Project Summary/Abstract Two-pore domain K+ channels (K2P) facilitate background leak K+ currents that regulate resting membrane potential. Fifteen K2P family members have been described in mammals; only two appear on the “Illuminating the Druggable Genome” list of understudied proteins, including KCNK12. As a fortuitous outcome of random mutagenesis screening in the roundworm Caenorhabditis elegans, we recently identified the C. elegans homolog of KCNK12, termed TWK-14, in a forward genetic screen for effectors of the mitochondrial unfolded protein response (UPRmt). Our group previously demonstrated how the UPRmt, which is restorative following acute stressors such as toxins, becomes dysregulated when experiencing chronic activation. Specifically, we showed that the intrinsically disordered protein, a-synuclein (a-syn), a primary pathologic factor in Parkinson’s disease (PD), chronically activates the UPRmt, resulting in the progressive neurodegeneration of C. elegans dopamine neurons (Martinez et al., 2017). Our discovery of twk-14 as encoding an inherently neuroprotective suppressor of dopaminergic neurodegeneration, establishes a physiologically relevant foundation for further investigation of the unresolved function of KCNK12. Like KCNK12, twk-14 expression is limited to neurons. The defined connectome map of C. elegans localizes TWK-14 to select sensory neurons of the head, in a postsynaptic position within the dopaminergic circuitry. In this R03 proposal, we will explore KCNK12/TWK-14 function using dopaminergic neurodegeneration in C. elegans as a phenotypic readout. In Aim 1, neuron- specific transgenic expression of KCNK12, in combination with a systematic genetic analysis of 30 candidate interactors of KCNK12/TWK-14 identified by database mining of Pharos and related resources, will be conducted to uncover modifiers of KCNK12-associated protection of C. elegans dopamine neurons. Outcomes of this analysis will illuminate a cell biological role(s) for this K2P, in vivo. In Aim 2, we will refine understanding of the newfound modulatory role of KCNK12/TWK-14 within the defined C. elegans dopaminergic circuitry. We explore a hypothesis whereby the loss of K2P channel regulation in the postsynaptic neurons of twk-14 mutants disrupts an inherently neuroprotective synaptic feedback loop required for proper neuropeptide signaling in the maintanence of dopamine homeostasis. Here we will parse the relative impact of putative components of this proposed mechanism through rigorous quantification of neurodegeneration, at the single neuron level, in isogenic populations of transgenic animals with diverse mutant backgrounds. In addition to the provisioning of new transgenic animals, vectors and mutant strains as deliverables, the broader impacts of this 1-year project have the potential to inform downstream research into KP2-associated drug discovery and therapeutic strategies for neurodegenerative diseases including PD or disorders where mitochondrial stress intersects with aberrant neurotransmission. Our approach exemplifies the utility of research with intact invertebrate models for expediting ascription of functions to understudied proteins.

Project Narrative KCNK12 is an understudied ion channel on the Illuminating the Druggable Genome list that we have identified as neuroprotective in an animal model of neurodegeneration associated with Parkinson’s disease. This movement disorder affects over 1 million Americans, yet therapies have largely eluded medical science. The purpose of this proposal is to functionally evaluate mechanisms associated with the neuroprotective role of KCNK12 using genetics and a whole animal model system.