Modifying the Internal Globus Pallidus (GPi) in Parkinson's Disease: Role of Glutamate in Restoration

黑质纹状体多巴胺（DA）丢失后，黑质纹状体内谷氨酸神经元的活性增加， 丘脑底核这些兴奋性神经元投射到内部苍白球[GPi，或脚内核 在啮齿类动物的神经核（EPN），它利用抑制性神经递质，GABA（伽马氨基丁酸）。 在帕金森病（PD）中，GPi的脑深部电刺激（DBS）可改善运动功能 并提供症状缓解。然而，在啮齿动物PD模型中，EPN/GPi的刺激没有导致 急性纹状体内输注6-羟基多巴胺后对运动缺陷或DA丢失的任何保护。 DBS也可能损害通道纤维。然而，直接减少GABA从 EPN/GPi神经元而不影响通道纤维可以回答这个问题。由于EPN/GPi神经元 利用囊泡GABA转运蛋白（VGAT）将GABA摄取到突触囊泡中，该基因的缺失 选择性地减少这些EPN/GPi神经元的GABA释放。 使用Cre/loxP重组酶基因技术，其中可以沉默特定基因，影响GABA 从EPN/GPi GABA神经元释放，可以通过删除特定的靶基因来实现。 通过将AAV-Cre注射到针对GABA转运蛋白VGAT（Vgatflox/flox）被floxed的小鼠中来观察脑区。到 确定GPi/EPN中Vgat基因的缺失是否可以对DA末端和细胞产生神经保护作用 损失使用神经毒素，MPTP（1-甲基-4-苯基-1，2，3，6-四氢吡啶），我们发现， 将AAV-Cre-GFP（绿色荧光蛋白）输注到EPN/GPi中标记了大约80-90%的那些 GABA神经元，如通过EPN/GPi神经元的GFP染色所确定的，并且增加GABA免疫胶体金 在运动丘脑的末端内标记（即，EPN/GPi投射到运动丘脑）。作为 通过酪氨酸羟化酶（TH）免疫反应性测定，这导致了双侧保护作用， 纹状体中的DA末端和改善的运动功能。对TH/DA有部分保护作用（~50%） 黑质神经元丢失。在神经干预后，使用单侧AAV- 将cre-GFP输注到EPN/GPi中，然后立即进行MPTP处理（MPTP/Cre），我们发现， MPTP/Cre组运动强度提高，纹状体和SNpc TH蛋白表达增加， MPTP组。使用另外的PD动物模型，其中AAV-α突触核蛋白（A-Syn）双侧表达。 输注到SNpc中，在用AAV-Cre删除EPN/GPi中的Vgat基因（即，保护）导致 与仅A-Syn组相比，SNpc中改善的运动强度和TH/DA细胞损失的阻断。 本项目的总体目标是确定Vgat基因的单侧缺失，在2 PD的动物模型，在EPN/GPi内，以下（即，神经修复，一个更简单的 相关模型）进行性施用MPTP或黑质内输注A-Syn，可 双侧逆转年轻和老年小鼠黑质纹状体通路内DA的丢失。一个 另一个目标是确定纹状体中谷氨酸在逆转DA耗竭中的作用， 到MPTP。本提案的具体目标是：1）确定Vgat基因的单侧缺失是否在 MPTP治疗4周后开始EPN/GPi治疗可逆转DA的丢失 纹状体/SN中的标志物并改善年轻和老年小鼠的运动功能，2.）确定是否单方面 双侧输注AAV-A-Syn后8周开始EPN/GPi内Vgat基因的缺失 （突变形式：A53 T）进入黑质后叶部可以双侧逆转多巴胺标记物的丢失， 纹状体/SN和改善年轻和老年小鼠的运动功能，以及3.）确定该机制是否 在MPTP处理的小鼠中，在纹状体内Vgat基因缺失后， EPN/GPi是由于纹状体谷氨酸增加。谷氨酸拮抗剂将被注入纹状体， 确定这是否会由于MPTP后EPN/GPi中Vgat基因的缺失而阻断DA恢复。

Following the loss of nigrostriatal dopamine (DA), there is increased activity of glutamate neurons within the subthalamic nucleus. These excitatory neurons project to the internal globus pallidus [GPi, or entopeduncular nucleus (EPN) in the rodent], which utilizes the inhibitory neurotransmitter, GABA (gamma aminobutyric acid). In Parkinson's disease (PD), deep brain stimulation (DBS) of the GPi results in improvement in motor function and provides symptomatic relief. However in a rodent PD model, stimulation of the EPN/GPi did not result in any protection against motor deficits or DA loss following acute intrastriatal infusion of 6-hydroxydopamine. DBS could also be damaging the fibers of passage. However, directly decreasing GABA release from the EPN/GPi neurons without affecting the fibers of passage could answer this concern. Since EPN/GPi neurons utilize the vesicular GABA transporter (VGAT) for uptake of GABA into synaptic vesicles, deletion of this gene would selectively decrease the release of GABA from those EPN/GPi neurons. Using the Cre/loxP recombinase gene technology where a specific gene can be silenced, effecting GABA release from the EPN/GPi GABA neurons, can be achieved through deleting a targeted gene in the specific brain area by injecting AAV-Cre into