Targeting glutamate carboxypeptidase in perinatal brain injury

以谷氨酸羧肽酶为靶点治疗围产期脑损伤 项目概要/摘要 由活化的星形胶质细胞和小胶质细胞介导的神经炎症和兴奋性毒性是主要的 围产期母体炎症性脑损伤的病理生理机制 这一时期，导致神经发育障碍，如脑瘫（CP）。我们假设， 这两种机制都将提供最大的神经保护并使正常发育成为可能。我们提出 为了实现这一目标，通过靶向酶谷氨酸羧肽酶II（GCPII），使用有效的，选择性 GCPII抑制剂2-（3-巯基丙基）戊二酸（2-MPPA），在我们建立的家兔模型的母体 炎症诱导CP。利用我们的新发现，GCPII在活化的细胞中选择性上调， 小胶质细胞在新生兔与CP，我们将提供2-MPPA使用树枝状纳米粒子（D-MPPA）， 特异性地针对这些细胞，从而提高功效并减少外周副作用。GCPII抑制 不仅能阻止神经肽中谷氨酸的水解，还能增加NAAG水平， 可以通过阻止突触前谷氨酸释放和通过 增加TGF-β1的合成和释放，从而促进正常的小胶质细胞功能，并发挥抗肿瘤作用。 炎症效应。我们的初步研究表明：（1）新生兔CP 脑谷氨酸水平，小胶质细胞“激活”与限制性小胶质细胞迁移，并减少TGF-β1在 （2）D-MPPA选择性定位于CP新生兔脑内活化的小胶质细胞和星形胶质细胞， （3）D-MPPA在降低细胞外谷氨酸和降低细胞内谷氨酸水平方面比单独使用D-MPPA更有效。 增加暴露于LPS的离体脑切片和混合胶质细胞培养物中的TGF-β1水平， 短期运动缺陷的兔与CP套件。基于这些有希望的结果，我们假设，抑制 通过D-MPPA特异性地在活化的小胶质细胞和星形胶质细胞中抑制GCPII， 小胶质细胞形态和迁移，并减少神经元和白色物质损伤，导致改善 在患有CP的新生兔子中，运动和认知缺陷持续到成年。为了验证这一点，我们将（1） 评估CP和对照兔试剂盒中的剂量依赖性药代动力学和药效学。（二） 确定D-MPPA和TGF-β1在改善离体脑中小胶质细胞形态和迁移中的作用 （3）评价静脉注射治疗CP的短期和长期疗效 CP雄性和雌性家兔试剂盒中的D-MPPA。这项工作是创新的，因为它是第一次研究 该研究专注于基于纳米技术的方法，专门针对活化的小胶质细胞中的GCPII 脑损伤后。它利用树枝状聚合物在炎症部位的选择性定位， 开发产后时期的治疗应用。这一多PI提案将为 GCPII在新生儿脑损伤中的作用，并将提供一种新的治疗方法， 在新生儿/儿科脑损伤、GCPII化学和药理学以及树枝状聚合物纳米医学方面的专业知识。

Targeting glutamate carboxypeptidase in perinatal brain injury Project summary/abstract Neuroinflammation and excitotoxicity, mediated by activated astrocytes and microglia, are major pathophysiological mechanisms implicated in maternal inflammation induced brain injury in the perinatal period, resulting in neurodevelopmental disorders such as cerebral palsy (CP). We hypothesize that targeting both these mechanisms will provide maximum neuroprotection and enable normal development. We propose to achieve this by targeting the enzyme glutamate carboxypeptidase II (GCPII), using the potent, selective GCPII inhibitor 2-(3-mercaptopropyl) pentanedioic acid (2-MPPA), in our established rabbit model of maternal inflammation induced CP. Capitalizing on our novel finding that GCPII is selectively upregulated in activated microglia in newborn rabbits with CP, we will deliver 2-MPPA using dendrimer nanoparticles (D-MPPA), specifically to these cells, thereby improving efficacy and reducing peripheral side effects. Inhibition of GCPII will not only prevent hydrolysis of glutamate from the neuropeptide, but will also increase NAAG levels which can independently exert neuroprotective effects by preventing pre-synaptic glutamate release and by increasing synthesis and release of TGF-β1, which in turn promotes normal microglial function and exerts anti- inflammatory effects. Our preliminary studies demonstrate that: (1) Neonatal rabbits with CP have increased brain glutamate levels, microglial `activation' with restrictive microglial migration, and decreased TGF-β1 in the brain, (2) D-MPPA selectively localizes in activated microglia and astrocytes in newborn rabbits with CP but not in controls (3) D-MPPA is more effective than the drug alone in decreasing extracellular glutamate and in increasing TGF-β1 levels in ex vivo brain slices and in mixed glial cultures exposed to LPS, and in improving short-term motor deficits in rabbit kits with CP. Based on these promising results we hypothesize that, inhibition of GCPII by D-MPPA specifically in activated microglia and astrocytes will decrease inflammation, improve microglial morphology and migration, and decrease neuronal and white matter injury, resulting in improvement in motor and cognitive deficits that persist until adulthood, in newborn rabbits with CP. To test this we will (1) Assess the dose-dependent pharmacokinetics and pharmacodynamics in CP and control rabbit kits. (2) Determine the role of D-MPPA and TGF-β1 in improving microglial morphology and migration in ex vivo brain slices from rabbits with CP and (3) Evaluate the short term and long term efficacy of treatment with intravenous D-MPPA in male and female rabbit kits with CP. The proposed work is innovative because it is the first study that is focused on a nanotechnology based approach to target GCPII specifically in activated microglia following brain injury. It takes advantage of the selective localization of dendrimers at sites of inflammation, to develop therapeutic applications in the postnatal period. This Multi-PI proposal will bring new insights to the role of GCPII in neonatal brain injury and will offer a novel therapeutic approach, bringing complementary expertise in neonatal/pediatric brain injury, GCPII chemistry and pharmacology, and dendrimer nanomedicine.