PROTEIN KINASE C SIGNALING IN
NEURODEGENERATION
A dissertation submitted
to Kent State University in partial
fulfillment of the requirements for the
degree of Doctor of Philosophy
By
Varun Kumar
May, 2016
© Copyright
All rights reserved
Except for previously published materials
Dissertation written by
Varun Kumar
B.Sc., University of Delhi, 2008
M.Sc., Devi Ahilya University, 2010
Ph.D., Kent State University, 2016
Approved by
Wen-Hai Chou , Chair, Doctoral Dissertation Committee
Srinivasan Vijayaraghavan , Members of Doctoral Dissertation Committee
Alexander L. Mdzinarishvili ,
Werner J. Geldenhuys ,
Gail C. Fraizer ,
Accepted by
Ernest J. Freeman , Director, School of Biomedical Sciences
James L. Blank , Dean, College of Arts and Sciences
ii TABLE OF CONTENTS……………………………………………………………………iii
LIST OF FIGURES………………………………………………………………………….vi
LIST OF ABBREVIATIONS……………………………………………………………….viii
ACKNOWLEDGEMENTS………………………………………………………………….x
CHAPTERS
1. Introduction……………………………………………………………………………...1
1.1 Protein Kinase C………………………………………………………………….1
1.2 Mode of PKC regulation………………………………………………………….2
1.3 Molecular targets in preconditioning……………………………………………..2
1. 4 PKC in ischemia………………………………………………………………...5
1.5 Activating Transcriptional factor 2………………………………………….…....6
1.6 Regulation of ATF2……………………………………………………………….7
1.7 Role of ATF2 in diseases…………………………………………………………10
1.8 The chemical-genetics approach…………………………………………….……11
1.9 Overview…………………………………………………………..…………….. 14
2. Determine the effect of PKCε deficiency following global cerebral ischemia………16
2.1 Introduction and rational……………………………………………………….....16
2.2 Material and methods……………………………………………………………..20
2.3 Results…………………………………………………………………………….25
2.3.1 Determine hippocampal neuronal degeneration in PKCε WT and KO mice
after global cerebral ischemia…………………………………………………25
2.3.2 Determine hippocampal neuronal cell death in PKCε WT and KO mice after
iii global cerebral ischemia………………………………………………………27
2.3.3 Determine the cerebrovascular anatomy in PKCε WT and KO mice………...29
2.3.4 Determine the regional cerebral blood flow before and after global cerebral
ischemia in PKCε WT and KO mice..………………………………….……. 31
2.3.5 Determine the physiological parameters for PKCε WT and KO mice………...33
2.4 Conclusion……………………………………………………………………..….36
3. Determine the molecular mechanisms of cell death after global cerebral ischemia..37
3.1 Introduction and rational..…………………………………………………………....37
3.2 Material and methods………………………………………………………………...40
3.3 Results………………………………………………………………………………..42
3.3.1 Determine the phosphorylation of ATF2 at Thr52 in mouse hippocampus……42
3.3.2 Determine temporal expression profile of PKCε in mouse hippocampus before
and after global cerebral ischemia……………………………..…………..……44
3.3.3 Determine temporal expression profile of ATF2 in mouse hippocampus before
and after global cerebral ischemia…………………………………..……….…46
3.3.4 Determine cytochrome c expression in mouse hippocampus after global cerebral
ischemia……………………………………………………………..………….48
3.4 Conclusion………………………………………………………………..………….50
4. Generation and characterization of ATP analog-specific PKCδ homology model in
silico…………………………………………………………………………………….51
4.1 Introduction and rational..…………………………………………………….……51
4.2 Material and methods………………………………………………………………52
iv 4.3 Results…………………………………………………………………………...... 54
4.3.1 Generate human PKCδ homology model using MOE software……………...54
4.3.2 Perform docking studies for substrates (N6-(benzyl)-ADP) and PP1 derived
inhibitors in the PKC homology model……………………………………..57
4.4 Conclusion…………………………………………………………………………65
5. Discussion and future directions……………………………………………………66
5.1 Discussion………………………………………………………………………….66
5.2 Future directions…………………………………………………………………...72
6. Bibliography………………………………………………………………………….74
v
LIST OF FIGURES
Figure 1. Schematic representation of regulation of ATF2 via PKC Figure 2. The chemical-genetics approach………………………………………………...... 13 Figure 3. Hippocampal neurodegeneration in PKCε+/+ and PKCε-/- mice after global cerebral
ischemia……………………………………………………………………………….26
Figure 4. Hippocampal neuronal cell death in PKCε+/+ and PKCε-/-
ischemia………………………………………………………………………………28
Figure 5. Assessment of cerebrovascular anatomy……………………………………………..30
Figure 6. Determination of regional cerebral blood flow (rCBF)…………………………...... 32
Figure 7. ATF2 (Thr52) phosphorylation by PKC in vivo……………………………………..43
Figure 8. Cytosolic PKC decreased in mouse hippocampus after global cerebral ischemia…..45
Figure 9. Temporal expression of ATF2 in mitochondria for PKCε+/+ and PKCε-/- mice after
global cerebral ischemia……………………………………………………………...47
Figure 10. Decreased release of cytochrome c in the cytosol for PKCε-/- mice after global
cerebral ischemia ………………………………………………………..…………..49
Figure 11. ATP representation in the nucleotide-binding pocket of WT and AS-PKCδ…...... 55
Figure 12. ATP interactions in the nucleotide-binding pocket of WT and AS-PKCδ…………..56
vi Figure 13. Comparison of N6-(benzyl)-ADP (BZ-ADP) interactions in the nucleotide-binding
pocket of AS and WT-PKCδ………………………………………………………..59
Figure 14. Comparison of 1 NA-PP1 interactions in the nucleotide-binding pocket of WT and
AS-PKCδ…………………………………………………………………………….60
Figure 15. Interactions of 1NM-PP1 and 2NM-PP1 with AS-PKCδ…………………………...61
Figure 16. Interactions of 1NA-PP1 and 1NM-PP1 with AS-PKA…………………………….62
Figure 17. Interactions of 2MB-PP1 with AS-PKCδ…………………………………………...63
Figure 18. Interactions of N6-(benzyl)-ATP and 1NA-PP1 with mAC…………………………64
Figure 19. Model for apoptosis after global cerebral ischemia………………………………….71
vii LIST OF ABBREVIATIONS
ACA anterior cerebral artery
AP1 activator protein 1
AS analog-specific
ATF2 activating transcription factor 2
ATP adenosine triphosphate
BCCAO bilateral common carotid artery occlusion bZIP basic leucine zipper domain
CREB c-AMP response element-binding protein
DAG diacylglycerol
ERK extracellular signal regulated kinase
FJC fluoro-jade c
HK1 hexokinase-1
HSP heat shock proteins
IPC ischemic preconditioning
IP3 inositol triphosphates
JNK c-Jun N-terminal kinase mAC mammalian adenylyl cyclase
MANT 2’(3’)-O-(N-methylanthraniloyl)
MAPK mitogen-activated protein kinase
MCA middle cerebral artery
2MB-PP1 1-(tert-butyl)-3-(2-methylbenzyl)-1H-pyrazolo[3,4-d]- pyrimidin-4-amine
1NA-PP1 1-(tert-butyl)-3-(1-naphthyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
viii 1NM-PP1 1-tert-butyl-3-(1-naphthalenylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
2NM-PP1 1-tert-butyl-3-(2- naphthalenylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine
NES nuclear export signal
OGD oxygen glucose deprivation
PcomA post-communicating artery
PDB protein data bank
PFA paraformaldehyde
PIP2 phosphatidylinositol 4,5-bisphosphate
PMA phorbol 12-myristate 13-acetate
PNBM para-nitrobenzyl mesylate
PP1 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine
RACK receptor for activated C kinase rCBF regional cerebral blood flow
ROS reactive oxygen species
TNF tumor necrosis factor
VDAC1 voltage-dependent anion-selective channel protein 1
ix ACKNOWLEDGEMENTS
First of all, I would like to express my sincere gratitude to my advisor, Dr. Wen-Hai
Chou, for his continuous guidance and encouragement during my time in the lab. He was always there for me when I needed help in my work. He constantly motivated me to be inquisitive and think independently in all my experiments. I am deeply indebted to his endeavors for providing a great research environment in the laboratory. I also appreciate his patience for other matters not related to research. It has really been a great time working in his laboratory.
I would also like to thank all my committee members: Dr. Alexander Mdzinarishvili, Dr.
Srinivasan Vijayaraghavan, Dr. Werner J. Geldenhuys and Dr. Gail C Fraizer for continuous support, constructive comments and encouragement during my PhD. I really appreciate their time and patience when needed during my stay at Kent State University. I also express my sincere gratitude to Dr. Tibor Kristian for his help in establishing mouse model of global cerebral ischemia.
My sincere thanks to Dr. Yi-Chinn Weng, who provided constructive insights into my experiments. She took care of everything when needed during my PhD. I doubt that I would be able to convey my appreciation fully, but I owe her my eternal gratitude. I would also like to thank my fellow lab mates, Guona Wang, Xiqian Han, JD, Isabella, Vivek, and Supreet for their support in my experiments.
Last but not the least, I would like to thank my parents, sisters, brother-in-law and all my cousins for their continuous support for what I do. I would like to express my special gratitude to my father, who always encourages and appreciates my work better than me. Words are not enough to express my gratitude fully to all my family members, who were always there for me.
x CHAPTER 1
Introduction
1.1 Protein Kinase C
Protein Kinase C (PKC) is a family of 10 serine-threonine kinases that regulate a broad spectrum of cellular functions (Newton and Messing, 2010; Steinberg, 2008). They are lipid-sensitive enzymes that are activated by growth factor receptors. These growth factor receptors stimulate phospholipase C (PLC), the enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate
(PIP2) to generate membrane-bound diacylglycerol (DAG) and inositol trisphosphate (IP3).
DAG activates PKC whereas, IP3 mobilizes intracellular calcium. Tumor-promoting phorbol esters such as phorbol 12-myristate 13-acetate (PMA) also activate PKCs.
All PKC family proteins contain a diverse regulatory domain in the amino-terminal region, followed by a flexible hinge and a conserved catalytic domain in the carboxyl-terminal tail. The catalytic domain is composed of two lobes. The amino-terminal lobe contains a glycine- rich loop of the consensus GXGXXGX sequence and an invariant Lys that positions ATP for phosphoryl transfer. The carboxyl-terminal lobe contains an activation loop that binds protein substrates for catalysis. The sequence linking these two lobes contains a large conserved hydrophobic residue called gatekeeper (Met or Ile in PKC isozymes). The gatekeeper amino acid residue limits the size of the hydrophobic region within the ATP binding pocket and confers selectivity for binding nucleotides and small molecule inhibitors (Steinberg, 2008). Based on
1 their amino-terminal structures and sensitivities to Ca2+ and DAG, the PKCs are classified into conventional PKCs (, I, II and ), novel PKCs (, , , and ), and atypical PKCs ( and /).
1.2 Mode of PKC regulation
According to the classical model of PKC activation, translocation to membrane indicates PKC activation (Chen et al., 1999). However, there have been other modes of PKC activation as well.
Growth factor receptors, pharmacological agents (PMA) (Tahara et al., 2009), lipid co-factors
(ceramide or arachidonic acid) (Huwiler et al., 1998) etc. activate PKCs. PKCs are also controlled through tyrosine modification (Konishi et al., 1997) or oxidative modifications
(Gopalakrishna and Jaken, 2000), phosphorylation (Keranen et al., 1995) on serine/threonine or tyrosine amino acid residues, which regulates their stability, protein-protein interaction, substrate specificity and subcellular targeting. They can also be cleaved by caspases, leading to their catalytically active form.
