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Functional analysis of KCNK12 in dopaminergic neuroprotection

Functional analysis of KCNK12 in dopaminergic neuroprotection
KCNK12在多巴胺能神经保护中的功能分析
批准号:
10665836
负责人:
Kim A Caldwell
金额:
$14.36万
依托单位国家:
美国
项目类别:
财政年份:
2023
资助国家:
美国
项目状态:
未结题
起止时间:
2023-06-01 至 2025-05-31
关键词:
AccelerationAcuteAddressAffectAfferent NeuronsAllelesAmericanAnabolismAnimal ModelAnimalsBioinformaticsBiologicalBiological AssayBiological ModelsCaenorhabditis elegansCell LineageCellsChemical ModifierChronicCommunitiesDatabasesDiagnosisDiseaseDopamineEtiologyEvaluationExperimental DesignsExperimental ModelsFailureFamily memberFeedbackFoundationsFunctional disorderGene TargetingGenesGeneticGenetic CrossesGenetic ScreeningGenetic TranscriptionGenomeGoalsGolgi ApparatusHeadHealthHomeostasisHomologous GeneHumanHumanitiesInvertebratesInvestigationIon ChannelLewy BodiesMammalsMapsMediatingMedicalMembrane PotentialsMicroscopicMiningMitochondriaMitochondrial DiseasesModelingMovement DisordersMutagenesisMutationNematodaNerve DegenerationNeurodegenerative DisordersNeuronsNeuropeptide ReceptorNeuropeptidesNuclearOutcomeParkinson DiseasePathologicPathologyPathway interactionsPhenotypePhysiologicalPopulationPositioning AttributePotassium ChannelProteinsRNA InterferenceRegulationResearchResourcesRestRoleScienceSeminalSensorySignal TransductionStressSymptomsSynapsesTestingTherapeuticToxinTransgenic AnimalsTransgenic Organismsactivating transcription factor 1alpha synucleinconnectomedopaminergic neurondrug discoveryexperiencegenetic analysisgenome resourcein vivoinnovationloss of functionmarginalizationmutantneuron lossneuronal survivalneuroprotectionneurotransmissionnoveloverexpressionpostsynapticpostsynaptic neuronspresynapticprogressive neurodegenerationresponsereuptakescreeningstressortraffickingtransgene expressionvector

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中文摘要
翻译
项目概要/摘要 双孔域 K 通道 (K2P) 促进调节静息膜的背景泄漏 K 电流 潜力。已在哺乳动物中描述了 15 个 K2P 家族成员;只有两个出现在“照明”上 “可药物基因组”的待研究蛋白质列表,包括 KCNK12。作为随机的偶然结果 在秀丽隐杆线虫中进行诱变筛选,我们最近鉴定了秀丽隐杆线虫 KCNK12 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)。我们的小组之前演示了 UPRmt(恢复性后续行动)如何 毒素等急性应激源在经历慢性激活时会变得失调。具体来说,我们 研究表明,本质上无序的蛋白质 a-突触核蛋白 (a-syn) 是帕金森病的主要病理因素 疾病(PD),长期激活 UPRmt,导致线虫进行性神经变性 多巴胺神经元(Martinez 等人,2017)。我们发现 twk-14 编码一种固有的神经保护作用 多巴胺能神经变性的抑制因子,为进一步的研究奠定了生理学相关基础 对 KCNK12 未解决的功能的研究。与 KCNK12 一样,twk-14 的表达仅限于神经元。 线虫的定义连接组图定位 TWK-14 以选择头部的感觉神经元, 多巴胺能回路内的突触后位置。在这个 R03 提案中,我们将探索 KCNK12/TWK-14 使用线虫中的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 的特异性转基因表达,结合 30 个候选者的系统遗传分析 通过 Pharos 和相关资源的数据库挖掘识别出的 KCNK12/TWK-14 交互者将被 旨在揭示 KCNK12 相关的线虫多巴胺神经元保护修饰因子。结果 该分析将阐明该 K2P 在体内的细胞生物学作用。在目标 2 中,我们将加深理解 KCNK12/TWK-14 在定义的线虫多巴胺能电路中新发现的调节作用。我们 探索 twk-14 突触后神经元 K2P 通道调节丧失的假设 突变体破坏了适当神经肽所需的固有神经保护性突触反馈回路 维持多巴胺稳态的信号传导。在这里,我们将解析假定的相对影响 该机制的组成部分通过对神经退行性变的严格量化,在单一 神经元水平,在具有不同突变背景的转基因动物的等基因群体中。除了 提供新的转基因动物、载体和突变株作为可交付成果,这一点的更广泛影响 为期 1 年的项目有可能为 KP2 相关药物发现和下游研究提供信息 神经退行性疾病的治疗策略,包括 PD 或线粒体应激障碍 与异常的神经传递相交叉。我们的方法体现了研究的实用性 无脊椎动物模型,用于加速将功能归因于未充分研究的蛋白质。
英文摘要
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.
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