Optogenetic dissection of EPHA1 missense mutations implicated in alzheimer's disease

The National Institute on Aging awarded East Carolina University $512,882 under the Aging Research program (CFDA 93.866) on September 1, 2026, to conduct optogenetic dissection of EPHA1 missense mutations implicated in Alzheimer's disease.

The research employs an optogenetic strategy to precisely control EPHA1 receptor tyrosine kinase activation in live cells, enabling rigorous investigation of how Alzheimer's-associated mutations (R791H, H888Y, and R926C) affect neuronal signaling and function. In the first aim, the team will engineer optogenetic EPHA1 constructs carrying each mutation and validate their biochemical activity and downstream signaling in HEK293 cells. The second aim expresses these constructs in mouse dissociated neuron cultures and human iPSC-derived neurons to examine how mutant EPHA1 alters canonical signaling pathways (MAPK/ERK, PI3K/AKT, and GSK3B) and to measure effects on neuronal morphology, synaptic density, and cell viability. The third aim uses multi-electrode array recordings to determine how mutant EPHA1 impacts network activity and synaptic function, and tests whether GSK3B inhibitors can reverse functional deficits caused by hyperactive signaling.

Performance occurs in Greenville, North Carolina, with project period from September 1, 2026, through August 31, 2029. This is a Project Grant, classified as assistance funding to a research institution.

Abstract

Alzheimer's disease (ad) is the leading cause of dementia, yet many of the molecular mechanisms linking genetic risk variants to neuronal dysfunction remain poorly understood. Recent genome-wide association studies have implicated EPHA1, a receptor tyrosine kinase, as a significant ad risk gene, and patient-derived sequencing has identified rare missense mutations (R791H, H888Y, and R926C) within and adjacent to its kinase domain. Preliminary studies suggest that these mutations alter receptor activity, trafficking, and downstream signaling, but the cellular consequences of these changes in neurons are unknown. This proposal uses an innovative optogenetic strategy to precisely control EPHA1 receptor tyrosine kinase activation in live cells, enabling a rigorous dissection of signaling and functional outcomes of ad-associated mutations. In aim 1, we will engineer optogenetic EPHA1 constructs carrying R791H, H888Y, or R926C mutations and validate their biochemical activity and downstream signaling in HEK293 cells. In aim 2, we will express these constructs in mouse dissociated neuron cultures and human ipsc-derived neurons to examine how mutant EPHA1 alters canonical signaling pathways (mapk/ERK, PI3K/AKT, and GSK3Β) and to measure effects on neuronal morphology, synaptic density, and cell viability. In aim 3, we will use multi-electrode array recordings to determine how mutant EPHA1 impacts network activity and synaptic function, and we will test whether GSK3Β inhibitors can reverse the functional deficits caused by hyperactive signaling. The expected outcome of this project is a mechanistic understanding of how distinct EPHA1 mutations-loss- of-function (R791H), gain-of-function (R926C), and potentially neutral (H888Y)-impact synaptic dysfunction and lead to neuronal network activity. These studies are significant because they address a major GAP in our understanding of how genetic risk factors contribute to ad pathogenesis. They are innovative because they employ optogenetics to achieve unprecedented temporal control of EPHA1 RTK signaling in neurons. By clarifying the mechanistic consequences of EPHA1 mutations, this work has the potential to identify novel therapeutic strategies targeting receptor signaling pathways in ad.