1.3 Molecular targets in preconditioning
PKC isoforms have been involved in many neurological diseases (Chou and Messing, 2008). Out of many neurological diseases, ischemic heart disease remains one of the leading causes of death in the western countries (Go et al., 2013). Cardiac arrest is one of the major ischemic heart diseases, which results in global cerebral ischemia (Kawai et al., 1992). The harmful effects after global cerebral ischemia are neurodegeneration (Onken et al., 2012) and the delayed neuronal cell death (Kirino, 2000). Out of many therapeutic approaches investigated yet, one of them is ischemic preconditioning (IPC), which is sufficient to protect the brain tissues from subsequent lethal ischemic insult (Blanco et al., 2006). Various lines of evidence suggest that PKCε is
2 activated during ischemic or pharmacological preconditioning (Blanco et al., 2006; Raval et al.,
2003; Dave et al., 2008; Kim et al., 2007; Lange-Asschenfeldt et al., 2004; Raval et al., 2007).
Activation of PKCε by peptide activator [ψεRACK] confers neuroprotection, possibly by PKCε phosphorylation of complex I and COX-IV, mitochondria K+ ATP channel (Kir6.2) (Raval et al.,
2007), dephosphorylation of complex III (Dave et al., 2008), and ERK1/2 activation (Kim et al.,
2007).
Several modes of IPC-mediated neuroprotection have been reported. One of them is through activation of glutamate receptors in preconditioning (Rothman and Olney, 1986; Sattler et al., 2000). In vivo studies in gerbils have suggested that inhibition of glutamate receptors with
MK-801 abrogates ischemic preconditioning-mediated neuroprotection (Kato et al., 1992). The other mode of IPC-mediated neuroprotection is through redistribution of PKCε to subcellular neuroprotective targets such as synaptic mitochondria, which further leads to the modulation of the phosphorylation status of mitochondria electron transport chain (Dave et al., 2008). The increased activity of mitochondria complexes elicited by PKCε activation after IPC improves the calcium buffering in mitochondria, thereby ameliorating glutamate-mediated excitotoxicity.
Furthermore, increased hyperpolarization after IPC may protect synaptic mitochondria by diminishing depolarization during ischemia (McCarthy et al., 2005). As described above, PKCε also phosphorylates mitochondria K+ ATP channel (Kir6.2), resulting in its opening during IPC
(Raval et al., 2007). This leads to influx of K+ ions leading to generation of reactive oxygen species (ROS), thereby activating phospholipase and PKC. This further amplifies the preconditioning effect.
PKCε activation during preconditioning also results in the functional modification of the
GABA synapses (increase of GABAA receptor-mediated miniature postsynaptic currents). This
3 leads to neuroprotection in the rat hippocampus after oxygen glucose deprivation (OGD)
(DeFazio et al., 2009). This study has a clinical application i.e. endogenous GABAA agonist may act as a potential therapeutic agent in vulnerable CA1 hippocampal pyramidal cells in order to ameliorate the severity of neurological outcome after stroke or cardiac arrest. Moreover, activation of GABAA receptors during lethal ischemia has been proposed as mechanism for acute isoflurane-mediated neuroprotection as well (Bickler et al., 2003). During IPC, PKCε also targets
Adenosine A1 receptors and NMDA receptors through PKC-ERK pathway in the organotypic hippocampal slices (Lange-Asschenfeldt et al., 2004).
Heat shock proteins (HSP) are induced under various stressful conditions like environmental stress (UV radiation, heavy metals), pathological conditions (infections or malignancies), physiological conditions (growth factors or cell differentiation). HSP70 is the most abundant HSP in cells. There have been reports of increased expression of HSP 70 after ischemic preconditioning (McLaughlin et al., 2003). Also, thermal preconditioning (42oC) modulates HSP70 expression and protects cerebellar granule cells of rats (Chen et al., 2004).
ERK phosphorylation implicated in preconditioning regulates many transcription factors, which in turn regulates HSP70 expression. Moreover, overexpression of HSP70 protects neuronal cells from subsequent lethal ischemic insult (Yenari et al., 1999). However, it is unknown how PKC might be modulating HSP70 during preconditioning.
Inflammatory cytokines (Interleukins-1, TNF-) are also implicated in ischemic preconditioning (Kariko et al., 2004). Tumor necrosis factor (TNF) plays a neuroprotective role after acute brain ischemic insults (Gary et al., 1998). Also, hypoxic preconditioning protects cultured neurons against hypoxic stress via TNF- (Liu et al., 2000). However, the role of PKC mediated preconditioning in the regulation of the inflammatory cytokines is not known yet.
4 These studies have contributed significantly in understanding the basic mechanism of preconditioning and the phosphorylation substrates of PKCε for neuroprotection. However, the use of preconditioning as a therapeutic approach has not become a standard clinical practice because the occurrence of cardiac arrest and cerebral ischemia is sudden and unpredictable.
Thus, therapeutic approaches that can be applied after ischemia have to be unraveled.
1.4 PKCε in ischemia
The role of PKCε after stroke has been controversial due to variable expression and activity of
PKCε (Reshef et al., 2000; Tauskela et al., 1999). Some studies suggest that PKCε is up- regulated after ischemia (Selvatici et al., 2003; Wang et al., 2004), while others do not suggest its up-regulation (Savithiry and Kumar, 1994; Harada et al., 1999). Differences in the ischemic insult duration, severity of different ischemia models, cells types involved and time points selected for assessing PKCε activity may have resulted in the conflicting role of PKCε after ischemia. Early PKCε activation via systemic delivery of [ψεRACK] (PKCε peptide activator) has a neuroprotective effect against subsequent focal cerebral ischemia in rats (Bright et al.,
2008). Moreover, post ischemic PKCε activation via [ψεRACK] is also neuroprotective against global cerebral ischemia via reducing the cerebral blood flow during the initial phase of reperfusion in rats (Della-Morte et al., 2011). PKCε role in the regulation of nitric oxide synthase associated with vascular tone may be responsible for this neuroprotective effect. However, the therapeutic time window for this study is limited to 30 minutes after ischemia.
PKCε has been implicated in hypothermia-mediated neuroprotection. A report suggests that the neuroprotective effect of hypothermia is mediated by preserving PKCε activity in the rat focal cerebral ischemia model (Shimohata et al., 2007). The mechanism in which hypothermia
5 inhibits PKCε cleavage and preserves its activity, is not well understood. However, PKCε cleavage is not mediated by caspase 3, but by other proteases which are unknown yet (Shimohata et al., 2007). However, the preservation of PKCε as secondary effect of hypothermia cannot be excluded. Moreover, it still remains elusive whether intra- or inter-ischemic hypothermia confers neuroprotection. Also, there are other activators of PKCε, which improves neurological outcome after stroke. Post-ischemic/hypoxia PKCε activation via bryostatin-1 prevents ischemia- induced changes in synaptogenesis, neurotropic activity and spatial learning and memory in adult rat brain (Sun et al., 2009). The therapeutics involving PKCε activation may actually extend the therapeutic time window for treatment of stroke.
In conclusion, the role of PKCε after ischemia is controversial and remains unclear.
Moreover, the effect of PKCε deletion after global cerebral ischemia has not been studied yet.
Therefore, we investigate the role of PKCε after global cerebral ischemia by using PKCε KO mice.
1.5 Activating Transcription Factor 2 (ATF2)
In order to fully assess PKCε as a therapeutic target after ischemia, PKCε phosphorylated substrates have to be identified. Over the years, several substrates such as mitochondria K+ ATP channel, (Raval et al., 2007), and ERK1/2 (Kim et al., 2007) have been investigated during preconditioning. However, direct phosphorylation substrates of PKCε after ischemia have not been reported. One of the potential substrates of PKCε is ATF2. The activation transcription factor (ATF: also known as cAMP-dependent transcription factor) belongs to activator protein 1
(AP1) transcription factor superfamily. The ATF2 gene is located on chromosome 2q32 and encodes a 505 amino acid protein. It is ubiquitously expressed and found abundantly in the brain
6 (Kara et al., 1990). Like other AP-1 transcription factors, ATF-2 protein has a basic leucine zipper domain (bZIP) within its C terminal (amino acids 350-414) that enables homo- or hetero- dimerization. The bZIP domain also contains nuclear localization and export sequences that facilitate trafficking of ATF2 out of the nucleus. Moreover, ATF-2 also contains N terminal zinc finger region and a transactivation domain, which regulates its transcriptional activity (Nagadoi et al., 1999). The transactivation domain of ATF2 binds to its own N terminal zinc finger region and blocks its own transcriptional activity through intramolecular autoinhibitory interaction (Li and
Green, 1996).
1.6 Regulation of ATF2
ATF2 is transcriptionally regulated via phosphorylation on Thr69 and Thr71 by JNK and p38.
(Livingstone et al., 1995). The phosphorylation-induced transcriptional activity may also be achieved by increasing the intrinsic histone acetylase activity of ATF2, thereby promoting DNA binding (Fuchs et al., 1997). This further prevents it from ubiquitination. Moreover, the transcriptional activity of ATF2 is also regulated by an intramolecular interaction between N- terminal transactivation domain and basic leucine zipper domain (Li and Green, 1996). ATF2 has two nuclear localization signal and one nuclear export signal in the basic region. Recently, a study suggests that a hydrophobic stretch in the N-terminal of ATF2 also acts as nuclear export signal (Hsu and Hu, 2012). While the mutation of both the nuclear export signal results in the predominant nuclear localization of ATF2, the mutation of only one nuclear export signal (NES) only partially increases its nuclear localization. Moreover, mutation of the N-terminal NES enhances the transcriptional activity of ATF2, which suggests that the novel NES negatively regulates the transcriptional potential of ATF2.
7 Moreover, ATF2 is a nucleocytoplasmic shuttling protein and its subcellular localization is regulated by dimerization with c-Jun (Li and Green, 1996). Homo- or Hetero-dimerization of
ATF2 is the key for its activation. ATF2 and c-AMP Response Element Binding Protein (CREB) proteins can homo- or hetero-dimerize with members of the activator protein 1 (AP1) transcription factor family proteins to form complexes. Like other basic leucine zipper transcription factors, it utilizes its basic region to bind DNA and its leucine zipper region to dimerize with its partner. ATF-2 binds the cAMP-response element, T (G/T) ACGTCA, as a homodimer or c-Jun as heterodimer. (Hai and Curran, 1991). ATF-2 homodimer or ATF-2 and c-
Jun heterodimer controls the transcription of a large set of genes involved in anti-apoptosis
(Salameh et al., 2010), cell growth (Shimizu et al., 1998), and DNA damage response (Dam,
1995).
Recent studies reveal that ATF2 also has transcriptional-independent functions such as
DNA damage response (Bhoumik et al., 2005), chromatin remodeling and mitochondria membrane organization, thereby highlighting the diverse location-dependent functions of this protein (Cho et al., 2001). PKCε phosphorylates ATF2 at Thr52, which promotes its nuclear localization and transcriptional activity. Upon genotoxic stress, PKCε activity is decreased which leads to translocation of ATF2 from the nucleus and its translocation to the mitochondria. ATF2 translocation to the mitochondria targets the Hexokinase 1-Voltage dependent anion channel 1
(HK1-VDAC1) complex, thereby compromising mitochondria outer membrane permeability and promoting mitochondria based cell death in Squamous cell carcinoma (SCC-9) cell line (Lau et al., 2012).
8
A B
Figure 1. Schematic representation of regulation of ATF2 via PKC (A) PKC
phosphorylates ATF2 at Thr52 residue and targets it to the nucleus. Higher
PKCactivity leads to the oncologic functions in melanoma. (B) Under genotoxic stress, phosphorylation of ATF2 by PKCdecreases, leading to ATF2 translocation to the mitochondria, thereby enhancing its pro-apoptotic functions.
