A genetically-encoded sensor to detect active metabolites linked to the pathogenesis of neurodegenerative diseases

Overexpression of the kynurenine pathway is implicated in Alzheimer’s disease and Huntington’s disease, due to the accumulation of the neurotoxic metabolites 3-hydroxykynurenine and quinolinic acid. Kynurenine sits at a pivotal point in the pathway, which can either branch off to generate the neuroprotectant kynurenic acid or proceed down the normal route via the generation of neurotoxic metabolites. An understanding of the spatiotemporal dynamics of the pathway and how it changes during disease is needed to develop novel therapeutics that halt the onset or progression of disease.

A genetically-encoded sensor to detect kynurenine in cells and monitor the real-time dynamics of kynurenine metabolism has been developed, comprising of a flavoenzyme that binds kynurenine, kynurenine 3-monooxygenase (KMO), fused to a blue-fluorescent protein, Azurite, as the reporter. Isothermal calorimetry was used to determine the dissociation constant of kynurenine from KMO, which was found to be 76.9 μM. Therefore, the sensor could determine concentrations of kynurenine between 10 μM and 1 mM. KMO reactions could be monitored in live cells by a ratiometric measurement of the fluorescence emission lifetime from the sensor and NADPH (a cofactor for the KMO-catalysed reaction). Separation of the decay components of KMO-Az and NADPH allowed us to determine kynurenine concentration relative to NADPH concentration. Multiphoton fluorescence lifetime imaging (FLIM) was validated as an alternative technique for imaging the sensor in bacterial cells expressing KMO-Az. Additionally, the sensor was transfected in HEK293T cells prior to live-cell FLIM experiments.

Novel cellular interactions of KMO were discovered by co-immunoprecipitating proteins that physically interact with KMO in HEK293T cells. Functional analysis revealed that KMO could play roles in the mitochondria, mitosis and microtubule-based processes. Several bona fide disease-causing (such as Huntington’s, Parkinson’s and Alzheimer’s disease) proteins were found to interact with KMO, further implicating KMO in the pathogenesis of neurodegenerative disease.