Regulation of Placental Autophagy by the Bcl-2 Family Proteins Myeloid Cell Leukemia Factor 1 (Mcl-1) and Matador/Bcl-2 Related Ovarian Killer (Mtd/Bok)

Home , Intermediate trophoblast

Manpreet Kalkat

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto

Regulation of Placental Autophagy by the Bcl-2 Family Proteins Myeloid Cell Leukemia Factor 1 (Mcl-1) and Matador/Bcl-2 Related Ovarian Killer (Mtd/Bok)

Manpreet Kalkat

Master of Science

Department of Physiology University of Toronto

2010 Abstract

The process of autophagy is defined as the degradation of cellular cytoplasmic constituents via a lysosomal pathway. Herein I sought to examine the regulation of autophagy in the placental pathologies preeclampsia (PE) and intrauterine growth restriction (IUGR). I hypothesized that the Bcl-2 family proteins Mcl-1L and MtdL regulate placental autophagy and contribute towards dysregulated autophagy in PE. My results demonstrate that Mcl-1L acts to repress autophagy via a Beclin 1 interaction, while MtdL induces autophagy when it interacts with Mcl-1L. My data indicate that while autophagy is elevated in PE, a pathology characterized by oxidative stress, it is decreased in IUGR, a hypoxic pathology. Treatment with sodium nitroprusside to mimic PE caused a decrease in Mcl-1L and an increase in MtdL levels in response to oxidative stress, thereby inducing autophagy. Overall, my data provide insight into the molecular mechanisms contributing to the pathogenesis of preeclampsia.

Acknowledgments

I would like to acknowledge the support my supervisor, Dr. Isabella Caniggia, who has provided me with many valuable lessons that have been instrumental in both my professional and personal growth in this early stage of my scientific career.

I would also like to express my gratitude for the guidance, feedback and support provided to me by the members of my student supervisory committee: Dr. Mingyao Liu, Dr. Jim Woodgett and Dr. Stephen Lye.

I'd like to extend my heartfelt thanks to all the members of the Caniggia Lab and the other labs of the sixth floor of TCP for their encouragement and moral support throughout the ups and downs of research. In particular, I would like to thank Julia Garcia for her scientific feedback, suggestions and excellent advice. I'd also like to thank Livia Deda, Tharini Sivasubramaniyam, Antonella Racano and Jocelyn Ray for their consistent scientific and emotional support, and importantly, for all the of the laughter and humour they have all brought to the lab every day.

Finally, I'd like to thank my family, who have not seen much of me in the last two years (for which I apologize) but without whom I would not have had the luxury to explore my intellectual pursuits.

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Contributions

I would like to acknowledge the contributions of the following people in the generation of the data described in this thesis:

Julia Garcia, for the generation of the HEK293-GFP and HEK293-GFPMtdL stably transfected cell lines and for the construction of the FlagMtdL, Mcl-1L and RFP-Mcl-1L vectors.

Doug Holmyard, for processing of placental tissue and HEK293 stably transfected cell lines for electron microscopy.

Mount Sinai Hospital Biobank (Toronto, Canada) and Dr. Tulia Todros (University of Turin, Italy) for supplying human placental tissue for immunoblot analysis and immunofluorescence staining.

Table of Contents

Acknowledgments ...... iii

Table of Contents ...... v

List of Figures ...... x

Chapter 1 ...... 1

1 Introduction ...... 1

1.1 The Placenta ...... 1

1.1.1 Early Placental Development ...... 1

1.1.2 Development of the Villous Structure ...... 4

1.1.3 Onset of placental perfusion and oxidative stress ...... 8

1.1.4 Preeclampsia ...... 8

1.1.5 Cell Death and Placentation ...... 9

1.1.6 Intrauterine growth restriction (IUGR) ...... 10

1.2 Autophagy ...... 11

1.2.1 Autophagic Induction ...... 12

1.2.2 Autophagic Nucleation and Expansion ...... 17

1.2.3 Autophagosome Fusion ...... 19

1.2.4 Mitophagy ...... 21

1.3 Bcl-2 family members ...... 21

1.3.1 Bcl-2 family regulation of apoptosis ...... 21

1.3.2 Bcl-2 Proteins and Mitochondrial Morphogenesis ...... 25

1.3.3 Bcl-2 proteins and non-apoptotic cell death ...... 25

1.3.4 Bcl-2 family members and autophagy ...... 26

1.3.5 Bcl-2 family proteins and the placenta ...... 27

1.4 Myeloid Cell Leukemia Factor 1 ...... 28

1.5 Matador/Bcl-2 related ovarian killer (Mtd/Bok) ...... 30

1.6 Human Placenta and Mtd/Mcl-1 ...... 32

1.7 Rationale and Hypothesis ...... 33

Chapter 2 ...... 34

2 Materials and Methods ...... 34

2.1 In Vitro Studies ...... 34

2.1.1 Cell Culture ...... 34

2.1.2 Protein Extraction ...... 35

2.1.3 Bradford Protein Assay ...... 35

2.1.4 Western Blot Analysis ...... 35

2.1.5 Immunoprecipitation studies ...... 36

2.1.6 Immunofluorescence staining ...... 37

2.1.7 Electron Microscopy ...... 39

2.1.8 Transient Transfection Experiments ...... 39

2.1.9 SNP Treatment ...... 40

2.1.10 Etoposide Treatment ...... 41

2.1.11 Rapamycin Treatment ...... 41

2.1.12 Glucose Starvation ...... 41

2.1.13 Oxygen Experiments ...... 41

2.2 Human Placental Tissue Experiments ...... 42

2.2.1 Collection and Sampling ...... 42

2.2.2 Protein Extraction ...... 44

2.2.3 Western Blotting ...... 44

2.2.4 Immunofluorescence Staining ...... 45

2.2.5 Electron Micrographs ...... 46

2.3 Statistical Analysis ...... 46 vi

Chapter 3 ...... 47

3 Results ...... 47

3.1 Mcl-1L is a repressor of autophagy ...... 47

3.2 Mcl-1L and MtdL interact ...... 54

3.3 MtdL is a novel inducer of autophagy ...... 58

3.4 MtdL decreases the endogenous interaction of Mcl-1L and Beclin 1 ...... 66

3.5 Autophagy is elevated in preeclampsia and decreased in IUGR ...... 69

3.6 Electron micrographs indicate increased presence of autophagosomes in PE...... 73

3.7 A model of oxidative stress induces autophagy via alterations in Mcl-1L/MtdL expression ...... 80

Chapter 4 ...... 87

4 Discussion ...... 87

4.1 Mcl-1 and MtdL are regulators of placental autophagy ...... 87

4.2 Oxidative stress induces autophagy in preeclampsia ...... 92

Chapter 5 ...... 97

5 Future Directions ...... 97

5.1 Is oxidative stress in trophoblast also sensed by Atg4? ...... 97

5.2 Are Vps34/PI3KIII complexes involved in MtdL induced autophagy? ...... 97

5.3 Is MtdL contributing to mitophagy? ...... 100

5.4 What is the functional effect of autophagy in trophoblast? ...... 100

References ...... 103

Appendices ...... 118

6 Appendix ...... 118

6.1 Rapamycin Treatment ...... 118

6.2 Low oxygen environment and autophagy ...... 120

6.3 Glucose Deprivation in JEG3 cells ...... 123

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6.4 Autophagy in Placental Development ...... 125

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List of Tables

Table 2.2.1 Clinical parameters of control, preeclamptic and IUGR patients...... 43

List of Figures

Figure 1.1.1 The blastocyst...... 3

Figure 1.1.2. Diagrammatic representation of floating and anchoring placental villi...... 5

Figure 1.1.3 EVT cells remodel maternal spiral arteries...... 7

Figure 1.2.1 Overview of the process of autophagy...... 14

Figure 1.2.2. Nutrient sensing and regulation of autophagy by mTOR...... 15

Figure 1.2.3 Regulation of autophagy by mTOR...... 16

Figure 1.2.4 Autophagosome formation...... 18

Figure 1.2.5 Process of autophagosome formation and fusion...... 20

Figure 1.3.1 Extrinsic and intrinsic pathways of apoptosis...... 23

Figure 1.3.2. Diagrammatic representation of protein domains in Bcl-2 family proteins...... 24

Figure 1.4.1. Diagrammatic representation of Mcl-1 isoforms...... 29

Figure 1.5.1. Schematic diagram of Mtd-L, Mtd-P and Mtd-S...... 31

Figure 3.1.1 Identification of LC3B-II via immunoblot analysis in JEG3 cells...... 48

Figure 3.1.2 Overexpression of Mcl-1L in JEG3 cells...... 50

Figure 3.1.3 Silencing of Mcl-1L in JEG3 cells...... 51

Figure 3.1.4 Effect of Mcl-1L silencing on lysosomal presence in JEG3 cells...... 52

Figure 3.1.5 Rapamycin treatment in HEK293 cells...... 53

Figure 3.2.1 Immunoprecipitation of Mcl-1L in JEG3 cells transfected with FlagMtdL...... 55

Figure 3.2.2 Colocalization of MtdL and Mcl-1L in HEK293 cells...... 57

Figure 3.3.1 Time and dose-course for doxycycline-mediated induction of MtdL...... 60

Figure 3.3.2 Immunoblot analysis of HEK293 cells stably expressing MtdL...... 62

Figure 3.3.3 Lysotracker® Red staining in HEK293 cells...... 63

Figure 3.3.4 Intracellular localization of MtdL in HEK293 cells...... 64

Figure 3.3.5 Electron micrographs of HEK293 cells stably expressing MtdL...... 65

Figure 3.4.1 Immunoprecipitation of Beclin 1 in HEK293 cells stably expressing MtdL...... 68

Figure 3.5.1 Expression of LC3B-II in normal and preeclamptic placentae...... 70

Figure 3.5.2 Expression of LC3B-II in normal and IUGR placentae...... 71

Figure 3.5.3 Spatial localization of Beclin 1 and Mcl-1 in normal and preeclamptic placentae. 72

Figure 3.6.1 Electron micrographs of the syncytium of normal and pathological placentae...... 75

Figure 3.6.2 Electron micropraphs of CT cells in normal and pathological placentae...... 77

Figure 3.6.3 Electron micrographs of the endothelium of normal and pathological placentae. .. 79

Figure 3.7.1 Expression of Mcl-1L and LC3B-II across time and dosage with SNP treatment in JEG3 cells...... 82

Figure 3.7.2 Expression of Mcl-1L, MtdL and LC3B-II in SNP treated JEG3 cells...... 84

Figure 3.7.3 Lysotracker® Red staining in SNP treated JEG3 cells...... 85

Figure 3.7.4 Spatial localization of Mtd in SNP treated JEG3 cells...... 86

Figure 4.1.1 Putative model of MtdL induced autophagy...... 90

Figure 4.2.1 Proposed model of oxidative stress induced autophagy in trophoblast cells...... 94

Figure 5.2.1 Co-immunoprecipitation for the association of Beclin 1 with Vps34/PI3KIII in HEK293-GFP and HEK293-GFPMtdL cells...... 99

List of Appendices

Appendix 6.1.1 Expression of p70S6K phosphorylated at Threonine 389 in JEG3, HEK293 and human placental lysate and rapamycin treatment in JEG3 cells...... 119

Appendix 6.2.1 Expression of Mcl-1L and LC3B-II in response to 20% and 3% oxygenation.122

Appendix 6.3.1 Expression of LC3B-II in JEG3 cells in response to glucose starvation...... 124

Appendix 6.4.1 Expression of LC3B-II in human placental development...... 126

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List of Abbreviations

3-MA 3-Methyladenine ACOG American College of Obstetrics and Gynaecology ALT alanine aminotransferase AMC age-matched control AMP adenosine monophosphate AMPK adenosine monophosphate kinase ATP adenosine triphosphate BOK Bcl-2 related ovarian killer

CO2 carbon dioxide CS caesarean section delivery CT cytotrophoblast D1 siRNA duplex 1 D2 siRNA duplex 2 DAPI 4',6-diamino-2-phenylindole DME Dulbecco's modified Eagle's medium ECL enhanced chemiluminescence EMEM Eagle's minimum essential medium EVT extravillous trophoblast FBS fetal bovine serum GFP green fluorescent protein HBSS Hank's buffered saline solution hCG human chorionic gonadotropin HELLP hemolysis, elevated liver enzymes, low platelets HIF-1 Hypoxia inducible factor 1 HRS hours ICM inner cell mass IP immunoprecipitation IUGR intra-uterine growth restriction LC3B microtubule associated light chain 3 Mcl-1 myeloid cell leukemia factor 1 mins minutes MOMP mitochondrial outer membrane permeabilization Mtd Matador mTOR mammalian Target of Rapamycin mvm microvillous membrane

O2 oxygen OMM outer mitochondrial membrane PBS phosphate-buffered saline

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PE preeclampsia PlGF placental growth factor PVDF polyvinylidene fluoride RFP red fluorescent protein RIPA radioimmunoprecipitation assay buffer SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM standard error of the mean sFlt-1 soluble fms-like tyrosine kinase 1 (VEGFR-1) siRNA small-interfering ribonucleic acid SS siRNA scramble sequence control ST syncytiotrophoblast TBST tris-buffered saline with Tween-20 TC term control TE trophectoderm TGFβ transforming growth factor β TNFα tumour necrosis factor α Tsc tuberous sclerosis complex Thr threonine TM transmembrane VEGF vascular endothelial growth factor VEGFR-1 vascular endothelial growth factor receptor 1 VD vaginal delivery WB western blot XIAP X-linked inhibitor of apoptosis

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Chapter 1

1 Introduction 1.1 The Placenta

The human placenta is a transient organ that serves as a conduit for the transfer of nutrients and oxygen from the mother to fetus, while acting as a protective barrier for the fetus from infectious agents. The placenta is an important source of hormones during pregnancy critical for maintaining pregnancy and proper embryo development, including human chorionic gonadotropin (hCG). hCG is a glycoprotein that maintains the corpus luteum thereby preventing the onset of menstruation (Muyan & Boime 1997). The placenta also produces progesterone and estrogen, hormones that stimulate uterine growth. Proper human placental development is a prerequisite for the growth of a normal and healthy fetus, and heavily relies upon the presence of an optimal intrauterine environment. The uterine environment changes dramatically from the first trimester to term due to a switch from a histiotrophic to a haemotrophic nutrition, as the result of the establishment of fetal-maternal blood flow (Burton et al., 2010). Up until 10 weeks of gestation, nutrition to the fetus is supported by endometrial glands, while the onset of fetal- maternal circulation occurs at about 10-12 weeks of gestation (Burton et al., 2002).

1.1.1 Early Placental Development

The fertilized zygote arrives in the uterine cavity within four to five days post conception. During this time the zygote undergoes a series of mitotic divisions and forms the blastocyst (Moore & Persaud 1998). At the blastocyst stage of development, the outer cell mass, termed the trophectoderm (TE), forms and surrounds a fluid filled interior, the blastocoel, and inner cell mass (ICM) (Figure 1.1.1). The trophectoderm forms the majority of the placental tissues, while the inner cell mass forms the embryo.

At the end of the first week of conception, the trophectoderm attaches to the uterine epithelial surface, the endometrium, forming a connection between the two apical surfaces of the trophectoderm and endometrium (Pijnenborg 1998). In humans, decidualization of the endometrium occurs under the influence of progesterone, resulting in uterine endometrial stromal

2 cells differentiating into decidual cells (Dimitriadis et al., 2010). Decidualization involves remodelling of the extracellular matrix in order to provide a receptive substrate for the invasion of trophoblasts. Trophoblasts secrete extracellular matrix molecules including laminins, and their receptor integrins, as well as matrix-remodeling metalloproteinases (Moore & Persaud 1998). At the site of attachment the trophoblast cells proliferate and differentiate into two cell types, namely the mitotically active cytotrophoblast (CT) cell layer and the multinucleated syncytiotrophoblast (ST) layer, which form as the result of cytotrophoblast fusion. As fusion proceeds, fluid-filled spaces form in the syncytium, termed lacunae, that perforate the syncytium and fill with maternal blood and secretions from endometrial capillaries and uterine glands, supporting placental histiotrophic nutrition (Moore & Persaud 1998; Burton et al., 2002).

Figure 1.1.1 The blastocyst.

At 4-5 days post-fertilization the blastocyst arrives in the uterus. The blastocyst is composed of trophectoderm, which will give rise to the placenta, and the inner cell mass that will become the embryo proper. At 8-9 days post-fertilization the blastocyst will implant into the uterine endometrium.

1.1.2 Development of the Villous Structure

1.1.2.1 Floating Villi

Development of the functional unit of the placenta, the chorionic villi, initiates immediately after implantation. Chorionic floating villi are the primary site for the exchange of gas and nutrients between the fetal and maternal circulations (Moore & Persaud 1998). Cytotrophoblast cells proliferate and extend into the syncytial mass forming sprouts that are termed primary villi. At about 5 weeks of gestation, mesoderm cells extend into the primary villi and transform them into secondary villi that contain a stromal core. The mesenchymal cells then differentiate and undergo the process of de novo vasculogenesis, marking the creation of tertiary villi (Demir et al., 2006). The tertiary villi undergo extensive branching which is characterized by expansion of the stroma and increased angiogenesis, which is mediated both by vascular endothelial growth factor-A (VEGF-A), an important factor that promotes the differentiation of mesenchymal cells into hemangioblastic stem cells, and placental growth factor (PlGF), a factor highly expressed by trophoblast cells (Demir et al., 2004; Burton et al., 2009).

Floating villi account for 90% of all chorionic villi and are suspended in the intervillous space where they are bathed in maternal blood. In their mature form, they are composed of an overlying syncytiotrophoblast layer that is maintained by the continual renewal by cytotrophoblast cells which divide and fuse with the overlying syncytium (Figure 1.1.2). In the second and third trimesters of gestation, the syncytium begins to aggregate and form syncytial knots. These knots are comprised of aging nuclei and are extruded into the maternal circulation, contributing to a pro-inflammatory response that is characteristic of normal pregnancy, and presumed to be cleared in the maternal lung by macrophages (Johansen et al., 1999; Burton & Jones 2009). The process by which cytotrophoblasts fuse to form syncytium and are extruded as knots is termed trophoblast turnover.

Figure 1.1.2. Diagrammatic representation of floating and anchoring placental villi.

Floating villi are comprised of an overlying syncytial layer and underlying cytotrophoblast progenitor cells. Syncytial knots are extruded nuclei produced by the syncytium. Anchoring villous columns connect the placenta to the uterus. They are composed of a proximal and distal region composed of migrating extravillous trophoblast cells that invade the maternal decidua and remodel spiral arteries. ST: syncytiotrophoblast layer; CT: cytotrophoblast, EVT: extravillous trophoblast.