Adapted from (Lau et al., 2012).
9 1.7 Role of ATF2 in diseases
ATF2 induction is generally regarded as an early stress-induced response (Hu et al., 1999). In the normal adult brain, ATF2 is ubiquitously found in the cerebral cortex, hippocampus, brain stem, substantia nigra and cerebellum. A study suggests that there is persistent ATF2 activation following transient cerebral ischemia in rat brain (Hu et al., 1999). ATF2 phosphorylation
(Thr71) has been found to be dramatically increased in apoptotic neurons in the 21-day-old rat brain following a unilateral hypoxic-ischemic insult (Walton et al., 1998). Moreover, ATF2 is also phosphorylated in the dying pyramidal cornu ammonis (CA1) neurons but not in surviving dentate granule (DG) neurons in the rat hippocampus following hypoxic-ischemic insult (Martin-
Villalba et al., 1998).
In contrast to normal cases, ATF2 is down-regulated in the hippocampus, substantia nigra, pars compacta and caudate nucleus for neurological diseases such as Alzheimer’s,
Parkinson’s and Huntington’s disease, respectively (Pearson et al., 2005). In the adult rat brain,
ATF2 is present in the nucleus of all neuronal cell populations, but not in the glial cells and its expression is decreased in axotomized neurons following nerve lesions (Ferrer et al., 1996).
ATF2 phosphorylation is also decreased by antidepressants and increased after prolonged stress specifically in the frontal cortex in rats (Laifenfeld et al., 2004). In the brain, cytoplasmic ATF-2 was observed in the cortical neurons of patients with Alzheimer’s disease (Yamada et al., 1997;
Yuan et al., 2009). ATF2 also plays roles in other diseases. Loss of ATF2 function leads to cranial motor neuron degeneration during embryonic mouse development (Ackermann et al.,
2011). ATF2 deficient mice has defects in endochondral ossification at epiphyseal plates called chondrodysplasia. These mice also show neurological deficit, decreased cerebellar purkinje cells,
10 atrophic vestibular sense organs, enlarged ventricles, ataxic gait, hyperactivity and decreased hearing (Reimold et al., 1996). One study suggests that there is concurrent up-regulation of c-
Jun/ATF-2 heteromer and also down-regulation of c-Fos expression in potassium deprivation- induced neuronal apoptosis. Based on all the above described evidences, ATF2 seems to a stress induced transcriptional factor, but its direct role in cerebral ischemia is not clearly understood.
Therefore, we investigated the role of ATF2 after global cerebral ischemia.
1.8 The chemical-genetics approach
ATF2 is involved in many diseases and might be a potential substrate of PKC as described above. Identification of substrates of PKC is important in order to further understand their role in neurological diseases. We used chemical-genetic approach to identify PKC substrates and focused on PKCδ. The reason we first focused on PKCδ because it is well studied novel PKC isozyme involved in ischemia. It also regulates behavioral responses to ethanol (Choi et al.,
2008) as well as promotes reperfusion injury after myocardial ischemia (Mochly-Rosen et al.,
2012). Using mice lacking PKCδ, Dr. Chou investigated the role of PKCδ in cerebral ischemia- reperfusion injury (Chou et al., 2004) and found that PKCδ KO mice show a significant reduction in the infarct size compared with WT mice. This outcome was associated with reduced neutrophil infiltration into infarcted brain tissue, as well as impaired neutrophil adhesion, migration, respiratory burst, and degranulation in vitro. To understand the molecular and cellular actions of PKCδ in physiological and pathophysiological states, it is important to generate a form of PKCδ that we can control and use it investigate the downstream signaling pathways of PKCδ.
A chemical-genetic approach has been developed to identify immediate phosphorylation substrates of kinases and to study results of kinase inhibition by selective, cell-permeable, small
11 molecule inhibitors (Bishop et al., 2001; Zhang et al., 2013b). This approach targets the structurally conserved ATP-binding pocket within all kinases to generate mutant alleles that can utilize specific ATP analogs in addition to ATP. The mutation creates a “cavity” by replacing a conserved bulky gatekeeper (methionine) with a smaller residue (alanine or glycine) in the ATP- binding pocket. The engineered ‘‘cavity’’ is located where the N6 amine of ATP usually sits, and thus allows for binding of structurally modified ATP analogs with bulky substitutions attached at the N6 position. Only the analog-specific (AS) mutant kinase, not the WT kinase, can efficiently use N6-substituted ATP analogs as phosphate donors. Therefore, only unique substrates of the
AS kinase are labeled by the ATP analogs.
The approach has been successfully used to identify direct substrates of several kinases, including JNK (Habelhah et al., 2001), v-Src (Shah and Shokat, 2002), ERK2 (Eblen et al.,
2003), cdk1 (Ubersax et al., 2003), Raf-1 (Hindley et al., 2004), cdk7 (Larochelle et al., 2006), and PKC (Chou et al., 2010; Wu et al., 2012). To facilitate the identification and purification of the direct substrates, an affinity tagging strategy to label the substrates that can be recognized by specific antibodies was developed (Allen et al., 2005). First, an AS-kinase mutant is used to thiophosphorylate the substrates with N6-(benzyl)-ATP-γS. The thiophosphate group tagged on the substrates is, then alkylated by para-nitrobenzyl mesylate (PNBM) to create thiophosphate ester epitopes that can be recognized by specific antibodies. The tagged substrates can be isolated by immunoprecipitation or immunoaffinity purification. These analog-specific mutations are designed to be functionally silent with respect to kinase activity and substrate specificity. The engineered AS-mutants are also uniquely sensitive to novel kinase inhibitors, such as analogs of
PP1 (Hanke et al., 1996).
12 Based on this approach, we generated WT and AS-PKCδ homology model in silico.
While AS-PKCδ can be regulated by specific ATP and PP1 analogs, WT can’t. In order to understand their mode of action, the specific interactions of analogs with residues in ATP binding pocket were revealed in silico. Our study provides valuable tools for the rational drug design against diseases, which involve PKC.
Figure 2. The chemical-genetics approach. An analog-specific (AS) kinase (shown in
blue) but not the wild-type (WT) kinases (shown in brown) can accept N6-substituted
ATP analogs (BZ-ATPγS) as phosphate donor and phosphorylates its unique substrate. In
the second step, alkylation with p-nitrobenzylmesylate (PNBM) forms thiophosphate
esters and thioethers. Only esterified analog-specific kinase substrates are recognized by
specific antibodies discriminating thiophosphate esters from thioethers.
Adapted from (Chou and Messing, 2008)
13
1.9 Overview
The neuroprotective roles of PKCε in preconditioning have been reported mainly using pharmacological agents, PKCε peptide activators and inhibitors. It is important to use additional methods such as genetic approaches to confirm and advance our understanding of how PKCε functions after global cerebral ischemia. ATF2, a recently identified PKCε substrate, is an attractive target to develop novel therapeutic interventions. In this proposal, we will use PKCε
KO mice to study the roles of PKCε and ATF2 after global cerebral ischemia using in vivo model. These studies, if positive, would provide the first insight into PKCε and ATF2 mediated signaling pathways after global cerebral ischemia. Our results will support the design of ATF2- based neuroprotective agents to reduce brain injury after cardiac arrest.
Identification of substrates of PKC has always been important to understand their cellular signaling. In the third specific aim, we have focused on PKC, a novel PKC isozyme which plays an essential role in cerebral ischemia. Using chemical-genetics approach, we will predict many potential PKC substrates and inhibitors by in silico studies. This study provides tools to identify substrates and investigate PKC-mediated pathways. These studies will be important to understand the role of PKC and PKC and its downstream substrates after global cerebral ischemia.
The project consists of three parts:
(i) Determine the effect of PKCε deficiency following global cerebral ischemia;
(ii) Determine the molecular mechanisms of cell death after global cerebral ischemia;
(iii) Generation and characterization of ATP analog-specific PKCδ homology model in
silico.
14 The first aim is to identify a potential phenotype between PKCε WT and KO mice after global cerebral ischemia. Since the phenotype of PKCε KO mice after global cerebral ischemia has not been investigated, it will a novel study to understand PKCε signaling in ischemia using a genetic mouse model. First, we will determine hippocampal neurodegeneration and cell death using Fluoro-Jade C (FJC) and cresyl violet staining. Subsequently, we will determine cerebral vascular anatomy, blood flow and physiological parameters (blood pH, glucose, temperature) using black ink perfusion, Laser Doppler Flowmetry and i-STAT system respectively.
The second aim is to understand the molecular mechanisms of hippocampal neuronal apoptosis after global cerebral ischemia. We will determine ATF2 (Thr52) phosphorylation, temporal expression profile of mitochondrial ATF2 and cytosolic PKC and cytochrome c release by immunoblotting.
The third aim is to predict potential substrates and inhibitors for PKC using in silico homology modeling and chemical genetics approach. Studies suggest that ATP analog-specific
(AS) substrates and PP1 derived inhibitors fit and maintain important interactions in the space created by mutation of the gatekeeper residue of the WT kinases and thus are potent substrates and inhibitors for AS kinases. We created in silico WT and AS-PKC homology model and docked and analyzed bulky substrates and inhibitors interaction within the ATP binding pocket using MOE, Chimera, Auto Dock Vina, and Discovery Studio softwares.
15 CHAPTER 2
Determine the effect of PKCε deficiency following global cerebral ischemia
2.1 Introduction and rational
Over the years, PKCε peptide activators such as ψεRACK or ψεHSP90 or PKCε peptide inhibitor such as εV1–2 have been used to study PKCε signaling after preconditioning and ischemia (Raval et al., 2003; Dave et al., 2008; Kim et al., 2007; Raval et al., 2007; Della-Morte et al., 2011). General PKCε activator peptide, ψεRACK or mitochondrial-selective PKCε activator, ψεHSP90 is neuroprotective against ischemia/reperfusion (Sun et al., 2013). However, the use of peptide activators or inhibitors might be transient and nonselective under in vivo and in vitro conditions. Therefore, we decided to use genetic animal model i.e. PKCε KO mice in order to understand the downstream substrates of PKCε after ischemia. Moreover, the use of genetic model will validate the specificity of the peptide activators or inhibitors used so far for the study of PKCε substrates.
In order to study forebrain ischemia, we need to develop a simple model of global cerebral ischemia, which could produce reproducible ischemia induced brain damage. Mouse models of forebrain ischemia have been complex to develop due to high variability in their vascular anatomy (Yang et al., 1997; Yonekura et al., 2004) and different ischemic insult sensitivities for
16 different strains of mice belonging to different genetic background (Sheldon et al., 1998;
Wellons et al., 2000). Many rat and mouse models for global cerebral ischemia have been developed such as three vessel occlusion model (Panahian et al., 1996), four vessel occlusion model (Pulsinelli et al., 1982), cardiocirculatory arrest model (Bottiger et al., 1999), and two vessel occlusion plus hemorrhagic hypotension model (Sheng et al., 1999). Most of the models described above are surgically challenging. Kristian and colleagues at University of Maryland have described a simple model of mouse forebrain ischemia (Onken et al., 2012), an example of two vessel occlusion plus systemic hypotension where the forebrain ischemia was induced by combination of bilateral common carotid artery occlusion (BCCAO) and isoflurane-induced hypotension. We have followed this model and induced forebrain ischemia in PKCε WT and KO mice.
