Investigation of the Zinc-Mitophagy Signaling in Hypoxic Cells a Dissertation Presented to the Faculty of the College of Arts An

Home , Pyrithione, Sodium dithionite

Investigation of the Zinc-Mitophagy Signaling in Hypoxic Cells

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Qiping Lu

May 2020

This dissertation titled

Investigation of the Zinc-Mitophagy Signaling in Hypoxic Cells

QIPING LU

has been approved for

the Department of Molecular and Cellular Biology

and the College of Arts and Sciences by

Yang V. Li

Professor of Biomedical Sciences

Florenz Plassmann

Dean, College of Arts and Sciences 3

ABSTRACT

LU, QIPING, Ph.D., May 2020, Molecular and Cellular Biology

Investigation of the Zinc-Mitophagy Signaling in Hypoxic Cells

Director of Dissertation: Yang V. Li

Zinc is one of the most essential trace elements in the body. The concentration of intracellular free zinc is strictly regulated. The abnormal zinc concentration has been implicated in numerous clinical manifestations including ischemic stroke. Zinc homeostasis is achieved by proteins and organelles which sequester zinc or release zinc.

Mitochondria are the power plants of the cell, the proper function of mitochondria is crucial for cellular metabolisms and physiological activities, their quality and quantity are regulated by mitophagy through selectively removing damaged mitochondria. Emerging evidence over the past decade has shown that zinc affects mitochondria in response to ischemia. It is progressively clear that zinc-mitochondrial interactions occur in and contribute to ischemic injury. Among the pathological effects of zinc accumulation on mitochondria, zinc induced release and accumulation of ROS draws special interest. ROS is a major cause of mitochondrial damage and initiates mitophagy. In addition, emerging evidence suggests that mitophagy plays critical roles in the pathophysiological process of cerebral ischemia. In this study I investigated the role of zinc and mitochondria in hypoxia-induced mitophagy, screened for and evaluated the cross talks between zinc and mitophagy under hypoxia condition. First, I studied the distribution of intracellular free zinc in multiple types of cell cultures, including HeLa cells, cortical neurons, and pancreatic HIT-T15 cells. The live cell fluorescent imaging with zinc specific probe 4 revealed that mitochondria, ER and the Golgi apparatus are potential zinc storage sites during cellular zinc homeostasis. Among them, mitochondria serve as a major organelle that sequesters zinc. I demonstrated the critical roles of zinc in stroke through my study in in vitro stroke model by showing that zinc chelation alleviates the hypoxia-triggered mitophagy activity, and zinc influx damages mitochondria and induces mitophagy. The interplay between zinc and mitophagy during hypoxia was then investigated by large- scale screening and fine-scale validation. The expression of superoxide dismutase 1

(SOD1) was elevated to defend the oxidative stresses during hypoxia and zinc influx, and during this process, spastic paraplegia 7 (SPG7) was high up-regulated, and possibly participate in the formation of mitochondrial permeability transient pore. In contrast,

SOD1 is not responsive to the mitochondrial uncoupler, Carbonyl cyanide-4-

(trifluoromethoxy) phenylhydrazone (FCCP), and superoxide dismutase 2 (SOD2) takes the major role in defending FCCP-induced oxidative stress defense. In conclusion, zinc chelation is effective in rescuing cells from stresses induced by stroke/hypoxia by reducing the damages to mitochondria, preserving the mitochondrial integrity and lowering the mitophagy activity. Furthermore, the concept of zinc and mitophagy crosstalk during hypoxia/stroke was demonstrated and investigated. Through the elucidation of zinc-mitophagy crosstalk, more promising interventions can be invented to target the zinc-mitophagy signaling during stroke.

DEDICATION

I dedicate this work to my father

Aijun Lu

Who had inspired me to chase a dream in life science

ACKNOWLEDGMENT

First and foremost, I would like to express the gratitude to my supervisor, Dr.

Yang V Li, for his constant guidance, strong support, valuable teaching, extraordinary patience through my doctorial training at Ohio University. He taught me how to fight through difficulties, solve problems, and maintain optimism. He has not only trained me so that I am capable with research in depth, but also quickly adapted to a past-paved environment with well-trained critical thinking ability.

I would also like to express my sincere gratitude to my committee, Dr. Robert

Colvin, Dr. Tomo Sugiyama, Dr. Zhihua Hua for maintaining the high standard and giving me valuable suggestions and being very supportive on my work. Moreover, I would like to thank Dr. Sarah Wyatt for her teaching in scientific writing and presentation, especially for her strong support and help in solving the difficulties I have faced.

I am grateful to everyone who was or is in the lab: Dr. Christian Stork, Kira

Slepchenko, Dr. Zhijun Shen, Dr. Xinge Yu, Yuli Hu, Zihui Wang, Hariprakash

Haragopal, and Katherine knies for their assistance, their instructive discussion during my study.

In addition, I would like to thank all my friends, Yunyi Feng, Panduan An,

Yanrong Qian, Xiaodan Zhao and many more, for their encouragement and support during the past years. Their intelligence and persistence have always inspired me to pursue a better outcome. 7

Last but not least, my deepest appreciation goes to my mother, Linge Liu, my brother and sister-in-law, Qiwei Lu and Yang Li, for their whole-hearted and everlasting support, care and love.

TABLE OF CONTENTS

Page

Abstract ...... 3 Dedication ...... 5 Acknowledgment ...... 6 List of Tables...... 11 List of Figures ...... 12 Abbreviations ...... 14 Introduction ...... 15 1. Zinc ...... 15 1.1 Zinc Content and its Roles ...... 15 1.2 Zinc Homeostasis and Trafficking ...... 16 1.3 Fluorescent Detection of Zinc ...... 17 1.4 Zinc in Cerebral Ischemia...... 18 2. Hypoxia ...... 19 2.1 Hypoxia Signaling Pathway ...... 19 2.2 Hypoxia in Stroke ...... 20 2.3 Hypoxia Inducing Chemicals...... 21 3. Mitochondria ...... 22 3.1 Mitochondrial Structure and Function ...... 22 3.2 Mitochondrial Quality Control ...... 23 3.3 Molecular Mechanisms of Mitophagy ...... 25 3.4 Mitophagy in Hypoxia ...... 26 4. The Potential Interplay between Zinc and Mitophagy during Hypoxia...... 27 Specific Aims ...... 29 Specific Aim 1: To Investigate the Intracellular Distribution of Free Zinc ...... 29 Specific Aim 2: To Investigate the Effect of Zinc on Mitophagy during Hypoxia by Modulating the Zinc Levels ...... 30 Specific Aim 3: To Investigate How Zinc Interplays with Mitophagy during Hypoxia by Screening and Validating the Candidate Genes involved in the Signaling Pathways ...... 31 Materials and Methods ...... 32 1.Cell Cultures ...... 32 9

2. Zinc Labeling ...... 32 3. Organelle and Zinc Co-labeling...... 33 4. Fluorescence Imaging ...... 34 5. in vitro Treatment to Induce or Alleviate Stresses ...... 34 6. Qualitative and Quantitative Analysis of Mitochondrial Morphology ...... 35 7. GEO Analysis for Large-scale Screening ...... 37 8. Quantitative Real-time PCR ...... 39 Results ...... 42 1. The Intracellular Distribution of Free Zinc in Cells ...... 42 1.1 Staining Pattern of Zinpry-1 in HeLa Cells is Concentration Dependent ...... 42 1.2 Zinpyr-1 Staining is Specific to Zinc ...... 43 1.3 Overview of Distribution of Intracellular Zinc in HeLa Cells ...... 45 1.4 Co-localization of Zinc and Mitochondria in HeLa Cells ...... 46 1.5 Co-localization of Zinc and Mitochondria in HIT-T15 Cells ...... 47 1.6 Mitochondria Observation in Primary Cortical Neurons ...... 49 1.7 Co-localization of Zinc and Mitochondria in Primary Cortical Neurons ...... 50 1.8 Co-localization of Zinc and Mitochondria in Individual Mitochondria ...... 51 1.9 Co-localization of Zinc and ER in HeLa Cells ...... 52 1.10 Co-localization of Zinc and ER in Pancreatic Beta Cells HIT-T15 ...... 53 1.11 Co-localization of Zinc and Golgi in HeLa Cells and HIT-T15 Cells ...... 54 1.12 Zinc was not Detectable in the Nucleus of Viable HeLa cells ...... 56 2. To Investigate the Effect of Zinc on Mitophagy during Hypoxia by Modulating the Zinc Levels ...... 58 2.1 Mitophagy Observation ...... 58 2.2 Hypoxia Induced Mitophagy Can be Alleviated by Zinc Chelator TPEN ...... 60 2.3 Zinc Induced Mitophagy is Alleviated by TPEN...... 64 2.4 Mitochondrial Morphology as an Indicator of Mitochondrial Wellness ...... 66 2.5 The Analysis of Mitochondrial Morphology under the Stresses of Hypoxia and Zinc Influx ...... 68 2.6 Qualitative and Quantitative Analysis of Mitochondrial Morphology by Evaluating Mitochondrial Roundness ...... 70 2.7 Qualitative and Quantitative Analysis of Mitochondrial Morphology by Evaluating Mitochondrial Circularity ...... 72 10

