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

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.

along with cytochrome c, maintaining a dynamic protein-protein interaction 14 . Cytochrome c (12 kDa), an important element of the electron transport chain in mitochondria is also involved in the initiation of apoptosis 15 . Cytochrome c is released into the cytosol and binds to the apoptotic protease activating factor-1 (Apaf-1) 16 . 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 . It has been demonstrated that this inhibition of IAPs allows for increased caspase activity, leading to cell death. Under physiological conditions, IAP is bound to caspases 3,7 and 9 resulting in their deactivation 15 . 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 . It has been previously demonstrated that HI results in increased expression of AIF in the cerebral cortex 20,21 .
Since the only clinically validated treatment for HI encephalopathy in term neonates is hypothermia but with only limited improvement in clinical outcomes, there is a need for the discovery of new signaling pathways for the development of therapeutic targets. In the term neonate affected by HI encephalopathy, the main areas of the brain affected are the hippocampus, sensory and motor cortex, basal ganglia, thalamus, and brain stem 22 . The HI insult may disrupt the formation of central motor pathways and alter the developmental plasticity of the newborn brain resulting in significant long term disabilities 23 .
Our previous work has indicated that HI activates the enzyme Src kinase [24][25][26] . 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.

Results
High Energy Phosphates. In order to ensure energy failure and significant HI, the levels of high energy compounds (ATP and PCr) were measured (μmoles/gr brain tissue). The ATP and PCr levels are shown in Table 1. The HI groups with and without PP2 were similar in the levels of ATP and PCr indicating that they were comparable in their degree of HI. Administration of Src kinase inhibitor (PP2) prior to HI did not restore ATP and PCr levels.
Neuropathology. The neuropathology mean score (M ± SD) was 0.33 ± 0.33 in Nx (p < 0.05 vs HI), 3.41 ± 0.34 in HI (p < 0.05 vs Nx) and 2.32 ± 0.23 in PP2 + HI (p < 0.05 vs HI). HI resulted in damaged neurons, pyknotic nuclei, ruptured or irregular bordered nuclear membranes and marked presence of apoptotic profiles. There was also pericellular edema and clustered inflammatory cells. However, Src kinase inhibition resulted in fewer damaged neurons, less karyorrhexis and pericellular edema, and thus attenuated neuronal cell injury compared to the group exposed to HI without Src kinase inhibition Fig. 1.

Mitochondrial Apoptotic Proteins.
In order to demonstrate the "leakage" of pre-apoptotic proteins from mitochondria to cytosol we measured the levels of cytochrome c [ Fig. 2], Smac/DIABLO [ Fig. 3] and AIF [Fig. 4] in both the cytosol and mitochondria [Fig. 5]. In mitochondria, compared to cytosol, the expression of cytochrome c, smac/DIABLO and AIF was increased during Nx and decreased during HI indicating a possible translocation of those proteins during HI from the mitochondria into the cytosol. However, inhibition of Src kinase did not result in any significant changes in the expression of the proteins inside the mitochondria. The results suggest that Src kinase has a regulatory role in preventing the "leakage" of the mitochondrial proteins into the cytosol. In the cytosolic compartment, cytochrome c, Smac and AIF expression was increased following HI compared to Nx (p < 0.05). However, administration of PP2 attenuated the HI-induced increased expression of each protein (PP2 + HI vs HI, p < 0.05; PP2 + HI vs Nx, p = NS).

Caspase-3 expression and activation.
We used immunohistochemistry to assess the expression of caspase-3 [ Fig. 6]. Normoxic animals stained negative for caspase-3 (Panel 6-A). HI was characterized by increased expression of caspase-3 (Panel 6-B). However, Src kinase inhibition resulted in attenuation of caspase-3 expression (Panel 6-C). The activity of Caspase-3 in the cytosolic fraction of the cerebral cortex of normoxic, hypoxic and hypoxic with PP2 is shown in Fig. 7. HI resulted in increased activity of Caspase-3 in the cytosolic fraction of the cerebral cortex of newborn piglets. Pretreatment with PP2 prevented the HI-induced increased activity of caspase-3 indicating that its activation is regulated by Src kinase Fig. 7.

