Unravelling the interplay between tau and the kynurenine pathway in neurodegenerative disease

Hypothesis: Altered tau expression leads to dysregulation of kynurenine pathway metabolism which in turns exacerbates cellular toxicity
Experimental Methods and Research Plan: To address this question in vivo we will employ a fly model of tauopathy which exploits the UAS/GAL4 bipartite expression system to drive expression of either wild-type tau or R406W mutant tau (linked to an autosomal dominant form of frontotemporal dementia) using a panneuronal driver (elavGAL4) [7]. This model recapitulates many phenotypes relevant to Alzheimer's disease and tauopathies, including neurodegeneration, aggregation/abnormal phosphorylation of tau, shortened lifespan, and altered mitochondrial dynamics. We have performed initial experiments with these flies in our laboratory and observed several phenotypes in line with the published work (Marti and Giorgini, unpublished). We will use transgenic RNAi lines to test the effects of KMO knockdown, as well as knockdown of TDO - the initial step in KP metabolism, on tau-dependent phenotypes in flies. In parallel, pharmacological inhibition of these enzymes will be conducted using inhibitors previously employed in our laboratory. Neuroactive KP metabolites will be fed to the flies to directly test causation and modulation of phenotypes. Several metrics will be employed to ascertain modulation of tau-dependent pathology, including lifespan analysis, assaying of neuronal apoptosis via TUNEL staining of nuclear DNA fragmentation and measurement of ROS in whole mount brains using the oxidation-sensitive probe DHE. We will monitor abnormal tau conformations/phosphorylation using appropriate antibodies (Alz50 or MC1 - abnormal tau conformations; AT8, AT100, or AT180 - phosphorylated tau in disease state), previously employed in this fly model [8]. Tau expression has been found to promote mitochondrial elongation in neurons of the adult fly brain via mislocalization of DRP, which offsets a critical balance in mitochondrial fission and fusion [7]. KMO may play a role in mitochondrial dynamics (Maddison and Giorgini, unpublished) and we thus propose to also study the effects of KP manipulation on mitochondrial length in Kenyon neurons of tau flies by confocal microscopy using co-expressed mitochondrially localized GFP (mitoGFP). Notably, we will measure a panel of key KP metabolites and neurotransmitters (via CRO BrainsOnline), which will allow correlation of the observed phenotypes with metabolite levels, and also ascertain effects on downstream neurotransmission. We have a great deal of experience implementing fly models of neurodegeneration providing a strong foundation for the proposed work.