We assessed the differences in neurodegeneration after global cerebral ischemia between
PKCε WT and KO mice. Various studies suggest that there is a delayed neurodegeneration in hippocampus, cortex, striatum after global cerebral ischemia (Murakami et al., 1998; Sheng et al., 1999; Wellons et al., 2000). However, hippocampus is the most susceptible part of the brain for the delayed neurodegeneration after global cerebral ischemia (Onken et al., 2012). So, we focused on the ischemia-induced delayed hippocampal neurodegeneration. In this model of global cerebral ischemia, the delayed neurodegeneration is observed 48 hour after ischemia and neurodegeneration and cell death matures at 72 hours after ischemia. So, we considered 72 hours as an appropriate time for detecting neurodegeneration using Fluoro-Jade C (FJC) staining. Since
FJC stains both degenerating neurons and dead neurons (Larsson et al., 2001), we have used cresyl violet stain to detect dead neurons.
17 Monitoring cerebral blood flow help in assessing how well the forebrain ischemia has been developed. Besides, we investigated whether the phenotypic differences between PKCε WT and KO mice after global cerebral ischemia is due to the differences in the cerebral blood flow.
So, we determined cerebral blood flow in PKCε WT and KO mice during the global cerebral ischemia. Posterior communicating artery (PcomA) connects anterior and posterior blood circulation of the brain and supplies blood flow from the posterior circulation of the body when bilateral common carotid artery is blocked for inducing global cerebral ischemia. Presence or absence of the posterior communicating artery can contribute to the variability in the histological outcome after ischemia. In order to assess the contribution of PcomA in ischemic histological outcome, we evaluated the development of PcomA in PKCε WT and KO mice.
High blood glucose can aggravate the post-ischemic neuronal damage (Martin et al.,
2006). Similarly, intra-ischemic hypothermia or post-ischemic hypothermia can be neuroprotective (Wellons et al., 2000). Blood pH differences might contribute to the variation in the post-ischemic histological outcome (Chopp et al., 1987). Therefore, we checked several physiological parameters for PKCε WT and KO mice under normal physiological conditions.
18 Specific aim. Determine the effect of PKCε deficiency following global cerebral ischemia.
1. Determine hippocampal neuronal degeneration in PKCε WT and KO mice after global cerebral ischemia
2. Determine hippocampal neuronal cell death in PKCε WT and KO mice after global cerebral ischemia
3. Determine the cerebrovascular anatomy in PKCε WT and KO mice
4. Determine the regional cerebral blood flow before and after global cerebral ischemia in PKCε
WT and KO mice
5. Determine the physiological parameters for PKCε WT and KO mice
19 2.2 Materials and methods
Animals
F2 generation hybrid PKCε WT and KO littermates (10-16 weeks) were produced by intercrossing F1 generation hybrid C57BL/6JXSV129SvJae for the study. PKCε KO mice were originally produced by homologous recombination in J1 recombinant stem cells (Khasar et al.,
1999). Mice were genotyped by PCR of tail digestion and all animal protocols were approved by the Animal care and Use Committee of Kent state University, following the National Institute of
Heath Guidelines for the care and use of Laboratory Animals.
Global cerebral ischemia
Animals were not fasted but provided free access to food and tap water before the surgery. The forebrain ischemia on two groups of mice strain (PKC WT and KO) was induced by combination of 10 minutes of BCCAO and 9 minutes of isoflurane-induced hypotension. The common carotid arteries were isolated for later clamping. BCCAO was preceded by increase of isoflurane from 1.5 % to 5 % in the respiratory gases [N2O and O2 (70:30)], which in combination results in fall of cerebral blood flow to 5 % of the baseline (100%). Animals were also kept on the heating pad (Fine Scientific tools) for three days at 37 °C and, then perfusion fixed for histology. The skull temperature was monitored using needle thermistor
(Thermoworks) placed subcutaneously adjacent to the skull. Blood glucose was always recorded for each animal used for the surgery using ACCU-CHEK Aviva glucose strips. Moreover, animals were allowed to recover in warm recovery chamber (T=36-37 °C) for two hours in order to prevent post-ischemic hypothermia induced neuroprotection (Wellons et al., 2000).
20 Fluoro-Jade C staining
Fluoro-Jade C (FJC) staining kit (Histo-Chem Inc.) was used for staining hippocampal neuronal degeneration. Three days following global cerebral ischemia, brains were perfused with 25 ml of
4% formaldehyde (PFA), transferred to 30 % sucrose overnight. 50 µm-thick brain sections were cut using cryostat, mounted on gelatin-coated slides and dried at 50-60 °C in the incubator (Type
37900 Culture Incubator) for 30 minutes. FJC staining was started by adding ethanol mixed in distilled water (9:1) on the slides and incubated for 5 minutes. The slides were, then transferred to 70% ethanol for 2 minutes and then, to distilled water for 2 minutes. They were further incubated with solution B (Potassium permanganate dissolved in distilled water (9:1) for 10 minutes and dipped in distilled water for a few seconds. Solution C ((Fluoro-Jade C stain mixed with solution D (DAPI) stain and distilled water (1:1:8)) was, then added on the slides and incubated for 10 minutes. The slides were, then rinsed for 1 minute in distilled water thrice and dried at 60 °C for 5 minutes, cleared by immersion in xylene for 5 minutes and, then cover slipped with mounting media.
Cresyl violet staining
Cresyl violet stain was prepared by adding 1.25 g of cresyl violet powder (Sigma-Aldrich) in 250 ml of warm distilled water containing 0.75 ml of glacial acetic acid. The solution was stirred over night and filtered next day. The sections were mounted on gelatin-coated slides and dried at
50-60 °C in the incubator for 30 minutes. Cresyl violet staining was started with the dehydration steps in 95%, 70% and 50% ethanol for 15,1,1 minute respectively. The slides were, dipped in distilled water for 2 minutes. After washing with distilled water, cresyl violet solution was put on the slides for 10 minutes, and then washed later with distilled water for 1 minute. The slides were
21 then, rehydrated with 50% ethanol, 70% acid ethanol (0.5 ml glacial acetic acid in 50 ml 70% ethyl alcohol), 95% and 100% ethanol for 1,2,2,1 minutes respectively, dried at 60 °C for 5 minutes, cleared by immersion in xylene for 5 minutes and then, cover slipped with mounting media.
Immunohistochemical analysis
All the FJC and cresyl violet stained sections used for quantification were from the same anatomical region (-1.79 from bregma). The method was modified as described (Wang et al.,
2012). The hippocampal degenerating and dead neuronal region were outlined and total number of stained pixels was measured in the outlined regions using a densitometric thresholding technique implemented with NIH ImageJ software. The threshold was set at a level just above that which would have counted background and nonspecific staining in areas outside the outlined region. The analysis was done in five sections and data were averaged. The staining analysis was done by a person blinded to the experimental groups.
Cerebrovascular anatomy
Higgins Black Magic waterproof ink (200–250 μl; Sanford Corp.) was injected into the left ventricle using a 26-gauge needle, and the right atrium was opened to release the effluent. Mice were decapitated and their heads were fixed in 10% neutral buffered formalin (Sigma-Aldrich) for 7 days. After 7 days, the brains were removed from the skulls and imaged using a Leica
EZ4HD stereomicroscope integrated with High Definition digital camera. The development of post-communicating artery (PcomA) was graded on a qualitative scale of 0 to 3 (Murakami et al.,
1997). Score 0 indicates no PcomA between anterior and posterior circulation; score 1 indicates
PcomA in capillary phase; score 2 indicates a small truncal PcomA; score 3 indicates well-
22 developed truncal PcomA. To visualize the territory of MCA, the peripheral branches of anterior cerebral artery (ACA) and MCA (middle cerebral artery) were traced and the points of anastomoses between ACA and MCA were identified and connected to establish a “line of anastomoses”. To assess the MCA territory, the distance from the midline to the line of anastomoses at coronal planes 2, 4, and 6 mm from the frontal pole was measured.
Regional cerebral blood flow (rCBF)
Regional cerebral blood flow (rCBF) was continuously monitored by Laser Doppler Flowmetry
(Perimed) with a flexible 0.8-mm fiber optic extension probe (Probe 407, Perimed). The tip of the probe was affixed to the intact skull over the right cortex at 2 mm posterior to the bregma and
4 mm lateral to the midline. The rCBF was recorded 5 minutes before ischemia to obtain a steady-state baseline (100%) and continued until 5 minutes after the ischemia. The percentage of the baseline rCBF (%) was calculated as the percentage relative to the baseline. Mice were excluded from further studies if sufficient occlusion (<30% of the baseline) and reperfusion
(>80% of the baseline) was not achieved based on the Laser Doppler Flowmetry.
Physiological parameters
Blood from common carotid artery was collected from both sham groups (PKCε WT and KO mice) and physiological parameters (pH, PaCO2, PaO2, sO2, Na, K) were measured by i-STAT system (Abbott Point of Care Inc.). During the ischemia, the body and skull temperature were maintained at 37.0 °C +- 0.5 using homoeothermic-heating pad and heating lamp respectively, in order to prevent hypothermia-induced neuroprotection (Wellons et al., 2000). The skull temperature was monitored and recorded using needle thermistor placed subcutaneously adjacent to the skull. Blood glucose was always recorded for each animal used for the surgery using
23 ACCU-CHEK Aviva glucose strips. Moreover, animals were allowed to recover in warm recovery chamber (T=36-37 °C) for 2 hours in order to prevent post-ischemic hypothermia induced neuroprotection (Wellons et al., 2000). Animals were also kept on the heating pad (37
°C) for 3 days after ischemia and, perfused with 4 % PFA for histology.
24 2.3 Results
2.3.1 Determine hippocampal neuronal degeneration in PKCε WT and KO mice after global cerebral ischemia
Fluoro-Jade C (FJC), a fluorescent anionic dye, is the most commonly used stain for detecting neurodegeneration in the brain (Schmued et al., 2005) which depicts greater morphological details than the other similar stains, Fluoro-Jade (Schmued et al., 1997) and Fluoro-Jade B
(Schmued and Hopkins, 2000). Using FJC staining, neurodegeneration was restricted to cornus ammonis 1 (CA1) sector of the hippocampus in PKCε KO mice 72 hours after global cerebral ischemia (Figure 3B). However, in PKCε WT mice, it was present in CA1, CA2, CA3 sectors of the hippocampus (Figure 3A). Moreover, dentate gyrus (DG) has few degenerating neurons in
PKCε WT while there was no neurodegeneration detected in DG for PKCε KO mice after ischemia. In conclusion, there was a significant difference in the number of degenerating neurons in the hippocampus between PKCε WT and KO mice 72 hours after global ischemia (Figure 3C).
This result suggests that PKCε is involved in ischemia-induced neurodegeneration.
25
Figure 3. Hippocampal neurodegeneration in PKCε+/+ and PKCε-/- cerebral ischemia. Fluoro-Jade C staining of (A) PKCε+/+ (B) PKCε-/- subjected to
10 min of forebrain ischemia and 3 days of recovery. The bright green stain highlighted the degenerating neurons. (C) Quantification of hippocampal neurodegeneration in
PKCε+/+ (n=5) and PKCε-/- mice (n=5) following 3 days of post-ischemic recovery (*p<
0.05) (two-tailed, unpaired t-test). Scale bars 500 µm.
26 2.3.2 Determine hippocampal neuronal cell death in PKCε WT and KO mice after global cerebral ischemia
FJC stain can detect degenerating as well as dead neurons (Larsson et al., 2001). Thereofore, we used cresyl violet staining to specifically detect only dead neurons. Our results indicate that neuronal cell death was restricted to CA1 sector of the hippocampus in PKCε KO mice after ischemia (Figure 4B) whereas, in PKCε WT mice it was found in all sectors (CA1, CA2, CA3,
DG) of the hippocampus (Figure 4A). Moreover, there was significant difference in the number of dead neurons in the hippocampus between PKCε WT and KO mice 72 hours after global cerebral ischemia (Figure 4C). This finding clearly demonstrates that hippocampal neuronal cell death is reduced in PKCε KO as compared with WT mice after ischemia.