1.1.2.2 Anchoring Villi

Anchoring villi comprise 10% of the chorionic villi, physically connect the placenta and embryo to the uterine wall, and are created as the result of trophoblast invasion into the maternal decidua (Moore & Persaud 1998). Cytotrophoblasts that escape the confines of the syncytial layer and form columns of nonpolarized cells are termed extravillous trophoblast cells (EVT) (Damsky et al., 1992). Villous cytotrophoblasts that are anchored to the basement membrane express α6β4 integrin subunits and laminins, while nonpolarized cells in the anchoring column along the invasive pathway express α5β1 integrin and a fibronectin matrix (Damsky et al., 1992). Growth factors and cytokines regulate trophoblast invasion, in particular interleukin-1β and epidermal growth factor have been demonstrated to stimulate invasion of trophoblast cells (Bass et al., 1994; Librach et al., 1994). In contrast, transforming growth factor β (TGFβ) signalling in response to hypoxia inducible factor 1 (HIF-1) inhibits the invasive capacities of trophoblast cells (Graham et al., 1992; Caniggia et al., 1997; Caniggia et al., 2000).

Trophoblast invasion follows two different pathways, the interstitial and the endovascular pathway (Figure 1.1.3). The interstitial pathway is controlled by EVT cells that migrate through the decidua and enter the inner myometrium at about 8 weeks of gestation and replace smooth muscle cells with a fibrinoid material. The endovascular pathway begins with the invasion of arterioles and arteries by the EVTs, and relining of the vessel endothelium. Invasion of the uterine wall occurs up to the first third of the myometrium and associated spiral arteries, and is maximal during the first trimester of gestation, peaking at about 10-12 weeks of gestation (Caniggia et al., 2000).

Conversion of the vessels and the replacement of the muscular wall by the EVTs results in the spiral arteries no longer being under the control of maternal neurovascular control mechanisms, resulting in a low resistance uterine network that efficiently increases the volume of maternal blood being delivered to the placenta (Pijnenborg 1998; Jauniaux et al., 2006). While fetal vessels appear in the developing villi at about 11-15 days following conception, a maternal- fetal circulation does not commence as endovascular plugs of EVT inhibit maternal blood flow until they regress at about 10-12 weeks of gestation. This restricted blood flow protects the fetus from teratogenesis mediated by reactive oxygen species and maintains a low level of metabolism in early blastocyst development (Jauniaux et al., 2000; Burton et al., 2010).

Figure 1.1.3 EVT cells remodel maternal spiral arteries.

In the interstitial pathway of remodeling, EVT cells degrade the smooth muscle wall of the spiral arteries and deposit fibrinoid material. In the endovascular pathway EVT cells re-line the endothelium and occlude the vessel until 10-12 weeks of gestation. EVT: extravillous trophoblast.

1.1.3 Onset of placental perfusion and oxidative stress

An early environment of low oxygen is recognized as an important factor in proper placental development, as low oxygen tension maintains trophoblast cells in a proliferative and non-invasive phenotype that is essential for early placentation events (Genbacev et al., 1996; Genbacev et al., 1997; Caniggia et al., 2000). Experiments using placental villous explants have demonstrated that maintaining the villous in low oxygen conditions of about 3% oxygen has a positive effect on the outgrowth capability of EVT cells, but nonetheless maintains these cells in a noninvasive phenotype (Genbacev et al., 1997; MacPhee et al., 2001). At 10-12 weeks of gestation, perfusion of the intervillous space results in a highly invasive EVT cell phenotype (Rodesch et al., 1992). The onset of maternal circulation results in a three-fold increase in intraplacental oxygen concentration. This was measured by oxygen electrode experiments that demonstrated an increase in oxygen tension from 15-20mmHg (2-3%) at 8 weeks of gestation to

55mmHg (8%) at 12 weeks of gestation, and a subsequent decrease to 6% O2 at term due to increased oxygen extraction by the fetus (Rodesch et al., 1992; Jauniaux et al., 2000). The syncytial layer of the placenta is especially exposed to damaging reactive oxygen species as it is directly bathed in maternal blood and contains low concentrations of antioxidant enzymes in the first trimester of pregnancy (Watson et al., 1997; Watson et al., 1998). From 8 weeks of gestation, a peak of markers of oxidative stress is observed in placental tissue, and is hypothesized to drive trophoblast differentiation, invasion and maturation (Genbacev et al., 1997; Caniggia et al., 2000; Jauniaux et al., 2000). Importantly, alterations in invasive properties have been associated with trophoblast pathology.

1.1.4 Preeclampsia

Preeclampsia (PE) is a devastating disease of placental origin, affecting 5-8% of pregnancies, and is a leading cause of fetal morbidity and mortality (2002). While many theories exist on the cause of PE, it is recognized that the placenta is central to this disorder, as the only symptomatic cure is the removal of the placenta. Hydatidiform moles, which develop in the absence of a fetus, can also lead to the development of PE, supporting the important role of the placenta in this pathology (Chun et al., 1964). Risk factors for PE include multiple gestations, first pregnancies, chronic hypertension, diabetes, and vascular disease. Early onset PE is characterized by onset of clinical symptoms of pregnancy-induced hypertension of greater than 140/90mmHg and proteinurea of ≥ 0.3g per 24 hrs after 20 weeks of gestation in a woman with

9 previously normal blood pressure and urine (2002). PE can also be characterized by other symptoms including edema, headaches, epigastric pains, and visual disturbances, and in severe cases, by hemolysis, elevated liver enzymes and low platelets (HELLP syndrome) (2002).

The etiology of preeclampsia is unknown; however some consensus exists on the origin of this disease of pregnancy. A general agreement has emerged that preeclampsia is the result of defects in early placental development and trophoblast invasion, resulting in shallow invasion, insufficient conversion of spiral arteries and reduced or abnormal placental perfusion (Robertson et al., 1985; Jauniaux et al., 2006). Histological examination of the placental bed from preeclamptic women has revealed that there is limited migration into the decidua and reduced invasion into the myometrial portions of the maternal spiral arteries as well as incomplete integrin switching as the trophoblast compartment remains α5 positive (Redline & Patterson 1995; Zhou et al., 1997). Ultrasound Doppler analysis of preeclamptic women demonstrated that they have reduced placental blood flow and increased uterine vascular resistance (Harrington et al., 1996). This altered perfusion leads to both a status of decreased oxygenation in preeclampsia and intermittent blood flow, leading to transient hypoxia/reoxygenation stress and nitrosative stress to the placenta (Myatt et al., 1996; Hubel 1999; Hung et al., 2004; Myatt & Cui 2004; Takagi et al., 2004; Soleymanlou et al., 2005). Preeclamptic placentae have been demonstrated to have a phenotype of immature cytotrophoblast cells with increased rates of proliferation and decreased invasive capabilities, as well as the presence of increased apoptotic cell death, all of which have been hypothesized to be the result of decreased oxygenation of the trophoblast (Genbacev et al., 1996; Soleymanlou et al., 2005; Soleymanlou et al., 2007; Ray et al., 2009).

1.1.5 Cell Death and Placentation

Apoptosis, a form of programmed cell death, has been recognized as an important regulator of normal placentation in order to maintain tissue homeostasis. Normal placentation is associated with some degree of trophoblast cell apoptosis. Caspase-8 has been determined to be an important factor in cytotrophoblast-syncytial fusion, as knockdown of caspase-8 activity in placental villous explants prevented fusion (Black et al., 2004). As well, nuclei of the syncytium that display chromatin condensation shed into the maternal circulation as syncytial knots, in a process that shares similarities with apoptosis (Jones & Fox 1991; Huppertz et al., 1998; Johansen et al., 1999; Heazell & Crocker 2008). As stated previously, this is thought to

10 contribute to a status of increased inflammatory response that is characteristic of normal pregnancy. However, in preeclampsia, apoptosis and shedding is increased, and this has been hypothesized to contribute to the excessive inflammatory response typical of this disease (Johansen et al., 1999; Goswami et al., 2006). This increased trophoblastic shedding may be the result of oxidative injury, as hypoxia and hypoxia-reoxygenation injury has been demonstrated to increase apoptosis in the syncytium of preeclamptic placentae (Burton & Jones 2009). The systemic inflammatory response and endothelial dysfunction in preeclampsia is thought to arise from factors in the maternal circulation as a result of excessive placental shedding. One factor of interest in preeclampsia is the soluble receptor for vascular endothelial growth factor, sVEGFR- 1, also termed soluble Fms-like tyrosine kinase 1 (sFlt-1), which has been reported to be secreted in response to hypoxia (Maynard et al., 2003; Nevo et al., 2008). When sFlt-1 is present in excess, it binds and inactivates VEGF-A, a key factor for endothelial survival and vasculogenesis, and is thought to induce systemic endothelial dysfunction, as evidenced by engorged glomerular capillary endothelial cells of the kidney (Maynard et al., 2003; Khankin et al., 2010).

1.1.6 Intrauterine growth restriction (IUGR)

The growth of a fetus is determined by both the genetic potential of the fetus and the nutritional and hormonal environment of the conceptus. Intrauterine growth restriction (IUGR) is defined as when the fetus fails to reach its potential growth and is generally accepted to be when the fetus falls below the 5th percentile for its gestational age (2001; Cetin & Alvino 2009). IUGR affects 7-15% of pregnancies and is a leading cause of perinatal mortality and morbidity (Alexander et al., 2003; Baschat 2004; Cetin & Alvino 2009). Many factors have been shown to contribute to low birth weight and can be grouped into maternal, fetal or placental factors (2001). Maternal contributors include smoking, extremes of reproductive age, malnutrition, diabetes, lung disease, and multiple gestation (2001). Placental factors include the presence of preeclampsia; however, IUGR can occur in the absence of maternal pregnancy induced hypertension. When IUGR is suspected, the diagnosis can be confirmed with ultrasound and Doppler analysis, with two sets of examinations 2 weeks apart (2001). However, the cause of most IUGR cases remains indeterminate, and share the common placental phenotype of placental insufficiency, the notion that the placenta is unable to sustain fetal growth (Sibley et al., 2005). While both preeclampsia and IUGR share the feature of decreased migration of the EVT cells

11 into the decidua, why some patients develop the symptoms of both preeclampsia and IUGR, while others are not affected by preeclampsia, remains to be established.

Very low birth weight from mid to late gestation has been correlated with an increased propensity towards adult disease such as coronary heart disease, obesity and diabetes later in life (the metabolic syndrome) (Barker 1995; Barker et al., 2010). In IUGR, both the fetus and placenta are small in size, however the relative ratio of placental mass to fetal mass is increased (Pardi et al., 2002). Many groups have characterized defects in placental metabolism and transport in IUGR, consistently demonstrating that there is decreased transport of amino acids to the IUGR fetus, and a decreased surface for the exchange of nutrients across the placenta (Jansson et al., 1993; Challis et al., 2000; Huppertz et al., 2006). Along with increased thickness of the villous structure, this reduces the diffusing capacity of the placenta by up to 60% (Huppertz et al., 2006). Severe IUGR pregnancies have been reported to exhibit reduced fetal- placental blood flow, and also to have a malformed and hypovascularized phenotype, suggesting IUGR placentae are hypoxic, and possibly contribute towards decreased nutrient exchange (Kingdom & Kaufmann 1999; Kingdom et al., 2000; Chen et al., 2002; Arroyo & Winn 2008; Nevo et al., 2008).

1.2 Autophagy

Autophagy is a cellular process that results in the degradation of cytoplasmic constituents via a lysosomal pathway by the engulfment of this material into double-membraned vacuoles termed autophagosomes (Deter et al., 1967; Melendez & Neufeld 2008). While there are three different kinds of autophagy (macroautophagy, microautophagy and chaperone-mediated autophagy), the term autophagy generally refers to macroautophagy. It is distinguishable from proteasome-mediated degradation processes in that autophagy is the only known cellular process that degrades entire organelles, including mitochondria, portions of the endoplasmic reticulum and ribosomes (Klionsky & Emr 2000; Kim et al., 2007).

Autophagy is a ubiquitous cellular process that has been characterized in many organisms from yeast to humans (Klionsky et al., 2007). While the process was identified more than 50 years ago, recent interest in this field of research has increased due to both the discovery of genes that control this process, the autophagy-related genes (Atg), and the understanding that autophagy has a large impact on human health and disease (Thumm et al., 1994; Harding et al.,

1995; Mizushima & Hara 2006; Melendez & Neufeld 2008; Mizushima et al., 2008). The best understood function of autophagy remains as an internal nutrient source for starved cells, however autophagy also has many other complex cellular functions. Overall, the process of autophagy can be divided into four distinct steps: induction, nucleation, expansion and fusion (Klionsky et al., 2007; Mizushima 2007).

1.2.1 Autophagic Induction

Autophagic induction is the upstream signaling event that results in autophagosome nucleation and formation (Figure 1.2.1). While autophagy is active at a basal level in most cells for protein turnover and organelle recycling, it can be rapidly activated in response to cellular stressors (Melendez & Neufeld 2008). The most potent activator of autophagy is nutrient deprivation of glucose and amino acids, which partially act by inactivation of the mammalian Target of Rapamycin, mTOR (Wullschleger et al., 2006; Melendez & Neufeld 2008). mTOR is a major nutrient sensor of the cell, and controls cell growth in response to nutrient availability, and is an important negative regulator of autophagy (Kim et al., 2002; Yu et al., 2010). It consists of two different complexes, entitled mTORC1 and mTORC2, of which only mTORC1 is sensitive to inhibitory rapamycin treatment and acts as the predominant nutrient sensor, while mTORC2 controls actin organization and possibly can also respond to the nutrient status of a cell (Wullschleger et al., 2006). The mTOR pathway can respond to growth factors via the PI3K class I pathway in response to insulin or insulin-like growth factors (IGFs) binding to their receptors, leading to the recruitment and activation of PI3K class I (Wullschleger et al., 2006). The tuberous sclerosis proteins Tsc1 and Tsc2 form a heterodimer that negatively transduces PI3K class I signaling to mTOR (Figure 1.2.2). Amino acid starvation, especially with leucine, results in rapid dephosphorylation of the downstream targets of mTOR, p70 ribosomal protein S6 kinase (p70S6K) and elongation factor 4 binding protein 1 (4E-BP1) (Kanazawa et al., 2004). While the exact mechanism of amino acid sensing is not known, it has been proposed that amino acid deprivation can by sensed via Tsc1/Tsc2 or via the Rheb GTPase (Gao et al., 2002; Garami et al., 2003).

mTORC1 senses the energy of a cell via AMP-activated protein kinase (AMPK), which is activated in response to low cellular energy levels, which is indicated by a high AMP to ATP ratio (Wullschleger et al., 2006; Luo et al., 2010). Activation of AMPK downregulates energy-

13 costly cellular events such as protein synthesis and results in increased ATP-generating processes including fatty acid oxidation and autophagic activation. Activated AMPK exerts its function by directly phosphorylating Tsc2 and leading to the inhibition of mTORC1 signaling (Inoki et al., 2003). mTOR also responds to cellular stress events, such as hypoxia, which results in decreased mTOR signaling in a mechanism that involves activation of Hypoxia Inducible Factor 1 (HIF-1) (Wullschleger et al., 2006). Prolonged hypoxia leads to ATP depletion and subsequent activation of AMPK.

The mechanism by which mTOR negatively regulates autophagy in mammalian cells is not entirely understood. However, in yeast and mammalian systems it is known that TOR inhibits the protein kinase Atg1, which is an early step in the activation of autophagy (Kamada et al., 2000; Scott et al., 2007). As well, in yeast TOR can hyperphosphorylate Atg13, reducing its binding affinity and activation of Atg1, and this event can be reversed by rapamycin treatment (Figure 1.2.3). Rapamycin inhibits mTOR kinase activity, resulting in downstream dephosphorylation and activation Atg13 (Peterson et al., 1999; Kamada et al., 2000).

Figure 1.2.1 Overview of the process of autophagy.

An isolation membrane forms from the endoplasmic reticulum and encircles cytoplasmic constituents forming the autophagosome. Subsequent fusion events with lysosomes result in the breakdown and recycling of the engulfed components.

Figure 1.2.2. Nutrient sensing and regulation of autophagy by mTOR.

Conditions of low ATP or hypoxia can be sensed by AMPK leading to mTOR inactivation. Low amino acid levels could inactivate mTOR either by sensing by Rheb or Tsc1/Tsc2. IGF and IGF-like signaling is sensed through PI3K class I stimulating the protein synthesis and cell growth. P: phosphorylation. Note: Arrows do not imply direct effects, pathways have been collapsed for clarity.

Figure 1.2.3 Regulation of autophagy by mTOR.

In conditions of activated mTOR, Atg13 is hyperphosphorylated thereby reducing its affinity for Atg1. When mTOR is inactivated by a variety of upstream signalling mechanisms, Atg13 is dephosphorylated and binds with high affinity to Atg1, thereby activating Atg1 and leading to the formation of the nucleation complex. P: phosphorylation.

1.2.2 Autophagic Nucleation and Expansion

The genes that are necessary for autophagy were first discovered in screens using yeast mutants that were defective in vacuole formation, and homologues for these genes have been discovered in mammalian systems (Harding et al., 1995). Autophagy genes control diverse aspects of autophagosomal formation, including expansion and fusion events.

A functional complex involved in vesicle nucleation consists of Beclin 1/Atg6/Vps30, Vps15/p150 and Atg14, and is termed Complex I (Itakura et al., 2008). This complex is present at the level of the isolation membrane, which is thought to be located at the endoplasmic reticulum or trans-Golgi network (Simonsen & Tooze 2009; Yen et al., 2010). Autophagosome formation has been suggested to result from the accumulation of Complex I and activity of the class III PI3K/Vps34 to produce phosphatidylinositol 3-phosphate (PI3P) (Hamasaki & Yoshimori 2010). PI3P production is thought to locally change the ER membrane curvature to create a site of autophagosome formation termed the autophagosome cradle (Figure 1.2.4). The autophagosome cradle develops to form the precursor structure to the autophagosome, the omegasome, which is still physically connected to the ER (Hamasaki & Yoshimori 2010).

Two conjugation systems that resemble ubiquitin-like protein conjugation are essential for the expansion of autophagosomes. In order for vesicle completion, Atg12 is activated by binding to Atg7 by a thioester bond. It is then transferred by the E2-like molecule Atg10 to Atg5 (Mizushima et al., 1998). A second ubiquitin-like system is used to anchor Atg8 (also known as microtubule-associated light chain 3, LC3) to the autophagosomal membrane, where it is present until autophagosome degradation (Melendez & Neufeld 2008). LC3 has two isoforms, one which is cytoplasmic and another form that is processed and associated with the autophagosomal membrane, termed LC3-II (Kabeya et al., 2000). Two LC3 bands, LC3-I and LC3-II can be detected via immunoblotting, at 18 and 16 kDa respectively. Expression levels of LC3-II are frequently used as a marker for autophagy, while LC3-I is particularly abundant in brain tissue (Mann & Hammarback 1996; Kabeya et al., 2000). LC3-II is conjugated to phosphatidylethanolamine by a series of reactions catalyzed by Atg4, Atg7 and Atg3, resulting an autophagosome dotted with LC3-II (Ichimura et al., 2000).

Figure 1.2.4 Autophagosome formation.

The process of autophagosome formation is thought to occur at the ER. PI3K class III activity produces PI(3)P which is thought to lead to deformation of the ER leading to the creation of the autophagosome cradle. The pre-autophagosomal structure is referred to as the omegasome.

