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 with HYPE activation levels in AD mouse models and human clinical samples, will aid in the development
of novel tools for retrospective analysis to better understand AD pathogenesis.