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

Panagiotis Kratimenos,Ioannis Koutroulis,Beamon Agarwal,Stamatios Theocharis,Maria Delivoria-Papadopoulos

doi:10.1038/s41598-017-16983-1

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.

Highlights

HI disrupts the mitochondrial membrane, with leakage of mitochondrial pre-apoptotic proteins into the cytosol, including the Second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) and cytochrome c and Apoptosis Inducing Factor (AIF)[12,13]
The present study aims to assess the effect of Src kinase inhibition on mitochondrial apoptotic proteins and further delineate cellular mechanisms of HI induced cerebral injury in newborn piglets
We showed that HI resulted in decreased levels of apoptotic proteins in the mitochondria while the cytosolic level of those proteins significantly increased

Summary

Introduction

HI disrupts the mitochondrial membrane, with leakage of mitochondrial pre-apoptotic proteins into the cytosol, including the Second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) and cytochrome c and Apoptosis Inducing Factor (AIF)[12,13]. Smac is released concurrently with cytochrome c from mitochondria into the cytosol during apoptosis and re-activates the initiator and effector caspases by deactivating the inhibitor of apoptosis protein (IAP) mediated inhibition[17]. Apoptosis-inducing factor (AIF) is released upon the loss of mitochondrial membrane potential and translocates to the nucleus resulting in cell death in a caspase-independent pathway[18] by binding to the death receptor[19]. In the present study we investigated the regulatory mechanism by which the pre-apoptotic proteins translocate from mitochondria to the cytosol during HI through the Src kinase in the cerebral cortex of HI newborn piglets

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