1.2.3 Autophagosome Fusion

Autophagosomes can fuse with endosomes forming a cellular compartment termed amphisomes, and the maturation of autophagosomes involves many sequential fusion events with vesicles from the endosomal and lysosomal cellular compartments, during which the autophagic compartment becomes more acidic (Figure 1.2.5) (Dunn 1990; Berg et al., 1998). The final stages of autophagy involve the transport of autophagosomes to lysosomes in a dynein- dependent manner, and membrane fusion of the two cellular compartments (Melendez & Neufeld 2008). The fusion products are then termed autophagolysosomes, and the contents are degraded by lysosomal hydrolases (Eskelinen 2005). Finally, the degradation products are returned to the cytoplasm and reused for cellular metabolism.

Figure 1.2.5 Process of autophagosome formation and fusion.

Early events in the autophagic process include nucleation and bending of the membrane to form the initiat autophagic vacuole. Fusion events with endosomes and lysosomes leads to maturation of the autophagosome and increased acidity of the organelle, thus creating the mature late stage degradative autophagolysosome.

1.2.4 Mitophagy

Mitochondria, via mitochondrial oxidative phosphorylation provide a large amount of energy to the cell, but this organelle also is the source of cellular reactive oxygen species (ROS) that can cause damage to lipids, DNA and proteins. Autophagy that is specific to the turnover of mitochondria has been termed mitophagy, and is the only known pathway that results in the clearance of mitochondria from the cytoplasm of a cell (Kim et al., 2007; Melendez & Neufeld 2008). Conditions that lead to mitochondrial stress have been reported to lead to mitophagic activation; however, the molecular mechanisms behind mitochondrial targeting are not well understood (Kanki et al., 2009). Studies on reticulocyte maturation have pointed to Nix (a Bcl-2 family member) and Parkin (a ubiquitin ligase), as proteins that are required for mitophagy in mammalian cells (Narendra et al., 2008; Ding et al., 2010). In yeast, mitophagy was found to be induced in response to nitrogen starvation, rapamycin treatment, and disturbances in mitochondrial membrane potential (Nice et al., 2002). Upon induction of mitophagy, Atg32 binds to Atg11, recruiting mitochondria into the pre-autophagosomal structure. However, in mammalian systems, the homologues to yeast Atg32 and Atg11 remain to be established (Nice et al., 2002).

1.3 Bcl-2 family members

The B cell lymphoma proteins (Bcl-2) are a group of promiscuous proteins with the capacity to hetero- and homodimerize with one another. The Bcl-2 family are important regulators of apoptotic cell death, but have also been characterized in the regulation of other cellular events, including cellular proliferation, mitochondrial morphogenesis, and autophagy. Bcl-2 family proteins have conserved Bcl-2 Homology (BH) domains that mediate the direct interactions between different members and govern how the proteins function. There are three subcategories for Bcl-2 proteins: BH3-only proapoptotic molecules, BH-multidomain proapoptotic molecules, and prosurvival molecules.

1.3.1 Bcl-2 family regulation of apoptosis

Apoptosis is the process of controlled cell death and is characterized by the morphological characteristics of nuclear and cytoplasmic condensation and cellular fragmentation (Kerr et al., 1972). Apoptotic cell death is mediated by a group of proteins termed

22 cysteine-aspartic acid proteases (caspases) that specifically cleave proteins after Asp residues (Salvesen & Dixit 1997). Caspases exist as inactive zymogens or proenzymes which are cleaved to generate two subunits that form a heterotetramer, the active form of the protein (Wang & Youle 2009).

There are two pathways through which apoptosis can occur, the extrinsic and intrinsic apoptotic pathways. The extrinsic pathway is also termed the death-receptor pathway, in which cell death is activated from outside the cell by ligand binding to cell death receptors such as Fas, TNF, TRAIL and DR3-6 (Figure 1.3.1.) (Wang & Youle 2009). Once the receptors are activated each receptor can form the death-inducing signaling complex (DISC) via the recruitment of Fas- associated death domain (FADD) and procaspase-8 and -10. Once caspase-8 and caspase-10 are activated, they directly cleave and activate caspase-3 and 7.

The intrinsic pathway is also called the mitochondrial pathway and involves regulation at the level of the mitochondria by the Bcl-2 antiapoptotic and proapoptic proteins. Bcl-2 antiapoptotic proteins include Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1/Bfl-1. Bcl-2 proapoptotic multidomain proteins include Mtd/Bok, Bax and Bak. These proteins regulate the cellular commitment to apoptosis by regulating the mitochondrial outer membrane potential (MOMP) (Wei et al., 2001). Once the MOMP is disturbed the cell is committed to death, and cytochrome c is released from the mitochondria. Cytochrome c binds apoptotic protease-activating factor 1 (APAF-1) which then undergoes oligomerization into the apoptosome. The apoptosome leads to caspase-9 activation, followed by caspase 3/7 which then dismantle the cell (Liu et al., 1996; Autret & Martin 2009; Wang & Youle 2009).

The BH3-only family members include Bid, Bad, Bim, Puma, Noxa, Hrk, Bmf, and Nbk/Bik (Figure 1.3.2). These proteins contain only one BH3 homology domain and are activated by cell death stimuli. Their BH3 domain is an amphipathic α-helix that can bind with the hydrophobic grooves formed by BH1 and BH2 domains of anti-apoptotic and proapoptotic Bcl-2 family proteins. BH3-only proteins operate upstream of the multidomain proteins, and when the multidomain proteins are deficient, BH3-only proteins cannot induce apoptosis (Wei et al., 2001; Zong et al., 2001). While Bcl-2 proteins share features with α-helical pore-forming proteins, a consensus has not been reached for the molecular mechanism of how MOMP actually is achieved.

Figure 1.3.1 Extrinsic and intrinsic pathways of apoptosis.

The extrinsic pathway of apoptosis is activated in response to death ligands. The intrinsic mitochondrial pathway relies on MOMP and cytochrome c release leading to caspase activation.

Figure 1.3.2. Diagrammatic representation of protein domains in Bcl-2 family proteins.

There are three classes of Bcl-2 family proteins, antiapoptotic, proapoptotic multidomain and proapoptotic BH3-only proteins. TM= transmembrane domain, BH= Bcl-2 homology domain.

There are three hypotheses regarding the activation of MOMP by proapoptotic Bcl-2 family proteins: direct activation, indirect activation and embedded together models (Leber et al., 2007; Lovell et al., 2008). In the direct activation model, BH3-only proteins are required to activate multidomain proteins by direct interaction, leading to MOMP. Evidence supports that with regards to Bax, MOMP relies upon activation of Bax by BH3-only proteins (Yin et al., 1994; Lovell et al., 2008). In the indirect activation model, proapoptotic proteins are stably bound to antiapoptotic proteins. Upon the induction of apoptotic stimuli, BH3-only proteins bind to the antiapoptotic proteins, releasing the proapoptotic molecules and leading to MOMP. The embedded together model proposes that MOMP does not occur until the multidomain proapoptotic molecules insert into the outer mitochondrial membrane (OMM) and undergo conformational changes prior to assuming the conformation needed to generate MOMP (Leber et al., 2007).

1.3.2 Bcl-2 Proteins and Mitochondrial Morphogenesis

Cells contain many long tubular mitochondria that can interconnect and form networks. These mitochondrial networks are dynamic and undergo remodeling through cycles of mitochondrial fission and fusion events (Autret & Martin 2009). Importantly, these networks can undergo remodeling in response to stress such as changes in energy demand and changing calcium levels. During apoptosis, mitochondria fragment via mitochondrial fission, which may promote cytochrome c release, or alternatively may be a consequence of apoptosis (Frank et al., 2001). Intriguingly, cells that lack Bax and Bak have mitochondria that are shorter with less extensive network formation and decreased rates of fusion, implying that besides their role in apoptosis, multidomain proapoptotic Bcl-2 family proteins play an essential role in regulating the morphology of mitochondria in healthy cells (Karbowski et al., 2006). Conversely, Bax and Bak have also been implicated in mitochondrial fission prior to cytochrome c release (Desagher & Martinou 2000).

1.3.3 Bcl-2 proteins and non-apoptotic cell death

Several studies have identified a form of non-apoptotic cell death. In mouse L929 fibroblast cells, the use of the pan-caspase inhibitor benzyloxycarbonyl-valyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD) directly induced cell death (Yu et al., 2004). Electron

26 micrographs of these cells demonstrated the presence of autophagic vacuoles. A similar phenomenon was observed in Bax/Bak-/- mouse embryonic fibroblasts (MEFs). This cell line was resistant to apoptosis induced by etoposide and staurosporine, but still underwent cell death (Shimizu et al., 2004). Electron micrographs of these cells revealed the formation of autophagosomes/autophagolysosomes, thus implicating autophagy in this alternative death scenario (Shimizu et al., 2004). Treatment of the cells with 3-methyladenine, an inhibitor of the class III PI3K complex resulted in improved cell viability, indicating that autophagy led to cellular demise.

1.3.4 Bcl-2 family members and autophagy

The connection between regulators of autophagy and Bcl-2 family members was first demonstrated with the discovery of Beclin 1 as a novel Bcl-2 interacting protein, and subsequent analyses demonstrated that Beclin 1 contained a BH3 domain (Liang et al., 1998; Oberstein et al., 2007). Beclin 1 is the homologue of yeast Atg6, and is an essential protein for the process of autophagy to occur. Knockout/knockdown experiments of Beclin 1 have demonstrated that Beclin 1 is required for autophagy and that decreased Beclin 1 protein is associated with an increased likelihood of tumorigenesis (Liang et al., 1999; He & Levine 2010). Unlike the other BH3-only Bcl-2 family proteins, Beclin 1 does not exhibit any proapoptotic activity; instead, overexpression of Beclin 1 induces autophagy (Pattingre et al., 2005; Maiuri et al., 2007).

Subsequent studies have determined that Beclin 1 can interact not only with Bcl-2, but also with Bcl-xL, Bcl-w and to a lesser extent with Mcl-1 via GST pull-down assays, structural analysis and fluorescence resonance energy transfer (FRET) assays (Pattingre et al., 2005; Erlich et al., 2007; Maiuri et al., 2007). However, Beclin 1 does not bind to the proapoptotic Bax, Bak, Bad, or tBid (Erlich et al., 2007). Importantly, the interaction of Bcl-2 and Beclin 1 was shown to be highly dependent on the relative abundance of each protein, and overexpression of Bcl-2 could inhibit Beclin 1-induced autophagy in mouse cardiac muscle (Pattingre et al., 2005). Subsequent experiments demonstrated that the interaction of Beclin 1 and Bcl-2 was mediated via the BH1 and BH2 domains of Bcl-2 and that overexpression of Bcl-2 resulted in decreased autophagy and inhibited the formation of the Beclin 1 and Vps34 complex (Pattingre et al., 2005). Intriguingly, only Bcl-2 that was targeted to the ER had this result on autophagic inhibition, while mitochondrial targeted Bcl-2 did not demonstrate the same effect. An

27 inhibitory brake needs to be retained on Beclin 1 to prevent complex formation with Vps34, as Beclin 1 mutants that were unable to bind Bcl-2 had a phenotype of constitutive autophagy and induced cell death (Pattingre et al., 2005).

Subsequently, the effects of BH3-only proteins in autophagic activation were studied, and it was discovered that BH3-only proteins could have an inductive effect on autophagic activation by disrupting the interaction of Bcl-2/Bcl-xL from Beclin (Erlich et al., 2007). Specifically, the interaction of Beclin 1 with the anti-apoptotic protein Bcl-xL was inhibited by coexpression with the BH3-only proteins tBid and Bad, but not by the multidomain proteins Bax and Bak (Erlich et al., 2007). Taken together, this implies that the activation of autophagy requires, to some extent, the release of Beclin 1 from its inhibitory complexes. Maiuri et al. demonstrated that under starvation conditions, continuously more Bcl-xL immunoprecipitated with Bad, indicating that Beclin 1 was complexing with Bcl-xL to inhibit autophagy, providing a greater functional understanding of autophagic regulation by Bcl-2 family members (Maiuri et al., 2007).

1.3.5 Bcl-2 family proteins and the placenta

In human placentation, a balance needs to be reached between trophoblast proliferation, differentiation and death to produce a functioning placenta and a healthy baby, while placental pathologies are characterized with alterations in these processes. In normal placentation, an increase in trophoblast apoptosis is observed in the third trimester; however, preeclamptic placentae are characterized by excessive trophoblast apoptosis. The human placenta expresses many factors of both the extrinsic and intrinsic apoptotic pathways. In terms of the extrinsic pathway, the placenta has been reported to express tumour necrosis factor (TNF) receptors, X- linked inhibitor of apoptosis protein (XIAP), Fas, Fas-ligand and caspase-8 (Gruslin et al., 2001; Kharfi et al., 2006). However, the cell damage present in placental diseases suggests that the intrinsic pathway may be more significant than the extrinsic (Pongcharoen et al., 2004; Kharfi et al., 2006; Heazell & Crocker 2008).

The intrinsic pathway of apoptosis is well studied in the placenta. Bcl-2 is present throughout gestation in the syncytium, and is absent in endothelial cells (Toki et al., 1999; Soni et al., 2010). Bcl-xL has been detected at the mRNA and protein level but its cellular localization has not been described. Bax/Bak have been described in third trimester, where they were found to be localized to the syncytium, and frequently observed in areas with damage,

28 likely contributing to the increased apoptosis observed in term placentae (Ratts et al., 2000; Cobellis et al., 2007). However, in studies using placental explants to model hypoxia/reoxygenation stress or altered oxygen conditions, no alterations in Bax or Bcl-2 expression were observed (Heazell et al., 2008). Both Mcl-1 and Mtd/Bok are highly expressed in reproductive tissues and appear to have very distinct and important roles in trophoblast homeostasis, and will be discussed in further detail (Hsu et al., 1997; Soleymanlou et al., 2005; Soleymanlou et al., 2007; Ray et al., 2009).

1.4 Myeloid Cell Leukemia Factor 1

Mcl-1L is a 37 kDa prosurvival member of the Bcl-2 family. It was first cloned as an early response gene in myeloid cells in response to cytokine treatment, but has been demonstrated to be expressed in a variety of cell types (Kozopas et al., 1993). Mcl-1 expression is important during development as Mcl-1 knockout blastocysts fail to undergo implant, and is also important for differentiation along the monocyte/macrophage pathway in ML-1 human myeloid leukemia cells (Kozopas et al., 1993; Rinkenberger et al., 2000). Mcl-1 has significant homology to Bcl-2 and in particular the C-terminus of Mcl-1 is similar to that of Bcl-2; however, it is unique in its N-terminal domain having PEST (proline, glutamic acid, serine, threonine) sequences, that are known to target proteins for rapid proteasomal turnover (Figure 1.4.1) (Kozopas et al., 1993; Day et al., 2005). The protein contains BH1-3 domains as well as a carboxy-terminal transmembrane (TM) domain (Yang et al., 1995). The N-terminal region of Mcl-1 plays an important role in both the turnover of Mcl-1 as well as its localization. Deletion of the first 79 amino acids of Mcl-1 impairs both its localization to the mitochondria and its antiapoptotic abilities (Germain & Duronio 2007).

Mcl-1 has several different isoforms. Mcl-1S is a splice variant lacking exon 2, and caspase-cleavage at Asp127 and Asp157 results in two different Mcl-1c products (Bae et al., 2000; Bingle et al., 2000; Herrant et al., 2004). Contrary to the prosurvival function of Mcl-1L, Mcl-1S, Mcl-1c127 and Mcl-1c157 are proapoptotic molecules (Bingle et al., 2000; Herrant et al., 2004). Mcl-1 does not bind to all BH3-only proteins with equal affinity; it can tightly bind to Bax, Bak, Mtd/Bok, Bim, Puma and Noxa and phosphorylated Bad, but its binding to Bik, Bmf and Hrk is weaker and it does not interact with antiapoptotic proteins (Leo et al., 1999; Day et al., 2005).

Figure 1.4.1. Diagrammatic representation of Mcl-1 isoforms.

Mcl-1L contains BH1-3 domains and a transmembrane domain as well as N-terminally located PEST sequences that promotes its rapid turnover. Caspase-cleavage sites at Asp127 and Asp157 results in two caspase cleavage products. Mcl-1S is the result of exon II skipping, resulting in a proapoptotic protein product that closely resembles BH3-only proteins. TM: transmembrane region; BH: Bcl-2 homology domain.

1.5 Matador/Bcl-2 related ovarian killer (Mtd/Bok)

Matador/Bcl-2 related ovarian killer (Mtd/Bok) is a multidomain proapoptotic Bcl-2 family member with BH1-3 domains and a C-terminal transmembrane domain (Hsu et al., 1997; Inohara et al., 1998). It was first characterized in a yeast two-hybrid screen using Mcl-1 as a bait in an ovarian fusion cDNA library (Hsu et al., 1997). However, unlike Bax and Bak, Mtd/Bok cannot bind to Bcl-2 or Bcl-xL, and also does not interact with Bax, Bak or Hrk (Inohara et al., 1998). To date, Mtd/Bok has only been reported to interact with Mcl-1, Bfl-1 and viral BHRF1 (Hsu et al., 1997). Several theories exist on the mechanism by which Mtd induces apoptosis independently of Bax and Bak. Firstly, it has been proposed that Mtd could bind and antagonize other prosurvival factors, namely Mcl-1 and Bfl-1. However, a mutant Mtd without a BH3 domain retains apoptotic activity. Alternatively, Mtd could induce apoptosis by directly forming pores in the OMM.

Bok/Mtd has 5 exons, and splicing of exon 3 results in a splicing variant termed Bok/Mtd-S (Figure 1.5.1) (Hsu & Hsueh 1998). Bok-S retains apoptotic activity, but is unable to dimerize with antiapoptotic proteins (Hsu & Hsueh 1998). Bok/Mtd also has a placental specific splice variant lacking exon 2, termed Bok/Mtd-P, that induces apoptotic death under conditions of reduced oxygenation and oxidative stress (Soleymanlou et al., 2005).

Figure 1.5.1. Schematic diagram of Mtd-L, Mtd-P and Mtd-S.

Full-length Mtd-L has all four BH domains and a transmembrane domain. Channel formation occurs in the region between BH1 and BH2 domains. Mtd-P forms as the result of exon II skipping, resulting in a truncated BH3 domain. Mtd-S results from exon III skipping resulting in a fused BH3 and BH1 domain. BH: Bcl-2 homology, TM: transmembrane domain.

1.6 Human Placenta and Mtd/Mcl-1

Mtd expression in the placenta has previously been reported to induce apoptosis of the trophoblast layers (Soleymanlou et al., 2005; Soleymanlou et al., 2007). Analysis of Mtd expression across placental development determined that MtdL and Mtd-S expression remained constant across the first trimester of gestation, while Mtd-P transcript levels increased in early first-trimester compared to later gestational periods (Soleymanlou et al., 2005). The expression of Mtd isoforms in early first trimester was predominantly localized to proliferating cytotrophoblast cells (Ray et al., 2009). Analysis of later first trimester (after 12 weeks of gestation) tissue revealed low Mtd immunoreactivity in cytotrophoblast cells and increased Mtd expression was observed in the apical membrane of the syncytiotrophoblast layers (Soleymanlou et al., 2005; Ray et al., 2009). Analysis of Mtd expression in preeclamptic placentae revealed increased expression of MtdL and Mtd-P in preeclampsia compared to age-matched control placentae and IUGR placentae (Soleymanlou et al., 2005).

