Effect of Src Kinase inhibition on Cytochrome c, Smac/DIABLO and Apoptosis Inducing Factor (AIF) Following Cerebral Hypoxia-Ischemia in Newborn Piglets

Abstract

We have previously shown that cerebral Hypoxia-ischemia (HI) results in activation of Src kinase in the newborn piglet brain. We investigated the regulatory mechanism by which the pre-apoptotic proteins translocate from mitochondria to the cytosol during HI through the Src kinase. Newborn piglets were divided into 3 groups (n = 5/group): normoxic (Nx), HI and HI pre-treated with Src kinase inhibitor PP2 (PP2 + HI). Brain tissue HI was verified by neuropathological analysis and by Adenosine Triphosphate (ATP) and Phosphocreatine (PCr) levels. We used western blots, immunohistochemistry, H&E and biochemical enzyme assays to determine the role of Src kinase on mitochondrial membrane apoptotic protein trafficking. HI resulted in decreased ATP and PCr levels, neuropathological changes and increased levels of cytochrome c, Smac/DIABLO and AIF in the cytosol while their levels were decreased in mitochondria compared to Nx. PP2 decreased the cytosolic levels of pre-apoptotic proteins, attenuated the neuropathological changes and apoptosis and decreased the HI-induced increased activity of caspase-3. Our data suggest that Src kinase may represent a potential target that could interrupt the enzymatic activation of the caspase dependent cell death pathway.

Epidermal Growth Factor Receptor Inhibition Reverses Cellular and Transcriptomic Alterations Induced by Hypoxia in the Neonatal Piglet Brain

“…Based on the biochemical results, we aimed to define the cellular effects of HX in the piglet cerebral cortex, and determined whether EGFRi pretreatment also affected HX-induced changes in cellular dynamics. First, we evaluated the overall cortical neuropathology following HX using H&E and a previously established and validated newborn piglet neuropathology score ( Hoque et al., 2014 ; Kratimenos et al., 2017a , 2017b ). The neuropathology score [median (interquartile range [IQR])] was 0 (0–1) in NX (n = 5) (p < 0.05 versus HX), 4 (3–4) in HX (n = 6) (p < 0.05 versus NX), and 2 (1–3) in EGFRi-HX (n = 7) (p < 0.05 versus HX) ( Figures 2 A–2C).…”

“…We aimed to explore whether EGFR interferes with specific signaling pathways activated by HX in the piglet brain ( Chakraborty et al., 2014 ; Chin et al., 1997 ; Ferriero, 2004 ; Jackson and Ceresa, 2017 ; Kratimenos et al., 2017a ; Soderling, 1999 ; Treda et al., 2016 ). As calcium plays a central role in cell signaling, we focused on the relationship between EGFR activation and intracellular Ca 2+ .…”

“…The activity of nuclear CaMKIV was enhanced by HX, but attenuated following EGFR inhibition, indicating that EGFR function also impacts nuclear function. In our previous studies, we demonstrated that inhibition of Src kinase following HX leads to attenuated neuropathological alterations and reduced activation of the nuclear enzymes ( Kratimenos et al., 2017a , 2017b ). Furthermore, decreased activation of CaMKIV is reflected in improved neuropathology in the neonatal brain ( Kratimenos et al., 2017b ).…”

Src Family Kinases Inhibition Ameliorates Hypoxic-Ischemic Brain Injury in Immature Rats

“…PP2 administration improved the motor, balance, and reflex functions and increased the distance traveled and frequency of exploratory behaviors of HI rats. In an HI model constructed using neonatal piglets, intravenous injection of PP2 reduced the cytoplasmic levels of preapoptotic proteins and apoptosis, ameliorated neuropathological changes, and reduced HI-induced caspase-3 activity, possibly reducing nerve cell death (Kratimenos et al, 2017). In another study using a neonatal piglet HI model, hypothermia and PP2 administration decreased apoptosis and attenuated neuropathology after HI (Kratimenos et al, 2018).…”

Aquaporin 4 and brain-related disorders: Insights into its apoptosis roles

“…Bcl-xL has a neuroprotective effect against brain ischemia (Cao et al, 2002[ 7 ]). Elevation of AIF, cytochrome c and Smac/DIABLO is also inherent to cerebral ischemia (Kratimenos et al, 2017[ 40 ]).…”