RESUMO
Background: Hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal morbidity and mortality worldwide. While the application of therapeutic hypothermia has improved neurodevelopmental outcomes for some survivors of HIE, this lone treatment option is only available to a subset of affected neonates. Src kinase, an enzyme central to the apoptotic cascade, is a potential pharmacologic target to preserve typical brain development after HIE. Here, we present evidence of the neuroprotective effects of targeting Src kinase in preclinical models of HIE. Methods: We performed a comprehensive literature search using the National Library of Medicine's MEDLINE database to compile studies examining the impact of Src kinase regulation on neurodevelopment in animal models. Each eligible study was assessed for bias. Results: Twenty studies met the inclusion criteria, and most studies had an intermediate risk for bias. Together, these studies showed that targeting Src kinase resulted in a neuroprotective effect as assessed by neuropathology, enzymatic activity, and neurobehavioral outcomes. Conclusion: Src kinase is an effective neuroprotective target in the setting of acute hypoxic injury. Src kinase inhibition triggers multiple signaling pathways of the sub-membranous focal adhesions and the nucleus, resulting in modulation of calcium signaling and prevention of cell death. Despite the significant heterogeneity of the research studies that we examined, the available evidence can serve as proof-of-concept for further studies on this promising therapeutic strategy.
RESUMO
BACKGROUND: Neonatal hypoxic brain injury is a major cause of intellectual and developmental disability. Hypoxia causes neuronal dysfunction and death in the developing cerebral cortex due to excitotoxic Ca2+-influx. In the translational piglet model of hypoxic encephalopathy, we have previously shown that hypoxia overactivates Ca2+/Calmodulin (CaM) signaling via Sarcoma (Src) kinase in cortical neurons, resulting in overexpression of proapoptotic genes. However, identifying the exact relationship between alterations in neuronal Ca2+-influx, molecular determinants of cell death, and the degree of hypoxia in a dynamic system represents a significant challenge. METHODS: We used experimental and computational methods to identify molecular events critical to the onset of excitotoxicity-induced apoptosis in the cerebral cortex of newborn piglets. We used 2-3-day-old piglets (normoxic [Nx], hypoxic [Hx], and hypoxic + Src-inhibitor-treatment [Hx+PP2] groups) for biochemical analysis of ATP production, Ca2+-influx, and Ca2+/CaM-dependent protein kinase kinase 2 (CaMKK2) expression. We then used SimBiology to build a computational model of the Ca2+/CaM-Src-kinase signaling cascade, simulating Nx, Hx, and Hx+PP2 conditions. To evaluate our model, we used Sobol variance decomposition, multiparametric global sensitivity analysis, and parameter scanning. RESULTS: Our model captures important molecular trends caused by hypoxia in the piglet brain. Incorporating the action of Src kinase inhibitor PP2 further validated our model and enabled predictive analysis of the effect of hypoxia on CaMKK2. We determined the impact of a feedback loop related to Src phosphorylation of NMDA receptors and activation kinetics of CaMKII. We also identified distinct modes of signaling wherein Ca2+ level alterations following Src kinase inhibition may not be a linear predictor of changes in Bax expression. Importantly, our model indicates that while pharmacological pre-treatment significantly reduces the onset of abnormal Ca2+-influx, there exists a window of intervention after hypoxia during which targeted modulation of Src-NMDAR interaction kinetics in combination with PP2 administration can reduce Ca2+-influx and Bax expression to similar levels as pre-treatment. CONCLUSIONS: Our model identifies new dynamics of critical components in the Ca2+/CaM-Src signaling pathway leading to neuronal injury and provides a feasible framework for drug efficacy studies in translational models of neonatal brain injury for the prevention of intellectual and developmental disabilities.
