Apoptosis-related mechanisms in the immature brain after hypoxia-ischemia

Abstract: Aims: To characterize the activation of caspases-3 and extracellular signal-regulated kinase (ERK) in the neonatal brain after hypoxia-ischemia (HI) injury and evaluate the neuroprotective effect of X-linked inhibitor of apoptosis (XIAP) protein, erythropoietin (EPO) and the non-erythropoietic derivative asialoEPO. Methods: Seven-day-old rats or 9-day-old mice were subjected to HI. At certain time points, animals were sacrificed and brains were collected for measurement of enzyme activity, immunoblotting, immunohistochemistry, immunoprecipitation, brain damage assessment and proteomic analysis.Results: In the normal control animals, basal caspase-3-like activity decreased 49% with age from postnatal day 10 to 13. After HI, the neonatal rat brain displayed a 25-fold increased caspase-3-like activity 24h post-HI. Transgenic overexpression of XIAP (TG-XIAP) produced significant neuroprotection after HI, reducing the brain tissue loss from 54.4 ± 4.1 mm3 (mean ± SEM) in wild type mice to 33.1 ± 2.1 mm3 in the TG-XIAP mice. Caspase-3-like and caspase-9-like activity was significantly reduced in the TG-XIAP mice after HI. ERK activation (P-ERK) was evident immediately after HI as judged by immunoblotting and immunohistochemistry in multiple brain regions, peaking at 30 min to 1 h post-HI. The P-ERK-positive cells co-localized with injury markers. Systemic administration (i.p.) of EPO or asialoEPO produced 52% and 55% reduction of brain infarct volume, respectively, although the plasma levels of asialoEPO had dropped below the detection limits (1 pM) at the onset of HI and those of EPO were in the nM range. In an attempt to identify the relevant molecular mechanisms responsible for this, up-regulation of synaptosomal-associated protein-25kDa (SNAP-25) was identified in the phosphoproteome, and ERK activation was significantly reduced in the asialoEPO treatment animals after HI.Conclusions: 1. Caspases and apoptotic mechanisms may be more important in the immature brain because of the higher levels of caspases during development. 2. Significant neuroprotection after neuronal XIAP overexpression indicates that administration of XIAP-related peptides might prove a useful strategy for neuroprotection after asphyxia in neonates. 3. ERK activation in neurons after HI in the neonatal brain occurred early, and mainly in cells displaying signs of damage. 4. EPO and the non-erythropoietic derivative asialoEPO both provided significant neuroprotection when administered 4 h prior to HI. Considering that EPO is already used in clinical, pediatric use, EPO-related compounds may quickly find their way into therapeutic applications aimed at neuroprotection.