mice that are floxed for the GABA transporter, VGAT (Vgatflox/flox). To determine if deletion of the Vgat gene in the GPi/EPN 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 unilateral infusion of AAV-Cre-GFP (green fluorescent protein) into the EPN/GPi labeled approximately 80-90% of those GABA neurons, as determined by GFP staining of EPN/GPi neurons, and increased GABA immuno-gold labeling within the terminals of the motor thalamus (i.e., the EPN/GPi projects to the motor thalamus). As measured by tyrosine hydroxylase (TH) immunoreactivity, this resulted in a bilateral protection from the loss of DA terminals in the striatum and improved motor function. There was partial protection (~50%) from TH/DA neuron loss in the substantia nigra pars compacta (SNpc). Following neurointervention, with unilateral AAV- cre-GFP infusion into the EPN/GPi followed immediately by MPTP treatment (MPTP/Cre), we find that there is improved motor strength and increased TH protein expression in the striatum and SNpc in the MPTP/Cre vs MPTP only group. Using an additional PD animal model, in which AAV-alpha synuclein (A-Syn) is bilaterally infused into the SNpc, prior deletion of the Vgat gene in the EPN/GPi with AAV-Cre (i.e., protection) resulted in improved motor strength and blockade of TH/DA cell loss in the SNpc compared to the A-Syn only group. The overall goal of this project is to determine whether unilateral deletion of the Vgat gene, in 2 animal models of PD, within the EPN/GPi, following (i.e., neurorestoration, a more translationally relevant model) either progressive administration of MPTP or intranigral infusion of A-Syn, can bilaterally reverse the loss of DA within the nigrostriatal pathway in both young and aged mice. An additional goal is to determine the role of glutamate in the striatum in reversing the DA depletion due to MPTP. The specific aims of this proposal are to: 1.) determine if unilateral deletion of the Vgat gene within the EPN/GPi initiated 4 weeks after progressive MPTP administration can bilaterally reverse the loss of DA markers in the striatum/SN and improve motor function in both young and aged mice, 2.) determine if unilateral deletion of the Vgat gene within the EPN/GPi initiated 8 weeks following bilateral infusion of AAV-A-Syn (mutant form: A53T) into the substantia nigra pars compacta can bilaterally reverse the loss of DA markers in the striatum/SN and improve motor function in both young and aged mice and 3.) determine if the mechanism behind the striatal TH/DA restoration in the MPTP-treated mice following deletion of the Vgat gene within the EPN/GPi is due to increased striatal glutamate. Glutamate antagonists will be infused into the striatum to determine if this will block the DA recovery due to deletion of the Vgat gene in the EPN/GPi following MPTP.