Functional analysis of KCNK12 in dopaminergic neuroprotection

项目概要/摘要 双孔结构域K+通道（K2 P）促进调节静息膜的背景漏K+电流 潜力在哺乳动物中已经描述了15个K2 P家族成员;只有两个出现在“Illuminating 药物基因组”未充分研究的蛋白质列表，包括KCNK 12。作为一个偶然的结果， 在线虫的诱变筛选中，我们最近鉴定了秀丽隐杆线虫。elegans KCNK 12的同源物，称为TWK-14，在线粒体未折叠效应子的正向遗传筛选中 蛋白质反应（UPRmt）。我们的小组先前演示了如何UPRmt，这是恢复以下 急性应激源如毒素在经历慢性激活时变得失调。我们特别 表明，本质上无序的蛋白质，a-突触核蛋白（a-syn），帕金森氏症的主要病理因素， 疾病（PD），慢性激活UPRmt，导致C. elegans 多巴胺神经元（Martinez等人，2017年）。我们发现twk-14编码一种内在的神经保护因子， 多巴胺能神经变性的抑制剂，建立了进一步的生理学相关基础， 对KCNK 12未解决功能的研究。与KCNK 12一样，twk-14的表达仅限于神经元。 构建了C.线虫定位TWK-14以选择头部的感觉神经元， 多巴胺能回路中的突触后位置。在此R 03提案中，我们将探索KCNK 12/TWK-14 使用C. elegans作为一个表型读数。在目标1中，神经元- KCNK 12特异性转基因表达及30个候选基因系统遗传分析 通过对Pharos和相关资源的数据库挖掘确定的KCNK 12/TWK-14的相互作用者，将被 进行揭示KCNK 12相关的C.多巴胺神经元。成果 该分析的结果将阐明该K2 P在体内的细胞生物学作用。在目标2中，我们将细化理解 新发现的KCNK 12/TWK-14在C.多巴胺能回路我们 探索一种假说，即在twk-14的突触后神经元中K2 P通道调节的丧失 突变体破坏了固有的神经保护突触反馈回路， 在维持多巴胺稳态的信号。在这里，我们将分析假定的 通过严格量化神经退行性变，在单个 神经元水平，在具有不同突变背景的转基因动物的同基因群体。除了有 提供新的转基因动物、载体和突变株作为交付物， 1-一年的项目有可能为下游研究提供KP 2相关药物发现的信息， 用于神经变性疾病的治疗策略，包括PD或其中线粒体应激 与异常的神经传递有关我们的方法证实了研究的效用， 无脊椎动物模型，用于加速未充分研究的蛋白质的功能归属。

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.