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.

Neuroprotective effect of Src kinase in hypoxia-ischemia: A systematic review

“…Two studies (n studies = 2, 10%) included both histologic and neurobehavioral outcomes, eleven studies (n studies = 11, 55%) only biochemical outcomes and six (n studies = 6, 30%) reported both biochemical and histologic outcomes. It is worth mentioning that one study (n studies = 1, 5%) utilized experimental data to create and validate a computational model of the critical intracellular signaling components of HI in neonatal brain (Kratimenos et al, 2022 ).…”

“…Our computational model indicated that Src-i disassociates Ca 2+ influx from Bax expression and modulates the interaction between the NMDAR and Src reducing Bax expression (Kratimenos et al, 2022 ). This model could provide a translational platform to design and screen drugs in neonatal hypoxic brain (Kratimenos et al, 2022 ).…”

“…Moreover, as therapeutic hypothermia is considered standard of care for HI in neonates, the additive effects of a combined hypothermia/Src kinase inhibition protocol should be further investigated (Kratimenos et al, 2018 ). Recently, our team validated a computational model with experimental measurements of critical intracellular signaling components and captured key molecular trends in this pathway (Kratimenos et al, 2022 ). Our computational model indicated that Src-i disassociates Ca 2+ influx from Bax expression and modulates the interaction between the NMDAR and Src reducing Bax expression (Kratimenos et al, 2022 ).…”

“…Recently, our team validated a computational model with experimental measurements of critical intracellular signaling components and captured key molecular trends in this pathway (Kratimenos et al, 2022 ). Our computational model indicated that Src-i disassociates Ca 2+ influx from Bax expression and modulates the interaction between the NMDAR and Src reducing Bax expression (Kratimenos et al, 2022 ). This model could provide a translational platform to design and screen drugs in neonatal hypoxic brain (Kratimenos et al, 2022 ).…”

“…Hossain et al demonstrated that Src kinase activation improves neuronal survival in primary cortical cell cultures (Paul et al, 2001 ; Haass and Mandelkow, 2010 ; Ittner et al, 2010 ; Liu and Sharp, 2011 ; Hossain et al, 2012 ), whereas several other studies have shown that Src kinase phosphorylation causes neuronal damage in ischemic stroke, intracerebral hemorrhage, and Alzheimer's disease (Haass and Mandelkow, 2010 ; Ittner et al, 2010 ; Liu and Sharp, 2011 ; Hossain et al, 2012 ). Porcine experimental models have been used to examine the deleterious effects of Src kinase in neonatal HI and have shown that it can induce the production of free radicals, causing secondary inflammation and excitotoxicity (Kratimenos et al, 2017 , 2018 , 2022 ).…”