27
Figure 4. Hippocampal neuronal cell death in PKCε+/+ and PKCε-/- global cerebral ischemia. Cresyl violet staining of (A) PKCε+/+ and (B) PKCε-/-
10 min of forebrain ischemia and 3 days of recovery. The dead neurons have a small lightly stained nuclei (marked with arrow). (C) Quantification of hippocampal neuronal cell death in PKCε +/+ (n=5) and PKCε-/- n=5) following 3 days of post-ischemic recovery (*p< 0.05) (two-tailed, unpaired t-test).
Scale bars 500 µm.
28 2.3.3 Determine the cerebrovascular anatomy in PKCε WT and KO mice
To investigate whether the attenuated neurodegeneration in PKCε KO mice was due to developmental defects in the cerebrovascular anatomy, we examined the distribution of cerebral arteries by black ink perfusion. There was no difference in the origins of the MCA (middle cerebral artery) or distribution of other major blood vessels of the circle of Willis (Figure 5A), the degree of posterior communicating arteries (PcomA) patency (Figure 5D), and the size of
MCA territory (Figure 5B and C) between PKCε WT and KO mice. Moreover, the patency of
PcomA is considered a critical factor in the development of neuronal injury after bilateral common carotid artery occlusion (BCCAO) in mice (Yang et al., 1997). Therefore, similar
PcomA development in PKCε WT and KO mice suggest that their phenotypic differences after
BCCAO between both genotypes were not due to the developmental defect in PcomA.
29
Figure 5. Assessment of cerebrovascular anatomy. (A) Shown are representative images of the brains from PKCε+/+ and PKCε-/- perfused with black ink. The major arteries in the circle of Willis (upper panels) and PcomA (indicated by arrowheads in lower panels) were identified. (B) Shown are the dorsal images of the
+/+ -/- brains from PKCε and PKCε mice perfused with black ink. The points of anastomoses were circled and connected to form the line of anastomoses. (C)
Distances from the line of anastomoses to the midline in PKCε+/+ (n = 3) and PKCε-/- mice (n = 3) mice were measured at coronal planes 2, 4, and 6 mm from the frontal pole. (D) Shown are the scores of PcomA plasticity in PKCε+/+ (n = 3) and PKCε-/- mice (n = 3).
30 2.3.4 Determine the regional cerebral blood flow before and after global cerebral ischemia in PKCε WT and KO mice
The cerebral blood flow pattern demonstrates how well the ischemia has been developed in
PKCε WT and KO mice. Before ischemia, cerebral blood flow was steadily maintained at the baseline (100%), dropped to 5% of the baseline during ischemia, reperfused well to the baseline after ischemia. Thus, there were no differences in the cerebral blood flow pattern between PKCε
WT and KO mice before, during and after ischemia (Figure 6). This demonstrates that ischemia has been well developed and complete for both genotypes. So, the phenotypic differences we observed between these two groups of mice after ischemia were not contributed by the differences in the blood flow pattern.
31
Figure 6. Determination of regional cerebral blood flow (rCBF). The rCBF during
BCCAO was measured continuously in PKCε+/+ (n=3-5) and PKCε-/- n=3-5) mice.
Steady-state rCBF before the BCCAO were used as baseline (100%), and the subsequent changes after the onset of ischemia were shown as the percentage relative to the baseline. rCBF dropped to 5% of the baseline and reperfused back to the baseline during and after surgery, respectively. There were no significant differences in the rCBF between PKCε+/+ and PKCε-/- mice during BCCAO.
32 2.3.5 Determine the physiological parameters for PKCε WT and KO mice
Early changes in the physiological parameters might affect the ischemic outcome (Wong and
Read, 2008). First of all, there was no significant difference in the weight of PKCε WT and KO mice before ischemia (Table 1). Blood glucose for both genotypes was found to be in the range
150-200 mg/dl before ischemia (Table 1). The ischemia surgery was not performed if blood glucose level was higher than 200 mg/dl because higher blood glucose exacerbates neuronal cell death after ischemia (Martin et al., 2006). Also, pre-ischemic, ischemic and post-ischemic head temperature was maintained at 37 °C in order to protect the brain from hypothermia-induced neuroprotection (Wellons et al., 2000) (Table 1) Moreover, under the normal physiological condition, there were no significant differences in pH, PaO2, PaCO2, sO2,Na, K in the blood for both genotypes (Table 2).
33
34
35
2.4 Conclusion
Our results indicated that hippocampal neurodegeneration and cell death were significantly reduced in PKCε KO mice compared with WT mice after global cerebral ischemia. This suggests that PKCε mediates hippocampal neurodegeneration and cell death after global cerebral ischemia. However, we do not exclude the possibility of contribution by other PKC isozymes in neurodegeneration and cell death after ischemia yet. Moreover, our results further suggested that phenotypic differences between the two genotypes were not due to their differences in cerebrovascular anatomy, cerebral blood flow or physiological parameters. This suggests that the minimal contribution of these parameters in the phenotypic differences between PKCε WT and
KO mice after global cerebral ischemia.
36 CHAPTER 3
Determine the molecular mechanisms of cell death after global cerebral ischemia
3.1 Introduction and rational
PKCε regulates the subcellular localization of ATF2 via Thr52 phosphorylation in melanoma
(Lau et al., 2012). During stress condition, decreased PKCε activity leads to the decline in Thr52
ATF2 phosphorylation resulting in translocation of nuclear ATF2 to mitochondria. This disrupts
HK1-VDAC1 (Hexokinase-1 Voltage dependent anion channel-1) mitochondrial complex, subsequently resulting in apoptosis (Lau et al., 2012). Thus, Thr52 ATF2 phosphorylation via
PKC is considered a master switch that regulates ATF2 subcellular localization, thereby mediating apoptosis in melanoma. This mode of mitochondria mediated apoptosis might also occur in neurons after ischemic stress provided that both ATF2 (Kara et al., 1990) and PKCε
(Roisin and Barbin, 1997) are ubiquitously expressed in the hippocampal neurons.
Proteins can be regulated by several means such as phosphorylation (Tsutakawa et al.,
1995), protein-protein interaction (Derry et al., 2006), intracellular targeting (Barradeau et al.,
2002). One of the basic mode of PKC activation is through its translocation from cytosol to membrane fraction (Mochly-Rosen and Gordon, 1998). One such novel PKC isoform, PKCε translocate to mitochondria as early as 24 hours after ischemic preconditioning and mediates neuroprotection (Dave et al., 2008). Similarly, mitochondrial-selective PKCε activator, ψεHSP90 is neuroprotective, if applied 15 minutes before focal cerebral ischemia (Sun et al., 2013).
37 Moreover, PKCε’s substrate, ATF2 translocates to mitochondria after stress and mediates apoptosis in melanoma (Lau et al., 2012). All these evidences suggest the importance of temporal expression profile of protein in order to use as a therapeutic target. The temporal expression profiles of PKCε and ATF2 are unknown in the hippocampus after global cerebral ischemia. Therefore, we would like to determine temporal expression profiles of PKCε and
ATF2 in the hippocampus of PKCε WT and KO mice after global cerebral ischemia.
Over the years, various reports suggest that release of cytochrome c in the cytosol mediates cell death after global cerebral ischemia (Endo et al., 2006; Sugawara et al., 1999).
Since, neurodegeneration and cell death occurs in delayed manner after global cerebral ischemia, we would like to determine the release of cytochrome c at 48 hours after global cerebral ischemia. Our study demonstrates that neurodegeneration and cell death are reduced in PKCε
KO mice as compared with WT mice (Figure 3 and 4). This is in contrast to what was expected because PKCε confers neuroprotection after ischemic or pharmacological preconditioning (Dave et al., 2008; Kim et al., 2007; Raval et al., 2007). Thus, neuronal cell death was expected to be increased in PKCε KO mice. One of the reasons can be the compensatory role by other isozymes in the absence of PKCε in the KO mice, which might mediate neuroprotection. Studies suggest that other PKC isozymes activity remain the same in the normal PKCε KO mice forebrain
(Hodge et al., 1999). However, in the hippocampus, the role of other isozymes after global cerebral ischemia is unknown and has to be investigated in the future.
38 Specific Aim: Determine the molecular mechanisms of cell death after global cerebral ischemia.
1. Determine the phosphorylation of ATF2 at Thr52 in mouse hippocampus
2. Determine temporal expression profile of PKCε in mouse hippocampus before and after global cerebral ischemia
3. Determine temporal expression profile of ATF2 in mouse hippocampus before and after global cerebral ischemia
4. Determine cytochrome c expression in mouse hippocampus after global cerebral ischemia
39 3.2 Materials and methods
Subcellular fractionation
Subcellular fractionation of the hippocampus was performed for PKCWT and KO mice before and at different time points (1, 4, 24, 48, 72 hours) after ischemia using mitochondria isolation kit (Thermo Scientific). Mouse hippocampal tissues were isolated, washed with 2-4 ml of PBS, cut into small pieces, and 800 μl of PBS was added to the minced tissues. Tissues were transferred in dounce homogenizer (Fisher Scientific) and 3-5 strokes were performed on ice in order to obtain homogenous suspension. The tissue homogenate was then centrifuged at 1,000 X g for 3 minutes at 4 °C. The supernatant was discarded and the pellet was suspended in 800 μl of
BSA/Reagent A solution, vortexed at medium speed for 5 seconds and then, incubated on ice for exactly 2 minutes. Ten ul of mitochondria isolation reagent B was added to the resuspended pellet. The resuspended pellet was then vortexed at maximum speed every minute for 5 minutes on ice. Eight hundred ul of mitochondria isolation reagent C was added to the tube after 5 minutes and inverted several times to mix (no vortex). After mixing, it was centrifuged at 700 X g for 10 minutes at 4 °C. The pellet obtained was the crude nucleus extract. The supernatant was then transferred to a new 2 ml tube and centrifuged at 12,000 X g at 4 °C. The pellet obtained is the mitochondria fraction and the supernatant is the cytosolic fraction. Five hundred μl of wash buffer was added to the mitochondria pellet and centrifuged at 12,000 X g for 5 minutes. The supernatant was discarded and mitochondria pellet was then resuspended in 50 μl of 1X RIPA buffer (Cell Signaling Technology). The resuspended mitochondrial pellet was sonicated quickly for 10 seconds at the minimum frequency (20 Hz), put on the ice for 15 minutes, centrifuged at
12,000 X g for 10 minutes at 4 °C. The supernatant after centrifugation was used as the
40 mitochondria fraction. Required amount of SDS sample buffer was added to the mitochondria and cytosolic fraction and protein concentration was measured using Bradford reagent for immunoblotting.
Immunoblotting
Nucleus, mitochondria and cytosol were isolated from the hippocampus using mitochondria isolation kit as described above. Subcellular fractions containing equal amount of proteins were separated by NuPAGE 4-12% Bis-Tris gels (Invitrogen), transferred to PVDF membranes and analyzed by Western blot using rabbit anti-Thr52 ATF2 (1:1000, Phosphosolution, Cat# p115-
52), rabbit anti-ATF2 (1:1000, Cell Signaling Technology, Cat# 9226), rabbit anti-Cytochrome
C (1:1000, Cell Signaling Technology, Cat# 11940), mouse anti-PKC (1:1000, BD, Cat#
610086), mouse anti-actin (1:1000, Sigma-Aldrich, Cat# A4700), rabbit anti-COX4I1 (1:1000,
Sigma Aldrich, Cat# AV42784). Immunoreactive bands were detected using enhanced ECL
(Pierce, Rockford, IL, USA), imaged using Luminescent Image Analyzer LAS-3000 (Fujifilm,
Edison, NJ, USA) and quantified using NIH ImageJ 1.37.
