Functional analysis of KCNK12 in dopaminergic neuroprotection

KCNK12在多巴胺能神经保护中的功能分析

基本信息

批准号：
10665836
负责人：
Kim A Caldwell
金额：
$ 14.36万
依托单位：
UNIVERSITY OF ALABAMA IN TUSCALOOSA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-06-01 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10665836
关键词：
Acceleration Acute Address Affect Afferent Neurons Alleles American Anabolism Animal Model Animals Bioinformatics Biological Biological Assay Biological Models Caenorhabditis elegans Cell Lineage Cells Chemical Modifier Chronic Communities Databases Diagnosis Disease Dopamine Etiology Evaluation Experimental Designs Experimental Models Failure Family member Feedback Foundations Functional disorder Gene Targeting Genes Genetic Genetic Crosses Genetic Screening Genetic Transcription Genome Goals Golgi Apparatus Head Health Homeostasis Homologous Gene Human Humanities Invertebrates Investigation Ion Channel Lewy Bodies Mammals Maps Mediating Medical Membrane Potentials Microscopic Mining Mitochondria Mitochondrial Diseases Modeling Movement Disorders Mutagenesis Mutation Nematoda Nerve Degeneration Neurodegenerative Disorders Neurons Neuropeptide Receptor Neuropeptides Nuclear Outcome Parkinson Disease Pathologic Pathology Pathway interactions Phenotype Physiological Population Positioning Attribute Potassium Channel Proteins RNA Interference Regulation Research Resources Rest Role Science Seminal Sensory Signal Transduction Stress Symptoms Synapses Testing Therapeutic Toxin Transgenic Animals Transgenic Organisms activating transcription factor 1 alpha synuclein connectome dopaminergic neuron drug discovery experience genetic analysis genome resource in vivo innovation loss of function marginalization mutant neuron loss neuronal survival neuroprotection neurotransmission novel overexpression postsynaptic postsynaptic neurons presynaptic progressive neurodegeneration response reuptake screening stressor trafficking transgene expression vector

项目摘要

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.

项目摘要/摘要两孔域K+通道（K2P）最喜欢的背景泄漏K+电流，调节静止膜潜在的。在哺乳动物中描述了15个K2P家庭成员；在“照明”上只有两个露面可毒品的基因组”清单的蛋白质清单，包括KCNK12。作为随机的偶然结果在rout虫秀丽隐杆线虫中，诱变筛查，我们最近确定了秀丽隐杆线虫 KCNK12的同源物，称为TWK-14，在正向遗传筛选中，用于线粒体展开的影响蛋白质反应（UPRMT）。我们的小组以前证明了如何修复的UPRMT 急性应激源（例如毒素）在经历慢性激活时会失调。具体来说，我们表明本质上受干扰的蛋白质A-核蛋白（A-Syn）是帕金森氏症的主要病理因素疾病（PD），长期激活UPRMT，导致秀丽隐杆线虫的进行性神经变性多巴胺神经元（Martinez等，2017）。我们发现TWK-14作为编码固有的神经保护作用多巴胺能神经变性的抑制剂，建立了与物理相关的基础，以进一步研究KCNK12的未解决功能。像KCNK12一样，TWK-14表达仅限于神经元。秀丽隐杆线虫的定义连接组图将TWK-14定位为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R03提案中，我们将探索KCNK12/TWK-14 秀丽隐杆线虫中使用多巴胺能神经退行性变性作为表型读数的功能。在AIM 1中，神经元 - KCNK12的特定转基因表达，结合30个候选者的系统遗传分析由Pharos和相关资源数据库挖掘确定的KCNK12/TWK-14的交互者将是进行揭示KCNK12相关的秀丽隐杆线虫多巴胺神经元的修饰符。结果该分析将阐明该K2P的细胞生物学作用，体内。在AIM 2中，我们将完善理解 KCNK12/TWK-14在定义的秀丽隐杆线虫多巴胺能电路中的新发现调节作用。我们探索一个假设，通过该假设，在TWK-14的突触后神经元中K2P通道调节的损失突变体破坏了适当的神经肽所需的固有神经保护突触反馈回路维持多巴胺稳态的信号。在这里，我们将解析推定的相对影响通过严格量化神经退行性的组成部分神经元水平，具有潜水员突变背景的转基因动物的同源性种群。除了提供新的转基因动物，向量和突变菌株作为可交付成果，这是更广泛的影响一年的项目有可能告知有关KP2相关药物发现的下游研究和神经退行性疾病的治疗策略，包括PD或线粒体压力的疾病与异常的神经传递相交。我们的方法表明了完整的研究实用性无脊椎动物模型，以加快功能声明以了解蛋白质。