This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator.
Aims and results: Matrix metalloproteinases (MMPs) have been linked to blood-brain barrier opening and neuronal death associated with ischemia. Although best known for their role in the proteolysis of extracellular protein targets, recent studies have revealed that MMPs are also localized to various intracellular sites including nucleus. We previously found increased active gelatinases (MMP-2/-9) in ischemic neuronal nuclei during early reperfusion. Intranuclear MMP-2 degraded nuclear DNA repair enzymes PARP-1 and XRCC1, suggesting a role for MMP-2 in nuclear matrix proteolysis and DNA repair after stroke. DNA base excision repair (BER) machinery is the main mechanism in neuronal nuclei to repair oxidized DNA. During BER and single strand break repair, PARP-1 is activated by DNA breaks and triggers XRCC1 recruitment to damage sites where it participates in BER. Our preliminary data showed that treatment with MMP inhibitor significantly attenuated accumulation of oxidized DNA bases in nuclei of ischemic rat brain neurons at 3 hrs reperfusion. Based on this finding, we propose a novel Hypothesis that activation of MMPs in the ischemic neuronal nucleus may degrade DNA repair enzymes and contribute to accumulation of oxidized DNA bases which trigger cell death in neurons. This project is focused on following studies: 1) Investigate the expression of other MMPs (MMP-9 and -3) in neurons and determine if they are capable of cleaving PARPs and nuclear BER enzymes. 2) Begin to investigate whether the degradation of PARPs and other BER enzymes contributes to accumulation of oxidative DNA damages and delayed neuronal death. In this aim, we also plan to determine if the inhibitors of MMPs can block the cleavage of PARPs and BER enzymes, and thereby improve survival of brain cells and be beneficial in recovery after ischemic insult. Infarct size, blood-brain barrier (BBB) permeability, and behavior test after stroke will be evaulated. 3) Set up primary neuron culture studies with OGD conditions. MMP-2 siRNA techniques will be employed in primary neuron cultures to investigate the roles of MMP-2 in DNA repair and neuronal survival following OGD. The in vitro study in this aim is to complement the in vivo studies above.
Showing the most recent 10 out of 118 publications