Analysis of Mcl-1 expression in preeclampsia compared to age-matched controls revealed increased expression of Mcl-1S and Mcl-1c and decreased expression of Mcl-1L in preeclampsia, contributing to increased trophoblast apoptosis (Soleymanlou et al., 2007). Mtd-L and Mtd-P overexpression in chinese hamster ovarian (CHO) and human choriocarcinoma BeWo cells resulted in the formation of apoptotic cells, expression of cleaved caspase-3 and increased cell death (Soleymanlou et al., 2005). Mtd-P expression was induced by hypoxia, and hypoxia- reoxygenation experiments in explants revealed increased expression of MtdL and Mtd-P and increased trophoblast cell death compared to control. The effect of increased cell death was reversed by the overexpression of Mcl-1 (Soleymanlou et al., 2005; Soleymanlou et al., 2007).

Additionally, MtdL was recently demonstrated to have an effect on trophoblast proliferation. MtdL specifically localizes to cells that are mitotically active in placental tissue, including the cytotrophoblast cells early in first trimester gestation (Ray et al., 2009). siRNA- mediated knockdown of MtdL expression resulted in decreased expression of cyclin E, a marker of the G1 phase of the cell cycle, and inducing increased amounts of MtdL using a stably transfected cell line resulted in increased cyclin E expression and BrdU incorporation, indicating enhanced cell cycling in trophoblast cells expressing MtdL (Ray et al., 2009). In preeclampsia, Mtd was localized to progenitor cytotrophoblast cells that coexpressed either cyclin E or the

33 proliferative marker Ki67, indicating that MtdL was also playing a role in the increased proliferation of cytotrophoblast cells present in preeclampsia and this was correlated to the hypoxic environment in preeclampsia (Ray et al., 2009).

1.7 Rationale and Hypothesis

Mtd and Mcl-1 play an important role in controlling trophoblast cell fate by regulating trophoblast apoptotic cell death and proliferation. Autophagy has recently been recognized as another important cellular response to stress, and in some cases has been reported to contribute to accelerated cell death. As well, autophagy is an important component of normal cellular metabolism and has been reported to be defective or altered in many human disease states and in response to stressors such as hypoxia, nutrient deprivation and oxidative stress.

Many reports have implicated oxidative stress and hypoxia in the pathogenesis of trophoblast-related disorders of pregnancy including PE and IUGR, and studies from our lab have demonstrated that Mcl-1 and Mtd are important mediators of trophoblast cell fate in response to oxidative stress. Therefore, I sought to evaluate whether autophagy is present in the human placenta, and if levels of autophagy are altered in preeclampsia and IUGR. Given the important role Bcl-2 has in regulating autophagy, I sought to examine if Mcl-1, a protein that shares homology with Bcl-2 and also interacts with Beclin 1, exerted an effect on placental autophagy, and whether its binding partner Mtd-L could also contribute to aberrant levels of autophagy in placental pathology. Overall my hypothesis was that Mcl-1 and Mtd are two Bcl-2 family proteins that regulate aberrant autophagy in placental pathology.

Chapter 2 2 Materials and Methods 2.1 In Vitro Studies

2.1.1 Cell Culture

JEG3 choriocarcinoma cells (ATCC, Manassas, VA, USA) were maintained at 20% oxygen tension in Eagle's Minimum Essential Media (EMEM) (ATCC, Manassas, VA, USA) with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA). JEG3 cells were passaged by washing with 10 mL of PBS without Ca2+/Mg2+ and trypsinizing with 0.25% trypsin (Invitrogen, Carlsbad, CA, USA). Cells were counted using trypan blue dye to exclude dead cells (Invitrogen, Carlsbad, CA, USA) and a hematocytometer and seeded overnight into 35mm x 6-well plate for experimentation at a concentration of 2x105 cells for 50% confluence or 4x105 cells for 100% confluence. In order to determine the optimal confluency for future experiments, cells were seeded at 2x105, 4x105 and 6x105 and collected for protein analysis as JEG3 cells have the capacity to proliferate beyond a monolayer.

Flp-In T-Rex human embryonic kidney HEK293 cells (gift of Dr. Gingras, Samuel Lunenfeld Research Institute, Toronto, Canada) were maintained at 20% oxygen tension in Dulbecco's Modified Eagle's (DME) high glucose medium with 10% tetracycline-free FBS (Invitrogen, Carlsbad, CA, USA) and 0.01% Blasticidin (Invitrogen, Carlsbad, CA, USA). The human MtdL gene was amplified from full-length cDNA hMtdL (Open Biosystems) by PCR using the forward primer 5'-GGCGCGCCAGAGGTGCTGCGGCGCTCCTCG-3' and the reverse primer 5'-CAGAGAGATGACCCGGATCCCG-3', as previously described (Ray et al., 2009). The PCR was digested with AscI/BamHI and cloned into pcDNA5/FRT/TO/GFP (gift of Dr. Gingras, Samuel Lunenfeld Research Institute, Toronto, Canada). HEK293-GFPMtdL and HEK293-GFP cell lines were generated by co-transfection of host HEK293-Flp Recombinase Target (FRT) cells with pOG44 vector, containing Flp recombinase gene, and pcDNA5FRT/TO/GFPMtdL vector or pcDNA5FRT/TO/GFP vector under the control of a hybrid human cytomegalovirus (CMV)/tetracycline promoter, and clones were selected for hygromycin resistance as previously described (Ray et al., 2009). HEK293-GFPMtdL and HEK293-GFP cells were maintained in DME high glucose media with 10% FBS, 0.01%

35 blasticidin and 0.4% hygromycin (Invitrogen, Carlsbad, CA, USA). Cells were washed with Ca2+/Mg2+ free PBS and trypsinized with 0.05% trypsin (Invitrogen, Carlsbad, CA, USA) and were seeded at a concentration of 4x105 cells/mL into 35mm 6-well plates or in 100mm plates for immunoprecipitation experiments. Expression of GFPMtdL or GFP was induced with doxycycline treatment (2.5ng/mL and 5ng/mL, Sigma-Aldrich Inc., St. Louis, MO, USA).

2.1.2 Protein Extraction

Cells were collected on ice in RIPA buffer (ddH2O, 3% 5M NaCl, 1.66% 3M Tris-HCl pH 7.5, 1% NP-40) with protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA). The lysate was vortexed and incubated on ice for 10 mins, then centrifuged for 10 mins at 14000xg at 4oC. For immunoprecipitation studies, HEK293-GFPMtdL and HEK293-GFP cells were cross- linked using 1% formaldehyde at room temperature for 10 mins, then rinsed in cold PBS and collected with 1% triton-x buffer (#9803, Cell Signaling Technology Inc., Danvers, MA, USA) containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Protein concentration was measured in duplicates using Bradford Protein Assay.

2.1.3 Bradford Protein Assay

Standards were created in duplicate in PBS at the following concentrations using a stock solution of 25µg/µL of BSA in PBS: 0, 1.25, 2.5, 5, 7.5, 10, 12.5 µg/µL protein. 1µL of each sample was added to 800µL of PBS and 200µL of Bradford Dye (Bio-Rad Laboratories, Hercules, CA, USA) was added to all standards and samples. The mixture was vortexed and transferred to plastic cuvettes and the protein concentration was measured using an absorbance spectrophotometer at 595nm.

2.1.4 Western Blot Analysis

Following Bradford protein analysis, 15 µg of protein from each sample was diluted with 15 µg of 2x sample buffer (10 mL of 1M tris pH6.8, 3.2g SDS, 16mL glycerol, 8mL β- mercaptoethanol, 4mL 0.1% Bromophenol blue, 1mL of ddH2O, diluted in a 1:1 ratio with ddH2O). The samples were seperated by SDS-PAGE. The SDS-PAGE gels were run at 100V with the following buffer:14.4g Tris base, 3.03g glycine, 0.1% sodium dodecylsulfate (SDS) in double-distilled (dd)H2O for a total volume of 1L. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes by hydrating the membranes in 100% methanol and transferred at

100V using the following transfer buffer: 14.4g Tris base, 3.03g glycine, 20% methanol in ddH2O for a total volume of 1L, for 1 hr at room temperature with an ice pack to prevent overheating. Anti-rabbit polyclonal Mcl-1L (12% gel, dilution 1:1000, S-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-rabbit polyclonal LC3B-II (15% gel, dilution 1:2500, ab48394, Abcam, San Francisco, CA, USA), anti-rabbit monoclonal PI3KIII/Vps34 (6% gel, dilution 1:1000, #3358, Cell Signaling Technology Inc., Danvers, MA, USA), anti-mouse monoclonal Beclin (10% gel, dilution 1:500, am 1818a Abgent Inc., San Diego, CA, USA), anti- goat polyclonal β-actin (dilution 1:1000, I-19 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-rabbit polyclonal Mtd/Bok (15% gel, dilution 1:200, AP1310a, Abgent Inc., San Diego, CA, USA) and anti-mouse monoclonal GFP (12% gel, dilution 1:500, B-2, Santa Cruz Biotechnology, Santa Cruz, CA, USA) membranes were blocked in 5% non-fat milk in Tris- buffered saline Tween-20 (TBST) for 1 hr at room temperature, with incubation of primary antibody in 5% non-fat milk at 4oC overnight. 3x10 min washes in TBST were performed and membranes were then incubated with horse-radish peroxidase (HRP) conjugated anti-rabbit, anti- mouse and anti-goat secondary antibodies (dilution 1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hr at room temperature in 5% non-fat milk diluted in TBST. Anti-rabbit monoclonal PI3KIII/Vps34 (6% gel, dilution 1:1000, #4263, Cell Signaling Technology Inc., Danvers, MA, USA), anti-rabbit monoclonal Phospho(Thr389)-p70S6K (8% gel, dilution 1:1000, #9234, Cell Signaling Technology Inc., Danvers, MA, USA) and anti-rabbit monoclonal Beclin (10% gel, dilution 1:1000, #3495, Cell Signaling Technology Inc., Danvers, MA, USA) membranes were blocked in 5% bovine serum albumin (BSA) in TBST for 1 hr, and the primary antibody was diluted in 5% BSA overnight at 4oC. The blots were subjected to 3x10 min washes in TBST, and incubated with HRP-conjugated secondary antibody (1:5000, Santa Cruz) at room temperature in 5% BSA for 1 hr. The membranes were then washed using 3x10 min washes in TBST. Detection of HRP-conjugated secondaries was performed using ECL plus chemiluminescent reagent (PerkinElmer Inc., Waltham, MA, USA) and imaged on x-ray film (GE Healthcare Limited, Pollards Wood, Buckinghamshire, UK) or using a VersaDoc gel- documentation system (Bio-Rad Laboratories, Hercules, CA, USA).

2.1.5 Immunoprecipitation studies

Following treatment, HEK293-GFPMtdL or HEK293-GFP cells were cross-linked using 1% formaldehyde diluted in PBS for 10 min at room temperature, then rinsed twice in cold PBS

37 and collected with 1% triton-x buffer (#9803, Cell Signaling Technology Inc., Danvers, MA, USA) with a protease inhibitor cocktail tablet (Roche, Indianapolis, IN, USA). The lysate was briefly sonicated on ice for 3x10 second pulses at medium speed and protein concentration was obtained using Bradford protein assay. Two hundred micrograms of cell lysate at a final concentration of 1 µg/µL was pre-cleared by rotating at 4oC for 3 hrs using 30 µL of protein A- agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The beads were removed from the sample by centrifugation of the sample at 4oC 3000xg for 5 min. The supernatant was transferred to a new Eppendorf tube. The lysate was incubated rotating at 4oC overnight with anti-rabbit monoclonal PI3KIII/Vps34 antibody (dilution 1:50, #3358 Cell Signaling Technology Inc., Danvers, MA, USA), anti-rabbit monoclonal Beclin (dilution 1:100, #3495, Cell Signaling Technology Inc., Danvers, MA, USA), anti-rabbit polyclonal Mcl-1 (dilution 1:20, S-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-rabbit normal IgG was used as a negative control (Santa Cruz Biotechnology, Santa Cruz, CA, USA). JEG3 cells were collected with RIPA buffer with protease inhibitor cocktail and precleared with 30µL of protein A-agarose beads, then incubated with anti-rabbit polyclonal Mcl-1 (dilution 1:20, S-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA) primary antibody overnight at 4oC.

After incubation with the primary antibodies, 30 µL of protein A-agarose beads were added to each sample and incubated at 4oC for 3 hrs slowly rotating. Samples were centrifuged at 40C at 3000xg and washed with 500 µL of buffer, and 2 washes of 500 µL of PBS. Following the washes, 25 µL of 3x sample buffer were added to the beads and the mixture was boiled and centrifuged at 14000xg. The immunoprecipitated lysates were subsequently immunoblotted with 20% of the sample loaded to immunoblot for the input, and 80% was loaded for the interaction of interest.

2.1.6 Immunofluorescence staining

For experiments involving HEK293 cells, glass coverslips were first coated with poly-L- lysine prior to plating of cells to enhance cell adherence. JEG3 cells were plated directly onto glass coverslips.

2.1.6.1 Formaldehyde Fixation

Cells were fixed using a 3.7% formaldehyde fixative. 37% formaldehyde was mixed with media to create a 3.7% fixative solution that was incubated with the cells for 15 mins at 37oC. Cells were then washed twice with PBS and refrigerated at 4oC prior to staining.

2.1.6.2 Lysotracker® Red Staining

Lysotracker® Red (Invitrogen, Carlsbad, CA, USA) was used to monitor lysosomal production as a surrogate marker for autophagy. Lysotracker® Red dye at a concentration of 1mM in DMSO was mixed with cell media to create a mixture at a final concentration of 50nM. This mixture was incubated with the cells for 1 hr at 37oC. Formaldehyde fixation was subsequently performed. The cells were washed 3x5 mins in PBS with gentle rotation, and nuclei were subsequently counterstained with 4',6-diamidino-2-phenylindole for 20 min (DAPI, Invitrogen, Carlsbad, CA, USA). Coverslips were mounted onto microscope slides using a drop of 50% glycerol in PBS as mounting medium, and adhered to the coverslip with nailpolish.

2.1.6.3 Co-immunofluorescence Staining Experiments

In experiments utilizing GFP- or RFP- tagged proteins, endogenous fluorescence was imaged by deconvolution microscopy. In order to visualize non-fluorescent proteins, following fixation cells were permeabilized with 0.2% Triton-X solution dissolved in PBS for 5 min with gentle rotation, and washed 2x5 min in PBS. Blocking was performed for 1 hr at room temperature with 5% normal donkey serum (NDS). The following primary antibodies were used overnight at 4oC: anti-rabbit polyclonal Mcl-1 (dilution 1:200, S-19 Santa Cruz), anti-rabbit polyclonal Mtd/Bok antibody (dilution 1:400, H-151 Santa Cruz), and anti-mouse monoclonal calreticulin (dilution 1:2000, ab22683 Abcam). Normal rabbit IgG and normal mouse IgG were used at the same concentration as the primary antibodies as negative controls (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The primary antibodies were diluted in a solution of antibody diluent (0.4% sodium azide, 0.625% gelatin in PBS filtered with 0.22mm syringe- driven filter) mixed at a 1:1 ratio with 5% NDS. Following incubation with the primary antibody, cells were washed 3x5 min with PBS on a rotating platform. Alexa Fluor® 594- conjugated and 488-conjugated donkey secondary antibodies were diluted into antibody diluent and used for 1 hr at room temperature (dilution 1:200, Invitrogen, Carlsbad, CA, USA). The cells were then washed with 3x5 min PBS washes and counterstained with DAPI diluted in PBS

39 for 20 mins. A drop of 50% glycerol in PBS was placed on the section and coverslips were adhered to the slide using nail polish. Images were subsequently captured using DeltaVision deconvolution microscopy with z-stacking (Applied Precision, LLC, Issaquah, WA, USA).

2.1.7 Electron Microscopy

HEK293-GFP and HEK293-GFPMtdL cells were plated at 70% confluence onto Thermanox™ plastic coverslips (Thermo Fisher Scientific, Roskilde, Denmark). The cells were treated with 2.5µg/mL of doxycycline for 24 hrs. The cells were rinsed with PBS and room temperature EM fixative (2% gluteraldehyde, 0.1M sodium cacodylate) was pipetted over the cells and incubated for 1 hr. The fixed cells were then stored at 4oC for 24 hrs prior to processing into semi-thin silver and thin gold sections by the Mount Sinai Hospital Electron Microscopy facility. Images were captured using a FEI Tecnai 20 Transmission Electron Microscope.

2.1.8 Transient Transfection Experiments

2.1.8.1 Overexpression of MtdL and Mcl-1L

pcDNAFlagMtdL vector was generated as previously described (Soleymanlou et al., 2005). The forward and reverse primers encoded a Kpnl and BamHI restriction site respectively for cloning into pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). Forward primer: 5'- CCCGGTACCACCATGATCCGGCCCAGCGTCTAC-3', reverse primer: 5'- CCCGGATCCGGGTCATCTCTCTGGCAACAACAGGAA-3'.

Human choriocarcinoma JEG-3 cells at 70% confluence were transfected with 3µg/35mm dish of either pcDNA3.1 vector, pcDNA3xFlag vector, pcDNAFlagMtdL or pcDNA3Mcl-1L vector (gift of Dr. Ruth Craig, Dartmouth Medical School, Hanover, NH) with 8 µL of Lipofectamine 2000® transfection reagent (Invitrogen, Carlsbad, CA, USA). Three micrograms of DNA were mixed with 250µL of OPTI-MEM low serum media (Invitrogen, Carlsbad, CA, USA) and 8µL of Lipofectamine 2000® were mixed with 250µL of OPTI-MEM and incubated at room temperature for 5 min. Subsequently, the DNA and Lipofectamine 2000® tubes were mixed and incubated for 20 mins and pipetted over the cells, then 1.5mL of EMEM with 10% FBS were added to each well. The medium was changed 4 hrs after transfection to

40 remove the lipofectamine, and protein extraction was performed 24 hrs and 48 hrs post- transfection.

2.1.8.2 Mcl-1L silencing experiments

Silencing of Mcl-1L protein in JEG3 cells was performed using 60 nM of 2 different siRNA Silencer® duplexes targeted against mRNA of Mcl-1L (Ambion Inc., Austin, TX, USA). Silencer® scramble sequence control siRNA (SS), which does not correspond to any known gene product was used as a control. Sixty nanomolars of siRNA and 6 µL of Lipofectamine 2000® were each mixed with 250 µL of OPTI-MEM, and incubated for 5 mins. The two tubes were subsequently mixed and incubated at room temperature for 20 mins and the mixture was pipetted over the cells and 1.5 mL of EMEM with 10% FBS was added to each well. Media was changed at 24 hrs post-transfection and protein was extracted 48 hrs post-transfection.

