Kynurenine 3-monooxygenase (KMO) and mitochondrial dynamics

We have played a central role in the establishment of kynurenine 3-monooxygenase (KMO) as a candidate therapeutic target in neurodegenerative disease. KMO is localized to the outer mitochondrial membrane and is a key regulatory enzyme in the kynurenine pathway of tryptophan degradation, which contains several neuromodulatory metabolites. KMO inhibition ameliorates disease henotypes in yeast, fruit fly, and mouse models of Huntington’s, Parkinson’s and Alzheimer’s disease. However, the cellular roles of this enzyme aside from kynurenine catalysis – as well as the physiological consequences of KMO inhibition - are poorly understood.
Indeed, it is not clear why KMO is localized to mitochondria, and if KMO plays a role in mitochondrial function. Provocatively, our initial work suggests that fruit flies lacking KMO exhibit impaired mitochondrial function and morphology. KMO-deficient fruit flies and mammalian cell models will be employed to address the role of KMO in mitochondria via the following aims:

1) Mitophagy and mitochondrial dynamics: Morphological studies will be performed via confocal microscopy using mitochondrial markers to test if KMO modulates mitochondrial morphology or total mitochondrial mass in mammalian cells. Parallel studies will be conducted using electron microscopy to ascertain mitochondrial ultrastructure. Key players in mitochondrial fission, fusion, biogenesis and mitophagy will be analysed and modulated in Kmo-deficient cells and flies. Fluorescent protein (e.g. GFP) tagging of endogenous KMO via CRISPR gene editing will be performed in mammalian cells and flies in order to monitor KMO association with mitochondria under the various experimental paradigms. The dependence (or not) of any observed effects on kynurenine pathway metabolism will also be explored via measurement and treatment/feeding of key metabolites.

2) Mitochondrial function: To correlate morphological data with mitochondrial function, high-resolution respirometry will be performed in KMO knockout mammalian cells generated using CRISPR gene editing. Mitochondrial membrane potential, ATP levels, and reactive oxygen species will also be determined. As KMO expression is cell-type specific in mammals, modulation of KMO activity in different cell types will be performed to understand the functional relevance of KMO in this context.

3) KMO protein interactors: To gain a better understanding of KMO function, we will perform proteomic analyses. Protein interactors of KMO will be identified via mass spectrometry, and validated by co-immunoprecipitation. Candidate interactors will also be selected and tested by this approach, which will also permit mapping of key interaction domains, and the cellular location of these interactions.