2.8 Qualitative and Quantitative Analysis of Mitochondrial Morphology by Evaluating Major Axis and Aspect Ratio ...... 74 3. Investigating the Genes Involved in Zinc-Mitophagy Interplay during Hypoxia by Large-scale Screening and Fine-scale Validation ...... 77 3.1 Gene Expression Omnibus Analysis to Reveal the Genes of Interest ...... 77 3.2 Identifying the Candidate Genes of in Hypoxic HeLa Cells by Comparing with Their Overlapping with Mitochondria Genes and Zinc Genes ...... 77 3.3 Identifying the Candidate Genes of in FCCP HeLa Cells by Analyzing their Overlapping with Mitochondrial Genes and Zinc Genes ...... 81 3.4 Candidate Genes Identified by Large-scale Screening and Literature Research ...... 84 3.5 The Superoxide Dismutase Multigene Family CuZn-SOD (SOD1) and Mn- SOD (SOD2) Act Differently in Response to FCCP, Hypoxia or Zinc Addition . 85

3.6 HIF-1a Gene Expression Level was not Altered by FCCP, Hypoxia or ZnCl2 Administrations ...... 89 3.7 The Expression of SPG7 is Highly Increased under Hypoxia and Exogenous Zinc Administration ...... 90 3.8 The Gene Expression of TRIM 14 is Elevated during Hypoxia, and Remains Stable during FCCP and Exogenous Zinc Stresses ...... 92 3.9 The Expression of Parkin was Elevated upon Mitochondrial Stresses, while PINK1 Expression Level Remained Unaffected ...... 94 3.10 PGC1A is Up-regulated in Response to Hypoxia and Zinc Influx ...... 96 3.11 Mitophagy Regulators NIX1 and FUNDC1 were Not Associated with the FCCP, Hypoxia or Zinc Influx Induced Mitophagy ...... 97 Discussion ...... 100 References ...... 114 Permissions of Reuse ...... 126

LIST OF TABLES

Page

Table 1. GEO records retrieved from NCBI GEO database ...... 38 Table 2. List of forward and reverse primers for genes tested by qPCR ...... 41 Table 3: List of 7 overlapped genes ...... 80 Table 4. List of 8 overlapped genes...... 83 Table 5. List of finalized candidate genes identified by large-scale screening and literature research ...... 84

LIST OF FIGURES

Figure 1. The staining pattern of Zinpyr-1 in HeLa cells is concentration dependent...... 43 Figure 2. Zinpyr-1 is specific to zinc...... 44 Figure 3. Cellular zinc distribution in HeLa cells shows a compartmentalized pattern. ... 46 Figure 4. Zinc is localized to mitochondria in HeLa cells...... 47 Figure 5. Co-localization of zinc and mitochondria in HIT-T15 cells...... 48 Figure 6. Mitochondria observation in primary cortical neurons...... 49 Figure 7. Zinc is localized to mitochondria in cortical neurons...... 51 Figure 8. The observation of zinc and mitochondria co-localization in single mitochondrion...... 52 Figure 9. Co-localization of zinc and ER in HeLa cells...... 53 Figure 10. Zinc is localized to the ER in HIT T15 cells...... 54 Figure 11. Zinc is localized to the Golgi apparatus in HeLa cells and HIT T15 cells...... 55 Figure 12. Absence of zinc staining in the nucleus...... 57 Figure 13. Mitophagy observation...... 59 Figure 14. Hypoxia induced mitophagy was alleviated by zinc chelation...... 61 Figure 15. Quantification of mitophagy levels in control, FCCP, hypoxia, hypoxia/TPEN groups...... 63 Figure 16. Zinc induced mitophagy was alleviated by zinc chelation...... 65 Figure 17. Quantitative analysis of the mitophagy level in zinc and zinc/TPEN groups .. 66 Figure 18. Representative images showing one of the typical morphologies of stressed mitochondria ...... 67 Figure 19. Mitochondrial morphology under the stress of hypoxia and exogenous zinc .. 70 Figure 20. Changes in mitochondrial roundness induced by hypoxia and exogenous zinc influx ...... 72 Figure 21. Changes in mitochondrial circularity induced by hypoxia and exogenous zinc influx ...... 74 Figure 22. The effects of hypoxia and exogenous zinc on mitochondrial major axis and aspect ratio ...... 76 Figure 23 The comparison of DEG with mitochondria related genes revealed 154 overlapped genes...... 78 Figure 24. The comparison of DEG (Hypoxia v.s. Normoxia) with zinc related genes revealed 97 overlapped genes ...... 79 Figure 25. The comparison of DEG from hypoxic HeLa cells with mitochondrial genes and zinc genes revealed 7 overlapped genes ...... 80 13

Figure 26. The comparison of DEG in FCCP treated HeLa cells with mitochondria related genes revealed 100 overlapped genes...... 82 Figure 27. The comparison of DEG in FCCP treated HeLa cells with zinc related genes revealed 74 overlapped genes...... 82 Figure 28. The comparison of DEG from FCCP treated HeLa cells with mitochondrial genes and zinc genes revealed 8 overlapped genes ...... 83

Figure 29. Hypoxia and ZnCl2 induces the expression of SOD1, FCCP induces the expression of SOD2 ...... 86

Figure 30. EC-SOD (SOD3) is not responsive to FCCP, Hypoxia or ZnCl2 stimuli ...... 88

Figure 31. mRNA level of HIF-1a is not altered by FCCP, Hypoxia or ZnCl2 stimuli .... 90 Figure 32. The expression of SPG7 is highly increased under hypoxia and exogenous zinc administration ...... 92 Figure 33. Hypoxia elevated the gene expression of TRIM14...... 93 Figure 34. The mRNA level of Parkin (PRNK) was elevated upon mitochondrial stresses...... 95 Figure 35. PGC1A was up-regulated in response to hypoxia and zinc influx...... 97 Figure 36. Mitophagy regulators NIX1 and FUNDC1 were not involved...... 98 Figure 37．Zinc and mitophagy interplay during hypoxia ...... 111

ABBREVIATIONS

CoCl2 cobalt chloride DEG Differentially expressed genes ER Endoplasmic Reticulum FCCP Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone GEO Gene expression omnibus GO Gene ontology HIF Hypoxia inducible factor HSP hereditary spastic paraplegia JACoP Just Another Colocalization Plugin MPTP mitochondrial permeability transition pore MT metallothionein Na2S2O4 sodium dithionite NaCN sodium cyanide NaN3 sodium azide NO Nitric Oxide OGD oxygen and glucose deprivation PCC Pearson’s correlation coefficient PHD prolyl-hydroxylase PINK1 PTEN-induced kinase 1 PTEN phosphatase and tensin homolog qPCR quantitative PCR ROS reactive oxygen species TPEN N,N,N’,N-tetrakis(2-pyridylmethyl)ethylenediamine TRIM tripartite motif TRIM14 Tripartite motif-containing 14 TSQ N-(6-methoxy-8-quinolyl)-4-methoxybenzenesulfonamide ZIPs Zrt-Irt-like proteins ZnTs zinc transporters

INTRODUCTION

1. Zinc

1.1 Zinc Content and its Roles

Zinc is the second most important trace elements in the body, only after iron. Zinc is essential for growth and development of mammals and other organisms. It is ubiquitous and found in all body tissues, 85% of total zinc in muscle and bone, 11% in the skin and the liver, and the remaining in the other tissues (Kawashima et al. 2011).

