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

Area of Science:

• Neuroscience
• Biochemistry
• Pharmacology

Background:

• Neurological disorders often involve inflammation and oxidative stress, leading to reduced glutathione (GSH) levels in the brain.
• Effective drug discovery for these conditions requires tools to monitor oxidative stress via cellular thiol reporters.

Purpose of the Study:

• To develop and validate a novel fluorescence visualization assay for detecting cellular thiol level changes.
• To assess the utility of the SemKur-IM probe in live neuronal cells for drug screening.

Main Methods:

• Developed a fluorescence assay using induced pluripotent stem cell (iPSC)-derived cortical glutamatergic neurons.
• Loaded neurons with the novel thiol-detection fluorescent probe, SemKur-IM (25 μM).
• Visualized changes in cellular thiol levels in response to N-acetyl-cysteine (NAC) using fluorescence microscopy.

Main Results:

• SemKur-IM successfully visualized changes in cellular thiol levels in neuronal somas and neurites.
• An increase in green fluorescence indicated changes in thiol levels, correlating with NAC exposure.
• The probe detected significant changes in thiol redox state, with minimal reduced thiols observed prior to NAC treatment.

Conclusions:

• The SemKur-IM probe is effective for detecting thiol levels in live cells and monitoring responses to chemical exposures.
• This assay is valuable for screening drugs that restore a reduced cellular state, relevant for neurological conditions.
• Potential applications include high-throughput screening of central nervous system (CNS) drugs impacting neuronal redox state, including Alzheimer's disease therapeutics like Posiphen.