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

改变帕金森病的内部苍白球 (GPi)：谷氨酸在恢复中的作用

• 批准号: 9898244
• 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: 2010
• 资助国家: 美国
• 起止时间: 2010-10-01 至 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898244
• 关键词: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Acute; Affect; Age; Animal Disease Models; Animal Model; Area; Bilateral; Brain; Butyric Acids; Caring; Carrier Proteins; Cell Nucleus; Cells; Clinical Research; Corpus striatum structure; Cre-LoxP; Data; Deep Brain Stimulation; Dependovirus; Disease; Disease model; Dopamine; Education; Electrodes; Excitatory Amino Acid Antagonists; Fiber; Funding; Gene Deletion; General Population; Genes; Genetic Transcription; Globus Pallidus; Glutamates; Goals; Green Fluorescent Proteins; Human; Hydroxydopamines; Incidence; Infusion procedures; Investigation; Label; Lateral; LoxP-flanked allele; Measures; Medial; Modeling; Motor; Motor Activity; Movement Disorders; Mus; N-Methylaspartate; Nerve; Neurons; Neurotoxins; Neurotransmitters; Outcome; Parkinson Disease; Pathway interactions; Population; Recovery; Reporting; Research; Research Priority; Research Project Grants; Rodent; Role; Site; Stains; Structure of subthalamic nucleus; Substantia nigra structure; Synaptic Vesicles; Technology; Thalamic structure; Tissues; Tyrosine 3-Monooxygenase; Vesicle; Veterans; aged; alpha synuclein; axon injury; density; dopaminergic neuron; endopeduncular nucleus; excitatory neuron; gamma-Aminobutyric Acid; immunoreactivity; improved; motor deficit; motor function improvement; mutant; neuron loss; neurorestoration; nigrostriatal pathway; pars compacta; prevent; protein expression; recombinase; restoration; uptake; vector; vesicular GABA transporter

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.

在黑质纹状体多巴胺(DA)丧失后，大脑中的谷氨酸神经元活动增强 丘脑底核。这些兴奋性神经元投射到苍白球内侧[GPI，或内眼 啮齿类动物的核团(EPN)，它利用抑制性神经递质GABA(伽马氨基丁酸)。 在帕金森病(PD)患者中，GPI的脑深部刺激(DBS)可改善运动功能 并提供症状缓解。然而，在啮齿动物帕金森病模型中，刺激EPN/GPI不会导致 对急性纹状体内注射6-羟基多巴胺后的运动障碍或DA丢失的任何保护。 星形胶质细胞也可能破坏通路的纤维。然而，直接减少GABA的释放 在不影响通路纤维的情况下，EPN/GPI神经元可以回答这一问题。由于EPN/GPI神经元 利用囊泡GABA转运体(VGAT)将GABA摄取到突触小泡中，该基因的缺失 会选择性地减少这些EPN/GPI神经元的GABA释放。 使用Cre/loxP重组酶基因技术，其中特定基因可以沉默，从而影响GABA 从EPN/GPI GABA神经元中释放，可以通过删除特定的靶基因来实现 通过将AAV-Cre注射到GABA转运体VGAT(Vgatflx/Flox)的小鼠体内，使其进入大脑区域。至 确定GPI/EPN中Vgat基因的缺失是否对DA终末和细胞具有神经保护作用 使用神经毒素mptp(1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine)，我们发现单侧 将AAV-Cre-GFP(绿色荧光蛋白)注入约80%-90%标记的EPN/GPI 通过对EPN/GPI神经元的GFP染色确定GABA神经元的数量，并增加GABA免疫金标记物 运动丘脑终末内的标记(即EPN/GPI投射到运动丘脑)。AS 通过酪氨酸羟化酶(TH)免疫反应性来衡量，这导致了对丢失的双侧保护 纹状体DA终末减少，运动功能改善。对TH/DA有部分保护(~50%) 黑质致密部神经元丢失(SNPC)。在神经干预后，单侧AAV- Cre-GFP注射入EPN/GPI后立即MPTP治疗(MPTP/Cre)，我们发现有 MPTP/CRE大鼠运动强度增强，纹状体和SNPC TH蛋白表达增加 仅MPTP组。使用另一种PD动物模型，其中AAV-α突触核蛋白(A-Syn)是双侧的 将带有AAV-CRE的EPN/GPI中的Vgat基因预先缺失(即保护)注入SNPC中，导致 与单纯A-Syn组相比，SNPC组的运动强度和阻断TH/DA细胞丢失的情况有所改善。 这个项目的总体目标是确定Vgat基因的单方面缺失是否会在2 帕金森病动物模型，在EPN/GPI内，遵循(即，神经恢复，更确切地说 相关模型)无论是MPTP的渐进给药还是黑质内注入A-Syn，都能 双侧逆转年轻和老年小鼠黑质纹状体通路中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 敬MPTP。这项建议的具体目的是：1.确定Vgat基因的单方面缺失是否在 进行性MPTP给药后4周开始的EPN/GPI可逆转双侧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，1.)确定是否为单边 双侧注射AAV-A-Syn后8周EPN/GPI内Vgat基因的缺失 (突变型：A53T)进入黑质致密部可逆转双侧脑内DA标志物的丢失 纹状体/SN，并改善幼年和老年小鼠的运动功能。确定该机制是否 MPTP处理的小鼠在纹状体TH/DA恢复背后的Vgat基因在 EPN/GPI是由于纹状体谷氨酸增加所致。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中的Vgat基因缺失而阻止DA恢复。