The average amount of Zn in the adult body is 2-3g, only about 0.1% of which is replenished daily, and human zinc requirement is estimated at 15mg/d (Maret and

Sandstead 2006).

In multicellular organisms, all Zn is intracellular, 30-40% is located in the nucleus, 50% in the cytoplasm, organelles and specialized vesicles, and the remainder in the cell membrane (Tapiero and Tew 2003). The total concentration of Zn in mammalian cells is thought to fall in the 100-500 µM range. Most of zinc is tightly bound with proteins. The cellular concentrations of free zinc range from picomolars in undifferentiated mammalian cells to ~0.3 mM in hippocampal synaptic vesicles (Budde et al. 1997).

Zinc is associated with a variety of biological processes. The functions of zinc are at multiple aspects, as a structural component in zinc fingers; a catalytic cofactor for many enzymes and transcription factors, and as a messenger in intracellular and intercellular signaling pathways (Kambe et al. 2015; MacDonald 2000; Truong-Tran et al. 2001; Murakami and Hirano 2008). 16

1.2 Zinc Homeostasis and Trafficking

The concentration of intracellular free zinc is strictly regulated, which is known as zinc homeostasis (Murakami and Hirano 2008). The maintenance of the zinc homeostasis is achieved generally through two main mechanisms: zinc transporting proteins and zinc- buffer system. The former contains two families of zinc transporter proteins: zinc transporters (ZnTs, SCL30) and Zrt-Irt-like proteins (ZIPs, SLC39) that actively transport zinc among cytosol, intracellular compartments, and extracellular medium (Cousins,

Liuzzi, and Lichten 2006). ZnT family proteins transport zinc from the cytoplasm into the organelle lumen or to the extracellular space (Eide 2006). High levels of intracellular zinc induce the expression of ZnTs (Eide 2006). ZIP family proteins function in the opposite way, which transport zinc from the extracellular space or from the lumen of cellular organelles to the cytoplasm (Eide 2006). Zinc buffering system is the second important part of zinc homeostasis that regulates zinc availability and accumulation in response to metabolic demand.

The majority of cellular zinc is sequestered by zinc binding proteins or into organelles. Among the zinc binding proteins, metallothionein (MT) plays a major role in intracellular trafficking (Cousins, Liuzzi, and Lichten 2006). Metallothioneins participate in the uptake, transport and regulation of intracellular zinc. One metallothionein monomer is capable of sequestering up to 7 zinc ions. Three in its beta domain and four in the alpha domain (Maret 2008). It is reported that cytosolic membrane-bound organelles such as mitochondria, ER and the Golgi apparatus contain free chelatable zinc

(Lu et al. 2016). 17

1.3 Fluorescent Detection of Zinc

To explore the distribution and physiological roles of biological zinc, especially the free zinc, a sensitive and non-invasive technique is desired. Fluorescent imaging stands out as a method of choice (Kimura and Koike 1998; Jiang and Guo 2004). The in vivo real-time monitoring of zinc in cells and subcellular compartments is achieved by fluorescent sensors. Zinc fluorophores recognize zinc and emit fluorescence upon zinc binding, which is a feasible method for real-time and real-space imaging of living cells without damaging them(Jiang and Guo 2004). The most commonly used zinc fluorescent probes include N-(6-methoxy-8-quinolyl)-4-methoxybenzenesulfonamide (TSQ),

Zinquin, Zinpyr and the ZnAF family (Thompson 2005).

Among them, Zinpyr-1 is feasible for intracellular exploration because it is cell permeable without prior modification. Zinpyr-1 is a fluorescein-based bright fluorescent probe for divalent zinc. It has a high quantum yield and excitation and emission wavelengths exceeding 490 nm. Zinpyr-1 is highly selective for zinc over other biological metals, and metal-dependent fluorescence occurs upon binding to zinc

(Walkup et al. 2000). Zinpyr-1 has been widely used for the investigation of zinc signals in plants, eukaryotic organisms, mammalian cells and other biological samples (Alker et al. 2019:1; Woodroofe et al. 2004; Sinclair et al. 2007).

Leveraging the use of zinpyr-1, zinc has been widely investigated in neuronal cell biology, where zinc appears as a neurosecretory product or cofactor and modulate neurotransmission (Frederickson et al. 2000), the dysregulation of zinc homeostasis in the central nervous system is associated with neuronal diseases, such as ischemic stroke, 18

Autism, Alzheimer’s disease, Parkinson’s disease (Sensi et al. 2011; Higashi et al. 2019;

Grabrucker and Grabrucker 2017; Baesler et al. 2019).

1.4 Zinc in Cerebral Ischemia

Stroke is a major cause of adult disability and one of the leading causes of death.

Cerebral ischemia is a common form of stroke. It occurs when the blood supply to the brain becomes blocked, reflecting the extraordinary vulnerability of the brain to loss of blood flow. Ischemic stroke is a devastating disease with a complex pathophysiology.

In the past decades, the understanding of ischemic neuronal death has been dominated by “Calcium-centric” hypothesis, which emphasized that calcium is one of the triggers involved in ischemic cell death through increased excitotoxicity (Choi, 1988).

Many studies have focused on the roles of calcium overload as an important link between neuronal toxicity and ischemic neurodegeneration. However, treatments or clinical trials targeted on calcium overload have met with limited success in reducing the volume or severity of neuronal damage (NINDS, 2002). Study from our lab questioned this

“calcium-centric” hypothesis by showing the abnormal accumulation of intracellular zinc during ischemia, stimulated by oxygen and glucose deprivation (OGD) in rat hippocampus (Stork and Li 2006). Further study from our lab indicates that zinc accumulation is a significant cause of ischemic neuronal death in rat hippocampus (Stork and Li 2009). The rapidly accumulated intracellular zinc in neurons during OGD, is taken up by mitochondria and contributes to consequent mitochondrial dysfunction, cessation of synaptic transmission, calcium deregulation and cell death in an acute slice OGD model of ischemia (Medvedeva et al. 2009). 19

The pathophysiological functions of zinc during cerebral ischemia are still very ambiguous, even though it has been thirty years since zinc was first purported to have a role in cerebral ischemia, as reported that zinc was involved in the selective death of dentate hilar neurons after cerebral ischemia (Tønder et al. 1990). Interestingly, emerging clues are showing the potent effects of zinc on mitochondrial function, making it progressively clear that zinc/mitochondrial interactions contribute to ischemic injury

(Shuttleworth and Weiss 2011; Dong et al. 2015; Zhao Yongmei et al. 2018; Ji et al.

2019; Qi et al. 2019).

2. Hypoxia

2.1 Hypoxia Signaling Pathway

Hypoxia refers to a condition in which oxygen is limited in organs, tissues, or cells. Limitation of oxygen availability participates in the development of pathological conditions such as stroke, heart infarction, cancer, aging and diabetes (Michiels 2004;

Krohn, Link, and Mason 2008). Most of cell responses to hypoxia are mediated by transcriptional regulator hypoxia-inducible factor 1 (HIF-1), which mediates adaptation to hypoxia through activation of a multitude of genes encoding proteins needed for improve tissue oxygen homeostasis, energy metabolism, and efficient management of hypoxia-induced toxic stress (Samaja and Milano 2015). In oxygenated conditions, HIF is hydroxylated by HIF prolyl-hydroxylases, allowing its recognition and degradation by the proteasome system (Maxwell et al. 1999). In hypoxic conditions, HIF is stabilized and translocated into the nucleus, where the HIFs function as transcription factor then 20 locate to the hypoxia-responsive elements (HREs) of its target genes, resulting in their transcriptional upregulation (Lee et al. 2019).

Notably, reperfusion is an effective way to limit hypoxic damage, but reperfusion itself may result in tissue injury. Reperfusion induces the oxidative damage central in the pathophysiology of hypoxia/reoxygenation injury and activates signaling mechanisms that in part synergize and in part oppose those induced by hypoxia (Samaja and Milano

2015).

