Computational analysis of cortical neuronal excitotoxicity in a large animal model of neonatal brain injury.

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 Ca-influx. In the translational piglet model of hypoxic encephalopathy, we have previously shown that hypoxia overactivates Ca/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 Ca-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, Ca-influx, and Ca/CaM-dependent protein kinase kinase 2 (CaMKK2) expression. We then used SimBiology to build a computational model of the Ca/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 Ca 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 Ca-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 Ca-influx and Bax expression to similar levels as pre-treatment.

CONCLUSIONS

Our model identifies new dynamics of critical components in the Ca/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.