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-?1, 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-?1 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-?1 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-?1 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.
Targeting glutamate carboxypeptidase in perinatal brain injury Project narrative: The proposed research is relevant to public health because it provides a better understanding of the role of maternal infection and inflammation in the pathogenesis of neurodevelopmental disorders such as cerebral palsy and autism that would enable the development of specific targeted therapy for prevention of these disorders. Injury to the developing brain results in neurodevelopmental disorders that are often chronic resulting in debilitating disabilities, with large life time health care costs and very little treatment options. Understanding the mechanisms of these diseases and designing targeted therapy using this knowledge will benefit a large number of affected children. The works uses a translational research approach using a multidisciplinary group. Therefore, this work is relevant to NIH's mission to ensure application of knowledge to enhance health and reduce illness and disability.