2.1.8.3 Transient Expression of RFP-Mcl1L

Mcl-1L was cloned into a pdsRed2 vector (gift of Dr. Andrea Jurisicova, Samuel Lunenfeld Research Institute, Toronto, ON, Canada) by the use of HindIII/BamHI restriction sites. Forward primer: 5'-CCCAAGCTTATGTTTGGCCTCAAAAGAAACGCGG-3', reverse primer:5'-CGCGGATCCCTTATTAGATATGCCAAACCAGCTCC-3'.

HEK293-GFP or HEK293-GFPMtdL were transfected with pdsRed vector or pdsRedMcl-1L using 6 µL of Lipofectamine 2000® for 24 hrs. Three micrograms of DNA and 6 µL of Lipofectamine 2000® were each mixed with 250 µL OPTI-MEM, incubated at room temperature for 5 mins, and mixed together for 20 mins prior to cell treatment. 1.5mL of DME media with 10% FBS were then added to each well. Media was changed 4 hrs after transfection and the cells were treated with 2.5ng/mL of doxycycline to induce either GFP or GFPMtdL expression. Formaldehyde fixation was performed 24 hrs post doxycycline treatment.

2.1.9 SNP Treatment

Sodium nitroprusside (SNP, Sigma-Aldrich Inc., St. Louis, MO, USA) crystals were diluted into EMEM media to create a 100mM stock that was subsequently aliquotted and frozen at -20oC. JEG3 cells at 70% confluence were treated with SNP at a concentration of 2.5 and 5

41 mM for 6, 12 and 24 hrs. After SNP treatment, either protein extraction, co-immunofluorescence staining or lysotracker red staining was performed.

2.1.10 Etoposide Treatment

JEG3 cells were treated with etoposide, a topoisomerase II inhibitor. Twenty-five, 50 and 100 uM of etoposide dissolved in EMEM medium with 10% FBS were pippetted over the cells and incubated for either 24 or 48 hrs. The cells were subsequently collected for protein analysis. Two microlitres of mouse brain extract (sc-2253, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as a positive control for the unlipidated LC3-I band.

2.1.11 Rapamycin Treatment

Rapamycin is an inhibitor of mTOR and rapidly induces autophagy (Peterson et al., 1999; Klionsky et al., 2007). Stock rapamycin (Sigma-Aldrich Inc., St. Louis, MO, USA) at a concentration of 2.5 mg/mL in dimethyl sulfoxide (DMSO) was diluted in medium to perform time-course and dosage experiments (0.1, 0.2, 0.3, 0.4 µM rapamycin) in JEG3 cells. JEG3 cells at 70% confluence were incubated either with 10% FBS or were starved of serum for 3 hrs prior to treatment with varying concentrations of rapamycin. DMSO was used as the vehicle control. Protein extraction was performed at 24 and 48 hrs after treatment.

As a positive control, HEK293 cells were treated with 1µM rapamycin and collected for protein or treated with lysotracker red and fixed 3 hrs after treatment.

2.1.12 Glucose Starvation

JEG3 cells were starved of glucose by washing cells with PBS and then providing cells at 70% confluence in 6-well 35mm plates with 2 mL Hank's Buffered Saline Solution (HBSS+). Cells were collected for protein extraction at several different timepoints.

2.1.13 Oxygen Experiments

The medium of JEG3 cells at 70% confluence in 35mm x 6-well plates was changed prior to the beginning of the experiment. Cells were transferred to a 3% oxygen incubator (3% O2, 5%

CO2, 92% N2) whereas control cells were maintained at 20% oxygenation (20% O2, 5% CO2,

75% N2). Cells were collected for protein extraction at 24 and 48 hr time-points.

2.2 Human Placental Tissue Experiments

2.2.1 Collection and Sampling

Tissue was collected with informed consent in accordance with the guidelines of participating institutional ethics guidelines (Faculty of Medicine, University of Toronto and Mount Sinai Hospital, Toronto, Canada). First-trimester human placental tissue were collected from elective pregnancy terminations by dilatation and curettage (5-9 weeks of gestation: n=18;10-15 weeks of gestation: n=21).

Placentae from pregnancies characterized by preeclampsia (PE) and intrauterine growth restriction (IUGR) were collected based on the American College of Obstetricians and Gynaecology (ACOG) criteria (2002). Preeclampsia (n=21) was defined using the criteria of blood pressure ≥ 140/90mmHg after 20 weeks of gestation in women with previously normal blood pressure and urinary excretion of ≥ 0.3g protein per 24 hrs (2002). Intra-uterine growth restriction without signs of preeclampsia (n=16) was defined using the criteria of normotensive blood pressure and fetal weight below the 5th percentile for gestational age. PE, IUGR, age- matched control (AMC, n=25), and term control (TC, n=20) samples were collected from random central and peripheral placental regions, rinsed in PBS and snap frozen in liquid nitrogen immediately after delivery. Patients with diabetes, infection, or kidney disease were excluded. Preterm and term control groups had no signs of placental pathology.. The clinical information for placental samples is summarized in Table 2.2.1.

Table 2.2.1 Clinical parameters of control, preeclamptic and IUGR patients.

Term (TC) Preterm/Age- PE (n=21) IUGR (n=16) (n=20) matched controls (AMC) (n=25)

Mean gestational age 38.20±0.21 30.97± 1.02 29.61± 32.88± 0.7922 at delivery (weeks) 0.6298

Blood Systolic 129.6±7.886 118.8± 4.239 162.5± 3.658 137.0± 12.38 pressure

(mmHg) Diastolic 74.71±6.664 75.17± 2.812 99.48± 2.182 85.83± 10.92

Proteinurea (g/24hr) Absent Absent 4.408± 2.079 Absent

Platelets (per L) 214.8± 16.23 244.8± 27.27 119.1± 11.41 218.0± 23.21

AST (U/L) 23.00± 3.464 14.33± 2.028 115.4± 30.19 21.67± 4.807

ALT (U/L) 11.50± 3.175 15.33± 4.333 108.3± 21.89 28.00± 15.10

Fetal sex M: 56% M: 31% M: 52% M: 44%

F: 44% F: 69% F: 48% F: 56%

Fetal weight 3177± 169.9 1822± 254.1 1725± 570.2 1246 ± 42.02

Mode of delivery CS: 100% CS: 62% CS: 81% CS: 100%

VD: 38% VD: 19%

Data are represented as mean ± SEM

AST: aspartate aminotransferase ALT: alanine aminotransferase CS: Caesarean section delivery VD: vaginal delivery

2.2.2 Protein Extraction

Protein extraction of human placental tissue was performed using a mortar and pestle cleaned with 100% methanol and on dry ice. Snap frozen placental chunks were placed in the mortar and ground with the addition of liquid nitrogen to create a powder. Tissue powder was transferred to an Eppendorf tube on dry ice. 1 mL of RIPA buffer (ddH2O, 3% 5M NaCl, 1.66% 3M Tris-HCl pH 7.5, 1% NP-40) with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) was added to each tube containing powdered placental tissue and a homogenizer was used in 2x45 second bursts followed by a 30 second waiting period. Samples were left standing at 4oC for 1 hr and then transferred to Eppendorf tubes. The lysate was vortexed and incubated on ice for 10 mins, then cold centrifuged for 10 mins at 14000xg at 4oC. Protein concentration was measured in duplicates using Bradford protein assay as described above.

2.2.3 Western Blotting

Thirty micrograms of human placental tissue was diluted with 2x sample buffer and was loaded on SDS-PAGE gels running at 100V with running buffer. Proteins were transferred onto PVDF membranes by hydrating the membranes in 100% methanol and transferred at 100V using transfer buffer for 1 hr at room temperature with an ice pack. Anti-rabbit polyclonal LC3B-II (ab48394 Abcam, 15% gel, dilution 1:2500) and anti-goat polyclonal β-actin (dilution 1:1000, I- 19 Santa Cruz) were blocked in 5% non-fat milk in Tris-buffered saline Tween-20 (TBST) for 1 hr at room temperature, with incubation of primary antibody at 4oC overnight. 3x10 min washes in TBST were performed and membranes were then incubated with horse-radish peroxidase (HRP) conjugated secondary antibody diluted in 5% non-fat milk (dilution 1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hr at room temperature. Anti-rabbit monoclonal Phospho(Thr389)-p70S6K (8% gel, dilution 1:1000, #9234, Cell Signaling Technology Inc., Danvers, MA, USA) was blocked in 5% bovine serum albumin (BSA) in TBST for 1 hr, and the primary antibody was diluted in 5% BSA overnight at 4oC. The blots were washed in TBST 3x10 min washes, and incubated with HRP-conjugated secondary antibody (dilution 1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature in 5% BSA for 1 hr. The membranes were washed using 3x10 min washes in TBST. Detection of HRP-conjugated secondaries was performed using ECL plus chemiluminescent reagent (PerkinElmer Inc.,

Waltham, MA, USA) and imaged on x-ray film (GE Healthcare Limited, Pollards Wood, Buckinghamshire, UK) or using the gel-documentation system VersaDoc (Bio-Rad Laboratories, Hercules, CA, USA).

2.2.4 Immunofluorescence Staining

Co-immunofluorescence staining in human placental sections was performed using sections cut from paraffin embedded blocks provided by the Mount Sinai Hospital Biobank (Toronto, Ontario, Canada). The sections were deparaffinized and hydrated by placement into xylenes for 3x5 min, 100% ethanol for 3x2 min, followed by 2 min in 95%, 90%, 85%, 80%,

70%, 50% ethanol in ddH20, then 2 min in ddH20. Sections were then placed in PBS gently rotating for 5 mins. In order to expose antigens for antibody binding, a solution was prepared by mixing 9 mL of citric acid (4.2g dissolved in 200 mL ddH20) and 41 mL of sodium citrate

(14.78g of crystal dissolved in 500mL ddH20) at pH6.0. The sections were placed into a plastic container filled with the solution and microwaved at power setting 4 for 5 mins, followed by a 15 min cool down with the lid on. The sections were then heated at power setting 4 for 3 mins followed by a 20 min cool down with the lid off. The sections were then placed in a glass container of PBS for 3x5 min washes gently rotating. Autofluorescence was quenched by placing the sections into 0.1% sudan black in 70% ethanol for 15 mins. Remaining sudan black was removed by 2x5 min PBS washes. The sections were then circled around with an immuno- pen (Invitrogen, Carlsbad, CA, USA) and subsequently blocked using 5% NDS for 1 hr at room temperature. Primary antibody was then used at a concentration of 1:100 for anti-mouse monoclonal Beclin (AM1818a, Abgent Inc., San Diego, CA, USA) and 1:200 for anti-rabbit polyclonal Mcl-1 (S-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in a 1:1 solution of 5% NDS and antibody diluent at 4oC overnight. Normal mouse and rabbit IgG were used at the same dilution as the primary antibody as a negative control. The sections were then washed 3x5mins in PBS with gentle rotation, and incubated with Alexa Fluor® 594-conjugated and 488-conjugated donkey secondary antibodies (dilution 1:200, Invitrogen, Carlsbad, CA, USA). The fluorophore-conjugated secondary antibodies were each used for 1 hr at room temperature and then washed 3x5 mins in PBS, and the sections were subsequently counterstained with DAPI for 15 mins. Coverslips were adhered to the slides using 50% glycerol in PBS as mounting media and sealed with nailpolish. Slides were refrigerated and imaged with

Deltavision deconvolution microscopy with z-stacking (Applied Precision, LLC, Issaquah, WA, USA).

2.2.5 Electron Micrographs

Placentae from consented patients were diced into 2mm pieces and rinsed with PBS to remove remaining blood within 10 mins of delivery. The chunks were then placed into room temperature EM fixative (2% gluteraldehyde, 0.1M sodium cacodylate). After 1 hr at room temperature, the tissue was maintained at 4oC for 24 hrs and processed into semi-thin silver and thin gold sections by the Mount Sinai Hospital Electron Microscopy facility. Images were captured using a FEI Tecnai 20 Transmission Electron Microscope.

2.3 Statistical Analysis

Densitometry for quantitating immunoblot experiments was performed using Image Quant 5.0 software (Molecular Dynamics, Piscataway, NJ, USA) and Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Expression of the protein of interest was normalized to β-actin. The coefficient of correlation (R) was calculated using Volocity software (PerkinElmer Inc., Waltham, MA, USA) to determine the degree of overlap between two different fluorophores by tracing around the outline of an individual cell.

Statistical analyses were performed using Graphpad Prism 5 software (San Diego, CA California). For comparisons with multiple groups, a one-way ANOVA with a Newman-Keuls post-hoc test or Kruskall Wallis test was used, and a t-test or Mann-Whitney U-test was used for comparisons between 2 groups. Significance was defined as p<0.05 and all data are represented as mean ± SEM. All experiments were performed with a minimum of 3 technical replicates.

Chapter 3 3 Results 3.1 Mcl-1L is a repressor of autophagy

Previous studies from our lab have addressed the importance of Mcl-1L as a key determinant of cell fate in trophoblast cells. It has been reported that Mcl-1L prosurvival protein levels are decreased in preeclampsia, contributing to increased trophoblast cell death in this pathology (Soleymanlou et al., 2007). Intriguingly, Mcl-1L has been reported to interact with Beclin 1, an essential protein for autophagic initiation (Erlich et al., 2007); however, the role of Mcl-1L in autophagic regulation has not been investigated.

To determine levels of autophagy, LC3B-II was used as a specific marker of autophagosomes. LC3B-II is present on the membrane of autophagosomes and is commonly used to detect autophagic induction by immunoblotting (Klionsky et al., 2007). To first determine if autophagy could be induced in JEG3 choriocarcinoma cells, a placental cell line of trophoblast origin, etoposide was employed. Etoposide is a topoisomerase type II inhibitor and has previously been reported to induce autophagy in a variety of cell lines including human glioma and cervical cancer cell lines (Katayama et al., 2006; Lee et al., 2007). A dose and time course for etoposide treatment was performed in JEG3 cells, for 24 and 48 hrs with either 0, 25, 50, or 100 µM of etoposide dissolved into EMEM media. Mouse brain extract was used as a positive control for the 18 kDa unlipidated LC3B-I, while etoposide mediated induction of LC3B-II was used to identify the lipidated 16 kDa band corresponding to LC3B-II (Figure 3.1.1a). In order to determine the effect of confluency on autophagic marker expression, cells were seeded to 50% confluence (2x105 cells per well), 100% confluence (4x105 cells per well) and 6x105 cells per well, as JEG3 cells have the capacity to proliferate beyond a monolayer. The cells were collected and immunoblotted for LC3B-II expression, demonstrating that confluency does not affect basal levels of autophagy in JEG3 cells (Figure 3.1.1b). β-actin immunoblotting was used as a loading control.

In order to determine if Mcl-1L is involved in autophagic regulation, loss and gain of function studies were performed in JEG3 cells. Mcl-1L was overexpressed by a transient transfection of pcDNA3.1Mcl-1L (+) or empty vector (control) for 24 and 48 hrs. While

Figure 3.1.1 Identification of LC3B-II via immunoblot analysis in JEG3 cells.

A. Representative immunoblot of JEG3 cells treated with etoposide and immunoblotted for LC3B. LC3B-1 was identified using a positive control (B), while LC3B-II was identified by using a known inducer of autophagy, etoposide. B = mouse brain extract, a positive control for LC3B-I. B. Representative immunoblot of JEG3 cells plated with 50% confluence, 100% confluence and 6x105 cells per well (150%) and immunoblotted for LC3B-II. β-actin was used as a loading control.

overexpression of Mcl-1L at 24 hrs did not have an effect on LC3B-II protein expression, overexpression of Mcl-1L for 48 hrs resulted in a more than 30% reduction in expression of LC3B-II (Figure 3.1.2). β-actin was used as a loading control in order to perform quantification using densitometric analysis, demonstrating that the 31.3±11.4% fold-decrease in LC3B-II expression was significant compared to control pcDNA3.1 transfected cells (Mann Whitney U- test, p<0.05, n=3).

To perform loss of function experiments, Mcl-1L expression was suppressed using two different siRNA sequences targeted against Mcl-1 mRNA in JEG3 cells (Figure 3.1.3, upper panel). Mcl-1L protein expression was quantified using densitometric analysis (Figure 3.1.3, left lower panel). Mcl-1L protein expression was significantly decreased at 48 hrs post-treatment by 72.7± 3.9% for duplex 1 (D1), and 61.2±8.0% for duplex 2 (D2) relative to scramble sequence (SS) control (one-way ANOVA with Newman-Keuls post-hoc test, p<0.01, n=4). LC3B-II protein levels were then analyzed by immunoblotting and densitometric analysis (Figure 3.1.3, lower right panel). LC3B-II protein levels were found to be significantly increased with both sequences (D1: relative fold increase of 1.79±0.32; D2: relative fold increase of 1.64± 0.19) in response to Mcl-1L silencing as compared to SS control (one-way ANOVA with Newman-Keuls post hoc test, p<0.05, n=4).

Lysosomal number is another marker of autophagy and is increased in end-stage autophagy, prior to lysosomal fusion with autophagosomes (Klionsky et al., 2007). Lysosomes were monitored using Lysotracker® Red, a tracer dye specific to the lysosomal compartment. To further verify the effect of Mcl-1L on autophagy, JEG3 cells were transfected with two siRNA sequences targeted against Mcl-1L mRNA (D1 and D2) and SS control siRNA, and lysosomal presence was monitored using Lysotracker® Red (Figure 3.1.4). Silencing of Mcl-1L with D1 and D2 resulted in increased lysosomal presence as compared to SS control, indicating that this increase in LC3B-II expression was associated with lysosomal activation, further indicating a role for Mcl-1L as an inhibitor of autophagy.

Autophagy is controlled by several upstream signaling pathways, many of which impinge upon the mammalian Target Of Rapamycin (mTOR) (Codogno & Meijer 2005). Under conditions of mTOR activation, autophagy is inhibited and can be monitored by the phosphorylation of p70S6K, a protein kinase downstream of mTOR. In response to mTOR

Figure 3.1.2 Overexpression of Mcl-1L in JEG3 cells.

Upper panel: Representative immunoblot of Mcl-1L overexpression in JEG3 cells C: control pcDNA3.1 transfected cells, +: pcDNA3.1Mcl-1L transfected cells. Lower panel: Densitometric analysis of the protein expression levels of LC3B-II normalized to β-actin and expressed as a fold change compared to control pcDNA3.1 transfected cells. Overexpression of Mcl-1L significantly decreased the expression of the autophagy marker LC3B-II at 48 hrs post- transfection (n=3, Mann-Whitney U-test, *p<0.05).

Figure 3.1.3 Silencing of Mcl-1L in JEG3 cells.

Upper panel: Representative immunoblot of Mcl-1L and LC3B-II protein levels in siRNA targeted silencing of Mcl-1L in JEG3 cells. D1: duplex 1; D2: duplex 2; SS: scramble sequence control. Lower left panel: Densitometric analysis of Mcl-1L protein expression normalized to β- actin and expressed as a fold change compared to SS control. Right panel: Densitometric analysis of LC3B-II protein expression levels normalized to β-actin and expressed as a fold change relative to scramble sequence control. Silencing of Mcl-1L resulted in significantly increased expression of LC3B-II at 48 hrs (n=4, one-way ANOVA with post-hoc Newman-Keuls multiple comparisons test, *p<0.05, **p<0.01).