Discussion
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. We also showed that HI resulted in translocation of pre-apoptotic proteins from the mitochondria into the cytosol. Inhibition of Src kinase prevented the increased cytosolic levels of cytochrome c, smac Study Groups ATP (μmoles/g brain) Phosphocreatine (μmoles/g brain) and AIF following HI suggesting that Src tyrosine kinase has a regulatory role in the protein trafficking of the mitochondrial membrane. The exact mechanism by which the aforementioned translocation occurs is a subject of current investigation although several theories have already been developed 12 .
In our laboratory we use the newborn piglet model of HI encephalopathy that shares comparative developmental ontogeny with the term newborn human brain. The distribution of brain injury following global HI in piglets is similar to the distribution of injury to human brain 27 . The newborn piglet is a reasonable model of neonatal encephalopathy due to its large size, presence of gyri and sulci, similar white/grey matter ratio and developmental age at term with human brain. Our group and other investigators [27][28][29][30][31][32][33][34][35][36][37][38] have used the newborn piglet model as it provides the advantage of real time measurements of physiologic parameters such as arterial blood gases and continuous blood pressure monitoring which allows titration of the FiO2 to achieve a precise and reproducible degree of HI 27,28,33,39 . Studies have shown that mitochondrial outer membrane permeabilization is the predominant form of apoptosis in the immature brain in comparison to the adult 40,41 . Mitochondria are implicated in numerous processes of the developing newborn brain (cellular powerhouse, programmed cell death, inflammation, formation of reactive oxygen species etc) 22  Photos were taken at ×200 and ×600 on the left, ×400 and ×600 in the middle panel and ×200 on the right panel.

Figure 2.
Representative cropped western blot image of cytochrome c levels expressed as optic density(OD/ mm 2 ) in the cytosol of the cerebral cortex of newborn piglets following 1 hour of HI and 2 hours of reoxygenation with and without pretreatment with PP2 (n = 5/group). HI resulted in increased expression of cytochrome c in the cytosol while pre-treatment with Src kinase inhibitor attenuated its expression.
cell death pathways and their regulation is crucial in order to identify targets and therapies to alleviate injury of the developing brain. Mitochondria are located in the cytosolic compartment and are embedded in the protein complexes of FAs system. The constantly changing morphology of the FAs enzymes leads to the opening of the mPTP and increased (mitochondrial outer membrane permeabilization(MOMP) 13 . Phosphorylation and activation of the Src kinase may promote the α9 dimerization, expand the mPTP, and allow for the leakage of apoptotic proteins into the cytosol. In contrast, when the enzyme is in its inactive form, Src kinase may block the opening of the mPTP, attenuate the swelling of the mitochondrial membrane and prevent the leakage of the mitochondrial proteins into the cytosol 7 . The increased level of AIF in neuronal cytosol is potentially due to the enhanced expression and translocation of the AIF from mitochondria to the cytosol. We therefore propose that Src kinase-mediated increased AIF expression in the cytosol during HI leads to increased DNA fragmentation and results in caspase-independent programmed neuronal cell death. Src kinase, by tyrosine phosphorylation of calmodulin at Tyr99, activates CaM kinase IV pathway and results to a CREB mediated expression of AIF.
The opening of the mitochondrial permeability transition pore (mPTP) through the inner and outer mitochondrial membrane, allows solutes, water and ions to enter the mitochondrial matrix 12,13 . The Bcl-2  family proteins may be either pro-apoptotic (Bax, BAD, Bak or Bok) or anti-apoptotic (Bcl-2, Bcl-xL, and Bcl-w). Specifically, activated Bax and Bak form pores at the outer mitochondrial membrane in giant unilamellar vesicles allowing transfer of larger molecular weight proteins such as Smac and cytochrome c 44 .
Protein tyrosine kinases, such as Src kinase, provide a multitude of intracellular signaling pathways including those related to cell survival and programmed cell death 45 . We have previously shown that Src kinase activation during HI occurs via an integrated feedback mechanism, possibly involving other kinases residing in the lipid membrane 46,47 . One of the fundamental initiating events in HI brain injury is the generation of nitric oxide (NO), free radicals and other reactive oxygen species that damage cell membranes, organelles, and even mitochondria 46 . Neuronal nitric oxide synthase (nNOS) is known to decrease the activity of phosphatases and would therefore promote the activation of kinases such as the Src kinase. This activation may further trigger caspase-mediated  programmed cell death via the release of mitochondrial protein in the HI newborn brain 16 , which in turn, can further activate caspase-independent pathways through AIF 48 . We propose that HI results in the activation of focal adhesion proteins such as Src kinase which regulates the leakage of the apoptogenic factors from the mitochondria into the cytosol. Subsequently, the cytochrome c, Smac and AIF lead to the activation of caspases -7,-9 and -3 which will result in DNA fragmentation and cell death Fig. 8.
In summary, the current study provides new insights into the regulation of trafficking of the mitochondrial apoptogenic factors by Src kinase. We showed that the HI-induced translocation of apoptotic proteins from the mitochondria into the cytosol is attenuated following inhibition of Src kinase. Together with our previous studies, these data further elucidate the effects of Src kinase on the apoptotic cascade, and identify Src kinase as a potential target to interrupt cell death pathways.  Pre-treatment with PP2 prevented the HI-induced increased activity of caspase-3 indicating that its activation is regulated by Src kinase. The caspase-3 activity is expressed as nmoles/mg protein/hr. HI studies were performed on Yorkshire piglets (Willow Glenn Farm in Strasburg, PA) on day of life 2 or 3. Anesthesia was used for all the procedures described below and all efforts were made to minimize pain and suffering. Newborn piglets were randomly divided into 3 groups (n = 5/group): normoxic, HI and HI pre-treated with PP2. The animals were anesthetized with Isoflurane (4%) and oxygen through a nostril cone and IV access was obtained within 5-10 minutes. Maintenance anesthesia was induced with Isoflurane 1.5%, Fentanyl 50 µg/ kg, Pancuronium: 0.3 mg/kg followed by oral endotracheal intubation. For the animals in HI + PP2 group, the PP2 was administered (1 mg/kg, IV) 30 minutes prior to HI in order to attenuate in vivo the Src activation in piglets 49 . The animals were ventilated for 1 hour under either normoxic (FiO2 = 0.21) or HI conditions; the later achieved by gradually lowering the FiO2 to 0.06 over 10 minutes. After 1 hour at FiO2 of 0.06, the HI piglets were subsequently re-oxygenated to Nx for two additional hours. Deep anesthesia was maintained throughout HI, recovery and brain harvesting. After the re-oxygenation period, the animals were either transcardially perfused with phosphate buffer saline (PBS) and paraformaldehyde for neuropathology studies or the brain was harvested and placed in liquid nitrogen at −80 °C for biochemical studies. There was a different process that was followed based on whether the animal would be used for biochemical or histopathology studies. For biochemical studies, at the conclusion of each experiment the cerebral cortex was harvested within five seconds following decapitation with a guillotine and a rapid midsagittal craniotomy with "scooping" of the brain parenchyma which was placed in liquid nitrogen. Then the brain was stored at −80° Celsius for subsequent biochemical analysis.

