Functional analysis of KCNK12 in dopaminergic neuroprotection

项目摘要/摘要 双孔结构域K通道(K2P)促进调节静息膜的背景泄漏K电流 潜力。在哺乳动物中已经描述了15个K2P家族成员；只有两个出现在 可药物基因组“未被研究的蛋白质清单，包括KCNK12。作为随机性的偶然结果 秀丽隐杆线虫的诱变筛选我们最近鉴定了秀丽线虫 KCNK12的同源物，称为TWK-14，在线粒体未折叠效应的正向遗传筛查中 蛋白质反应(UPRmt)。我们的小组之前演示了UPRmt是如何恢复以下内容的 急性应激源，如毒素，在经历慢性激活时会变得失调。具体来说，我们 研究表明，帕金森病的主要病理因素a-突触核蛋白(a-syn)是一种内在紊乱的蛋白质 疾病(PD)，慢性激活UPRmt，导致线虫进行性神经退化 多巴胺神经元(Martinez等人，2017年)。我们发现TWK-14编码一种固有的神经保护作用 多巴胺能神经退行性变的抑制者，为进一步 KCNK12未解功能的研究。与KCNK12一样，TWK-14的表达仅限于神经元。 线虫已定义的连接体图谱定位TWK-14以选择头部的感觉神经元，在 突触后在多巴胺能回路中的位置。在这份R03提案中，我们将探索KCNK12/TWK-14 在线虫中使用多巴胺能神经变性作为表型读数的功能。在目标1中，神经元- KCNK12基因的特异性转基因表达及对30例候选基因的系统遗传分析 通过对航标和相关资源的数据库挖掘确定的KCNK12/TWK-14的相互作用者将是 旨在揭示KCNK12相关保护线虫多巴胺神经元的修饰物。结果 这一分析将阐明这种K2P在体内的细胞生物学作用(S)。在目标2中，我们将完善理解 新发现的KCNK12/TWK-14在线虫多巴胺能回路中的调节作用。我们 探讨TWK-14突触后神经元K2P通道调节丧失的假说 突变体扰乱正常神经肽所需的固有神经保护性突触反馈环路 维持多巴胺动态平衡的信号传导。在这里，我们将分析假定的 这一建议的机制的组成部分通过严格量化神经变性，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中。除 提供新的转基因动物、载体和突变株作为可交付物，这将产生更广泛的影响 为期一年的项目有可能为下游对KP2相关药物的发现和研究提供信息 包括帕金森病或线粒体应激障碍在内的神经退行性疾病的治疗策略 与异常的神经传递相交。我们的方法举例说明了使用完整的 加速研究不足的蛋白质功能归属的无脊椎动物模型。

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.