2.2 Hypoxia in Stroke

Brain is particularly vulnerable to hypoxic conditions. Hypoxia following stroke is common and associated with severe pathological consequences, including functional and behavioral deficits in neuronal system, as a result of the neuronal cell death in brain tissue (Brose, Golovko, and Golovko 2016). Neuronal cell death from hypoxia/stroke is a mixed type of apoptosis and necrosis which is determined by time, region and potentially by patterns of neuronal connectivity (Northington et al. 2001). The detailed mechanisms have not been well elucidated, but previous studies have suggested that hypoxia induced neuronal cell death involves in multiple interacting pathophysiological processes including excitotoxicity, mitochondrial dysfunction and oxidative stress.

Excitotoxicity is the specific type of neurotoxicity mediated by glutamate, it is a primary mechanism of neuronal injury following hypoxia, it requires influx of calcium ion through the N-methyl-D-aspartate receptor (NMDA receptor), NMDA receptor promotes neuronal death or survival depending on what’s downstream (Lai, Zhang, and

Wang 2014). 21

Mitochondrial dysfunction plays a role as determinant of neuronal death/survival after hypoxia/stroke. The cell death signaling begins with the release of mitochondrial cytochrome c in the hypoxic brain. Cytochrome c is translocated from the mitochondria to the cytosolic in brain slices that are subject to hypoxia-ischemia, followed by caspase 9 activation and then caspase 3 activation, and triggers the death of cells by apoptosis

(Chan 2005; Chan 2006). Mitochondria also contributes to nitric oxide (NO) in the development of hypoxic damage (Sims and Anderson 2002). Nitric oxide synthesis is increased in the brain during hypoxic-ischemic as a physiological response to compensate the deficiency in oxygen and substrate for neural cells (Bolaños and Almeida 1999; Akira et al. 1994). The toxic effects of NO are mainly due to the production of nitrates and the release of free radicals, which directly damage mitochondrial enzymes and genetic materials (Chen et al. 2017; Eliasson et al. 1999).

Oxidative stress and the production of reactive oxygen species (ROS) play a pivotal role in the pathogenesis of apoptotic cell death in ischemic stroke. Oxidative stress after cerebral ischemia damages cellular lipids, proteins, and compromises the integrity of the genome, resulting in DNA lesions, cell death in neurons, and impairments in neurological recovery after stroke (Li et al. 2018; Zhao et al. 2016).

2.3 Hypoxia Inducing Chemicals

To characterize the hypoxia response at cellular level, it is necessary to establish hypoxia models in cell culture. Hypoxia conditions can be achieved by physical hypoxia or chemical hypoxia. Physical hypoxia is the use of hypoxia chamber or a CO2 incubator with regulated oxygen levels. Alternatively, hypoxia can also be induced by chemical 22 reagents. The common chemical hypoxia inducing reagents include cobalt chloride

(CoCl2), sodium cyanide (NaCN), sodium azide (NaN3) and sodium dithionite (Na2S2O4).

Cobalt in CoCl2 can replace the ferrous ion in hemoglobin and stabilize hypoxia inducible factors (HIF) 1 and 2 and mimic the hypoxia-induced changes in gene expression, but CoCl2 can damage cell membrane and fail to keep the degree of hypoxia at a constant state. NaCN induced chemical hypoxia is associated with altered gene expression in stress-related genes and caspase-3 cellular activity (Li et al. 2018). NaN3 blocks the electron transduction chain by inhibiting cytochrome c oxidase, further blocks oxidative phosphorylation of cells (Tuboly et al. 2013), but NaN3 exhibits high cytotoxicity which limits its application. Na2S2O4, also known as sodium hydrosulfite or hyposulfite, is one of the most used chemical methods to establish hypoxic models. It is a strong reductant which reduces oxygen in in aqueous solutions rapidly, it consumes dissolved oxygen in solution and produce hypoxic environment (Bright and Ellis 1992;

Lin, Tsai, and Wu 2014; Chen et al. 2007).

3. Mitochondria

3.1 Mitochondrial Structure and Function

Mitochondria are fundamental organelles in eukaryotic cells, which evolved from an ancient aerobic bacterium that took up residence inside the cytoplasm of an anaerobic host cell (the endosymbiont theory). Depending on the cell type, mitochondria have a very different overall structure, from individual, bean-shaped organelles to a highly branched, interconnected tubular network. Mitochondria are dynamic organelles capable of dramatic changes in morphology, due to mitochondrial fission and fusion. The balance 23 between fusion and fission is a major determinant of mitochondrial number, length, and degree of interconnection. Mitochondria divide using their own circular strand of DNA and the number of mitochondria per cell varies widely. In cells where there is high energy demand, large numbers of mitochondria are found. In heart muscle cells about 40% of the cytoplasmic space is taken up by mitochondria. In liver cells the figure is about 20-25% with 1000 to 2000 mitochondria per cell.

The best-known function of mitochondria is the production of ATP via oxidative phosphorylation. In addition to energy metabolism, mitochondria are involved in diverse activities, such as β-oxidative of fatty acids, the biosynthesis of substances, including certain amino acids and heme groups. The synthesis and maturation of iron-sulfur clusters, which are essential cofactors for many proteins, occur in the mitochondria (Lill and Mühlenhoff 2008). Mitochondria along with the endoplasmic reticulum also play a vital role in Ca2+ homeostasis and phospholipids biogenesis (Hayashi et al. 2009;

Kornmann et al. 2009). Besides that, mitochondria have also been shown to play important roles in aging and apoptosis (Chan 2006; Karbowski and Youle 2011). Given its fundamental roles in cellular metabolism, mitochondrial dysfunction causes a wide range of human diseases, including neurodegenerative diseases and cancer

(Mandemakers, Morais, and Strooper 2007; Patten et al. 2010; Schapira 2006).

3.2 Mitochondrial Quality Control

Mitochondria are highly dynamic organelles and their proper function is crucial for the maintenance of cellular homeostasis and function. Mitochondrial quality must be well controlled to avoid cell death. As the site of oxidative phosphorylation in mammal 24 cells and a major site for the production of reactive oxygen species, mitochondria are under risk of damages from free radicals. Although mitochondrial antioxidants scavenge and neutralize these free radicals, mitochondria are still a primary target of oxidative stress-mediated damage (Toyokuni 1999). In addition to ROS, stimuli such as inflammation, radiation, toxic chemicals and reperfusion will also damage mitochondria

(Balaban, Nemoto, and Finkel 2005).

Eukaryotes have evolved several quality control mechanisms to preserve mitochondrial homeostasis and prevent cellular damage. Mitochondria contain their own proteolytic system to monitor and degrade misfolded or unfolded proteins inside mitochondrial compartments (Baker and Haynes 2011; Matsushima and Kaguni

2012). Furthermore, the proteasome system is involved in the elimination of damaged outer mitochondrial proteins and proteins that fail to be imported (Karbowski and Youle

2011; Yoshii et al. 2011). In addition to the mitochondrial proteolytic system and the proteasome, evidence suggests that mitochondrial-derived vesicles (MDVs) engulf selected mitochondrial cargos and deliver them to lysosomes or peroxisomes for degradation (Soubannier et al. 2012). Mitochondria are dynamic organelles that constantly undergo fission and fusion to regulate the expansion and morphology of the mitochondrial network. Through fission and fusion mitochondria also repair damaged components by segregating or exchanging material (Bliek, Shen, and Kawajiri 2013).

To prevent the accumulation of damaged mitochondria, mammalian cells execute mitochondrial autophagy, or mitophagy, to achieve the controlled elimination of damaged mitochondria. Mitophagy is triggered in the presence of severely damaged or superfluous 25 mitochondria to remove selectively damaged mitochondria and maintain mitochondrial homeostasis. During mitophagy, entire mitochondria are sequestered in double- membrane vesicles, known as autophagosomes, and are delivered to lysosomes for degradation (Youle and Narendra 2011).

3.3 Molecular Mechanisms of Mitophagy

3.3.1 The PINK1/Parkin Pathway The cytosolic E3 ubiquitin ligase Parkin and the mitochondrial phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), which are associated with an autosomal recessive form of parkinsonism (Valente et al. 2004), have been found to work in the same pathway to initiate mitophagy (Greene et al. 2003). PINK1 is selectively recruited to damaged mitochondria that have lost their membrane potential and becomes stabilized on the outer mitochondrial membrane in response to mitochondrial damage and recruits Parkin (Lazarou et al. 2012; Narendra et al. 2010; Narendra et al. 2008). After

Parkin translocation and activation, Parkin mediates the ubiquitylation of several outer mitochondrial membrane proteins, which in turn recruit other proteins to mitochondria, resulting in the fragmentation and isolation of impaired mitochondria from the healthy mitochondrial pool (Gegg et al. 2010; Yoshii et al. 2011; Youle and Narendra 2011).

