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A Molecular Target of Hypoxic-Ischemic Injury in Developing Brain and Cerebral Palsy
Brain damage due to hypoxia-ischemia (HI) at perinatal/neonatal stages is the most common cause of cerebral palsy in children, for which currently there is no promising therapy. The molecular mechanism(s) of perinatal hypoxic-ischemic brain injury and its implications in developing cerebral palsy has not been fully explored. Therefore, it is of utmost importance to better understand the mechanisms involved in the hypoxic-ischemic brain damage in neonates.
The central hypothesis of this proposal is that induction of neuronal pentraxin 1 (NP1), a novel neuronal protein and a member of a newly recognized subfamily of “long pentraxins”, is part of the molecular cascade of neuronal death program in neonatal brain injury triggered by hypoxia-ischemia (HI). To test our hypothesis we will perform the following Specific Aims:
Aim 1, We will use NP1 knockout (KO), neuronal pentraxin (NP)-triple KO (NP-TKO) and wildtype (WT) neonatal mice model of HI. First, we will examine NP1 induction and the magnitude of injury in different brain areas of WT vs. NP1 KO mice at various time periods post-HI. Proposed experiments will determine the temporal and regional pattern of NP1 induction specific to injury in different brain regions particularly in motor areas of the brain. Results will directly determine the link between the NP1 induction and its specific involvement in the brain injury mechanism. Next, because of the structural and functional similarities between NP1 and the family member NP2 (also called Narp), we will use NP-TKO neonatal mice (NP1, NP2 and NP receptor are knocked down) and subjected to HI. Comparisons of WT, NP1KO and NP-TKO will also allow us to delineate NP1 function from NP2, and will address if NPs can compensate for one another in the injury mechanism. Our results will reveal a causal role of NP1 in a, yet unstudied, mechanism of hypoxic-ischemic injury in the developing brain.
Aim 2, We will use WT, NP1 KO and NP-TKO primary cortical and hippocampal neuronal cultures to mechanistically link NP1 induction to hypoxic-ischemic neuronal death. First, we will examine NP1 and NP2 induction in WT (WT vs. KO) cultured primary cortical and hippocampal neurons exposed to different time periods of oxygen and glucose deprivation (OGD) conditions. Next, to demonstrate the specific requirement for NP1 induction in neuronal death, we will use a gain-of-functions strategy by infecting KO cells with lentivirus constructs expressing NP1 (pLenti6v5-Nptx1) to reintroduce NP1 into the NP1 KO and NP-TKO cells. Similarly, we will overexpress NP2 in NP1 KO cells or reintroduce NP2 into TKO cells and exposed to OGD to elucidate the role of NP2 in neuronal injury/death. Results will reveal if NP1 alone or both NP1 and NP2 participate in the injury mechanism. Comparison of these results will demonstrate the specific requirement of NP1 in the neuronal death program.
Aim 3, Cerebral damage occurring with HI has been attributed to excitotoxicity. We will examine whether altering NP1 function alters AMPA-and NMDA-receptor-mediated excitotoxicity in brain. We will use NP1 KO and NP-TKO mice in our established neonatal animal model of excitotoxicity in vivo. Next, we will perform NP1 gain-of-function experiments using KO neuronal cultures as described in Aim 2, and will examine the specific involvement of NP1 relative to NP2 in NMDA- and AMPA-induced excitotoxicity. Results will explain how NP1 is involved in the brain injury triggered by HI.
Our findings will have a major impact towards understanding a new, yet unstudied, mechanism of hypoxic-ischemic brain damage in neonates. Our findings will ultimately contribute to stimulate new strategies for effective therapeutic applications, and will provide the opportunities to meet the challenges for cerebral palsy and other neurological disabilities in children.
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