Targeting glutamate carboxypeptidase in perinatal brain injury

靶向谷氨酸羧肽酶在围产期脑损伤中的作用 项目摘要/摘要 由激活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。 围产期母体炎症所致脑损伤的病理生理机制 时期，导致神经发育障碍，如脑瘫(CP)。我们假设目标是 这两种机制都将提供最大限度的神经保护，并使正常发育得以实现。我们建议 通过靶向谷氨酸羧基肽酶II(GCPII)来实现这一点，使用有效的、选择性的 GCPII抑制剂2-(3-巯基丙基)戊二酸(2-MPPA)，在我们建立的兔母体模型中 炎症诱发慢性胰腺炎。利用我们的新发现，GCPII在激活后选择性上调 在新生CP兔的小胶质细胞中，我们将使用树枝状大分子纳米粒(D-MPPA)传递2-MPPA， 特别针对这些细胞，从而提高疗效并减少外周副作用。GCPII的抑制作用 不仅会阻止谷氨酸从神经肽中水解，还会增加NAAG水平，从而 可通过阻止突触前谷氨酸释放而独立发挥神经保护作用 增加转化生长因子-β1的合成和释放，进而促进正常的小胶质细胞功能，发挥抗肿瘤作用。 发炎效应。我们的初步研究表明：(1)新生兔脑性瘫痪的发病率增加 脑谷氨酸水平、小胶质细胞活化和限制性小胶质细胞迁移，以及降低转化生长因子-β1。 脑，(2)D-MPPA选择性地定位于新生兔慢性胰腺炎后活化的小胶质细胞和星形胶质细胞 不在对照组(3)D-MPPA在降低细胞外谷氨酸和胰岛素方面比单独用药更有效 增加体外脑片和混合胶质细胞培养中转化生长因子-β-1的水平，并改善 慢性阻塞性肺疾病兔模型的短期运动障碍。基于这些有希望的结果，我们假设，抑制 D-MPPA特异性地在激活的小胶质细胞和星形胶质细胞中表达GCPII将减轻炎症，改善 小胶质细胞形态和迁移，减少神经元和脑白质损伤，从而改善 持续到成年的运动和认知障碍，在患有CP的新生兔子身上。为了测试这一点，我们将(1) 评价CP和对照兔试剂盒中的剂量依赖性药代动力学和药效学。(2) D-MPPA和转化生长因子-β-1在改善小胶质细胞形态和迁移中的作用 (3)评价静脉注射治疗慢性胰腺炎的近期和远期疗效 D-MPPA在雄性和雌性CP兔试剂盒中的表达。这项拟议的工作具有创新性，因为这是第一项研究 重点是基于纳米技术的方法，特别是在激活的小胶质细胞中靶向GCPII 在脑损伤之后。它利用树枝状大分子在炎症部位的选择性定位， 在出生后阶段开发治疗应用。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，带来互补 在新生儿/儿童脑损伤、GCPII化学和药理学以及树枝状大分子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.