Role of the subthalamic nucleus and TrkB following dopamine loss

 描述（由申请人提供）： 随着黑质纹状体多巴胺（DA）的丧失，丘脑底核（BTH）内谷氨酸神经元的活性增加。在动物模型和患有帕金森病（PD）的人类中，帕金森病的损伤或高频刺激/脑深部刺激（DBS）都导致运动功能的改善并提供症状缓解。然而，DBS和小脑损伤都可能影响通道纤维。在不影响通道纤维的情况下，直接使轴突神经元失活可以回答这个问题。由于突触神经元利用囊泡谷氨酸转运体2（VGLUT 2）将谷氨酸摄取到突触囊泡中，因此该基因的缺失将选择性地使那些突触谷氨酸神经元失活并阻断谷氨酸释放。 使用Cre/loxP重组酶基因技术，其中特定基因可以被沉默，通过将AAV-Cre注射到针对VEGF 2被floxed的小鼠（VEGF 2flox/flox）中，可以通过在特定脑区域中缺失靶基因来实现谷氨酸神经元的失活。为了确定是否VEGF 2基因的删除可以对DA末端和细胞损失的神经保护使用神经毒素，MPTP（1-甲基-4-苯基-1，2，3，6-四氢吡啶），我们发现，AAV-Cre-GFP（绿色荧光蛋白）的单侧输注到海马失活的约80%的这些海马谷氨酸神经元，通过GFP标记的海马神经元。酪氨酸羟化酶（TH）免疫反应性测定，这导致了双边保护的DA终端的损失在纹状体和DA神经元在黑质的黑质pars延髓（SNPC）后逐步管理MPTP相比，野生型小鼠处理的毒素。与溶剂处理组相比，MPTP后黑质（SN）内活化酪氨酸激酶受体B（Trk B）降低35%。如果在MPTP治疗（即神经干预）开始后发生VGLUT 2缺失，我们发现纹状体神经末梢恢复到载体治疗组的近80%。由于MPTP导致SN中激活的TrkB减少，因此我们发现在SN中导致细胞外谷氨酸减少的VAP 2基因的缺失将允许SN中激活的TrkB恢复到对照水平并保护SNpc神经元。为了解决这种保护的可能机制，我们发现在MPTP治疗开始后2周全身施用TrkB激动剂7，8-二羟基黄酮（即，神经干预）导致阻断了由于持续MPTP治疗引起的纹状体内TH的任何进一步损失。 该项目的总体目标是确定在以下情况下是否启动了VEGF 2基因的缺失（即，恢复）MPTP的渐进施用或 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的丢失。我们还将确定TrkB在阻断或逆转MPTP引起的DA耗竭中的作用。本提案的具体目标是：1）确定在渐进性MPTP施用后4周开始的纹状体内VEGF 2基因的缺失是否可以逆转年轻和老年小鼠中纹状体/SN和运动功能中DA标志物的丧失，2.）确定在将6-OHDA输注到背外侧纹状体中后12天开始的纹状体内的VEGF 2基因的缺失是否可以逆转年轻和老年小鼠中纹状体/SN和运动功能中DA标志物的损失，以及3.）确定在缺失SN内的VkB 2基因后对MPTP的神经保护是否是由于通过每日将TrkB激动剂输注到SN中来维持SN中活化TrkB的水平。

 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.