Sunday, May 18, 2025
5/18/2025
Analytical Tools for Light-Initiated Zn2+ Signaling in Neurodegenerative Disease - PROJECT SUMMARY Overwhelming evidence has implicated oxidative stress and zinc dysregulation as mechanisms that underlie neurodegeneration, especially in Alzheimer’s disease (AD) and Parkinson’s disease (PD). In its free, ionic state, Zn2+ undergoes co-release with glutamate in selected brain regions and interacts with conserved binding sites on regulatory proteins, such as transporters, receptors, and intracellular signaling proteins. These interactions directly influence the release and uptake of multiple neurotransmitters, including dopamine (DA), a catecholamine that plays important roles in learning and memory. However, chemical modification of amino acid residues, including those involved in Zn2+ binding, occur in AD and PD due to formation of reactive oxygen species (ROS). These modifications hamper Zn2+’s ability to modulate the function of proteins that control the release and uptake of DA and other signaling molecules. The resulting imbalances in neurotransmitter levels can impair neuronal function, compromise neuronal health, and impede cognitive processes. Unfortunately, studies of how Zn2+ release and ROS production interact to regulate sub-s neurotransmitter signaling have been neglected because the analytical methodology is underdeveloped, resulting in knowledge gaps that hinder the development of therapeutic strategies aimed at normalizing metal signaling. Thus, there is a critical need to identify and quantify these functional and structural changes in neurodegenerative disease. Our overarching goal is to develop analytical approaches to determine how Zn2+’s ability to modulate DA release and uptake on the sub-s timescale is affected in AD and PD. Our central hypothesis is that oxidative modification of histidine residues at binding sites impede Zn2+’s regulatory function and is a common mechanism in AD and PD model organisms. We have previously combined fast-scan cyclic voltammetry (FSCV) with light-induced release of Zn2+ from custom-designed, photoreactive cages, and showed that treatment of zebrafish with rotenone impeded the inhibitory effect of Zn2+ over DA uptake, yet potentiated this effect over release. We will further develop our FSCV/photorelease technique to determine how disease state alters Zn2+ signaling in genetic models of AD and PD. Targeted proteomic analyses of brains will then identify sites of oxidative modifications on specific amino acids involved in Zn2+ binding. Our specific aims, which are complementary, yet independent, are: 1) Determine how extracellular Zn2+ transients affect DA release and uptake with sub-s and micron-scale precision, 2) Determine how alterations in intracellular Zn2+ levels influence DA release/uptake and ROS generation, and 3) Correlate specific protein amino acid modifications with functional alterations of Zn2+ on DA release/uptake. Importantly, our approach is adaptable to measuring how other biologically active metals, such as Fe3+, Cu2+, and Mn2+, affect other electroactive signaling molecules, such as norepinephrine and histamine, with high spatiotemporal resolution. Thus, there is the promise that researchers can identify specific metal-binding sites that have been modified in AD, PD, and other diseases, and design selective drug therapies to target them.
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