Subsequently, impaired mitochondria are recognized and degraded by the autophagosomes and lysosomes.

3.3.2 BNIP3- and BNIP3L/NIX-Mediated Mitophagy

Mitochondria can also be cleared via a Parkin-independent pathway, in which pathway autophagy receptor proteins on the mitochondria interact directly with microtubule-associated proteins 1A/1B light chain 3B (LC3) on the autophagosome. In 26 mammalian cells, mitochondrial NIX/BNIP3L and BNIP3 serve as autophagy receptors for the selective clearance of mitochondria (Hanna et al. 2012; Novak et al. 2010). BNIP3 and BNIP3L/NIX are BCL2-related proteins that are localized to outer mitochondrial membrane. NIX and BNIP3 target mitochondria for autophagy by directly binding to

LC3 on the autophagosome.

BNIP3L/NIX was identified as a mediator of mitochondria elimination in immature red blood cells (Novak et al. 2010; Sandoval et al. 2008). Studies of erythrocyte differentiation indicate that BNIP3L/NIX is required for the engulfment of mitochondria by autophagosomes, but not required for the induction of mitophagy

(Hanna et al. 2012; Novak et al. 2010). Studies in cardiac myocytes showed that in the process of BNIP3-regualted mitochondrial autophagy, dynamin 1-like (Drp1) protein was triggered to translocate to mitochondria resulting in mitochondrial fission, which then recruits Parkin and activates Parkin-dependent mitophagy (Lee et al. 2011).

3.4 Mitophagy in Hypoxia

In response to hypoxia, HIF1 regulates the transcription of hundreds of genes, including a series of molecular mechanisms designed to maintain energy and redox homeostasis (Manalo et al. 2005). HIF inhibits mitochondrial biogenesis and respiration, which is considered critical for metabolic adaptation to hypoxia (Manalo et al. 2005).

Hypoxia induces mitophagy, which is an adaptive metabolic response that promotes survival of cells under prolonged hypoxia condition. This process requires the HIF- dependent expression of BNIP3 and the autophagy machinery (Zhang et al. 2008). BNIP3 and BNIP3L/NIX mediates mitophagy, a metabolic adaptation to hypoxia that is able to 27 control ROS production and DNA damage and thus promotes cell survival; in contrast, severe hypoxic conditions or anoxia induces a HIF-independent autophagic response, which should be seen as the outcome of failed adaptation (Kim et al. 2006).

Hypoxia induces mitochondrial fragmentation and mitophagy, but it is unclear about the mechanisms linking mitochondrial fragmentation and mitophagy under hypoxic conditions. Studies showed that FUNDC1, an outer mitochondrial membrane protein, is involved in the mitochondrial clearance in response to hypoxic conditions (Wu et al.

2014:1). FUNDC1 acts as receptor and mediates mitophagy by interacting with autophagosomal protein LC3 (Liu et al. 2012; Wu et al. 2014). FUNDC1 was also found specifically required for mitochondrial fragmentation by recruiting DRP1 to drive mitochondrial fission in response to hypoxic stress (liu et al. 2016).

4. The Potential Interplay between Zinc and Mitophagy during Hypoxia

Emerging evidence over the past decade has shown that zinc affects mitochondria in response to ischemia (Shuttleworth and Weiss 2011). It was progressively clear that the zinc-mitochondrial interactions occur in and contribute to ischemic injury, however, it is uncertain regarding the role and activity of mitochondria that associated with ischemic relevant zinc accumulation. Pathological zinc accumulation induces swelling and release of reactive oxygen species (ROS) from mitochondria (Weiss, Sensi, and Koh 2000). Zinc overload causes loss of mitochondrial membrane potential and reduced cellular ATP levels. Excessive zinc is taken up by mitochondria and induces mitochondrial permeability transition (Dineley, Votyakova, and Reynolds 2003). Studies also showed that endogenous zinc contributes to mitochondrial dysfunction. Zinc chelation by 28

CaEDTA blocked mitochondrial release of cytochrome c and downstream caspase 3 activation (Calderone et al. 2004). Endogenous zinc accumulates rapidly in neurons during brain slice oxygen and glucose deprivation (OGD), and contributes to the irrecoverable mitochondrial dysfunction by loss of the mitochondrial membrane potential

(Medvedeva et al. 2009).

Study from our group showed that NADPH oxidase may mediate zinc-induced

ROS accumulation, which suggested a mechanism of crosstalk between zinc and mitochondrial ROS through positive feedback processes that eventually causes excessive free zinc and ROS accumulation during the course of ischemic-stress (Slepchenko, Lu, and Li 2017). As ROS is the major cause of mitochondrial injury, an interesting question is proposed to investigate possible interplays between zinc and mitophagy in ischemic hypoxia.

SPECIFIC AIMS

Overall hypothesis: Intracellular zinc is sequestered within multiple types of intracellular organelles, majorly within mitochondria. The cytosol zinc level is much lower than organellar zinc. During hypoxia/stroke, there is a rapid elevation of zinc level in the cytosol, which contributes to ROS generation. The mitochondria damaged by ROS are cleared by mitophagy. On the other hand, the damaged mitochondria releases zinc and aggravates the toxicity induced by zinc overload. Zinc removal by zinc specific chelator could alleviate the damage to mitochondria and the activity of mitophagy.

Furthermore, I expect to observe the reduction of mitophagy activity by zinc chelation during hypoxia. I hypothesize that there is an interplay between zinc and mitophagy during hypoxia. The existence of the zinc-mitophagy interplay during hypoxia will be revealed, and the pathway will be investigated by large-scale screening and fine-scale validation.

Overall objective: To investigate the intracellular distribution of zinc and confirm the zinc-sequestering organelles in multiple types of cells. To evaluate the effect of zinc in hypoxia-induced mitophagy by investigating that changes in mitophagy activity through zinc level modulation. To determine the interplay of zinc and mitophagy during hypoxia. To explain the interplay of zinc and mitophagy during hypoxia by screening and validating the genes involved in the process.

Specific Aim 1: To Investigate the Intracellular Distribution of Free Zinc

Objective: Zinc is fundamental for numerous cellular functions. The homeostasis and distribution of intercellular zinc is strictly regulated. However, the exact distribution 30 of free zinc within live cells remains elusive. The objective of specific aim 1 is to evaluate the intercellular distribution of free zinc.

Hypothesis: Cellular organelles, mainly mitochondria, but also include ER and the

Golgi apparatus, are the major storage sites for intracellular free zinc. The organelles sequester zinc and maintains zinc homeostasis under physiological conditions. Also, the level of the intracellular zinc in cytosol or nucleus are so low and not observable.

Specific Aim 2: To Investigate the Effect of Zinc on Mitophagy during Hypoxia by

Modulating the Zinc Levels

Objective: Study from our lab has shown that zinc accumulate under hypoxia condition, and the accumulation of zinc plays important roles in cellular dysfunction and death associated with hypoxia (Slepchenko, Lu, and Li 2016; Stork and Li 2006; Stork and Li 2009). Moreover, we determined the role of zinc in initial ROS accumulation during hypoxia and showed that zinc accumulation triggers mitochondrial ROS production during hypoxia (Slepchenko, Lu, and Li 2016). Further study from us

(Slepchenko, Lu, and Li 2017) showed that damaging mitochondria induced a significant elevation in intracellular zinc level. The objective of specific aim 2 is to evaluate the effect of zinc in hypoxia-induced mitophagy by investigating that changes in mitophagy activity through modulating intracellular zinc levels.

