Targeting glutamate carboxypeptidase in perinatal brain injury

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Project summary/abstract Despite significant advances in neonatal care and implementation of therapeutic hypothermia, term and preterm survivors of neonatal encephalopathy are at high risk for developing cognitive and learning deficits even in the absence of functional motor deficits. Recent studies have implicated cerebellar dysfunction with the long-term learning disorders seen in these children. Impaired Purkinje cell development has been linked to impairment in cerebellar associative learning. Microglia have been shown to play a major role in the development of Purkinje cells, the primary output neurons of the cerebellar cortex. Intrauterine inflammation leads to microglial activation that results in maldevelopment of the Purkinje cells and cerebellar learning deficits. Activated microglia in the cerebellum overexpress the enzyme glutamate carboxypeptidase II (GCPII), which hydrolyzes the abundant neuropeptide N-acetylaspartylglutamate (NAAG, a specific agonist of the metabotropic glutamate receptor, mGluR3) to N-acetyl-aspartate and glutamate. Reduced NAAG levels have been linked to impaired cognition. This study proposes to specifically target microglial GCPII enzyme using dendrimer conjugated to the nanomolar potent but poorly brain penetrating GCPII inhibitor 2PMPA (2- (phosphonomethyl) pentane-1,5-dioic acid) (D-2PMPA). The central hypothesis is that normal microglial function and dynamics play a critical role in cerebellar development, and inhibiting upregulated GCPII in activated microglia with D-2PMPA will lead to increased NAAG in the cerebellum enabling normal Purkinje cell development and improving cerebellar learning and memory in juvenile rabbits exposed to low dose endotoxin in utero. This will be tested by (1) Evaluating the effects of low dose intrauterine endotoxin exposure on Purkinje cell development, cerebellar microglial response and cerebellar learning (tested by classical eyeblink conditioning reflex) in a rabbit model of maternal inflammation induced brain injury. (2) Evaluate efficacy of D- 2PMPA mediated microglial GCPII inhibition on Purkinje cell development and cerebellar learning at 6-8 weeks of age in rabbits exposed to intrauterine inflammation and (3) Determine mechanisms of cerebellar neuroprotection by D-2PMPA mediated by NAAG induced activation of mGluR3. This study will provide a better understanding of how low-grade maternal-fetal inflammation in the perinatal period can lead to cerebellar learning deficits long term. The proposed work is innovative because it uses a novel and potent D- 2PMPA conjugate to target microglial GCPII for addressing cognitive and learning deficits and enable normal development of the cerebellum in a rabbit model of perinatal brain injury. This Multi-PI proposal will bring synergistic expertise in perinatal/neonatal brain injury, neuropharmacology/GCP2 and nanomedicine, and clinical translation to accomplish these aims. This work can lead to the development of novel therapies to address learning deficits commonly seen with neonatal brain injury.