Figure 3.1.4 Effect of Mcl-1L silencing on lysosomal presence in JEG3 cells.

JEG3 cells were silenced with two different siRNA sequences, D1 and D2, targeted against Mcl- 1L mRNA. Forty-eight hours post-treatment the cells were stained with Lysotracker® Red to visualize lysosomes, demonstrating increased lysosomal presence in Mcl-1L silenced cells compared to SS. Chromatin is stained with DAPI (blue). SS: scramble sequence control; D1: Duplex 1; D2: Duplex 2 (n=3).

Figure 3.1.5 Rapamycin treatment in HEK293 cells.

A. HEK293 cells were treated with 1uM rapamycin for 3 hrs and immunoblotted for the expression of p70S6K phosphorylated at Threonine 389. Rapamycin treatment dephosphorylated p70S6K in conditions of 3 hrs of serum starvation and in nonstarved cells. st: starved; nst: non-starved; R: rapamycin treated B. As a positive control for lysosomal induction, HEK293 cells were treated with 1uM rapamycin for 3 hrs resulting in increased lysosomal induction, as visualized by the use of Lysotracker® Red dye. Nuclei are counterstained in DAPI (blue), (n=3).

activation, p70S6K is phosphorylated at Thr389 (Peterson et al., 1999). Human embryonic kidney cells (HEK293) were either starved of serum for 3 hrs (st), not-starved (nst) or treated with rapamycin (R). Rapamycin treatment resulted in dephosphorylation of p70S6K (Figure 3.1.5a). In order to determine the specificity of Lysotracker® Red dye in monitoring lysosomal activation, HEK293 cells were treated with 1uM rapamycin for 3 hrs and were stained with Lysotracker® Red dye. Compared to DMSO control treated cells, lysosomes were induced in response to rapamycin treatment in HEK293 cells (Figure 3.1.5b).

3.2 Mcl-1L and MtdL interact

Bcl-2 family proteins exert their effects on a variety of different cellular processes dependent upon their specific interaction partners. While Mcl-1L and MtdL have been reported to interact in a yeast two-hybrid system, I sought to examine if these proteins interact in JEG3 choriocarcinoma cells and HEK293 cells (Hsu et al., 1997).

To demonstrate that Mcl-1L and the multidomain proapoptotic molecule MtdL interact in trophoblast cells, co-immunoprecipitation studies were conducted in JEG3 cells. JEG3 cells were transfected with pcDNA3.1Flag vector and pcDNA3.1FlagMtdL and immunoblotted to confirm transfection (Figure 3.2.1a). Immunoprecipitation of Mcl-1L specifically pulled-down associated FlagMtdL in cells transfected with FlagMtdL (Figure 3.2.1b). To further confirm this association in a HEK293 cells, co-immunofluorescence staining was performed. HEK293 cells stably expressing GFP (HEK293-GFP) and GFPMtdL (HEK293-GFPMtdL) under the regulation of a hybrid human cytomegalovirus (CMV)/tetracycline promoter, which express GFP and GFPMtdL in response to doxycyline treatment were utilized. Expression of GFP or GFPMtdL in HEK293-GFP and HEK293-GFPMtdL cells were induced by treatment of 2.5 ng/mL doxycycline for 24 hrs, and immunostained using an antibody for endogenous Mcl-1. In HEK293-GFPMtdL cells, MtdL depicted in green, and Mcl-1 in red, colocalized in apoptotic cells with condensed nuclei, as indicated by yellow. Nuclei are shown in blue and normal rabbit IgG was used as a negative control (Figure 3.2.2a).

To confirm the association of specifically the long isoform of Mcl-1 with MtdL, cells were transfected with Mcl-1L tagged with red fluorescent protein (RFP). Expression of GFP or GFPMtdL was induced in HEK293-GFP and HEK293-GFPMtdL cells with 2.5 ng/mL

Figure 3.2.1 Immunoprecipitation of Mcl-1L in JEG3 cells transfected with FlagMtdL.

A. Representative immunoblot analysis of JEG3 cells transiently transfected with either control pcDNA3.1Flag or pcDNA3.1FlagMtdL vector and immunoblotted 24 hrs after treatment. B. Representative immunoprecipitation of endogenous Mcl-1L in JEG3 cells transfected with FlagMtdL for 24 hrs. Immunoprecipitation revealed an association of Mcl-1L with FlagMtdL 24 hrs post-transfection. Neg: Negative IgG control, (n=3).

Figure 3.2.2 Colocalization of MtdL and Mcl-1L in HEK293 cells.

A. HEK293-GFP and HEK293-GFPMtdL cells treated with 2.5ng/ml doxycycline for 24 hrs were stained for endogenous Mcl-1 (red) revealing association between MtdL and Mcl-1 (yellow). Nuclei were counterstained with DAPI (blue). B. Immunofluorescence staining of HEK293-GFP and HEK293-GFPMtdL cells treated with 2.5 ng/mL doxycycline for 24 hrs and transfected with pdsRed or pdsRedMcl-1L vector. Nuclei were counterstained with DAPI (blue).

doxycycline for 24 hrs and the cells were transfected with either pdsRed vector (control) or pdsRedMcl-1L (Figure 3.2.2b). Cells transfected with pdsRedMcl-1L and expressing GFPMtdL demonstrated colocalization of MtdL and Mcl-1L in a perinuclear cellular location. Chromatin was counterstained with DAPI (blue).

3.3 MtdL is a novel inducer of autophagy

To determine if MtdL was affecting autophagy, gain of function studies with MtdL were performed in stably transfected HEK293 cells. Stably transfected HEK293 cells were used due to the highly cytotoxic properties of MtdL, a protein that rapidly induces apoptosis when present at high concentrations (Hsu et al., 1997). In order to examine the effects of the expression of MtdL at low-levels, the stably transfected cells were used and treated with low concentrations of doxycycline to induce low-level GFPMtdL expression. Initially, to elucidate the appropriate concentration of doxycycline and timepoint for further experiments a timecourse for induction of GFPMtdL was performed for 12, 24 and 48 hrs using 0, 2.5 and 5 ng/mL of doxycycline (Figure 3.3.1a). Densitometric analysis of GFPMtdL expression in response to doxycycline treatment demonstrated that MtdL was significantly elevated in response to 2.5 and 5 ng/mL doxycycline treatment at 24 hrs of treatment, and the two dose levels did not differ significantly (Figure 3.3.1b, one-way ANOVA with Newman-Keuls post-test, p<0.001, n=3). Densitometric analysis of LC3B-II expression following treatment with 2.5 and 5 ng/mL doxycycline for 24 hrs demonstrated that both concentrations of doxycycline elevated LC3B-II expression (2.5ng/mL: 4.47± 1.02; 5ng/mL: 6.15± 1.11 fold increase, one-way ANOVA with Newman-Keuls post-test, p<0.05, n=3) (Figure 3.3.1c). However, the two treatments did not produce significantly different levels of LC3B-II protein expression. Therefore, further experiments were performed with 2.5ng/mL of doxycycline for 24 hrs of treatment.

To determine the specific effect of GFPMtdL compared to control GFP expressing cells, HEK293-GFP and HEK293-GFPMtdL cells were induced with 2.5 ng/mL of doxycycline and immunoblotted for LC3B-II and GFP to confirm expression (Figure 3.3.2, upper panel). Expression of GFPMtdL resulted in significantly increased levels of LC3B-II expression with a fold increase of 4.59± 1.03 compared GFP-expressing cells, as quantified by densitometric analysis (Figure 3.3.2, lower panel; one-way ANOVA with Neuman-Keuls post-hoc test, p<0.001, n=3). HEK293-GFP and HEK293-GFPMtdL cells were subsequently stained with

Lysotracker® Red, revealing increased lysosomal formation in cells expressing GFPMtdL as compared to GFP control cells and cells not treated with doxycycline (Figure 3.3.3). Together, these findings demonstrate that MtdL exerts an opposing effect to that of Mcl-1L in autophagic induction.

Figure 3.3.1 Time and dose-course for doxycycline-mediated induction of MtdL.

A. Immunoblot analysis for the expression of GFPMtdL and LC3B-II protein in response to 0, 2.5 and 5 ng/mL doxycycline treatment at 12, 24 and 48 hrs post-treatment. B. Densitometric analysis of GFPMtdL expression at 24 hrs post-treatment, normalized to β-actin relative to 0 doxycycline treatment. C. Densitometric analysis of LC3B-II expression at 24 hrs post- treatment, normalized to β-actin relative to 0 doxycycline treatment. (n=3, one-way ANOVA with Newman-Keuls post-hoc test, *p<0.05, ***p<0.001).

Figure 3.3.2 Immunoblot analysis of HEK293 cells stably expressing MtdL.

Upper panel: Representative immunoblot for GFP, GFPMtdL and LC3B-II expression in stably transfected HEK293 cells in response to treatment of 2.5 ng/mL of doxycycline. Lower panel: Densitometric analysis of the expression of LC3B-II in response to doxycyline treatment normalized to β-actin and expressed as a fold change relative to 0 doxycycline treatment. LC3B- II expression was significantly elevated 4.47±1.02-fold with GFPMtdL expression compared to GFP induced cells (n=3, one-way ANOVA with Newman-Keuls Multiple comparison test, ***p<0.001).

Figure 3.3.3 Lysotracker® Red staining in HEK293 cells.

HEK293-GFP and HEK293-GFPMtdL cells were induced to express either GFP or GFPMtdL using 2.5 ng/mL doxycycline. Twenty-four hrs post-induction, Lysotracker® Red staining was performed to visualize lysosomes. Nuclei are counterstained with DAPI (blue). Increased lysosomal formation was observed in cells expressing GFPMtdL (n=4).

Figure 3.3.4 Intracellular localization of MtdL in HEK293 cells.

HEK293-GFP and HEK293-GFPMtdL cells were induced with 2.5 ng/mL of doxycycline to express GFP and GFPMtdL. Twenty-four hrs after induction, the cells were immunostained for calreticulin, a marker of the ER. GFPMtdL localizes to the ER membrane in cells with condensed nuclei. Correlation of overlap (R) = 0.93 ± 0.01 for apoptotic cells and 0.59 ± 0.03 for healthy cells. Green: GFP; red: calreticulin; blue: nuclei; (n=6).

Figure 3.3.5 Electron micrographs of HEK293 cells stably expressing MtdL.

HEK293-GFP and HEK293-GFPMtdL cells were induced to express GFP and GFPMtdL using 2.5 ng/mL doxycycline for 24 hrs. Autophagosome formation was observed in cells expressing GFPMtdL, as indicated by the black arrows. *: mitochondria, n: nucleus.

To determine if MtdL exerts a stimulatory effect on autophagy at the level of the endoplasmic reticulum (ER), where other Bcl-2 proteins have been implicated in autophagic induction (Wada et al., 1995; Pattingre et al., 2005), staining for the ER was performed using calreticulin (shown in red) as a specific organelle marker (Figure 3.3.4). GFP and GFPMtdL was induced with 2.5 ng/mL of doxycycline for 24 hrs and subsequently immunostained for calreticulin and the nuclei were counterstained with DAPI (blue). Normal mouse IgG was used as a negative control. GFPMtdL (green) localized to the ER and the coefficient of correlation was measured using Volocity software. The correlation coefficient (R) for MtdL and calreticulin was calculated to be 0.93±0.01 in cells with condensed nuclei, indicating that MtdL does translocate to the ER (n=3). Interestingly, in cells that were not condensed, the correlation factor was determined to be 0.59 ± 0.03 (n=3). This indicates that in cells that are not rounded, a subset of MtdL translocates to the ER membrane, however in cells that are apoptotic, as identified by condensed nuclei and cytoplasm, MtdL extensively colocalizes with a marker of the ER.

Consequently, HEK293-GFP and HEK293-GFPMtdL cells that were treated with 2.5 ng/mL of doxycycline for 24 hrs were processed for electron micrograph images to visualize the presence of autophagosomes. Autophagosomes were identified by characteristic features of a double-membraned structure present in the cytoplasm of the cell, containing cellular constituents (Klionsky et al., 2007). The presence of autophagosomes was confirmed for cells expressing MtdL, as indicated by the black arrow (Figure 3.3.5).

3.4 MtdL decreases the endogenous interaction of Mcl-1L and Beclin 1

Beclin 1 is an important component in upstream autophagic signaling, at the level of autophagosome nucleation, that has been reported to interact with several prosurvival Bcl-2 family members (Erlich et al., 2007). To determine if Mcl-1L associates with Beclin 1, co- immunoprecipitation studies for the association of Beclin 1 and Mcl-1L were performed in stably transfected HEK293 cells (Figure 3.4.1a). Co-immunoprecipitation of Beclin 1 and Mcl-1L demonstrated that in baseline conditions, Mcl-1L does interact with Beclin 1, leading to low levels of autophagy, as previously shown in Figure 3.3.2. However, in conditions of MtdL induction with 2.5 ng/mL doxycycline for 24 hrs, the interaction between Mcl-1L and Beclin 1 is reduced, as demonstrated by co-immunoprecipitation for Beclin 1 and immunoblotting for

Figure 3.4.1 Immunoprecipitation of Beclin 1 in HEK293 cells stably expressing MtdL.

A. HEK293-GFP and HEK293-GFPMtdL cells were induced to express GFP and GFPMtdL using 2.5 ng/mL doxycycline. Immunoprecipitation for endogenous Beclin in HEK293-GFP and HEK293-GFPMtdL cells revealed decreased association with Mcl-1L with Beclin following 24 hrs of MtdL induction. B. Left panel: Representative immunoblot for Mcl-1L expression in the lysates (input) immunoprecipitated for Beclin 1. Right panel: Densitometric analysis of the input normalized to β-actin. C. Left panel: Representative immunoblot from the supernatant of lysates immunoprecipitated for Beclin 1. Right panel: Densitometric analysis of the supernatant normalized to resulted in increased levels of Mcl-1L in cells expressing GFPMtdL. D. Relative abundance of Mcl-1L in the supernatant of lysates immunoprecipitated for Beclin 1 normalized to the expression of Mcl-1L in the input lysate. GFPMtdL expression resulted in a 1.25± 0.02 - fold increase in Mcl-1L presence in the supernatant relative to cells expressing GFP alone (n=3, paired t-test, **p<0.01).

69 associated Mcl-1L (Figure 3.4.1a). In order to quantify this change in association, the lysate was probed for the relative amounts of Mcl-1L before (input) and after (supernatant) immunoprecipitation of Beclin 1. HEK293 input lysates expressing GFP and GFPMtdL were probed prior to immunoprecipitation for the expression of Mcl-1L (Figure 3.4.1b, left panel). Densitometric analysis and normalization to β-actin revealed a trend towards increased Mcl-1L expression in response to GFPMtdL expression (Figure 3.4.1b, right panel). The supernatant remaining after immunoprecipitating for Beclin 1 was subsequently probed for Mcl-1L expression (Figure 3.4.1c, left panel). Densitometric analysis and normalization to β-actin revealed a trend towards increased Mcl-1L present in the supernatant of cells immunoprecipitated for Beclin 1 and expressing MtdL (Figure 3.4.1c, right panel). In order to determine the relative amount of Mcl-1L remaining in the supernatant after immunoprecipitation of Beclin 1, the quantity of Mcl-1L in the supernatant was normalized to the amount of Mcl-1L present prior to immunoprecipitation of Beclin 1 (Figure 3.4.1d). A significantly increased amount of Mcl-1L protein was determined to be present in the supernatant of cells expressing MtdL compared to cells expressing GFP, as determined by a paired t-test (n=3, p<0.01). This indicates that MtdL expression is leading to a decreased Mcl-1L and Beclin interaction, contributing to increased autophagy.

3.5 Autophagy is elevated in preeclampsia and decreased in IUGR

Preeclampsia is characterized by increased oxidative stress, leading to the activation of cellular stress responses. Additionally, our lab has reported that there are decreased levels of Mcl-1L and increased expression of the pro-death MtdL protein in preeclampsia contributing towards increased trophoblast cell death (Soleymanlou et al., 2007). As autophagy is an important stress defense mechanism and is regulated by MtdL expression, I sought to examine the status of autophagy in preeclamptic tissue.

To ascertain the levels of autophagy in human placental tissue, immunoblotting was performed using LC3B-II as a marker of autophagy (Figure 3.5.1). Immunoblotting for LC3B-II expression in severe PE placentae compared to AMC and TC placentae revealed significantly increased levels of autophagy in PE (n=14), with a fold increase of 2.05±0.47

Figure 3.5.1 Expression of LC3B-II in normal and preeclamptic placentae.

Immunoblot analysis for LC3B-II in PE, AMC and TC placentae reveals increased expression in severe PE vs AMC and TC (PE n=14, AMC n=8, TC n=4, One-way ANOVA with Newman- Keuls post-test, **p<0.01, ***p<0.001).

Figure 3.5.2 Expression of LC3B-II in normal and IUGR placentae.

Representative Immunoblot of IUGR, AMC and TC placentae probed for LC3B-II expression (Kruskall Wallis Test, IUGR n=12, AMC n=12, TC n=7, *p<0.05).

Figure 3.5.3 Spatial localization of Beclin 1 and Mcl-1 in normal and preeclamptic placentae.

Human PE and AMC placentae were immunostained for the localization of Mcl-1 (red) and Beclin (green), revealing increased coexpression in AMC placentae. CT= cytotrophoblast, ST=syncytiotrophoblast. Nuclei are counterstained in DAPI (blue), (n=3).

73 compared to AMC (p<0.01, n=8), and a fold increase of 7.54±0.82 compared to TC (n=4, p<0.001, one-way ANOVA with Newman-Keuls post-test). Conversely, immunoblotting for LC3B-II expression in IUGR placentae (n=12) demonstrated a fold decrease of 70.8±13.5% compared to AMC (n=12) and a fold decrease of 58.6±5.7% compared to TC placentae (n=7), (p<0.05, Kruskall Wallis test, Figure 3.5.2).

To determine the spatial localization of Mcl-1 and Beclin 1 in vivo, co- immunofluorescence staining experiments were performed using human placental tissue (Figure 3.5.3). In PE placentae, Mcl-1, shown in red, was primarily localized to the nuclei of cytotrophoblast cells, with minimal cytoplasmic localization. Beclin 1, depicted in green, is widely expressed in the syncytial layer and cytotrophoblastic cells, as well as in the stroma. Nuclei are shown in blue (DAPI). In contrast in AMC the expression of Mcl-1 is less restricted to cytotrophoblast nuclei and is more diffuse and cytoplasmic in the trophoblastic layers. The spatial localization of these proteins suggests that in PE, Beclin 1 is not coexpressed with Mcl-1, while there is a higher degree of coexpression in the same cell layers in AMC placentae.