Hypothesis: Zinc influx by exogenous zinc administration will damage mitochondria and induce mitophagy. Zinc influx induced mitophagy can be alleviated by applying zinc chelator. Hypoxia-induced mitophagy can be alleviated by zinc chelation. 31

Specific Aim 3: To Investigate How Zinc Interplays with Mitophagy during Hypoxia by

Screening and Validating the Candidate Genes involved in the Signaling Pathways

Objective: To investigate the genes involved in zinc-mitophagy interplay during hypoxia by large-scale screening and fine-scale validation. To perform large-scale screening by leveraging the public achieved high-throughput gene expression data in

National Center for Biotechnology Information (NCBI) Gene expression omnibus (GEO) database. To analyze, evaluate the data and generate a list of candidate genes involved in zinc-mitophagy interplay during hypoxia. To validate the shortlisted genes by quantitative real-time PCR. To explain the zinc-mitophagy crosstalk during hypoxia by stating the changes and possible roles of the genes involved in the process.

Hypothesis: The genes involved in the zinc-mitophagy crosstalk during hypoxia may function in the following aspect: oxidative stress defense system, mitochondrial fission and fragmentation, mitophagy induction and development.

MATERIALS AND METHODS

1.Cell Cultures

HeLa cell culture: HeLa cells were cultured in EMEM (Gibco) supplemented with 4% FBS at 37°C in a 5% CO2, 95% air. Cells were cultured on a petri dish with a glass bottom of 35 mm diameter (MatTek Corporation, Ashland, MA, USA). Freshly cultured cells with confluency of 70%-80% were used for live-cell imaging.

HIT-T15 cell culture: pancreatic β-cell line HIT-T15 were purchased from ATCC

(Manassas, VA, USA). HIT-T15 cells were maintained in RPMI 1640 (Gibco, Grand

Island, NY, USA) supplemented with 10% fetal bovine serum and 5% dialyzed horse serum at 37°C in a 5% CO2, 95% air. Cells were cultured on a petri dish with a glass bottom of 35 mm diameter (MatTek Corporation, Ashland, MA, USA). Freshly cultured cells with confluency of 70%-80% were used for live-cell imaging.

Primary cortical neuron cells: Seven-day-old rat primary cortical neuron cells were gift from Dr. Robert Colvin (Ohio University). Cells were cultured on a petri dish with a glass bottom of 35 mm diameter (MatTek Corporation, Ashland, MA, USA).

2. Zinc Labeling

To detect intracellular labile zinc, I used Zinpyr-1 (Sigma-Aldrich, St. Louis, MO,

USA), a selective fluorescent sensor for zinc that is membrane permeable (Walkup et al.

2000). Fluorescent probes for zinc typically consist of a zinc-chelating unit and a fluorescent reporter. Zinpyr-1 has the di-2-picolylamine (DPA) ligand as the zinc- chelating unit. DPA ligand has no measurable affinity for Ca2+ or Mg2+. Zinpyr-1 easily permeates through the membrane making it an ideal candidate to study Zn2+ in live cells. 33

Zinpyr-1 has zinc sensitivity in nM range (significantly lower than binding affinity of most proteins to zinc) and its fluorescence increases three fold upon zinc binding; the Kd for the Zinpyr-1Zn2+ complex is 1 nM (Walkup et al. 2000). Cells were incubated with

2.5-10 μm Zinpyr-1 for 10-30 min at 37°C in 5% CO2. After incubation, cells were washed three times with Hank’s Balanced Salt Solution (HBSS, Life Technologies,

Grand Island, NY, USA), and then visualized by fluorescence microscopy.

3. Organelle and Zinc Co-labeling

Mitochondria-zinc co-labeling: Labeling of mitochondria was performed by incubating cells with MitoFluor Red 589 (Molecular Probes, Eugene, OR, USA). HeLa cells and 7-day-old primary cortical neurons were incubated with 1 ml of dye solution

(250 nM of MitoFluor Red 589 and 10 μM of Zinpyr-1 dissolved in HBSS) at 37°C, 5%

CO2 for 20 min. After incubation, the cells were washed three times with HBSS.

ER-zinc co-labeling: I used ER Tracker Red (Molecular Probes, Eugene, OR,

USA) to label the ER in HeLa cells. HeLa cells were incubated with 1 ml of dye solution

(1 μM of ER Tracker Red and 10 μM of Zinpyr-1 dissolved in HBSS) at 37°C, 5%

CO2 for 20 min. After incubation, the cells were washed three times with HBSS.

Golgi-zinc co-labeling: BODIPY TR Ceramide (Molecular Probes, Eugene, OR,

USA) was used to visualize the Golgi apparatus. Freshly cultured HeLa cells and HIT-

T15 cells were incubated with 1.5 μM BODIPY TR Ceramide at 4°C for 30 min. Cells were then washed with ice-old HBSS and incubated with 2.5 μM Zinpyr-1 for further 20 min at 37°C. Cells were washed three times before imaging. 34

Nucleus-zinc co-labeling: Labeling of the nucleus was performed with SYTO Red

64 (Molecular Probes, Eugene, OR, USA). HeLa cells were first incubated with 10 μM

Zinpyr-1 for 10 min and then with 2.5 μM SYTO Red 64 for 10 min at 37°C, 5% CO2.

The cells were washed and imaged in HBSS.

4. Fluorescence Imaging

Fluorescence microscopy: Fluorescent signals were detected using a customized fluorescence microscope, Zeiss LSM 510 confocal microscope and Nikon A1R confocal microscope. For the customized fluorescence microscope, all the images were captured using ImagePro Plus software (Media Cybernetics, Silver Spring, MD, USA) at exposures of about 1 s (For excitation: blue filter: 470/70 nm, green filter: 517/30 nm; for emission: green filter-517/30 nm, red filter-620/40 nm; Filters were purchased from

Chroma). ImagePro plugins were used to enhance contrast, adjust the brightness and improve the resolution of the images. Regions of interest were marked and cropped using

ImageJ software. Fluorescence intensities were calculated using ImageJ. Co-localization was measured with ImagePro as Pearson’s correlation value. 3D images were collected using Zeiss LSM 510 confocal microscope (Oberkochen, Germany) with 100X magnification, and analyzed with Zeiss imaging software. Co-localization of mitochondria and lysosomes were observed using Nikon A1R confocal microscope and analyzed with Nikon imaging software.

5. in vitro Treatment to Induce or Alleviate Stresses

Chemical hypoxia: Chemical hypoxia was induced using 4 mM final concentration of sodium dithionite (DT) in oxygen and glucose deprived (OGD) HEPES 35 buffer. To achieve OGD, nitrogen gas was bubbled through the physiological buffer for at least 10 min before the experiment. This OGD and 4 mM sodium dithionite buffer was added by pipetting as 2x concentrated solution into the petri dish holding the cells to induce rapid and reliable hypoxic condition.

FCCP: Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) is uncoupler which damages mitochondria. It was administered in the live cells at a concentration of 4 µM as indicated.

Exogenous zinc: Zinc influx was induced by pipetting 2x concentrated solution of

HEPES buffer with final concentrations of 50 µM ZnCl2 and 10 µM Na-pyrithione, a zinc ionophore, which allows zinc ions to enter the cells.

TPEN: N,N,N’,N-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) is a membrane permeable zinc chelator, it was administered in the live cells at a concentration of 33 µM as indicated.

6. Qualitative and Quantitative Analysis of Mitochondrial Morphology

Mitochondrial staining: Freshly cultured HeLa cells at a confluency of 70% were stained by MitoTracker Red, the mitochondria specific fluorescent indicator. HeLa cells are flat and lie on the monolayer in the cell dish and have a visible mitochondrial network with mitochondrial staining. This allows for visualization and evaluation of the mitochondria inside of the living cells.

Mitochondrial morphology observation: The HeLa cells stained by MitoTracker

Red were observed with the customized fluorescence microscope. Images were randomly captured. To measure the mitochondria, the process of the cell was evaluated for 36 mitochondrial morphology and the nucleus and perinuclear regions were avoided, the reasons are as following. First, organelles such as Golgi apparatus and ER were around the nucleus; second, the cluster of mitochondria near the nucleus would make it difficult to distinguish the true morphology of the mitochondria; finally, it is easier to establish the mitochondrial network in the processes of the cell.

Mitochondrial morphology analysis: To analyze these images, an ImageJ macro called Mito-Morphology was used (Dagda et al. 2009). First, a line was drawn around the perimeter of the area of cell to be measure. Then, this part of the cell was transferred to a blank image with a black background. This did not cause changes in the cellular and mitochondrial parameters. The same threshold was used and applied to cover all the mitochondria in the selected part of the cell. Finally, the mitochondria of the selected part of the cell were measured using the Mito-morphology macro. The mitochondrial circularity, mitochondrial roundness, major axis, and aspect ratio were determined by

Mito-morphology macro.

