Role of the subthalamic nucleus and TrkB following dopamine loss

多巴胺丢失后丘脑底核和 TrkB 的作用

• 批准号: 9339486
• 负责人: Charles Kenneth Meshul
• 金额: --
• 依托单位: PORTLAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9339486
• 关键词: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Acute; Address; Affect; Age; Agonist; Animal Disease Models; Animal Model; Animals; Area; Bilateral; Brain; Caring; Cell Death; Cells; Clinical Research; Corpus striatum structure; Cre-LoxP; Deep Brain Stimulation; Disease; Dopamine; Education; Fiber; Frequencies; Funding; Gene Deletion; Gene Targeting; General Population; Genes; Glutamates; Goals; Green Fluorescent Proteins; Human; Hydroxydopamines; Incidence; Infusion procedures; Injectable; Intervention; Investigation; Label; Lesion; LoxP-flanked allele; Measures; Medical center; Motor; Movement Disorders; Mus; Nerve; Neurons; Neurotoxins; Parkinson Disease; Pharmacology; Phosphotransferases; Population; Receptor Protein-Tyrosine Kinases; Research; Research Priority; Research Project Grants; Role; STN stimulation; Structure of subthalamic nucleus; Substantia nigra structure; Synaptic Vesicles; Technology; Toxin; Tyrosine 3-Monooxygenase; Veterans; aged; dopaminergic neuron; excitotoxicity; extracellular; immunoreactivity; motor deficit; motor function improvement; neuroprotection; neurorestoration; nigrostriatal pathway; pars compacta; public health relevance; receptor; recombinase; restoration; uptake; vesicular glutamate transporter 2

 DESCRIPTION (provided by applicant): Following the loss of nigrostriatal dopamine (DA), there is an increase in activity of glutamate neurons within the subthalamic nucleus (STN). In animal models and in humans with Parkinson's disease (PD), both a lesion or high frequency stimulation/deep brain stimulation (DBS) of the STN results in improvement in motor function and provides symptomatic relief. However, both DBS and a lesion of the STN could be affecting the fibers of passage. Direct inactivation of the STN neurons without affecting the fibers of passage could answer this concern. Since STN neurons utilize the vesicular glutamate transporter 2 (VGLUT2) for uptake of glutamate into synaptic vesicles, deletion of this gene would selectively inactivate those STN glutamate neurons and block glutamate release. Using the Cre/loxP recombinase gene technology where a specific gene can be silenced, inactivation of the STN glutamate neurons can be achieved through deleting a targeted gene in the specific brain area by injecting AAV-Cre to mice that are floxed for Vglut2 (Vglut2flox/flox). To determine if deletion of the Vglut2 gene in the STN can be neuroprotective against DA terminal and cell loss using the neurotoxin, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), we find that a unilateral infusion of AAV-Cre-GFP (green fluorescent protein) into the STN inactivated approximately 80% of those STN glutamate neurons, as determined by GFP labeling of STN neurons. As measured by tyrosine hydroxylase (TH) immunoreactivity, this resulted in a bilateral protection from the loss of DA terminals in the striatum and DA neurons in the substantia nigra pars compacta (SNpc) following progressive administration of MPTP compared to the wildtype mice treated with the toxin. There was a 35% decrease in activated tyrosine kinase receptor B (TrkB) within the substantia nigra (SN) following MPTP compared to the vehicle treated group. If VGLUT2 deletion occurred following the initiation of MPTP treatment (i.e, neurointervention), we find striatal nerve terminals recovered to nearly 80% of the vehicle treated group. Since MPTP results in a decrease in activated TrkB in the SN, deletion of the Vglut2 gene within the STN, which we found resulted in a decrease in extracellular glutamate in the SN, would allow activated TrkB in the SN to return to control levels and protect the SNpc neurons. To address a possible mechanism of this protection, we find that systemic administration of the TrkB agonist, 7,8-dihydroxyflavone, 2 weeks after the initiation of MPTP treatment (i.e., neurointervention) resulted in a blockade of any further loss of TH within the striatum due to continued MPTP treatment. The overall goal of this project is to determine whether deletion of the Vglut2 gene within the STN initiated following (i.e., restoration) either progressive administration of MPTP or intrastriatal infusion of 6-hydroxydopamine (6-OHDA) can reverse the loss of DA within the nigrostriatal pathway in both young and aged mice. We will also determine the role of TrkB in blocking or reversing the DA depletion due to MPTP. The specific aims of this proposal are to: 1.) determine if deletion of the Vglut2 gene within the STN initiated 4 weeks after progressive MPTP administration can reverse the loss of DA markers in the striatum/SN and motor function in both young and aged mice, 2.) determine if deletion of the Vglut2 gene within the STN initiated 12 days following infusion of 6-OHDA into the dorsolateral striatum can reverse the loss of DA markers in the striatum/SN and motor function in both young and aged mice, and 3.) determine if the neuroprotection from MPTP following deletion of the Vglut2 gene within the STN is due to maintained levels of activated TrkB in the SN by daily infusion of a TrkB agonist into the SN.

