Mechanisms of Inhibition of L-Dopa Induced Dyskinesia (LID) by GPCR Smoothened Activation.

GPCR 平滑激活抑制左旋多巴诱发的运动障碍 (LID) 的机制。

基本信息

批准号：
10260380
负责人：
Andreas H Kottmann
金额：
$ 19.63万
依托单位：
CITY COLLEGE OF NEW YORK
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-09 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10260380
关键词：
Acute Affinity Chromatography Agonist Alleles Amino Acids Animals Attenuated Basal Ganglia Basal Ganglia Diseases Binding Biological Assay Bradykinesia Brain Cells Chemistry Chimera organism Chimeric Proteins Clinic Clinical Communities Complement Complication Corpus striatum structure Cyclic AMP Cytoplasmic Tail Data Dependovirus Development Disease model Dopamine Dose Drug Targeting Engineering Event Exhibits Exposure to Functional disorder G-Protein-Coupled Receptors GTP-Binding Proteins Generations Genetic Glutamates Goals Harvest Human Hydrolysis Hypersensitivity Hypokinesia In Vitro Interneurons Kinetics L-DOPA induced dyskinesia Label Levodopa Light Link Lipase Lipids Macaca Mass Spectrum Analysis Metabolic Methionine Methionine-tRNA Ligase Methods Modeling Modification Molecular Mus Nerve Degeneration Neurons Neurotransmitters Oncogenic Optics Parkinson Disease Pathway interactions Patients Pharmaceutical Preparations Pharmacology Photophobia Play Point Mutation Post-Translational Protein Processing Primates Proteins Proteome Reagent Research Resources Role SHH gene Sampling Signal Pathway Signal Transduction Sonic Hedgehog Pathway Structure-Activity Relationship Symptoms Synaptosomes System Testing Therapeutic Tissues aphakia mice attenuation biophysical properties cell type cholinergic clinical investigation cyclopamine design and construction dopamine replacement therapy dopaminergic neuron drinking water druggable target effective therapy experimental study expression vector extracellular fluorophore gamma-Aminobutyric Acid high reward high risk in vivo in vivo Model melanopsin mouse model multimodality nerve supply new therapeutic target nonhuman primate optogenetics protein biomarkers recruit response side effect smoothened signaling pathway sonic hedgehog receptor tool

项目摘要

Dopamine (DA) substitution therapy by L-DOPA effectively ameliorates many Parkinson's Disease (PD) symptoms caused by DA neuron (DAN) degeneration but leads to the formation of debilitating L-DOPA induced dyskinesia (LID) in most patients after several years of treatment. This treatment complication severely curtails the therapeutic window of L-DOPA and finding strategies that overcome LID formation remains a pressing clinical need. We recently found that DAN not only release dopamine but also the secreted cell signaling factor Sonic Hedgehog (Shh) which they use to signal selectively to cholinergic (CIN) and fast spiking (FS) interneurons among all dopaminergic targets. CIN have become implicated in LID independently via multiple lines of research and clinical observations. Thus our findings suggested that LID might emerge because CIN and FS are exposed to high DA but low Shh signaling during DA replacement therapy. Consistent with this hypothesis we found that stimulation of the Shh pathway through agonists of the Shh effector GPCR Smoothened (Smo) in addition to L-DOPA attenuates LID in mouse- and non-human primate-models of PD. While our primate studies suggest that augmenting Shh/Smo signaling together with L-DOPA might attenuate LID in PD it is unlikely that agonists of Smo are viable drugs in humans because of their severe oncogenic liability. Instead we aim to identify druggable events downstream of Shh/Smo signaling in CIN and FS in in vivo models of PD and LID. The hope is that emerging targets might be more selective and their pharmacological manipulation more tolerable than stimulating Smo signaling. To enable this goal we propose here to adapt and validate methods that allow (1) optic control over Shh/Smo signaling in the basal ganglia (BG) of LID expressing animals and (2) neuron subtype specific metabolic tagging and identification of proteins and their post-translational modifications in response to Smo activation in LID. In aim1 we will produce chimeric Melanopsin:Smoothened proteins (Mel:Smo) and determine their light sensitivity in vitro. Light responsive Mel:Smo chimeras will then be expressed in vivo in a Cre dependent manner from Adeno-Associated Viruses (AAV) in CIN of the dorso-lateral striatum of the LID sensitive Aphakia mouse line. We will test whether light activation of Mel:Smo chimeras attenuates LID and will quantify that effect relative to Smo agonists that activate non recombinant Smo. These reagents will test the hypothesis that Smo signaling in CIN causes LID attenuation. In aim2 we will identify proteins that become modified in response to Smo signaling selectively in either CIN or FS interneurons of mice that were acutely injected with either agonists or antagonists of Smo. This approach is enabled by mice expressing Cre in CIN or FS interneurons and that carry a conditional methionyl-tRNA synthetase L274G point mutation (MetRS) allele which causes the incorporation of azidonorleucine instead of methionine into de novo synthesized proteins. Tagged proteins and their modifications will be identified by mass spectrometry upon affinity purification. These tools will allow the mechanistic study of an emerging key signaling pathway at the molecular level in the healthy BG of behaving animals and might identify drug targets in LID and other BG diseases.