Mitochondrial circularity: The circularity was determined by Mito-Morphology.

The circularity value represents how closely the measured mitochondria represents a circle (Wiemerslage and Lee 2016). The value of circularity ranges from 0.0 to 1.0. A circularity of 1.0 indicates that the mitochondria is a perfect circle.

Circularity = 4π (mean area / mean perimeter2)

Mitochondrial roundness: the mitochondrial roundness shows how much the mitochondria represents a perfect circle compared to an elongated or uneven shape. The 37 value of 1.0 shows that the mitochondria is a perfectly round circle. The value approaching 0.0 shows a more uneven shape.

Roundness = (4 * area) / (π * major axis2)

Mitochondrial major axis: the major axis is the length of the mitochondria.

Aspect ratio: the aspect ratio is the ratio of major axis/minor axis (Mortiboys et al.

2008).

Aspect Ratio = major axis / minor axis

Statistical analysis: These parameters were statistically analyzed. The significance between non-treated and treated cells were analyzed using Student’s unpaired t-test with Welch’s correlation. The significance between different biological treatments was measured with single-factor ANOVA. P-values under 0.05 were considered significant. The software used was Prism.

7. GEO Analysis for Large-scale Screening

Gene Expression Omnibus (GEO) database: GEO database is an international public repository that archives and freely distributes high-throughput gene expression and other functional genomics datasets, it is supported and launched on the National Center for Biotechnology Information (NCBI) (Clough and Barrett 2016).

GEO record retrieval: To find the most relevant data, the structured and filtered queries were used with GEO DataSets on https://www.ncbi.nlm.nih.gov/geo/ to identify data of interest. Table 1 listed the three GEO records that are most relevant to my specific aims. The three GEO records are studies that employed in HeLa cells to investigate the gene expression profiles under hypoxic condition (GSE79069 (Bourseau-Guilmain et al. 38

2016)), zinc addition (GSE49657 (Homma et al. 2013)), or FCCP (GSE84631 (Quirós et al. 2017)). Each GEO record has information profile to describe the study including title, study summary, organism, citation, and platform and series accession numbers upon which the DataSet is based.

Table 1. GEO records retrieved from NCBI GEO database

GEO record analysis: The initial GEO record analysis is based on curated

DataSet records, which provide both visualization and data analysis tools for normalized, array-based gene expression studies stored in GEO. Customizable data analysis tools are used to assist with identification of genes of interest within that DataSet.

GEO analysis with GEO2R: GEO2R is a web-based and user-driven analysis tool for GEO data. GEO2R is accessed by clicking the text “Analyze with GEO2R” on the

Series record, then on the GEO2R page with a table of all Samples in the Series complete with Sample accession numbers, cell types and other attributes. The analysis was performed to obtain the top differentially expressed genes cut-off at P-value <0.01. The expression pattern of each gene can be visualized to depict expression profile graphs. The complete set of ordered results is retrieved (Clough and Barrett 2016).

Mitochondrial or zinc related genes: The gene ontology (GO) resource

(http://geneontology.org/) was used to identify the mitochondrial related genes. The GO 39 knowledgebase is the source of information on the functions of genes, it provides the foundation for large-scale gene analysis. Through the filtered search, 1286 mitochondrial related genes in Homo Sapiens were identified. Through the filtered search on the GO resource, total 1719 zinc related genes were identified in Homo Sapiens.

8. Quantitative Real-time PCR

Treatment to cells: Freshly-cultured HeLa cells were treated with 4 mM final concentration of sodium dithionite (DT) in oxygen and glucose deprived HEPES buffer for 2 hours to induce chemical hypoxia, the treated HeLa cells were then harvested for

RNA extraction. Freshly-cultured HeLa cells were treated with FCCP for 2 hours to uncouple mitochondria, the treated HeLa cells were then harvested for RNA extraction.

Freshly-cultured HeLa cells were treated with 50 µM ZnCl2 and 10 µM Na-pyrithione for

2 hours to induce a zinc influx, the treated HeLa cells were then harvested for RNA extraction.

Control group: The chemicals for treatment were prepared in HEPES buffer, for this reason, the control group was done by administration freshly cultured cells with the

HEPES solution only.

RNA extraction: Total RNA was extracted using the NucleoSpin RNA (Clontech

Laboratories, Inc., Mountain View, CA, USA) protocol. The concentration and integrity of total RNA was determined with a ND-1000 Spectrophotometer (NanoDrop

Technologies, Inc., Wilmington, DE, USA). 40

cDNA synthesis: cDNA was synthesized from 100 ng RNA using the High-

Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific Inc., Waltham, MA

USA).

quantitative PCR: Each qPCR reaction contained 5 uL SYBR Green PCR Mix

(Life Technologies, Waltham, MA USA), 0.5 uL of 10 µM forward and reserve primers and 1 ul diluted cDNA. The total volume was made up to 10 ul with nuclease free water.

The CT values were determined using the BioRad CFX Connect Real-Time System (Bio- rad., Hercules, CA USA) with the following thermocycler: initial denaturation 95oC for 2 min, followed by 40 PCR cycles, each cycle consisting of 95 oC for 5s, 60 oC for 30s, and

95 oC for 2 min, and SYBR green fluorescence emissions were monitored after each cycle.

Data analysis: The fold changes in gene expression were calculated by applying

-ΔΔCT the 2 method where ΔΔCT = ΔCTsample − ΔCTcontrol. Statistical analysis was performed on three independent biological replicates, each consisting of three technical repeat measurements. Housekeeping gene RPS18 was used for normalization purposes.

Table 2. List of forward and reverse primers for genes tested by qPCR

RESULTS

1. The Intracellular Distribution of Free Zinc in Cells

1.1 Staining Pattern of Zinpry-1 in HeLa Cells is Concentration Dependent

To visualize intracellular zinc ions, I used Zinpyr-1, a fluorescein-based sensor for divalent zinc that is cell permeable. Zinpyr-1 displays a highly zinc-selective fluorescence response. (Walkup et al. 2000) The commonly used cell line, HeLa cell, is chosen for live cell imaging and zinc observation. HeLa cells are adherent cells that firmly attached to the glass-bottom cell plates and spread thinly into a single layer with a diameter of 40 µM but only a few micrometers in height. Moreover, HeLa cells have been used to study zinc response for decades. However, there is lack of comprehensive study to show the distribution of intracellular zinc in HeLa cells.

First I investigated the optimal staining conditions of Zinpyr-1 in HeLa cells. It was stated that the fluorescence of the metal-free ligand changes with pH. A consistent physiological ionic strength and pH was used for all the following experiments. (zinpyr-1 staining buffer) The HeLa cells were stained with different concentrations of Zinpyr-1 for

30min at 37oC.

The range of Zinpyr-1 concentrations used for HeLa cell staining was from 0.5

µM to 10 µM. HeLa cells incubated with different concentrations of Zinpyr-1 were showing different staining patterns. As we can see in Figure 1, incubation of HeLa cells with 0.5 µM Zinpyr-1 for 30min and subsequent fluorescence microscopic imaging indicated that Zinpyr-1 stained HeLa cells had bright perinuclear punctate pattern with minimal staining of the remaining cell soma. Similar staining pattern was observed with 1 43

µM, 1.5 µM and 2 µM Zinpyr-1 incubation, respectively. When HeLa cells incubated with 2.5 µM Zinpyr-1, in addition to the bright perinuclear punctate pattern, the cellular compartments were stained as well. With the increase of zinpyr-1 concentration, more and more cellular compartments were showing up. When the concentration of Zinpyr-1 used to dye HeLa cell was more than 10 µM, the staining pattern did not change in my observations.

Figure 1. The staining pattern of Zinpyr-1 in HeLa cells is concentration dependent. Freshly cultured HeLa cells were stained with different concentrations of Zinpyr-1 for 30 min. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. Scale bars indicate 10 µm.