41 3.3 Results
3.3.1 Determine the phosphorylation of ATF2 at Thr52 in mouse hippocampus
Under normal physiological condition, PKCε phosphorylates ATF2 at Threonine 52 (Thr52) residue in the melanoma and targets it the nucleus (Lau et al., 2012). However, It is unknown whether PKCε phosphorylates ATF2 at Thr52 in the mouse hippocampus. We checked the expression of ATF2 (Thr52) phosphorylation in the nuclear extract of PKCε WT and KO mice hippocampus. Our Western blotting result indicated that ATF2 (Thr52) phosphorylation was significantly reduced in the nuclear extract of PKCε KO mice hippocampus compared with WT mice (Figure 7 A and B). This suggests that PKCε phosphorylates ATF2 at Thr52 residue in vivo under normal physiological condition. However, we did not observe any significant differences in the total ATF2 expression between the two control groups (Figure 7C). This suggests that the absence of PKCε does not affect the total ATF2 expression under normal condition.
42
Figure 7. ATF2 (Thr52) phosphorylation by PKC in vivo. (A) A representative
Western blot of ATF2 (Thr52) in the hippocampus of PKC+/+ and PKC-/-mice. (B)
Quantitative analysis of pATF2 (T52) / Actin showed a significant decrease of ATF2
(Thr52) in the control PKC-/- n=4) compared to control PKC+/+ mice (*p<
0.05) (two-tailed, unpaired t-test). (C) Quantitative analysis of ATF2 / Actin showed no significant difference in ATF2 expression between PKC-/-mice (n=4) and PKC-/- mice (n=4). Samples were normalized to -actin.
43
3.3.2 Determine temporal expression profile of PKCε in mouse hippocampus before and after global cerebral ischemia
The role of PKCε after cerebral ischemia has been controversial due to variable expression and activity of PKCε reported previously (Reshef et al., 2000; Tauskela et al., 1999). Some studies suggest upregulation of PKCε after ischemia (Selvatici et al., 2003; Wang et al., 2004), while others does not (Savithiry and Kumar, 1994; Harada et al., 1999). In order to understand the role of PKCε after global cerebral ischemia, we checked the expression of PKCε in the cytosolic fraction of the hippocampus before and after ischemia. Our Western blotting result indicated that
PKCε decreased significantly in the cytosol at 24, 48 and 72 hours after global cerebral ischemia compared with the control condition (Figure 8). This suggests that the attenuation of PKCε expression may initiate cell death signaling pathways during late reperfusion hours.
44
Figure 8. Cytosolic PKC decreased in mouse hippocampus after global cerebral ischemia. (A) A representative Western blot of PKC expression in the hippocampus of the
PKC+/+ mice (n=5) after ischemia. (B) Quantitative analysis of PKC/Actin showed a significant decrease of PKCprotein at 24 hours (**p<0.005), 48 and 72 hours
(***p<0.0005) after ischemia compared with control (one-way ANOVA, Newman-Keuls post hoc test).
45 3.3.3 Determine temporal expression profile of ATF2 in mouse hippocampus before and after global cerebral ischemia
PKCε phosphorylates ATF2 at Thr52 residue, which is indicated by the significant reduction of
ATF2 phosphorylation in the hippocampus nucleus extract of PKCε KO mice (Figure 7). This suggests us that there might be change in total hippocampal ATF2 expression after ischemia. Our
Western blotting results demonstrated that the total mitochondrial ATF2 was slightly higher, however not significant, in the hippocampus of control PKCε KO mice as compared with control
WT mice (Figure 9A). This suggests that PKCε partially restricts ATF2 in the nucleus and prevents it from translocation to mitochondria to some extent under normal physiological condition.
In PKCε WT mice, ATF2 started translocating to mitochondria as early as 1 hour and continued till 48 hours after ischemia as compared with control. However, in PKCε KO mice,
ATF2 translocated to mitochondria from early 1 hour till 24 hours after ischemia. At 48 hours, there was significant reduction in ATF2 mitochondrial translocation for PKCε KO mice after ischemia (Figure 9). So, the persistent translocation of ATF2 to mitochondria till 48 hours after ischemia might be one of the reasons for increased cell death in PKCε WT mice. However, early translocation of ATF2 to mitochondria till 24 hours after ischemia in PKCε KO might be just a stress response, which might not be sufficient to cause extensive neuronal cell death in the hippocampus in PKCε KO mice compared with WT.
46
Figure 9. Temporal expression of ATF2 in mitochondria for PKCε+/+ and PKCε-/- mice after global cerebral ischemia. (A) A representative Western blot of ATF2 translocation to mitochondria at 1, 4, 24, 48 and 72 hours after global cerebral ischemia in
PKCε+/+ and PKCε-/- mice. COXIV was used as a mitochondrial loading control. (B)
Quantitative analysis of ATF2/COXIV demonstrates a significant induction of ATF2 in mitochondria for PKCε+/+ mice (n=4-5) compared with the control (n=4-5) at 24 and 48 hours after global cerebral ischemia while in PKCε-/- mice (n=4-5), it was only at 24 hours after ischemia compared with its control (***p<0.0005, one-way ANOVA with Newman-
Keuls post hoc test). (C) Mitochondria purity from cytosolic contamination was determined using antibodies against mitochondria marker protein COXIV.
47 3.3.4 Determine cytochrome c expression in mouse hippocampus after global cerebral ischemia
The release of cytochrome c in the cytosol is an indictor of cell death during late reperfusion hours after global cerebral ischemia (Sugawara et al., 1999). In order to assess the cytochrome c release after global cerebral ischemia, we checked its expression in the cytosol at 48 hours after global cerebral ischemia, which is a critical time point in our model because cell death matures at this time (Onken et al., 2012). Our Western blot results indicated that the cytosolic cytochrome c expression was significantly reduced in PKCε KO mice compared with WT mice 48 hours after ischemia (Figure 10). This confirms our finding that increased cytochrome c release had contributed to more hippocampus cell death in PKCε WT as compared to KO mice during late reperfusion hours after ischemia.
48
Figure 10. Decreased release of cytochrome c in the cytosol for PKCε-/- mice after global cerebral ischemia. (A) A representative Western blot showing the cytochrome c release in the cytosol of PKCε+/+ and PKCε-/- mice at 48 hours after global cerebral ischemia. -actin was used as a loading control. (B) Quantitative analysis of cytochrome c/Actin showed a significant decrease of cytochrome c release in PKCε-/- mice (n=3) compared with PKCε+/+ mice (n=3) (*p<0.05, two-tailed, unpaired t test).
49 3.4 Conclusion
Our results indicated that ATF2 phosphorylation (Thr52) was significantly decreased in the nuclear fraction of PKCKO mice compared with WT mice. This suggests that
PKCphosphorylates ATF2 in vivo. Also, the expression of PKC was decreased significantly at
24, 48 and 72 hours after global cerebral ischemia. Moreover, persistent delayed translocation of
ATF2 to mitochondria till 48 hours in PKCWT mice and 24 hours in the KO mice suggested a role of ATF2 in mitochondria-mediated cell death. In PKCWT mice, the persistent translocation of ATF2 to mitochondria for 48 hours after ischemia might be sufficient to cause extensive neuronal cell death in the hippocampus. Importantly, 48 hours is the critical time point at which differences in ATF2 mitochondrial translocation may contribute to differences in cell death between two genotypes after global cerebral ischemia. This is further confirmed by the significant differences in cytochrome c release between the two genotypes at 48 hours after ischemia. Moreover, cell death matures till 48 hours after this model of global cerebral ischemia
(Onken et al., 2012).
50 CHAPTER 4
Generation and characterization of ATP analog-specific PKCδ homology model in silico
4.1 Introduction and rational
PKCδ has been implicated in the regulation of cerebral ischemia-reperfusion injury (Mochly-
Rosen et al., 2012; Chou et al., 2004) and behavioral responses to ethanol (Choi et al., 2008). In order to further understand its role in neurological diseases, it is important to generate a form of
PKCδ that we can control and use it to investigate the downstream signaling pathways of PKCδ.
A chemical-genetic approach has been developed to identify immediate phosphorylation substrates of kinases and to study results of kinase inhibition by selective, cell-permeable and small molecule inhibitors (Bishop et al., 2001; Zhang et al., 2013b). This approach targets the structurally conserved ATP-binding pocket within all kinases to generate mutant alleles that can utilize specific ATP analogs in addition to ATP. The mutation creates a “cavity” by replacing a conserved bulky gatekeeper (methionine) with a smaller residue (alanine or glycine) in the ATP- binding pocket. The engineered ‘‘cavity’’ is located where the N6 amine of ATP usually sits, and thus allows for binding of structurally modified ATP analogs with bulky substitutions attached at the N6 position. Only the analog-specific (AS) mutant kinase, not the WT kinase, can efficiently use N6-substituted ATP analogs as phosphate donors. Therefore, only unique substrates of the
AS kinase are labeled by the ATP analogs.
51 The approach has been successfully used to identify direct substrates of several kinases, including JNK (Habelhah et al., 2001), v-Src (Shah and Shokat, 2002), ERK2 (Eblen et al.,
2003), cdk1 (Ubersax et al., 2003), Raf-1 (Hindley et al., 2004), cdk7 (Larochelle et al., 2006),
PKC (Chou et al., 2010; Wu et al., 2012; Kumar et al., 2015; Weng et al., 2015). These analog- specific mutations are designed to be functionally silent with respect to kinase activity and substrate specificity. The engineered AS-mutants are also uniquely sensitive to novel kinase inhibitors, such as analogs of 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine
(PP1) (Hanke et al., 1996).
4.2 Materials and methods
Homology modeling
PKCδ homology models (amino acid 341 to 669) were generated using MOE version 2011.10 software and PKC with ATP bound in the active kinase domain (Protein Data Bank (PDB) code
3A8W) (Takimura et al., 2010) as the reference structure. During the homology modeling procedure in MOE, ten models were developed and the final model was used for the studies. The homology model developed by MOE did not have the ATP or bound waters for correct ATP positioning in the binding pocket. We therefore transferred the ATP and binding pocket waters from PKC into the binding pocket of the PKC homology model. The complex of PKC WT and ATP/waters, were then energy minimized and the pH of the system was set at pH 7.4 to correlate with experimental conditions. After the PKC model was developed, we mutated the
Met-427 into Ala using the side chain mutation function in MOE with energy minimized. AS-
PKA was generated by mutating Met-120 into Ala with energy minimized in the crystal structure of PKA (PDB code 1ATP) (Zheng and Guan, 1993). The interactions of ATP and inhibitors with
52 the residues of nucleotide-binding pocket were analyzed using the two-dimensional ligand- receptor interaction diagrams of Discovery Studio 3.5.
Docking studies
Docking of N6-(benzyl)-ADP (derived from PDB entry 1KSW) and 1NM-PP1 (PubChem code
5154691) was performed using the MOE software, which utilizes an induced fit model of the ligand and receptor for docking. Docking of 1NA-PP1 (PubChem code 4877), 2MB-PP1
(modified from the co-crystal structure of Src-AS1/3MB-PP1 (PDB code 4LGG)) was performed using AutoDock Vina (Trott and Olson, 2010) integrated with Chimera version 1.7 (Pettersen et al., 2004). The pH of induced fit docking procedure used in the MOE was set at 7.4. The top 10 poses returned from the docking studies were evaluated by visual inspection.
N6-(benzyl)-ATP (derived from PDB entry 1KSW) was docked in the soluble catalytic core of membrane-bound mammalian adenylyl cyclase (VC1:IIC2, PDB code 1TL7) (Mou et al.,
2005) using AutoDock Vina (Trott and Olson, 2010) integrated with Chimera. The top 10 poses were evaluated by visual inspection. 2’(3’)-O-(N-Methylanthraniloyl)-guanosine 5’-triphosphate
(MANT)-GTP, forskolin and the chain C-containing GTP in the VC1:IIC2 crystal structure were deleted before docking. The adenine ring of 1NA-PP1 (PubChem code 4877) was aligned with the guanine ring of MANT-GTP in VC1:IIC2.