L-DOPA 的多巴胺 (DA) 替代疗法可有效改善多种帕金森病 (PD) 由 DA 神经元 (DAN) 变性引起的症状，但会导致衰弱的左旋多巴 (L-DOPA) 的形成大多数患者在治疗几年后会出现诱发运动障碍（LID）。这种治疗并发症严重缩短 L-DOPA 的治疗窗口并寻找克服 LID 形成的策略仍然是迫切的临床需求。我们最近发现DAN不仅释放多巴胺，还释放分泌的多巴胺。细胞信号因子 Sonic Hedgehog (Shh)，用于选择性地向胆碱能 (CIN) 和快速信号发出信号所有多巴胺能靶标中的尖峰（FS）中间神经元。 CIN 已独立地与 LID 相关通过多方面的研究和临床观察。因此我们的研究结果表明 LID 可能会出现因为在 DA 替代治疗期间，CIN 和 FS 暴露于高 DA 但低 Shh 信号。与这一假设一致，我们发现通过Shh激动剂刺激Shh通路在 PD 小鼠和非人类灵长类动物模型中，效应器 GPCR Smoothened (Smo) 与 L-DOPA 一起可减弱 LID。虽然我们的灵长类动物研究表明，与 L-DOPA 一起增强 Shh/Smo 信号传导可能会减弱 PD 中的 LID，但 Smo 激动剂不太可能成为人类可行的药物，因为它们的作用严重的致癌责任。相反，我们的目标是识别 Shh/Smo 信号下游的可药物事件 PD 和 LID 体内模型中的 CIN 和 FS。希望新兴目标可能更具选择性和它们的药理学操作比刺激 Smo 信号传导更容易耐受。为了实现这一目标，我们在此提出调整和验证允许（1）对基底层中的Shh/Smo信号进行光学控制的方法 LID 表达动物的神经节 (BG) 和 (2) 神经元亚型特异性代谢标记和鉴定 LID 中响应 Smo 激活的蛋白质及其翻译后修饰。在 Target1 中，我们将生产嵌合黑视蛋白：平滑蛋白 (Mel:Smo) 并测定其光体外敏感性。光响应 Mel:Smo 嵌合体随后将在体内以 Cre 依赖性表达 LID 敏感无晶状体背外侧纹状体 CIN 中腺相关病毒 (AAV) 的方式鼠标线。我们将测试 Mel:Smo 嵌合体的光激活是否会减弱 LID，并对其进行量化相对于激活非重组 Smo 的 Smo 激动剂的作用。这些试剂将检验以下假设： CIN 中的 Smo 信号传导导致 LID 衰减。在 Target2 中，我们将识别被修饰的蛋白质急性注射 Smo 信号的小鼠的 CIN 或 FS 中间神经元选择性响应 Smo 的激动剂或拮抗剂。这种方法是通过在 CIN 或 FS 中表达 Cre 的小鼠实现的中间神经元，携带条件性甲硫氨酰-tRNA 合成酶 L274G 点突变 (MetRS) 等位基因这导致叠氮正亮氨酸而不是甲硫氨酸掺入从头合成的蛋白质中。标记的蛋白质及其修饰将在亲和纯化后通过质谱法进行鉴定。这些工具将允许在分子水平上对新兴的关键信号通路进行机制研究。行为动物的健康 BG，并可能确定 LID 和其他 BG 疾病的药物靶点。