Red and Far-Red Fluorescent Biosensors for Compartmentalized Redox Signaling

University Of Virginia, Charlottesville VA

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Abstract

ABSTRACT Alzheimer?s disease (AD) has become a global challenge but its exact cause is still unknown. Oxidative stress is a popular hypothesis in AD. Even in other AD hypotheses, oxidative stress still plays important roles in cascade events that can lead to neuron death. Because of the importance of oxidative stress in the etiology and pathogenesis of AD, research tools that can conveniently evaluate oxidative stress in AD models are expected to greatly catalyze and accelerate research on AD. Currently, researchers may use synthetic fluorescent indicators to image oxidative stress, but those small- molecule-based indicators often have limited specificity and it is difficult to extend their use into live animals. The objective of our parental R01GM129291 project is to develop red and far-red fluorescent indicators to image compartmentalized, thiol-based redox signaling. Our genetically encoded fluorescent redox indicators (GEFRIs) can monitor clearly defined redox processes. Moreover, by using viral vectors, cell-type-specific promotors, and organelle-targeting sequences, methods are well-established to express these genetically encoded indicators in particular subcellular compartments and cell types in the brains of live animals. In line with the parental project, we now request an administrative supplement with an objective to extend our fluorescent indicators to image redox signaling in live cells, tissues, and in vivo mouse models related to AD. We will validate GEFRIs first in AD cell models, and next in acute and organotypic AD brain slices, and finally in the brains of live AD mice. Moreover, we will integrate GEFRIs with a spectrally compatible fluorescent Ca2+ indicator (GCaMP6), electrophysiological patch-clamp recoding, and a chemogenetic hydrogen peroxide generator (DAAO) to explore the connections between oxidative stress and neuronal excitability and plasticity in AD brains. The proposed research will lead to a novel capability of optically recording (and chemogenetically manipulating) redox signaling in awake behaving animals, extending the in vivo measurement of specific neuronal activities beyond Ca2+, voltage, and a few neurotransmitters. We expect that these novel, powerful research tools will benefit the AD research community.