53 4.3 Results
4.3.1 Generate human PKCδ homology model using MOE software.
To understand molecular basis for differences in binding affinity and specificity of ATP and PP1 analogs in WT and AS-PKCδ, we generated PKCδ models in silico. Because the X-ray crystal structure of PKCδ is not available, PKCδ homology models were created based on the crystal structure of human PKCι (PDB code 3A8W) (Takimura et al., 2010). The root-mean-square deviation (RMSD) for the overlaid structure of WT and AS-PKCδ is 0.279 Å (Figure. 11A), suggesting the M427A mutation did not significantly change the three-dimensional structure of
PKCδ (Carugo and Pongor, 2001). The position of ATP was nearly identical in the nucleotide- binding pockets of WT and AS-PKC, with the adenine ring pointing towards the gatekeeper residues (Figure 11B). In both WT and AS-PKCδ, the adenine ring of ATP formed hydrogen bonds with Glu-428 and Leu-430 near the gatekeeper. The ATP phosphate groups were positioned by the charged interactions with invariant Lys-378, and by hydrogen bonds with Ser-
359, Phe-360, and Gly-361 in the glycine-rich loop (Figure 12A, B). These interactions are similar to those reports in previous studies of the crystal structure of ATP bound PKA (PDB code 1ATP) (Chatrchyan et al., 2012; Kornev et al., 2006; Zheng and Guan, 1993) PKC (PDB code 3A8W) (Takimura et al., 2010), PKC (PDB code 3PFQ) (Raval et al., 2011), suggesting the accuracy of PKC homology models. ATP interacted with the same residues in WT and AS-
PKCδ, but the electrostatic interaction with the gatekeeper residue (Met-427) in WT-PKCδ was substituted with weaker van der Waals interaction with Ala-427 in AS-PKCδ.
54
Figure 11. ATP representation in the nucleotide-binding pocket of WT and AS-PKCδ.
(A) The homology model of WT-PKCδ (gray) was superimposed with the AS-PKCδ model
(orange). Gatekeeper residue Met-427 in WT-PKCδ is highlighted in green. (B) ATP
(yellow) docked in WT-PKCδ was superimposed with ATP (colored by atom type, Nitrogen
(blue), Carbon (black), Oxygen (red) and Phosphorus (orange)) docked in AS-PKCδ.
55 A
B
Figure 12. ATP interactions in the nucleotide-binding pocket of WT and AS-PKCδ.
Shown are ATP-interacting residues in the nucleotide-binding pocket of (A) WT and
(B) AS-PKCδ. Residues involved in electrostatic (hydrogen-bond, charge, or polar), van der Waals, covalent bond, water, and metal interactions with the ligand are shaded in pink, green, magenta, aquamarine, and dark gray, respectively. Hydrogen-bond interactions with amino acid main-chains and amino acid side-chains residues are represented by green and blue dashed arrows directed towards the electron donor.
Charged interaction is shown by a pink dashed arrow with heads on both sides. Pi interaction is representative by an orange line with, indicating the interaction.
56 4.3.2 Perform docking studies for substrates (N6-(benzyl)-ADP) and PP1 derived inhibitors in the PKC homology model.
Because N6-(benzyl)-ADP has been crystalized with an analog-specific allele of c-Src (T338G)
(Witucki et al., 2002), we extracted N6-(benzyl)-ADP from the co-crystalized structure (PDB code 1KSW) and docked it in PKCδ homology models. N6-(benzyl)-ADP and ATP docked at similar positions within the nucleotide-binding pocket of AS-PKCδ (Figure 13A). The benzyl ring of N6-(benzyl)-ADP was positioned deeper in the nucleotide-binding pocket, and placed in a space enlarged by mutating Met-427 into Ala-427. This change allowed the phosphate groups of
N6-(benzyl)-ADP to interact with the glycine-rich loop for potential phosphoryl-transfer (Figure
13C). In contrast, N6-(benzyl)-ADP was shifted outward in the nucleotide-binding pocket of
WT-PKC (Figure 13B), preventing its phosphate groups to interact with the glycine-rich loop
(Figure 14D). Moreover, the hydrogen bonds formed between ATP and Glu-428 and Leu-430 in
AS-PKC (Figure 12B) were lost with N6-(benzyl)-ADP (Figure 13C).
To investigate the basis for specific 1NA-PP1 inhibition of AS-PKC, we compared docking of 1NA-PP1 and ATP in AS-PKC (Figure 14A) and WT-PKC (Figure 14B). The adenine rings of 1NA-PP1 and ATP were aligned in AS-PKC, but not in WT-PKC.
Specifically, the adenine rings of 1NA-PP1 and ATP formed hydrogen bonds with the same residues (Glu-428 and Leu-430) in AS-PKC (Figure 12B and Figure 14C). Moreover, the naphthyl ring of 1NA-PP1 fits in the space created by M427A mutation, forming interactions with the invariant Lys-378 in AS-PKC (Figure 14C). Neither of these interactions was found with 1NA-PP1 docked in WT-PKC (Figure 14D) or with 1NM-PP1 and 2NM-PP1 in
AS-PKC (Figure 15).
57 PKA, another member of the AGC kinase family, has been engineered following the same approach (Niswender et al., 2002). The AS-PKA mutant (M120A) is sensitive to both
1NA-PP1 and 1NM-PP1 inhibitors. To investigate the structural basis for PP1 inhibition in AS-
PKA as well as to compare the mechanism with AS-PKC, we docked 1NA-PP1, 1NM-PP1 and
ATP in AS-PKA. The adenine rings of 1NA-PP1 and 1NM-PP1 aligned with the adenine ring of
ATP in AS-PKA, with the naphthyl and naphthylmethyl rings situated in the engineered cavity
(M120A) (Figure 16A and B). Interestingly, the adenine rings of 1NA-PP1 and 1NM-PP1 also formed hydrogen bonds with Glu-121 and Val-123 (corresponding to Glu-428 and Leu-430 in
PKC (Figure 16 C and D).
To assess whether the in silico AS-PKC model can be used to screen potential inhibitors, we docked novel PP1 analogs (Zhang et al., 2013a) and found 2MB-PP1 (Figure 17A) is better aligned with the adenine ring of ATP than 1NA-PP1 (Figure 17B). 2MB-PP1 interacts with the same residues of AS-PKC as 1NA-PP1, but the interaction is further strengthened by an additional interaction with Phe-633 (Figure 17C). This stacking interaction has been implicated in stabilizing nucleic acid-protein complexes (Boehr et al., 2002; Burley and Petsko,
1985). We investigated one possible off-target family of proteins, membrane-bound mammalian adenylyl cyclases (mAC), which possess large hydrophobic pockets in their catalytic domains that accommodate bulky substituents in nucleotides, such as MANT (Mou et al., 2005; Seifert et al., 2012). Our docking studies suggested that N6-ATP and PP1 analogs are unlikely to be specific ligands for mAC (Figure 18). Taken together, we conclude that PP1 analogs inhibit AS-
PKC and AS-PKA by competing with ATP for interaction with Lys, Glu, Leu/Val, and Phe residues within the purine binding pockets of these mutant kinases.
58
Figure 13. Comparison of N6-(benzyl)-ADP (BZ-ADP) interactions in the nucleotide- binding pocket of AS and WT-PKCδ. Shown are the superimposed structures of BZ-
ADP (colored by atom type) and ATP (yellow) in (A) AS (B) WT-PKCδ. Ala-427 is highlighted in purple in AS-PKCδ and Met-427 in green in WT-PKCδ. Shown are the BZ-
ADP interacting residues in the nucleotide-binding pocket of (C) AS and (D) WT-PKCδ.
ATP interacts with the residues in the glycine-rich loop (red *) and the invariant Lys-378
(red arrow head). The gatekeeper is indicated by a red arrow.
59
Figure 14. Comparison of 1 NA-PP1 interactions in the nucleotide-binding pocket of WT and AS-PKCδ. Superimposed structure of 1NA-PP1 (colored by atom type) and
ATP (yellow) in (A) AS and (B) WT-PKCδ. Ala-427 is highlighted in purple in AS-
PKCδ, and Met-427 is highlighted in green in WT-PKCδ. 1NA-PP1- interacting residues in the nucleotide-binding pocket of (C) AS and (D) WT-PKCδ. 1NA-PP1 forms hydrogen bonds with Glu-428 and Leu-430 (red number arrow), interaction with Lys-378 (red arrowhead). The gatekeeper is indicated by red arrow.
60
Figure 15. Interactions of 1NM-PP1 and 2NM-PP1 with AS-PKCδ. (A)
Superimposed structures of 1NM-PP1 (colored by atom type) and ATP (yellow) in AS-
PKCδ. (B) Superimposed structures of 2NM-PP1 (colored by atom type) and ATP
(yellow) in AS-PKCδ. The mutated gatekeeper (Ala-427) in AS-PKCδ is highlighted in purple. (C) 1NM-PP1- (D) 2NM-PP1-interacting residues in the nucleotide-binding pocket of AS-PKCδ. Glu-428 and Leu-430 are indicated by red number symbols.
61
Figure 16. Interactions of 1NA-PP1 and 1NM-PP1 with AS-PKA. (A) Superimposed structures of 1NA-PP1 (colored by atom type) and ATP (yellow) in AS-PKA. (B)
Superimposed structures of 1NM-PP1 (colored by atom type) and ATP (yellow) in AS-
PKA. The mutated gatekeeper (Ala-120) in AS-PKA is highlighted in purple. (C) 1NA-
PP1- and (D) 1NM-PP1- interacting residues in the nucleotide-binding pocket of AS-
PKA. The gatekeeper, invariant Lys, and residues forming hydrogen bonds with ligand are indicated by red arrows, arrowheads, and red number symbols, respectively.
62
Figure 17. Interactions of 2MB-PP1 with AS-PKCδ. (A) Chemical structure of
2MB-PP1. (B) Shown are the superimposed structure of 2MB-PP1 (colored by atom type) and ATP (yellow) in AS-PKCδ. Ala-427 is highlighted in purple in AS-PKCδ.
(C) 2MB-PP1- interacting residues in the nucleotide-binding pocket of AS-PKCδ.
2MB-PP1 forms hydrogen bonds with Glu-428 and Leu-430 (red number arrow),
interaction with Lys-378 (red arrowhead), and interaction with Phe-633
(red dagger). The gatekeeper is indicated by red arrow.
63
Figure 18. Interactions of N6-(benzyl)-ATP and 1NA-PP1 with mAC. (A)
Superimposed structures of N6-(benzyl)-ATP (yellow) and MANT-GTP (colored by atom type) in the catalytic site of mAC. The residues (Thr-408, Ala-409, Gln-410, and, Glu-411) in the hydrophobic pocket of mAC facing the MANT group are highlighted in green. The Trp-1020 residue close to the guanine ring of MANT-GTP is highlighted in magenta. (B) Superimposed structures of 1NA-PP1 (yellow) and
MANT-GTP (colored by atom type) in the catalytic site of mAC. The naphthyl ring of 1NA-PP1 forms a steric clash with one of hydrophobic residues (Trp-1020) highlighted in magenta.