3.6 Electron micrographs indicate increased presence of autophagosomes in PE

To further confirm immunoblotting data regarding increased autophagy in PE, electron micrographs (EM) of severe PE placentae and control placentae were imaged for the presence of autophagosomes. Autophagosomes were identified by their characteristic double-membraned appearance and cytoplasmic contents contained within a vacuole (Klionsky et al., 2007). In Figure 3.6.1, the syncytial layer of placentae was found to be highly vacuolated in TC, AMC and PE placentae, while it had a condensed appearance in IUGR placentae. TC and AMC placentae retained an organized syncytial appearance with a well-retained microvillous membrane (mvm). In contrast, in PE, the syncytial layer was observed to be disrupted and the mvm was highly disorganized in appearance. Evidence for increased autophagy was found in PE placentae. Autophagosomal structures are indicated with a black arrow while mitochondria are indicated with an asterisk (*).

Electron micrographs for the cytotrophoblast layer revealed that the CT cells of TC, AMC and IUGR placentae appeared similar, with minimal presence of vacuoles (Figure 3.6.2).

Syncytial Layer

Figure 3.6.1 Electron micrographs of the syncytium of normal and pathological placentae.

Electron micrographs of the syncytium of PE, IUGR, AMC and TC placentae (TC n=3, AMC n=3, PE n=3, IUGR n=4. Black arrows denote autophagosomes, n:nuclei, *:mitochondria, mvm: microvillous membrane.

Cytotrophoblast Cells

Figure 3.6.2 Electron micropraphs of CT cells in normal and pathological placentae.

Electron micrographs of cytotrophoblast of PE, IUGR, AMC and TC placentae (TC n=3, AMC n=3, PE n=3, IUGR n=4. Black arrows denote autophagosomes, CT: cytotrphoblast cell ST: syncytium, n:nuclei, *:mitochondria, mvm:microvillous membrane, ECM: extracellular matrix.

Endothelial Cells

Figure 3.6.3 Electron micrographs of the endothelium of normal and pathological placentae.

Electron micrographs of the endothelium of PE, IUGR, AMC and TC placentae (TC n=3, AMC n=3, PE n=3, IUGR n=4. Black arrows denote autophagosomes, ST: syncytium, n:nuclei, *:mitochondria, ECM: extracellular matrix, L: vessel lumen.

In PE placentae, however, evidence for mitophagy was found in the majority of CT cells, as indicated by the black arrows and was not observed in AMC, TC or IUGR placentae.

Electron micrographs of the endothelial layer of placental blood vessels demonstrated that this cell layer is very thin and elongated (Figure 3.6.3). In PE placentae however, this layer appears disorganized in appearance and has the unique appearance of autophagosomes encapsulating mitochondria, as indicated by the black arrows. Mitophagic vacuoles were frequently identified in PE in all cell layers examined, indicating that the increase in autophagy in PE in part results in increased turnover of mitochondria. This phenomenon was not observed in IUGR, AMC or PE placentae.

3.7 A model of oxidative stress induces autophagy via alterations in Mcl-1L/MtdL expression

As PE placentae are characterized by a status of increased oxidative stress, I evaluated the response of Mcl-1L, MtdL and autophagy in response to adverse oxidative conditions (Hubel 1999; Hung & Burton 2006). Sodium nitroprusside (SNP) is a compound that results in the release of reactive NO species and OH- radicals leading to a status of intracellular nitrile and oxidative stress. To determine if autophagy in PE may be the result of increased oxidative stress in trophoblast cells, SNP was used to generate reactive oxygen species in JEG3 choriocarcinoma cells. Time-course experiments were performed to determine the concentration of SNP (0, 2.5 and 5mM) and time-point (6, 12 and 24 hrs) for autophagic activation (Figure 3.7.1). Following time-course experiments, 6 hrs of treatment with 2.5 and 5 mM of SNP were chosen for further experiments.

Subsequent treatment of JEG3 cells with 2.5 and 5 mM of SNP for 6 hrs, resulted in significantly decreased Mcl-1L expression levels (2.5mM fold decrease: 17.7±4.3%, 5mM fold decrease: 30.5±7.2% ; p<0.05, one-way ANOVA, n=6), as depicted in Figure 3.7.2a. Conversely, MtdL levels increased with 2.5 mM of SNP (Figure 3.7.2b; fold increase: 21.0±7.0%; p<0.01, n=4, 1-way ANOVA), but not with 5mM of SNP. These changes in Mcl-1L and MtdL expression in response to SNP were correlated with significantly increased LC3B-II expression in response to both 2.5 (Figure 3.7.2c; fold increase: 2.43±0.33) and 5mM (fold increase: 2.68±0.35) of SNP (one-way ANOVA, p<0.001, n=6). Autophagic induction in response to SNP treatment was further confirmed with Lysotracker® Red dye, revealing

81 increased lysosomal formation in cells treated with 2.5 and 5mM SNP for 6 hrs (Figure 3.7.3). Nuclei were counterstained in DAPI (blue).

To determine if SNP induced oxidative stress was causing a change in MtdL localization along with autophagic induction, co-immunofluorescence staining was performed for MtdL and calreticulin, an ER marker, in JEG3 cells. Staining for the localization of MtdL, depicted in green, and calreticulin (red) demonstrated a change in MtdL intracellular distribution in response to SNP treatment (Figure 3.7.4). In control untreated JEG3 cells, MtdL is distributed throughout the cytoplasm of the cell, and also has strong nuclear staining, as previously reported (Ray et al., 2009). SNP treatment altered the morphology of JEG3 cells, which gained the appearance of condensed and abnormally shaped nuclei. SNP treatment resulted in MtdL localization into aggregates surrounding a peri-ER region, while the ER reflected a vacuolated appearance. Normal mouse IgG was used as a negative control. Taken together, the presented data indicates that Mcl-1L and MtdL exert opposing functions on autophagic activation following oxidative stress in a trophoblast cell model.

Figure 3.7.1 Expression of Mcl-1L and LC3B-II across time and dosage with SNP treatment in JEG3 cells.

Representative immunoblot of JEG3 cells treated with 2.5 and 5 mM of SNP for 6, 12 and 24 hrs and immunoblotted for Mcl-1L and LC3B-II protein expression.

Figure 3.7.2 Expression of Mcl-1L, MtdL and LC3B-II in SNP treated JEG3 cells.

Representative immunoblot of JEG3 cells treated with 2.5 and 5mM of SNP for 6 hrs. A. SNP treatment resulted in decreased Mcl-1L expression with both 2.5 and 5 mM of SNP (n=6, one- way ANOVA with Newman-Keuls Multiple Comparisons test,*p<0.05, **p<0.01. B. MtdL levels increased with treatment of 2.5 mM of SNP for 6 hrs (n=4, one-way ANOVA with Newman-Keuls Multiple Comparisons test,**p<0.01. C. LC3B-II expression increased with both 2.5 and 5 mM of SNP. (n=6, one-way ANOVA with Newman-Keuls Multiple Comparisons test,***p<0.001).

Figure 3.7.3 Lysotracker® Red staining in SNP treated JEG3 cells.

JEG3 cells were treated with 2.5 and 5 mM SNP for 6 hrs and stained for lysosomes using Lysotracker® Red, demonstrating increased lysosomal formation in response to SNP treatment compared to control. Chromatin are stained with DAPI (blue).

Figure 3.7.4 Spatial localization of Mtd in SNP treated JEG3 cells.

JEG3 cells treated with 2.5 mM SNP for 6 hrs were stained for MtdL (green) and calreticulin (red). MtdL forms aggregates and localizes to peri-endoplasmic location in response to SNP treatment. Chromatin are stained with DAPI (blue).

Chapter 4 4 Discussion

Preeclampsia is a devastating disorder of placental origin affecting 5-7% of all human pregnancies, and is diagnosed by the onset of the clinical symptoms of maternal pregnancy- induced hypertension and proteinurea, with the only known symptomatic intervention being delivery of the fetus and associated placenta (2002; Hung & Burton 2006). While the cause of preeclampsia remains unknown, the placenta plays a central role in its genesis and it is established that the placenta of preeclamptic pregnancies are characterized by a status of oxidative stress, leading to a hyperproliferative phenotype of the progenitor cytotrophoblast cells, and increased death of the trophoblastic cell layers, which extrude and are shed in excess as debris into the maternal circulation (Hubel 1999; Soleymanlou et al., 2005; Ray et al., 2009). This debris in the maternal circulation is believed to contribute to a status of generalized endothelial dysfunction, leading to the clinical symptoms of preeclampsia (Roberts 1998; Roberts & Cooper 2001). While it has been recognized that in preeclampsia, apoptosis is a major component of trophoblastic cell death, the contribution of autophagy to this placental pathology has yet to be determined.

4.1 Mcl-1 and MtdL are regulators of placental autophagy

My results indicate that autophagy is highly elevated in all cell layers of preeclamptic placentae, and I present novel findings that in a trophoblast cell model, Mcl-1L is a repressor of autophagy, while its binding partner MtdL is an inducer of this lysosomal degradation pathway. Moreover, I present evidence to support that this regulation of autophagy is at the level of the ER, where several key players in autophagic regulation have previously been shown to reside (Heath-Engel et al., 2008), and mediated by changes in the interaction of Mcl-1L with Beclin 1.

Recent publications have demonstrated that crosstalk exists between regulators of apoptosis and autophagy, which is mediated by the haploinsufficient tumour suppressor protein Beclin 1. Beclin 1 mobilizes from the trans Golgi network to ER membranes when autophagy is induced and forms a complex involving the lipid kinase Vps34/PI3KIII leading to autophagic membrane nucleation (Heath-Engel et al., 2008; Levine et al., 2008; He & Levine 2010). Beclin 1 is a Bcl-2 family protein containing only a BH3 domain, exerts no apoptotic effects, and has

88 been reported to interact with Bcl-2, Bcl-xL, Bcl-w and to a lesser extent with Mcl-1 (Oberstein et al., 2007; Levine et al., 2008). Specifically, the interaction with Bcl-2 was shown to inhibit starvation-induced autophagy via the sequestration of Beclin 1 from its autophagy activating complex with Vps34/PI3KIII (Pattingre et al., 2005). Conversely, pro-apoptotic BH3-only proteins of the Bcl-2 family including Noxa, Puma, BimEL and Bad have been shown to induce autophagy by binding to and sequestering the prosurvival protein Bcl-2 from interacting with Beclin 1 (Levine et al., 2008).

While Bcl-2 has been extensively characterized as an inhibitor of autophagy, the involvement of Mcl-1 has remained neglected in the literature due to its relatively weaker interaction with Beclin 1. However, in the human placenta, Mcl-1 and Mtd are the key Bcl-2 proteins mediating trophoblast cell survival and proliferation, necessitating the question of whether they are also involved in the regulation of placental autophagy. Previous reports have indicated that the expression levels of prosurvival Bcl-2 and proapoptotic Bax are unaltered in response to oxidative stress in human placental explants, likely not contributing to or accounting for the increased trophoblastic cell death present in preeclamptic placentae (Heazell & Crocker 2008; Heazell et al., 2008). Importantly, studies from our laboratory have previously described that the proapoptotic Bcl-2 family protein MtdL and its placental specific isoform MtdP are elevated in preeclamptic placenta in response to oxidative stress, contributing to increased trophoblastic proliferation and apoptosis typical of preeclamptic placentae. As well, the prosurvival Mcl-1L protein has decreased expression in preeclampsia, further promoting cell death (Soleymanlou et al., 2005; Soleymanlou et al., 2007; Ray et al., 2009). Taken together, this information suggests that Bcl-2 family proteins exert their functions in a tissue specific context.

Previous reports have indicated that prosurvival Bcl-2 and Bcl-xL inhibit Beclin 1 dependent autophagy at the level of autophagosomal nucleation (Pattingre et al., 2005). My results indicate, by the use of loss and gain of function experiments in a trophoblast cell lineage, that Mcl-1L also has a function in regulating autophagy, and specifically inhibits the process via an interaction with Beclin 1. Silencing Mcl-1L resulted in the rapid induction of markers of autophagy, indicating that in baseline cellular conditions, Mcl-1L maintains autophagy at low basal levels. I also demonstrated that the autophagosomal formation caused by MtdL induction is due to a sequestration of Mcl-1L from its interaction with Beclin 1, as indicated by my

89 immunoprecipitation and overexpression studies. I propose that this event releases the repressive brake by Mcl-1L on Beclin 1. This allows for autophagic induction, presumably via increased Beclin 1 interaction with Vps34/PI3KIII as indicated by other studies involving Bcl-2 (Pattingre et al., 2005) (Figure 4.1.1). Along with MtdL localization to the ER, this provides evidence for an important role of MtdL in autophagic regulation, and further supports the notion that Bcl-2 family proteins mediate their functional effects as the result of their specific interaction partners and localization (Pattingre et al., 2005; Heath-Engel et al., 2008; Ray et al., 2009). Intriguingly, Mtd/Bok is a multidomain BH3 family member, and is the first multidomain proapoptotic Bcl-2 family protein to be directly implicated in autophagic induction and this is the first report to demonstrate MtdL localization to the ER.

Bcl-2 family proteins have been reported to localize to a variety of cellular compartments and thereby influence different cellular processes. The most common and well understood localization is the mitochondria. The Bcl-2 family proteins constitutively localize to the mitochondria, and the mobilization of Bax from the cytoplasm to the mitochondria is a key event in the induction of apoptotis (Desagher & Martinou 2000; Soleymanlou et al., 2005; Bhatt et al., 2008; Ray et al., 2009). MtdL and MtdP mitochondrial mobilization has been reported in response to oxidative stress in human preeclamptic placentae (Soleymanlou et al., 2005). Conversely, nuclear localization of MtdL in the human placenta has been reported to induce progression through S-phase of the cell cycle (Ray et al., 2009). A nuclear localization for Mcl- 1L has been reported to play a role in inhibiting cell cycle progression through the S-phase, via an interaction with proliferating cell nuclear antigen (PCNA) and at G2 phase via an interaction with cyclin dependent kinase 1 (Cdk1) (Fujise et al., 2000; Jamil et al., 2005). Bcl-2 family proteins including Bax,, Bak, Bcl-2, and Mcl-1 have been reported to localize to the ER, where they have been reported to play a role in regulating cellular Ca2+ homeostasis and signalling (Yang et al., 1995; Demaurex & Distelhorst 2003; Lao & Chang 2007). An ER localization has been implicated in autophagic regulation in the early steps of autophagosome membrane formation (Pattingre et al., 2005; Heath-Engel et al., 2008; Hamasaki & Yoshimori 2010).

Figure 4.1.1 Putative model of MtdL induced autophagy.

In baseline conditions, Mcl-1L directly interacts with Beclin 1 to inhibit autophagosome formation. When MtdL expression is induced, MtdL complexes with Mcl-1L thereby allowing Beclin 1 to interact with Vps34/PI3KIII at the level of the ER thereby induction autophagosome formation.

Studies on cell death in preeclamptic placentae have reported on apoptosis and necrosis as mechanisms for trophoblastic shedding into the maternal circulation, but have not yet examined the relative contribution of autophagy towards trophoblastic cell death. In this study, I provide evidence that autophagy is significantly elevated in severely preeclamptic placentae, corroborating findings from a previous study reporting the presence of autophagosomes in the trophoblast cells layers of preeclamptic placentae (Oh et al., 2008). Herein, I provide evidence for the presence of autophagy in the trophoblast cell layers and in the endothelium of placental blood vessels via electron microscopy. The syncytiotrophoblast layer is the first layer exposed to oxidative damage as it is directly bathed in maternal blood. In preeclampsia, the placenta experiences aberrant blood flow, and consequently is exposed to hypoxic injury and oxidative stress (Jauniaux et al., 2000; Soleymanlou et al., 2005; Jauniaux et al., 2006; Heazell et al., 2008; Hung et al., 2008). The highly vacuolated and disorganized appearance of the syncytiotrophoblast layer of PE placentae indicates that autophagy is exacerbated in this cell layer, likely in response to oxidative stress, and may be contributing to the observed excessive cell death in the syncytium. In stark contrast, the trophoblastic layer in AMC and TC placentae retains an organized appearance with a well-defined syncytium and microvillous membrane.

Placentae from fetuses with IUGR had an entirely distinguishable phenotype from PE placentae, supporting the notion that IUGR and PE are two different disorders of pregnancy stemming from different placental insults (Burton et al., 2009). Via electron microscopy, IUGR placentae were easily identifiable by a compact syncytial phenotype with minimal vacuolization. Little evidence was found supporting autophagic activation in IUGR, via both western blot analysis for LC3B-II and electron micrographs. While IUGR placentae have been reported to be in a condition of general hypoxia, the status of oxidative stress in IUGR has not been extensively examined (Nevo et al., 2008; Zamudio et al., 2010). Several reports indicate that IUGR placentae are not under oxidative stress, while in contrast, placentae from patients with PE and IUGR have elevated expression of markers for placental oxidative stress and decreased antioxidant defense mechanisms, possibly indicating that PE placentae uniquely experience oxidative stress while IUGR placentae are simply hypoxic (Sahlin et al., 2000; Takagi et al., 2004; Zamudio et al., 2010).

The IUGR fetus has been reported to be nutrient deprived due to both decreased placental to fetal amino acid transport via System A activity, and a decrease in villous surface area for

92 nutrient exchange to occur (Jansson et al., 2002; Pardi et al., 2002; Regnault et al., 2005). While amino acid transport is decreased from the placenta to the fetus, glucose transport remains unaltered, despite many reports of the fetus being in a state of hypoglycemia in utero (Jansson et al., 1993; Jansson et al., 2002). Although there is no change in the abundance of placental glucose transporters, it has been reported that IUGR placentae have increased glycolytic metabolism compared to control placentae, thus contributing to fetal hypoglycemia (Challis et al., 2000). Recently, it has been proposed that in response to decreased oxygen availability in IUGR placentae, the placenta undergoes reprogramming, switching it from oxidative to anaerobic metabolism, leading to increased glucose metabolism and decreased ROS formation via mitochondrial respiration (Illsley et al., 2010). This indicates that IUGR placentae are not in a state of nutrient deprivation but contribute to fetal undernutrition and subsequent fetal growth reductions via abnormal metabolism in response to a low oxygen environment (Illsley et al., 2010). The build-up of placental amino acids due to decreased transport to the fetus would also have a powerful inhibitory effect on autophagy, via mTOR signaling (Codogno & Meijer 2005). These findings are corroborated by my data that the level of autophagy, as assessed by the marker LC3B-II and electron microscopy, is significantly decreased in IUGR placentae relative to controls, supporting the notion that IUGR placentae are not in a state of starvation or oxidative stress.

4.2 Oxidative stress induces autophagy in preeclampsia

It is widely accepted that preeclampsia is associated with an increased status of both oxidative and nitrile stress in the placenta(Hubel 1999; Burton 2004; Myatt & Cui 2004; Burton et al., 2009). In my study, SNP treatment was used to generate oxidative stress to model PE in JEG3 cells. SNP has the ability to release NO, resulting in a status of nitrile stress, and donates hydroxyl radicals via Fenton reaction, resulting in downstream Bcl-2 family activation (Cardaci et al., 2008). Our lab has previously utilized SNP treatment in JEG3 cells and found that it induced trophoblast cell death via mobilization of Mtd/Bok from the cytoplasm to the mitochondria, and death could be reversed by the overexpression of Mcl-1L (Soleymanlou et al., 2007; Ray et al., 2009). In this study, I report that SNP treatment in JEG3 cells resulted in increased autophagy and this effect is correlated with decreased Mcl-1L expression, increased MtdL expression and altered MtdL localization (Figure 4.2.1).

