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)丢失后，丘脑底核(STN)内谷氨酸神经元的活动增加。在动物模型和人类帕金森氏病(PD)模型中，损毁或高频刺激/脑深部刺激(DBS)STN都能改善运动功能，并提供症状缓解。然而，DBS和STN的损伤都可能影响通路的纤维。在不影响通路纤维的情况下直接灭活STN神经元可以回答这个问题。由于STN神经元利用囊泡性谷氨酸转运体2(VGLUT2)将谷氨酸摄取到突触小泡中，该基因的缺失将选择性地灭活这些STN谷氨酸神经元并阻止谷氨酸的释放。利用Cre/loxP重组酶基因技术，可以沉默特定基因，通过将AAV-Cre注射到Vlux 2(Vlux/Flox)小鼠体内，删除特定脑区的靶向基因，从而实现STN谷氨酸神经元的失活。为了确定在短臂旁核中缺失Vlu2基因是否对使用神经毒素MPTP(1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine)，的DA末梢和细胞丢失具有神经保护作用，我们发现，通过绿色荧光蛋白标记发现，单侧向短串联核内注入AAV-Cre-GFP(绿色荧光蛋白)可使大约80%的短串联核内的谷氨酸神经元失活。通过酪氨酸羟化酶(TH)免疫反应测定，与野生型小鼠相比，进行性给予MPTP后，这对纹状体DA终末和黑质致密部(SNPC)DA神经元的丢失具有双侧保护作用。与赋形剂治疗组相比，MPTP治疗后黑质(SN)中活化的酪氨酸激酶受体B(TrkB)减少了35%。如果在MPTP治疗(即神经干预)开始后发生VGLUT2缺失，我们发现纹状体神经末梢恢复到赋形剂治疗组的近80%。由于MPTP导致SN中激活的TrkB的减少，我们发现STN内的Vlos2基因的缺失会导致SN中细胞外谷氨酸的减少，从而使SN中激活的TrkB恢复到控制水平，并保护SNPC神经元。为了探讨这种保护的可能机制，我们发现，在MPTP治疗(即神经干预)开始2周后，全身应用TrkB激动剂7，8-二羟基黄酮会阻止由于继续MPTP治疗而导致的纹状体内TH的进一步丢失。这个项目的总体目标是确定在MPTP或MPTP的渐进性给药之后(即恢复)STN内是否启动了Vher2基因的删除 纹状体内注射6-羟基多巴胺(6-OHDA)可逆转幼年和老年小鼠黑质纹状体通路中DA的丢失。我们还将确定TrkB在阻断或逆转MPTP引起的DA耗竭方面的作用。这项建议的具体目的是：1.(1.确定在递增MPTP给药4周后开始删除STN内的Vher2基因是否可以逆转年轻和老年小鼠纹状体/SN中DA标记的丢失和运动功能的丧失，2)确定在向背外侧纹状体注射6-OHDA后12天开始删除STN内的Vather2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标志物的丢失，以及3.)确定在STN内VGlu2基因缺失后MPTP的神经保护作用是否由于每天向SN中注入TrkB激动剂而维持SN中激活的TrkB水平。