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。

• 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
• 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

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.

左旋多巴（L-DOPA）替代多巴胺（DA）治疗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，但导致形成衰弱的L-DOPA 在大多数患者中，在治疗数年后诱发运动障碍（LID）。这种治疗并发症 严重缩短了左旋多巴的治疗窗口，并找到了克服LID形成的策略， 仍然是一个迫切的临床需求。我们最近发现，DAN不仅释放多巴胺， 细胞信号传导因子Sonic Hedgehog（Shh），用于选择性地向胆碱能（CIN）和快 在所有多巴胺能靶点之间的尖峰（FS）中间神经元。CIN与LID独立相关 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CIN和FS在DA替代治疗期间暴露于高DA但低Shh信号。 与这一假设一致，我们发现通过Shh受体激动剂刺激Shh通路， 除L-DOPA外，效应物GPCR Smoothened（Smo）减弱小鼠和非人灵长类PD模型中的LID。虽然我们的灵长类动物研究表明，增加Shh/Smo信号与左旋多巴， 可能减弱PD中的LID，但是Smo的激动剂不太可能是人类中可行的药物，因为它们 严重的致癌倾向。相反，我们的目标是确定Shh/Smo信号下游的药物事件， PD和LID体内模型中的CIN和FS。人们希望，新出现的目标可能更具选择性， 它们的药理学操作比刺激Smo信号传导更耐受。为了实现这一目标，我们 在此建议调整和验证允许（1）基底膜中Shh/Smo信号传导的光学控制的方法， 神经节（BG）和（2）神经元亚型特异性代谢标记和鉴定 蛋白质及其翻译后修饰响应于LID中的Smo激活。 在aim 1中，我们将产生嵌合黑视素：平滑蛋白（Mel：Smo），并确定它们的光 体外敏感性然后，光响应性Mel：Smo嵌合体将在体内以Cre依赖性表达。 腺相关病毒（AAV）在LID敏感性无晶状体眼背外侧纹状体CIN中的作用 鼠标线。我们将测试Mel：Smo嵌合体的光激活是否减弱LID，并将量化其 相对于激活非重组Smo的Smo激动剂的作用。这些试剂将测试以下假设： CIN中的Smo信号传导导致LID衰减。在aim 2中，我们将鉴定在细胞中被修饰的蛋白质。 在急性注射Smo的小鼠的CIN或FS中间神经元中选择性地响应Smo信号传导， Smo的激动剂或拮抗剂。这种方法通过在CIN或FS中表达Cre的小鼠实现 中间神经元和携带条件性甲硫氨酰-tRNA合成酶L274 G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鉴定。 这些工具将允许在分子水平上对新出现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。