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

Abstract

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.

Keywords: Calcium/calmodulin; Computational modeling; Excitotoxicity; Neonatal brain injury; Nuclear calcium; SimBiology; Src kinase.