Materials and Methods
The piglets that were utilized for histopathology analysis, were perfused with paraformaldehyde and then decapitated. The brain was carefully dissected and placed on ice and then, areas of motor and sensory cortex, hippocampus, putamen and caudate nucleus were dissected, prepared and fixed.
Neuropathology. HI injury was confirmed histologically, by Hematoxylin and Eosin (H&E) stain and evaluation. Upon brain removal, mid-sagittal sections comprising of cerebrum, brainstem and cerebellum en block were fixed immediately by immersion in 10% neutral formalin, processed according to standard neurohistological procedures, and embedded in paraffin. Parasagittal sections corresponding to hippocampus were stained with H&E. Neuropathology was assessed by two independent pathologists and scores ranged from 0 (normal) to 4 (injured neurons >75% of high power field) as described by Hoque et al. 50 . Briefly, sections from motor cortex, somatosensory cortex, putamen, hippocampus, caudate nucleus and thalamus were used to evaluate for viable neurons, apoptotic profiles, pericellular edema, pyknotic nuclei and vascular thickening. Cell counting was performed by two different investigators blinded to treatment and clinical course in two specific areas identified using a pig brain atlas namely the CA1 region of ventral hippocampus and the putamen 51 . Morphological cell counting was carried out under light microscopy at 600× magnification using oil immersion lens. In the hippocampus, cells were counted in an ocular field centered on the pyramidal layer of sector CA1 and extending into the stratum oriens and stratum radiatum. In each animal, viable neurons were counted in 8 neighboring, non-overlapping ocular fields along the CA1 region. In the putamen, viable neurons were counted in 8 ocular fields selected randomly in a raster pattern starting in the dorsolateral region of the putamen and ending in the ventral putamen 50 .
Normal neurons were evaluated and counted using morphological criteria. Neurons with well demarcated round nuclei and containing a nucleolar profile were counted. Features of ischemic injury included nuclear hyperchromasia, nuclear pyknosis, cytoplasmic eosinophilia, cytoplasmic shrinkage, cytoplasmic micro-vacuolation, and cell homogenisation 50 .