 描述（由申请人提供）： 黑质纹状体多巴胺（DA）丧失后，丘脑底核（STN）内谷氨酸神经元的活性增加。在患有帕金森病 (PD) 的动物模型和人类中，STN 的损伤或高频刺激/深部脑刺激 (DBS) 均可改善运动功能并缓解症状。然而，DBS 和 STN 损伤都可能影响通道纤维。直接灭活 STN 神经元而不影响通道纤维可以解决这个问题。由于 STN 神经元利用囊泡谷氨酸转运蛋白 2 (VGLUT2) 将谷氨酸摄取到突触小泡中，因此删除该基因将选择性地使这些 STN 谷氨酸神经元失活并阻止谷氨酸释放。 利用可以沉默特定基因的 Cre/loxP 重组酶基因技术，通过向 Vglut2 (Vglut2flox/flox) 小鼠注射 AAV-Cre，删除特定脑区的目标基因，从而实现 STN 谷氨酸神经元失活。为了确定 STN 中 Vglut2 基因的缺失是否可以通过神经毒素 MPTP（1-甲基-4-苯基-1,2,3,6-四氢吡啶）对 DA 末端和细胞损失起到神经保护作用，我们发现单侧输注 AAV-Cre-GFP（绿色荧光蛋白）到 STN 中大约 80% 的 STN 失活 谷氨酸神经元，通过 STN 神经元的 GFP 标记确定。通过酪氨酸羟化酶（TH）免疫反应性测量，与用毒素处理的野生型小鼠相比，在渐进性施用 MPTP 后，这导致了纹状体中 DA 末端和黑质致密部（SNpc）中 DA 神经元损失的双边保护。与媒介物治疗组相比，MPTP 后黑质 (SN) 内活化的酪氨酸激酶受体 B (TrkB) 减少了 35%。如果在开始 MPTP 治疗（即神经干预）后发生 VGLUT2 缺失，我们发现载体治疗组的纹状体神经末梢恢复到近 80%。由于 MPTP 会导致 SN 中激活的 TrkB 减少，因此我们发现 STN 内 Vglut2 基因的删除会导致 SN 中细胞外谷氨酸减少，从而使 SN 中激活的 TrkB 返回到控制水平并保护 SNpc 神经元。为了解决这种保护的可能机制，我们发现在开始 MPTP 治疗（即神经干预）后 2 周全身施用 TrkB 激动剂 7,8-二羟基黄酮，可阻止纹状体中由于继续 MPTP 治疗而导致的任何进一步的 TH 损失。 该项目的总体目标是确定 STN 内 Vglut2 基因的删除是否在逐步施用 MPTP 或后开始（即恢复）。 纹状体内输注 6-羟基多巴胺 (6-OHDA) 可以逆转年轻和老年小鼠黑质纹状体通路内 DA 的损失。我们还将确定 TrkB 在阻断或逆转 MPTP 引起的 DA 消耗中的作用。该提案的具体目的是：1.) 确定在进行性 MPTP 给药后 4 周开始删除 STN 内的 Vglut2 基因是否可以逆转年轻和老年小鼠纹状体/SN 和运动功能中 DA 标记的损失，2.) 确定在将 6-OHDA 输注到背外侧纹状体后 12 天开始删除 STN 内的 Vglut2 基因是否可以逆转损失 3.) 确定 STN 内 Vglut2 基因缺失后 MPTP 的神经保护作用是否是由于每天向 SN 中输注 TrkB 激动剂以维持 SN 中激活的 TrkB 水平。