Oxidative DNA damage (ODD) arises rapidly after cerebral ischemic/reperfusion injury and leads to apurinic/apyrimidinic (AP) sites and strand breaks. If DNA repair is insufficient, ODD arrests gene transcription, suppresses neuronal activity, and culminates in cell death. Base excision repair (BER) evolved as the predominant endogenous mechanism for repairing ODD in the brain. Our previous studies have unequivocally established that BER has remarkable potential to promote cell survival and long-term functional recovery after stroke injury. Both short-patch (SP) and long-patch (LP) BER pathways are necessary to repair ODD. The SP- BER pathway repairs base damage and intact AP sites with unmodified deoxyribose phosphate (dRP) residues. However, oxidized AP sites (with oxidized dRP residues), the most prominent lethal oxidative lesion in the ischemic brain, cannot be repaired satisfactorily with SP-BER. The newly characterized LP-BER pathway is responsible for the complete repair of oxidized AP sites. Structure-specific flap endonuclease 1 (FEN1) is an essential BER enzyme that controls LP-BER by acting as bi-endonuclease/exonuclease and recruiting partner BER enzymes to the lesion site. However, the role of FEN1-dependent LP-BER in CNS injury is completely unknown. Therefore, we are the first group to examine FEN1 in the context of stroke. Our pilot studies show that: 1) FEN1-dependent LP-BER function critically determines stroke outcomes; overexpression of FEN1 robustly protects against ischemic injury and improves neuronal function, whereas conditional deletion of FEN1 exacerbates injury, resulting in a dramatic increase in neuron and oligodendrocyte death in stroke brains. 2) Cyclin-dependent kinase (CDK) 5 is a novel endogenous inhibitor of FEN1 in neurons and OLs. Following ischemia, activated CDK5 phosphorylates FEN1 at Ser187, thereby disabling FEN1- dependent LP-BER. 3) Administration of TAT peptides to block the CDK5/FEN1 interaction restores LP-BER function and reduces ischemic injury. Given these observations, we propose three specific aims to test the overall hypothesis that activation of FEN1-dependent LP-BER improves long-term stroke outcomes by promoting neuron and oligodendrocyte survival and white matter recovery following ischemia/reperfusion.
AIM 1 : Test the hypothesis that FEN1-dependent LP-BER promotes functional recovery following ischemic brain injury. Transient focal ischemia (tFCI, 30/60min MCAO) will be induced in mice of both genders with tamoxifen-inducible conditional knockout or overexpression of FEN1.
AIM 2 : Test the hypothesis that ischemia- induced activation of CDK5 disables LP-BER by phosphorylating FEN1 at Ser187.
AIM 3 : Test the hypothesis that administration of a cell-permeable peptide blocking FEN1 phosphorylation (by CDK5) enhances LP-BER activity and improves stroke outcomes in male or female, young and aged mice.
Recent research has found that complete repair of oxidative DNA lesions in brain cells replies on the newly discovered long-patch base excision repair (LP-BER) pathway, controlled by the essential BER enzyme flap endonuclease 1 (FEN1). This proposal will test the hypothesis that enhancement of FEN1-dependent DNA repair activity improves long-term neurological outcomes after cerebral ischemia by promoting the repair process of injured neurons and white matter. Positive results from this proposal may help identify a new neurorestorative mechanism for the prevention of permanent disability after stroke.
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