Histopathology and Apoptotic Cell Identification. HI injury was confirmed histologically, by
Hematoxylin and Eosin (H&E) stain and the caspase-3 expression was determined by immuno-staining. Upon brain removal, mid-sagittal sections comprising brainstem and cerebellum en block were fixed immediately by immersion in cold (4 °C) 10% neutral formalin, processed according to standard neurohistological procedures, and embedded in paraffin. Parasagittal sections corresponding to the paravermis were stained with hematoxylin and eosin (H&E) and cresyl violet (CV), and serial (5 μm) adjacent sections, numbered in sequence, were used for immunostaining. Immunohistochemistry was performed on paraffin embedded material, using antibodies directed to caspase-3, using the three-step immunoperoxidase technique (Biocare Medical, Walnut Creek, CA, USA) as previously described 52 . Sections were counterstained either with methyl green or CV stains.
Biochemical Studies. Isolation of the Cytosolic Fraction. Cerebral tissue mitochondrial fraction was isolated by homogenizing one gram of cerebral cortical tissue by a Dounce-type glass homogenizer (seven strokes with pestle clearance 0.15 mm and seven strokes with pestle clearance 0.07 mm) in 30 ml of fresh isolation medium containing 0.32 M sucrose, 1 mM EDTA and 20 mM Tris-HCl buffer, pH 7.1. The homogenate was centrifuged for 3 min at 1500 × g and the resulting supernatant was centrifuged at 15,000 × g for 10 min to provide the crude mitochondrial pellet. The 15,000 × g supernatant was centrifuged at 100,000 × g for 60 min to obtain the cytosolic fraction 53 , and the protein concentration was determined 54 .
Determination of ATP and Phosphocreatine. Cerebral tissue HI was confirmed by determining the levels of high energy phosphates, ATP and PCr as previously described 55,56 . Briefly, frozen cortical tissue was powdered under liquid nitrogen, as previously described in 6% weight by volume perchloric acid 56 . The extract was thawed on ice and centrifuged at 2,000 g for 15 min at 4 °C. The supernatant was neutralized to a pH of 7.6 using 2.23 M K 2 CO 3 /0.5 M triethanolamine/50 mM EDTA buffer and then centrifuged at 2,000 g for 15 min at 4 °C. Supernatant (300 μL) was added to 1 ml of buffer (50 mM triethanolamine, 5 mM MgCl 2 , 2 mM EDTA, 2 mM glucose, pH 7.6) and 20 μL l NADP. The NADP concentration was 10 mg/ml in 50 mM Triethanolamine (TRA)-HCl buffer. Glucose-6-phosphate dehydrogenase (10 μL) was added and the samples were incubated and read after 8 min. Hexokinase (10 μL) was then added, absorbance readings were taken until steady state was reached and ATP concentration was calculated from the increase in absorbance at 340 nm. Next, ADP (20 μL) and creatine kinase (20 μL) were added to the solution. The samples were read every 5 min for 60 min and phosphocreatine concentrations were calculated from the increase in absorbance at 340 nm.
Caspase 3 activity. The activity of caspase-3 was determined spectrofluorometrically at 37 °C using specific fluorogenic substrate for caspase-3 (Ac-DEVD-AMC) obtained from Enzo Life Sciences. Continuous recording was performed at excitation 360 nm/emission 435 nm wave lengths. The activity was determined from the initial slope where the rate of reaction is linear and calculated using the amino-methyl-coumarin (AMC) as standard. The enzyme activity was expressed as nmoles/mg protein/hr. Statistical Analysis. Data on ATP, PCr, caspase-3 activity and expression, cytochrome c, Smac/DIABLO, AIF expression and neuropathology were analyzed using one way analysis of variance (ANOVA). A p value < 0.05 was considered significant. Pair-wise comparisons between the groups utilized Tukey-test. All values are presented as mean ± standard deviation (SD). The mean neuropathology scores were compared using Kruskal-Wallis One Way ANOVA on Ranks.

Limitations
The study was performed in 2-or 3-day old newborn piglets because of the practical barriers to obtain 1-day-old newborn piglets. It was not deemed feasible to perform all studies (biochemical assays and histopathology) on the same animals. Biochemical studies such as high energy compounds and enzyme assays require deep freezing of brain tissue to −80 °C at the time of harvest. Conversely, transcardial perfusion and tissue fixation is required for neuropathology. To overcome this limitation, we performed the same experimental process on a larger number of animals in each group in order to perform both histopathologic and biochemical studies. While the pretreatment with Src kinase inhibitor prior to HI may not readily translate to the clinical scenario of neonatal HI injury, the aim of the current study is to delineate the effect of Src kinase on the mitochondrial pre-apoptotic proteins. Future work will focus on the effect of such small molecule inhibitors when administered following the HI event.
Data Availability. The datasets generated during and/or analysed during the current study are available from Dr. Maria Delivoria-Papadopoulos' Laboratory at Drexel University College of Medicine.