J. Regino Perez-Polo

Neonatal hypoxia-ischemia (HI) affects 60% of low birth weight infants and up to 40% of preterm births. Cell death and brain injury after HI have been shown to cause long-lasting neurological deficits. Two motor coordination tests on rats that had been exposed to HI on postnatal day 7 (P7) showed that HI in the P7 rat is associated with significant motor coordination impairment. These results call attention to the risks associated with perinatal ischemia and the need for proper treatment to reverse HI-induced deleterious effects.

Animal and human studies show that in‐utero exposure to preeclampsia alters fetal programming and results in long‐term adverse cardiovascular outcomes in the offspring. Human epidemiologic data also suggest that offspring born to preeclamptic mothers are also at risk of adverse long term neurodevelopmental outcomes. Pravastatin, a hydrophilic lipid‐lowering drug with pleiotropic properties, was found to prevent the altered cardiovascular phenotype of preeclampsia and restore fetal growth in animal models, providing biological plausibility for its use as a preventive agent for preeclampsia. In this study, we used a murine model of preeclampsia based on adenovirus over‐expression of the anti‐angiogenic factor soluble Fms‐like tyrosine kinase 1, and demonstrated that adult offspring born to preeclamptic dams perform poorly on assays testing vestibular function, balance, and coordination, and that prenatal pravastatin treatment prevents impairment of fetal programming.

The genotoxic, extracellular accumulation of amyloid β (Aβ) protein and subsequent neuronal cell death are associated with Alzheimer's disease (AD). APE1/Ref‐1, the predominant apurinic/apyrimidinic (AP) endonuclease and essential in eukaryotic cells, plays a central role in the base excision repair (BER) pathway for repairing oxidized and alkylated bases and single‐strand breaks (SSBs) in DNA. APE1/Ref‐1 is also involved in the redox activation of several trans‐acting factors (TFs) in various cell types, but little is known about its role in neuronal functions. There is emerging evidence for APE1/Ref‐1's role in neuronal cells vulnerable in AD and other neurodegenerative disorders, as reflected in its nuclear accumulation in AD brains. An increase in APE1/Ref‐1 has been shown to enhance neuronal survival after oxidative stress. To address whether APE1/Ref‐1 level or its association with other proteins is responsible for this protective effect, we used 2‐D proteomic analyses and identified cytoskeleton elements (i.e., tropomodulin 3, tropomyosin alpha‐3 chain), enzymes involved in energy metabolism (i.e., pyruvate kinase M2, N‐acetyl transferase, sulfotransferase 1c), proteins involved in stress response (i.e., leucine‐rich and death domain, anti‐NGF30), and heterogeneous nuclear ribonucleoprotien‐H (hnRNP‐H) as being associated with APE1/Ref‐1 in Aβ(25–35)‐treated rat pheochromocytoma PC12 and human neuroblastoma SH‐SY5Y cell lines, two common neuronal precursor lines used in Aβ neurotoxicity studies. Because the levels of some of these proteins are affected in the brains of AD patients, our study suggests a neuroprotective role for APE1/Ref‐1 via its association with those proteins and modulating their cellular functions during Aβ‐mediated neurotoxicity.

Hypoxic brain injury during fetal or neonatal development leads to damaged immature neurons and can result in cognitive or behavioral dysfunction. Hyperoxia therapy (treatment with oxygen) is commonly applied to infants with signs of perinatal hypoxia-anoxia. Both hypoxia and hyperoxia have been shown to result in apoptosis in the brains of rats in several animal models. One determinant of cellular commitment to cell death is the differential expression of the Bcl-2 family of proteins in response to trauma. Here, we characterize cell death and the expression of Bcl-2 homologous proteins in 7-day-old neonatal rat cerebral cortex after hypoxia (5% O(2) for 40 min) and/or hyperoxia (>95% O(2) for 2 h after hypoxia). The expression of Bcl-2 and Bcl-X(L), two anti-apoptotic proteins, decreased at 24 h after hypoxia. Bcl-X(L) increased after either hyperoxia or hypoxia+hyperoxia. We did not detect significant changes in the cytoplasmic levels of pro-apoptotic protein Bax after any of these three treatments. Using cell death ELISA and DNA FragEL assays, we observed increased cell death at 24h after hypoxia, hyperoxia or hypoxia+hyperoxia treatments. At 24 h after either hypoxia, hyperoxia or hypoxia+hyperoxia, caspase 3 activity also increased significantly. Our results suggest that both hypoxia and hyperoxia alone can induce cell death. The Bcl-2 --> cytochrome c --> caspase 3 pathway played a role in hypoxia-induced cell death, while other pathways may be involved in hyperoxia-induced cell death.

Signal transduction pathways that mediate neuronal commitment to apoptosis involve the nuclear factor κB (NF‐κB) transcription factor. Bcl‐X_L is a potent regulator of apoptosis in the CNS and is highly expressed in the developing and adult brain. We identified three putative NF‐κB DNA binding sequences clustered upstream of the brain‐specific transcription start site in the upstream promoter region. Recombinant p50/p50 and NF‐κB proteins from nuclear extracts bound to these sites as determined by electrophoretic mobility shift assay and biotin‐oligonucleotide/streptavidin affinity assays. NF‐κB overexpression, coupled with bcl‐x promoter/reporter assays using a series of murine bcl‐x promoter and deletion mutants, has identified the downstream 1.1 kb of the bcl‐x promoter as necessary for basal promoter activity and induction by NF‐κB. The mutagenic removal of NF‐κB binding sites individually or in combination revealed altered response patterns to p49/p65 and p50/p65 overexpression. These results support the hypothesis that NF‐κB can act to enhance Bcl‐X_L expression via highly selective interactions, where NF‐κB binding and bcl‐x promoter activation are dependent on both DNA binding site sequence and NF‐κB subunit composition. Our data suggest that molecular events associated with NF‐κB promote regulation of neuronal apoptosis in the developing or injured CNS.