64 4.4 Conclusion
Based on the crystal structure of human PKCPDB code 3A8W), PKC homology models were developed. The mutation of the gatekeeper residue did not significantly change the three- dimensional surface PKC. ATP was found aligned in the nucleotide-binding pockets of WT- and AS-PKCwith the adenine ring pointing toward the gatekeeper residues. ATP formed all the important interactions for the phosphoryl transfer and its stability. N6-(benzyl)-ADP and ATP docked at similar positions within the nucleotide-binding pocket of AS-PKCsuggesting the mutation of the gatekeeper residue created sufficient space to accommodate large bulky groups in AS-PKCowever, this could not happen in WT-PKCThus, N6-(benzyl)-ADP can be a potent substrate for AS-PKCbut not WT-PKCSimilarly, large bulky PP1 inhibitors such as
1NA-PP1 and 2MB-PP1 were predicted to be potent inhibitors for AS-PKCbecause their adenine rings were found aligned with the adenine ring of ATP, thereby competing with ATP for interaction with Lys, Glu, Leu, Val, and Phe. However, this was not true for WT-PKC
Based on these results, AS-PKChomology model can be used to screen potential substrates and inhibitors.
65 CHAPTER 5
Discussion and future directions
5.1 Discussion
Our study demonstrated that neuronal degeneration and cell death were significantly reduced in
PKCε KO as compared with PKCε WT mice after global cerebral ischemia (Figure 3 and Figure
4). We also demonstrated that ATF2 translocated to mitochondria, which might mediate cell death after global cerebral ischemia (Figure 9). However, in PKCε KO mice, it translocated more during early reperfusion time after global cerebral ischemia (Figure 9).
PKCε activation confers neuroprotection during ischemic or pharmacological preconditioning as previously described (Blanco et al., 2006; Raval et al., 2003; Dave et al.,
2008; Kim et al., 2007; Lange-Asschenfeldt et al., 2004; Raval et al., 2007). Based on the previous studies, in the absence of PKCε, neuronal cell death was expected to be increased in
PKCε KO mice. However, the results we obtained was rather unexpected. Several reasons can be attributed to the reduced neuronal cell death in the KO mice. One of the reasons can be the compensatory role by other PKC isozymes in the PKCε KO mice after global cerebral ischemia, which mediate neuroprotection. However, a study suggests that most of the PKC isozymes expression remain unchanged in the PKCε KO mice forebrain (Hodge et al., 1999). But, the role of other PKC isozymes is unknown in PKCε KO mouse hippocampus after cerebral ischemia and has to be investigated in the future.
66 We also asked the question whether there was any difference in ATF2 translocation pattern in PKCε WT and KO mice, which might have contributed to differences in the cell death between them. We found that mitochondrial ATF2 was slightly more in the KO mice as compared with WT mice under physiological condition (Figure 9). This suggests that mitochondria in the KO mice have been more exposed to ATF2 from early development, which might have made them resistant to apoptosis.
We also reported that ATF2 translocated out more at 1, 4 and 24 hours after ischemia in
PKCε KO mice as compared with WT mice (Figure 9). This early translocation of ATF2 till 24 hours might be just a stress response and insufficient to initiate cell death. In fact, this might create a preconditioning like effect, which could be neuroprotective in the KO mice. Moreover, persistent ATF2 translocation to mitochondria till 48 hours in PKCε WT mice might alleviate preconditioning like neuroprotective effect as seen in the KO mice. The detailed mechanisms have to be elucidated in the future. We also showed that ATF2 translocation to mitochondria decreased significantly at 48 hours in PKCε KO as compared with WT mice after ischemia
(Figure 9). We also found significant difference in cytochrome c release at 48 hours after ischemia (Figure 10). Thus, 48 hours is the critical time for delayed cell death after this global cerebral ischemia model. The importance of this particular time point after global cerebral ischemia is also supported by a study, which demonstrates that cell death is not detected as early as 48 hours after ischemia (Onken et al., 2012). This also suggests that significant decrease of
ATF2 in mitochondria at 48 hours in PKCε KO mice might confer neuroprotection, which could not occur in PKCε WT mice. The above study is also supported by a recent finding that estrogen confers neuroprotection in rat global cerebral ischemia by reducing ATF2 activity in mitochondria at 48 hours after global cerebral ischemia (Han et al., 2015). The information about
67 ATF2 and PKCε in neuronal cells will help us to better understand the diverse PKCε signaling in neurons.
Another reason for the difference of cell death for PKCε WT mice and KO mice after ischemia can be the differences between preconditioning and global cerebral ischemia studies.
Preconditioning is a sub-lethal dose of ischemia lasting only for 2 minutes, while global cerebral ischemia is more severe and can proceed for at least 10 minutes. Ischemic or pharmacological preconditioning activates PKCε and confers neuroprotection (Blanco et al., 2006; Raval et al.,
2003; Dave et al., 2008; Kim et al., 2007; Lange-Asschenfeldt et al., 2004; Raval et al., 2007).
This might not happen in our study because 10 minutes of global ischemia has decreased PKCε activity to a significant level, which can’t be further activated. This is the reason why most studies activate PKCε before ischemia in order to confer neuroprotection (Raval et al., 2003;
Dave et al., 2008; Kim et al., 2007). A study also suggests that PKCε activation confers neuroprotection, if activated 30 min before global cerebral ischemia (Della-Morte et al., 2011).
Moreover, it can’t mediate neuroprotection, if activated 3 minutes after or 24 hours before focal cerebral ischemia (Bright et al., 2008). These studies suggest that PKCε can confer neuroprotection only if activated within a certain therapeutic window before ischemia. Previous studies have suggested that PKCε decreases after ischemia in vitro (Selvatici et al., 2003; Wang et al., 2004) and in vivo (Shimohata et al., 2007), which is also supported by our study.
Furthermore, some studies also suggest that PKCε is not activated within 24 hours after ischemia in vivo (Savithiry and Kumar, 1994; Harada et al., 1999). These discrepancies may be attributed to severity of ischemic insult, mouse species difference, as well as time of post-ischemic observation.
68 We were also interested in determining and predicting substrates of other PKC isozymes.
We studied PKC first because it is another member of PKC isozyme involved in ischemia- neuronal cell death (Chou et al., 2004). We generated ATP analog-specific (AS)
PKChomology models based on chemical-genetics approach to determine potential
PKCsubstrates and inhibitors. Placement of the benzyl ring of N6-(benzyl)-ATP in the cavity created by the M427A mutation allowed stable interaction with the glycine-rich loop for efficient phosphoryl transfer. The naphthyl and 2-methylbenzyl rings of 1NA-PP1 and 2MB-PP1 were similarly accepted in the cavity, permitting 1NA-PP1 and 2MB-PP1 to compete with ATP for stable interactions with Glu-428 and Leu-430 as well as the invariant Lys-378 and Phe-633 of
AS-PKC (Figure 14 and Figure 17). In contrast, the insensitivity of WT-PKC for PP1 (Figure
15) and N6-ATP analogs (Figure 13B) could be explained by steric clash of the naphthyl ring of
1NA-PP1 and the benzyl ring of N6-(benzyl)-ATP with the large Met-427 gatekeeper.
The glycine-rich loop (G356 KGSFGK), invariant Lys-378, gatekeeper Met-427, two residues near the gatekeeper (Glu-428 and Leu-430), and Phe-633 are associated with 1NA-PP1,
2MBPP1, and N6-(benzyl)-ATP binding in AS-PKC These residues are in the amino-terminal lobe of the PKC catalytic domain. The catalytic domain for substrate phosphorylation is highly conserved in the AGC family (Steinberg, 2008; Kornev et al., 2006), and the phosphoryl transfer event has been well characterized in PKA (Taylor et al., 2013; Kornev et al., 2006). The invariant Lys interacts with and phosphates, and the glycine-rich loop interacts with and
phosphates to orient ATP for catalysis. Thus, disrupting these interactions would be predicted to impede ATP binding and substrate phosphorylation.
The gatekeeper lies in the middle of the catalytic domain and is a key residue for determining the selectivity of inhibitors (Steinberg, 2008). This residue is conserved as a large
69 hydrophobic amino acid. No kinases with a small Gly or Ala gatekeeper residue have been found in the human kinome (Dar and Shokat, 2011). The side chain of the gatekeeper controls the relative accessibility of a hydrophobic pocket located adjacent to the N6 amine of ATP.
Moreover, the gatekeeper residue may also regulate the autocatalytic activity of ERK2 (Emrick et al., 2006) and the flexibility of PKA (Schauble et al., 2007).
In summary, ATP analog-specific substrates and PP1 inhibitors are potent molecules in
AS PKCbecause they can easily fit and maintain all important interactions for their stability in the kinase pocket due to the extra space created by mutation of the large gatekeeper residue to a smaller residue. Moreover, the more neuronal cell death in PKCε WT mice may be contributed by the persistent translocation of ATF2. However, in PKC KO mice, less neuronal cell death may be the result of early translocation of ATF2 to the mitochondria, which might be a stress response and creates preconditioning-like neuroprotective effect. The study helps us to understand the diverse PKCε and ATF2 signaling and design potential ATF2 and PKCε mediated drug target after ischemia.
70
Figure 19. Model for apoptosis after global cerebral ischemia (A) Under normal physiological condition, PKC phosphorylates ATF2 at Thr52 and targets it to the nucleus in PKC WT mice. (B) In PKC WT mice, global cerebral ischemia causes decrease in ATF2 T52 phosphorylation and PKC expression and increase in ATF2 leakage to mitochondria, thereby causing delayed apoptosis. (C) Under normal physiological condition, ATF2 T52 phosphorylation is attenuated in PKC KO mice. (D) In PKC KO mice, global cerebral ischemia may cause phosphorylation of ATF2 at Thr52 by other PKC isozymes and restricts it to the nucleus. Thus, ATF2 leakage to mitochondria is decreased, thereby causing less apoptosis.
71 5.2 Future directions
Cardiac arrest affects approximately 400,000 people annually in the United States (Go et al.,
2014). Those who survive, suffer from post-ischemic brain damage, which is a huge burden to their family and society. Our research project aims to ameliorate the post-ischemic brain damage by elucidating the molecular pathway involved in ischemia-induced delayed brain damage, which can be used to identify related therapeutic targets. Over the years, the research over the neuroprotective effect of preconditioning via PKCε activation has contributed significantly for the pre-ischemic therapeutic target in cerebral ischemia field (Blanco et al., 2006; Raval et al.,
2003; Dave et al., 2008; Kim et al., 2007; Lange-Asschenfeldt et al., 2004; Raval et al., 2007). but the unpredicted nature of cerebral ischemia emphasizes our study to look for the post- ischemic therapeutic targets.
If PKCε is considered to play a significant role in ischemia induced delayed neurodegeneration, the temporal expression profile of PKCε after ischemia would provide insights into the therapeutic window during which PKCε activator can be used as a therapeutic agent to ameliorate ischemia induced neurodegeneration. However, specificity of PKC isoform inhibitor is always an issue due to similarity of functions of PKC isoforms (Mochly-Rosen et al.,
2012). Our study also suggests that ATF2 might mediate mitochondria-mediated apoptosis after global cerebral ischemia. If ATF2 can be blocked efficiently from being transported out from nucleus to the mitochondria, this might prove to be a useful therapeutic target after ischemia.
However, specific ATF2 nuclear export inhibitor could be difficult to obtain due to its multiple nuclear export signals (Hsu and Hu, 2012). Moreover, PKCε phosphorylates ATF2 at Thr52 residue in vivo. The functional significance of this finding in relation to ATF2 transcriptional activity needs to be investigated.
72 A chemical-genetics approach has been developed to identify immediate phosphorylation substrates of kinases and to study results of kinase inhibition by selective, cell-permeable, small molecule inhibitors (Habelhah et al., 2001; Shah and Shokat, 2002; Kumar et al., 2015). It provides not only a method to specifically interrogate the role of individual kinases in cell signaling, but also a method to reveal kinase-specific sets of phosphorylated substrates. The AS-
PKC models herein will not only be useful in identifying PKCspecific substrates and designing modulators in silico to manipulate PKCkinase activity but will also provide a foundation for future application of this chemical-genetics approach to other protein kinases.
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