurotoxic in primary neurons. 2: Dissect the molecular mechanisms by which HYPE promotes neuroprotection in healthy neurons and neurodegenerative pathways in diseased neurons. Aim 3: Determine the temporal correlation between HYPE's deregulation with various disease phenotypes in AD mouse models and clinical tissues. Innovation: The proposal is based on our discovery of HYPE as a critical player and AMPylation as a key mechanism that regulate AD pathogenesis. HYPE was identified as a novel Cdk5 substrate using an innovative chemical genetic approach. These studies revealed a unique mechanism that leads to intense Cdk5 deregulation. We have identified several direct targets of HYPE, which will be used to unravel the molecular mechanisms by which HYPE promotes various physiological and pathological events in neurons and in vivo. In addition, several innovative cellular tools have been developed that will be used in the proposed studies. Significance: Identification of HYPE as a potential target and its interplay with Cdk5 unlocks a powerful mechanism for therapeutic targeting of this highly debilitating disease. Analysis of AMPylation of specific targets, coupled...