While my results enhance the understanding of autophagic regulation in the human placenta, the functional role of autophagy in pathological and normal human placentae has yet to be definitively established. Autophagy has been demonstrated to be a cell death mechanism independently of apoptosis. The extensive vacuolization and lysosomal activation that some dying cells exhibit has necessitated the classification of Type II (autophagic) cell death, in which the dying cell exhibits features of autophagy, while the exact mechanism of cellular demise is not entirely characterized (Gozuacik & Kimchi ; Erlich et al., 2007). In mouse L929 fibroblast cells, oxidants, ceramide and radiation have been reported to induce caspase-independent cell death (Fiers et al., 1999). In L929 cells, caspase inhibition by use of the pan-caspase inhibitor benzyloxycarbonyl-valyl-alanyl-aspartic acid (O-methyl) fluoro-methylketone (zVAD) also directly induced cell death, which could be inhibited by RNAi directed against Atg7 and Beclin 1 (Yu et al., 2004). Additional data in cancer cell lines reveals that the knockdown of autophagy machinery can lead to tumour progression, and that Beclin 1 is a haploinsufficient tumour suppressor protein, further supporting a role for autophagy in cell death (Liang et al., 1999). In Drosophila, autophagy has been identified as a cell death inducer in salivary glands, and in contrast, as a cell survival mechanism in the larval fat body (Scott et al., 2004; Berry & Baehrecke 2007). Remarkably, an engulfment receptor termed Draper (Drpr) has been discovered to specifically regulate death-inducing autophagy in Drosophila salivary glands, but not starvation-induced autophagy in the larval fat body, suggesting that distinct factors regulate autophagy in different cellular contexts (McPhee et al., 2010).

It has also been proposed that autophagy is a cytoprotective response in stressed cells in order to prolong cell survival. This is supported by studies that have demonstrated increased cell death in cells that lack the genes essential for autophagy to occur (Levine & Yuan 2005). The prosurvival capacity of autophagy is most frequently observed in response to nutrient insufficiency. In autophagy deficient yeast and amoeba, starvation conditions rapidly induce cell death, and mice that lack Atg5 die during the neonatal period due to starvation (Otto et al., 2003; Kuma et al., 2004; Levine & Yuan 2005). In human HeLa cells, inhibition of autophagy has been demonstrated to induce apoptosis, further supporting the cytoprotective role of autophagy (Boya et al., 2005). Indeed, the autophagy observed in preeclamptic placentae may be defensive,

Figure 4.2.1 Proposed model of oxidative stress induced autophagy in trophoblast cells.

In response to oxidative stress, cellular ROS formation leads to decreased levels of Mcl1-L and increased levels of MtdL. Both decreased Mcl-1L and increased levels of MtdL contribute to freeing Beclin 1 protein, which can then induce autophagy at the level of the ER.

95 in order to turnover damaged organelles in response to hypoxia reoxygenation injury in the trophoblast layer. This notion is supported by my finding of mitophagy in all trophoblastic layers, suggesting autophagy is recycling damaged mitochondria.

Other reports have indicated that autophagic activation precedes apoptotic cell death and may predispose cells towards a death phenotype (Yousefi et al., 2006). Autophagy is frequently discussed as an independent event distinct from apoptosis with the absence of apoptotic features (Klionsky et al., 2007). However recent reports have identified that autophagic regulation is intricately connected to that of apoptosis, via the factors Atg5 and Beclin 1, and likely many other proteins (Luo & Rubinsztein 2007). High levels of autophagy in trophoblast cells that are dying, and the absence of concomitant cell survival in PE indicates that in preeclampsia, some of the observed trophoblast cell death may be the result of increased autophagic levels, which in trophoblast are regulated by the Mcl/Mtd system.

The data presented in this thesis supports that in response to MtdL, both cellular processes of autophagy and apoptosis are occuring in the same cell. This observation is consistent with my data regarding increased autophagic activation in PE placental tissue and previous reports of elevated trophoblast apoptosis in PE (Myatt & Cui 2004; Soleymanlou et al., 2005). In the data presented herein, extensive colocalization was seen between MtdL and the ER membrane in apoptotic cells, which were identified by condensed nuclei, suggesting that autophagy and apoptosis are occuring in the same cell, possibly at the same time. As well, extensive Mcl-1L and MtdL colocalization was also observed in apoptotic cells, which may be related to their apoptotic functions as well as their autophagic regulation.

In summary, alterations in the balance between Mtd and its prosurvival binding partner Mcl-1L is responsible for many of the characteristics of PE placentae, including elevated autophagy, excessive proliferation of cytotrophoblast progenitor cells and apoptosis of the trophoblast layers. Mtd induction in PE is likely the result of increased oxidative insult to the placenta. Many studies have attempted to ameliorate the symptoms of PE by increasing the dosage of antioxidants consumed by women early in pregnancy, including vitamins C and E. While antioxidants have profound effects on reversing oxidative damage in vitro, success in large-scale clinical trials has been very limited (Tannetta et al., 2008; McCance et al., 2010;

Roberts et al., 2010; Sibai 2010). Therefore, targeting downstream effectors of oxidative stress, such as Mtd, might provide more promising future interventions for this disease of pregnancy.

Understanding the precise mechanisms regulating autophagic induction in PE and the functional response in trophoblast tissue to autophagy is essential in order to understand future therapeutic targets for PE. Therefore future studies in regards to autophagy in the human placenta should be targeted to elucidating these concepts more fully.

Chapter 5 5 Future Directions 5.1 Is oxidative stress in trophoblast also sensed by Atg4?

In the data presented herein, a status of oxidative stress was shown to activate autophagy through changes in Mcl-1L and MtdL expression levels, thus resulting in increased levels of autophagy. However, reactive oxygen species (ROS) have also been reported to activate autophagy independently through oxidation of the protein product of autophagy-related gene 4 (Atg4) (Scherz-Shouval et al., 2007). Atg4 is an essential part of the autophagy cascade after Atg5 conjugation to Atg12, and must become inactive following the initial cleavage of LC3B-I into LC3B-II to ensure LC3B-II conjugation to the autophagosomal membrane (Hemelaar et al., 2003). ROS serve as signalling molecules for many cellular processes, and not surprisingly have also been implicated in autophagic activation in response to nutrient deprivation. However, it remains to be established whether a cellular insult of ROS could also independently activate autophagy via an Atg4 response. To evaluate in JEG3 choriocarcinoma cells whether SNP treatment can inhibit Atg4, the following approach can be undertaken. The activity of Atg4 can be measured by assessing the relative cleavage of LC3B-I to LC3B-II in lysates treated with SNP, with and without the reducing agent dithiotheitrol (DTT). As previously reported, DTT would recover Atg4 activity to baseline conditions if it had been oxidized by ROS in response to SNP (Scherz-Shouval et al., 2007). This would provide valuable insight into other possible mechanisms by which adverse oxidative stress conditions are sensed and relayed in trophoblast cells, and the relative importance different response mechanisms independent of Bcl-2 family activation have upon autophagic transduction.

5.2 Are Vps34/PI3KIII complexes involved in MtdL induced autophagy?

The class III phosphatidylinositol 3-kinase (PI3KIII) is a regulator of intracellular membrane trafficking, and is the orthologue to yeast Vacuolar Protein Sorting 34 (Vps34) (Schu et al., 1993). Vps34/PI3KIII forms a complex involving several other proteins including the protein kinase hVps15, leading to subsequent Vps34 activation (Yan et al., 2009). In mammalian cells, Beclin 1 forms two different PI3KIII complexes, one with Atg14, termed

98 complex I, and another with UV irradiation resistance-associated gene (UVRAG), termed complex II, providing evidence that PI3KIII and Beclin 1 can form multiple complexes that are both involved in autophagic activation (Itakura et al., 2008). In the present study, the association of Beclin 1 with Vps34/PI3KIII was examined via co-immunoprecipitation analysis, providing inconclusive results pending further investigation (Figure 5.2.1). Alternatively if the interaction between Vps34/PI3KIII and Beclin 1 is not altered due to MtdL, the activity of Vps34/PI3KIII could be altered. Future experiments could confirm whether Vps34/PI3KIII complexes have enhanced activity in response to MtdL expression, thus leading to autophagosome nucleation and formation.

Figure 5.2.1 Co-immunoprecipitation for the association of Beclin 1 with Vps34/PI3KIII in HEK293-GFP and HEK293-GFPMtdL cells.

Preliminary co-immunoprecipitation for the association of Beclin 1 with Vps34/PI3KIII in HEK293-GFP and HEK293-GFPMtdL cells indicates no change in assocation. Cells were treated with 0 or 2.5 ng/mL doxycycline for 24 hr, immunoprecipitated for Vps34/PI3KIII and immunoblotted for Beclin 1 association (n=3). Neg: negative IgG control; Pos: positive control.

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5.3 Is MtdL contributing to mitophagy?

Bax-interacting factor 1 (Bif-1) has been reported to be an essential protein for autophagy, and interacts with complex II, which consists of Beclin 1, UVRAG and Vps34/PI3KIII, and has been hypothesized to deform membranes leading to membrane curvature due to its N-BAR domain (Frost et al., 2009; Simonsen & Tooze 2009). Additionally, Bif-1 is localized to mitochondria where it has been reported to directly interact with Bax and has been demonstrated to activate both Bax and Bak leading to apoptotic induction (Takahashi et al., 2005). As Bax activation has been demonstrated to lead to mitochondrial fragmentation, Bif-1 has been postulated to be a candidate protein in targeting mitochondria for mitophagic events (Takahashi et al., 2009). It remains an intriguing possibility whether, if due to the homology and functional similarities between MtdL and Bax, if MtdL can undergo an interaction with Bif-1. This is a putative secondary mechanism by which MtdL could be contributing to increased the mitophagy evidenced in PE.

5.4 What is the functional effect of autophagy in trophoblast?

While the work presented in this thesis has examined the regulatory mechanisms behind enhanced autophagic activation in response to oxidative stress, the functional outcome of increased autophagy in trophoblast has not been fully answered in this study. It is apparent from both the data herein, and from reports of other groups, that PE is characterized by excessive autophagy and apoptosis within the syncytium (Soleymanlou et al., 2007; Oh et al., 2008; Burton & Jones 2009). My data supports the proposition that in trophoblast, autophagy may be priming the cells for subsequent apoptotic events. Several experiments could shed further insight into the possible roles of MtdL induced-autophagy and the functional effect of autophagy in trophoblast tissue. Firstly, live-cell imaging experiments should be performed in HEK293-GFPMtdL cells to determine when autophagy is induced and when signs of apoptosis can be detected within a single cell. This would shed further light on whether MtdL is inducing apoptosis and autophagy concurrently or whether autophagy precedes apoptosis in this cell line.

Autophagy-related protein 5 (Atg5) was first discovered in yeast and is involved in the conjugation steps of autophagosome formation (Mizushima et al., 1998). While full-length Atg5 is required for autophagosome formation, it has also been reported that a cleavage product of

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Atg5 can prime cells towards apoptosis (Yousefi et al., 2006). In response to death stimuli, calpain-mediated cleavage of Atg5 results in a pro-apoptotic 24kDa isoform of Atg5 that is mitochondrial targeted and complexes with Bcl-xL, leading a transition from autophagy to apoptotic cell death via Bax displacement and oligimerization (Yousefi et al., 2006).

In this study, it was observed that MtdL was having an inductive effect on both apoptosis, as previously reported (Inohara et al., 1998; Soleymanlou et al., 2005), and an independent effect on autophagic stimulation via an interaction with Mcl-1L. However, whether MtdL is directly triggering both of these events, or if the apoptotic transition is enhanced by Atg5 cleavage was not addressed. The status of Atg5 in these cells should be determined in order to address if Atg5 cleavage is also partially responsible for the apoptotic phenotype present in the HEK293- GFPMtdL cell line in response to low levels of MtdL induction. Alternatively, if Atg5 cleavage is indeed occurring in these cells, it would be interesting to determine whether MtdL could possibly be encouraging or exacerbating calpain-mediated cleavage of Atg5. As Atg5-cleaved toxicity can be rescued by Bcl-2 overexpression, whether Mcl-1L overexpression could as well rescue the apoptotic switch also remains to be addressed. As preeclamptic placentae are characterized by a phenotype of both excessive apoptosis and autophagy, it is likely that these cellular processes are activated concurrently by a variety of cell signalling pathways, including MtdL functions and possibly via Atg5 cleavage.

Once the dynamics of Mcl-1L/MtdL regulated apoptosis and autophagy are better understood, functional experiments in placental explants can be carried out to better understand the effect of oxidative stress on placental autophagy. While cell lines provide a model for trophoblast cells, human villous placental explants provide a model of the functional chorionic unit, including the two trophoblast layers and vasculature. Placental explants from healthy placentae can be exposed to SNP treatment as well as hypoxia/reoxygenation experiments. Electron micrographs can be taken of the tissue to see if it develops the preeclamptic phenotype of a vacuolated syncytium with increased evidence for mitophagy.

Finally, explants of PE placentae can be treated with 3-methyladenine (3-MA), an inhibitor of PI3K with greater affinity for class III proteins. The tissue and media can be subsequently monitored to detect if more or less shedding of syncytium occurs, indicating whether cell death has increased or decreased in response to inhibited autophagy (Seglen &

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Gordon 1962). These experiments would more fully answer the question of the role of autophagy in placenta and if the elevated autophagy seen in PE is aiding the tissue to cope with a deleterious environment or further accelerating cell death in response to oxidative stress.

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Appendices 6 Appendix 6.1 Rapamycin Treatment

When mTOR is activated, autophagy is inhibited and can be monitored by phosphorylation status of p70S6K, a protein kinase downstream of mTOR. In response to mTOR activation, p70S6K is phosphorylated at Thr389. P(Thr398)p70S6K was used as an additional marker of the autophagy status in JEG3 cells, HEK293 cells and placental lysates from three different gestational ages. JEG3 cells were either starved for 3 hrs of FBS or not starved, and HEK293 cells were treated with 1µM rapamycin for 3 hrs and cultured in the presence of either serum (nst) or serum starved (st). Placental lysate from 7 and 12 weeks of gestation and term were also used. The above lysates were immunoblotted for

P(Thr389)p70S6K expression, and expression was detectable in placental tissue and in HEK293 cells but not in JEG3 cells. In HEK293 cells, P(Thr389)p70S6K expression was abrogated in response to rapamycin treatment (Appendix 6.1.1a). Therefore, P(Thr389)p70S6K was not further used as marker of autophagic levels in JEG3 cells. In order to determine if autophagy could be activated by rapamycin treatment in JEG3 cells a dose and time course for rapamycin treatment was performed. No significant changes in LC3B-II expression were found at either 24 or 48 hrs of treatment with differing concentrations of rapamycin compared to vehicle DMSO control (Appendix 6.1.1b). Rapamycin was not found to induce autophagy in JEG3 choriocarcinoma cells.

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Appendix 6.1.1 Expression of p70S6K phosphorylated at Threonine 389 in JEG3, HEK293 and human placental lysate and rapamycin treatment in JEG3 cells.

A. Immunoblot for P-Thr 389 p70S6K reveals expression in HEK293 cells and placental lysate and was diminished in response to 1 µM rapamycin treatment for 3 hrs. St: FBS starved for 3 hrs, nst: nonstarved, R: rapamycin treated, w: weeks of gestation. B. JEG3 cells were treated for 24 and 48 hrs with 0.1-0.8 µM rapamycin and immunoblotted for LC3B-II, C: control, V: vehicle.

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6.2 Low oxygen environment and autophagy

As trophoblast pathologies are characterized by a hypoxic environment, experiments were performed in JEG3 cells to determine if a relatively hypoxic environment of 3% oxygen could induce LC3B-II expression. A timecourse was performed for 24, 48 and 72 hrs in both

20% and 3% oxygen tensions and protein expression levels for Mcl-1L and LC3B-II were monitored. In response to low oxygen, Mcl-1L levels decreased after 24 hrs, with more variable results at 48 and 72 hrs of treatment, despite reports in the literature that Mcl-1 is a HIF-1 responsive gene (Appendix 6.2.1a) (Piret et al., 2005). LC3B-II expression levels consistently decreased in response to hypoxic stimulus at all timepoints (Appendix 6.2.1b).

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Appendix 6.2.1 Expression of Mcl-1L and LC3B-II in response to 20% and 3% oxygenation.

Timecourse of JEG3 cells cultured in 3% and 20% oxygen conditions for 24, 48 and 72 hrs. A. Immunoblot analysis for Mcl-1L expression at 3% and 20% oxygenation. B. Immunoblot analysis for LC3B-II expression at 3% and 20% oxygenation (n=3).

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6.3 Glucose Deprivation in JEG3 cells

The most powerful inducer of autophagy is glucose starvation which activates autophagy independently of apoptosis (Kuma et al., 2004; Codogno & Meijer 2005). The previous interventions presented, including etoposide and SNP treatment have the disadvantage of also inducing apoptosis concurrently with autophagic activation. In order to determine the effect of glucose deprivation upon a trophoblast cell model, JEG3 cells were starved of glucose by using

Hank's Buffered Saline Solution (HBSS+) for 3, 6, and 24 hrs and then immunoblotted for

LC3B-II expression (Appendix 6.3.1). However, no significant changes in LC3B-II expression were observed in response to glucose starvation in JEG3 cells, indicating that JEG3 cells do not respond to glucose starvation within the first 24 hrs of treatment (n=3).

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Appendix 6.3.1 Expression of LC3B-II in JEG3 cells in response to glucose starvation.

JEG3 cells were starved of glucose using HBSS+ for 3, 6 and 24 hrs and immunoblotted for LC3B-II expression (n=3).

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6.4 Autophagy in Placental Development

Early placental development occurs at a low oxygen environment of about 3% O2 which is essential for CT proliferation and to stimulate EVT growth at the distal ends of the villous tips (Genbacev et al., 1996; Genbacev et al., 1997; Caniggia et al., 2000). A low oxygen environment is maintained until the maternal circulation opens to the developing placenta as the result of EVT spiral artery remodeling, at about 10-12 weeks of gestation, resulting in a rise in oxygen tension to about 8% O2. In order to understand the effect of oxygen upon placental autophagy, the levels of LC3B-II expression were immunoblotted in first trimester human placental samples (Appendix 6.4.1). While there is significantly more LC3B-II expression in first trimester development compared to term placentae, no significant difference was observed in autophagic activation from placentae of 5-9 weeks of gestation compared to placentae from 10-15 weeks of gestation.

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Appendix 6.4.1 Expression of LC3B-II in human placental development.

Upper panel: Representative immunoblot of LC3B-II expression in human placental lysate. Lower panel: Densitometric analysis of LC3B-II protein expression normalized to β-actin relative to TC. (5-9 weeks of gestation n=18, 10-15 weeks of gestation n=21, TC n=5). Kruskall-Wallis test with Dunn's multiple comparisons, *p<0.05, **p<0.01.