Real-time thiol detection in iPSC-derived neuron cultures using SemKur-IM, a novel fluorescent dithio probe

Abstract

Neurological disorders associated with inflammation and oxidative stress show reduced glutathione (GSH) levels in the human brain. Drug discovery efforts and pharmacological studies would benefit from tools (e.g. chemical probes) that detect changes to oxidative stress, from the perspective of physiologically-relevant reporters like cellular thiols, including GSH. To this end, we have developed a fluorescence visualization assay using iPSC-derived cortical glutamatergic neurons that were loaded with 25 μM of a novel thiol-detection fluorescent probe, SemKur-IM. This probe enables visualization of cellular thiol level changes in the neuronal somas and neurites, in response exposure to N-acetyl-cysteine (NAC). Cellular thiol redox state was observed to change, based on an increase in green fluorescence (485 nm excitation maximum; 525 nm emission maximum) due to changes in thiol levels, from 0 to 40 mM. Interestingly, prior to treatment with NAC, cells did not appear to have significant levels of reduced thiols. Our studies demonstrate the utility of SemKur-IM in the detection of thiol levels in live cells in response to chemical exposures, such as from drugs that return the cell to a healthier reduced state. An initial application to screening the effects of an Alzheimer's disease drug candidate, Posiphen, using fluorescence cell sorting is presented. Other potential applications include high throughput screening of central nervous system (CNS) drugs thought to work by affecting cellular redox state in neurons.