1.2 Zinpyr-1 Staining is Specific to Zinc

When Zinpyr-1 was invented by Dr. Tsien (Walkup et al. 2000), it was stated by the author that Zinpyr-1 displays a highly Zinc selective fluorescence response, only Zn2+ and Cd2+ have been observed to enhance Zinpyr-1 fluorescence, and other transition 44 metal ions can quench fluorescence (Walkup et al. 2000). Here I tested the specificity of

Zinpyr-1 to Zn2+ by testing the changes in fluorescence upon exogenous zinc addition or zinc chelation.

Figure 2. Zinpyr-1 is specific to zinc. The bright perinuclear punctate staining increases upon the addition of the zinc ionophore Zn2+/pyrithione and can be reversed by treatment with TPEN, indicating the fluorescence is zinc-induced. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. Scale bars indicate 10 µm.

Fresh HeLa cells stained with 5 µM Zinpyr-1 was shown in Figure 2. Exogenous zinc can be absorbed by cells through zinc transporters which locate on the cell membrane. Furthermore, the solubility and uptake of zinc was facilitated by ionophore.

Here I prepared ZnCl2 and Ionophore solution mixture right before the experiment. A final concentration of 10 µM ZnCl2 and 10 µM Ionophore was applied to the pre-stained 45

HeLa cells. The fluorescence signal was observed and recorded, as shown in Figure 2, here was a significant increase in the level of fluorescence upon exogenous zinc addition.

Furthermore, a zinc remover, N,N,N′,N′-tetrakis (2-pyridinylmethyl)-1,2- ethanediamine (TPEN) was applied to the cells. TPEN is a membrane permeable zinc

2+ chelator that would result in zinc deficiency. TPEN has a high affinity to Zn , with Kd =

0.26 fM. Upon TPEN administration, the fluorescent signal was quickly quenched.

Residual fluorescence after TPEN addition remained in the compacted cellular compartments, a possible reason is that TPEN did not get into the matrix of this cellular compartments yet, so the free zinc was preserved in this site. Other than that, most of the intracellular zinc was chelated. The changes in zinpyr-1 fluorescent signals resulted from exogenous zinc addition and zinc removal indicate that Zinpyr-1 is a specific indicator to zinc.

1.3 Overview of Distribution of Intracellular Zinc in HeLa Cells

As shown in Figure 3, after staining with Zinpyr-1, a selective fluorescence zinc indicator, HeLa cell showed an uneven distribution of zinc-dependent fluorescence throughout the entire cell, with high amounts of free zinc detected in what looked like cellular compartments that encircle the nuclear. The staining also proceeded to cellular processes. On the other hand, cytosol and nucleus had nearly no detectable zinc fluorescence. Next, to further study zinc distribution, I stained the cells with organelle specific fluorescence indicators to determine whether free zinc was present in the membrane bound organelles, which were evidenced by the co-localization of fluorescence zinc signals and organelle specific fluorescence signals. 46

Figure 3. Cellular zinc distribution in HeLa cells shows a compartmentalized pattern. Images of HeLa cells labeled with fluorescent zinc indicator Zinpyr-1. 2D images were captured using a customized fluorescence microscope equipped with a 100X/1.1NA objective lens. 3D images were captured using a Zeiss LSM510 confocal microscope equipped with a 100X/1.3NA objective lens and analyzed using Zeiss imaging software. Scale bars indicate 10 µm.

1.4 Co-localization of Zinc and Mitochondria in HeLa Cells

First, I assessed whether mitochondria were one of the storage sites for intracellular free zinc. HeLa cells were co-stained with Zinpyr-1 and MitoFluor Red 589 for 20 min. Mitochondria in HeLa cells resemble squiggly lines occupying the major portion of cytosol. The red fluorescence from MitoFluor Red 589 highly co-localized with the green fluorescence from Zinpyr-1. This suggests that mitochondria are involved in the storage of intracellular free zinc (Figure 4). HeLa cells contain more than thousands of mitochondria which, in live imaging, tend to appear as mitochondrial clustering. Therefore, while HeLa cells can provide an overall picture for the co- 47 localization of mitochondria and zinc, they cannot be used to visualize individual mitochondria.

Figure 4. Zinc is localized to mitochondria in HeLa cells. HeLa cells were dual-stained with 15 µM Zinpyr-1 and 250 nM MitoFluor Red 589. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. Scale bars indicate 10 µm.

1.5 Co-localization of Zinc and Mitochondria in HIT-T15 Cells

In the body, the highest amount of total zinc content is stored in the mammalian pancreas, which can reach millimolar levels in the interior of the dense-core granule. In the mammalian pancreas, zinc plays important roles in insulin crystallization, two zinc ions coordinate six insulin monomers to form the hexametric-structure on which maturated insulin crystals are based (Slepchenko, James, and Li 2013). In addition to be a structural component, zinc functions in the accurate processing, storage, secretion and action of insulin in beta cells. Considering the importance of zinc structurally and 48 functionally, and especially the abundance of zinc in this type of cells, I evaluated the zinc distribution in pancreatic beta cells, HIT-T15.

For live-cell imaging, HIT-T15 is more challenging than HeLa cell, because HIT-

T15 cells are only one-tenth the size of HeLa cells. I freshly cultured the pancreatic HIT-

T15 cells and stained them with 5 µM Zinpyr-1 and 500 nM MitoFlour Red 589 simultaneously. The results were shown in Figure 5. In the left image of Figure 5, Zinpyr-

1 staining showed that the zinc was compartmentalized into organelles, some of the organelles were mitochondria, as proved by co-staining with Mitochondria indicators in the right panel of Figure 5. HIT-T15 cells also showed much higher zinc signal outside the mitochondria, this may suggest that other cellular compartments are storing zinc as well.

Figure 5. Co-localization of zinc and mitochondria in HIT-T15 cells. Freshly cultured HIT-T15 cells were dual-stained with 5 µM Zinpyr-1 and 500nM MitoFluor Red 589 for 25 min. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. Scale bars indicate 10 µm.

1.6 Mitochondria Observation in Primary Cortical Neurons

For both HeLa cells and pancreatic HIT-T15 cells, hundreds of mitochondria cluster together, therefore it is not feasible to observe individual mitochondrion using these two cell lines. To visualize single mitochondrion, I utilized primary seven-day-old cortical neurons from rat. Neuronal axons are typically 1µM in diameter but can be as long as 60 µM. In neuronal cells, mitochondria arise mainly in the neuronal cell body, but they can be delivered to and retrieved from the axon, and mitochondria retain their identity as discrete organelles through a long transit (Santel and Fuller 2001). Therefore, it is expected to observe discrete mitochondria in the axons.

Figure 6. Mitochondria observation in primary cortical neurons. 50

Seven-day-old rat primary cortical neurons were stained with 250nM MitoFluor Red 589 for 30 minutes. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. The cells were observed and recorded under 40X and 100X objective lens, respectively. Scale bars indicate 10 µm.

The primary cortical neurons were stained with the mitochondrial indicator,

MitoFluor Red 589, to visualize mitochondria. Cells were observed and recorded, as shown in Figure 6, the fluorescence signals emitted from mitochondria. The highest fluorescence signals were observed from the soma, indicating that the high density of mitochondria in the cortical neuronal cell body. In addition to cell body, fluorescence signals were also detected in axons and collaterals.

1.7 Co-localization of Zinc and Mitochondria in Primary Cortical Neurons

After a successful observation of discrete mitochondria in primary cortical neurons, I further evaluated the co-localization of zinc and mitochondria in this type of cells by co-staining them with Zinpyr-1 and MitoFluor Red 589, the results were shown in Figure 7. Highest fluorescence signals from both dyes were observed in neuronal cell body, in contrast, the fluorescence intensity was relatively low in the axons. In order to have a comprehensive observation of the whole cortical neurons, the stained cells had to be exposed for longer time to capture the fluorescence signal from axons, this was why there was over-saturation of fluorescence signal in soma under 40X objective lens (top images in Figure 7). Discrete mitochondria in axon were observed under 100X objective lens, as indicated by the arrows in the bottom images of Figure 7. 51

Figure 7. Zinc is localized to mitochondria in cortical neurons. Rat primary cortical neurons were dual-stained with 10 µM Zinpyr-1 and 250 nM MitoFluor Red 589. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. The cells were observed and recorded under 40X and 100X objective lens, respectively. Scale bars indicate 10 µm.

1.8 Co-localization of Zinc and Mitochondria in Individual Mitochondria

To observe the discrete mitochondria, dual-stained primary cortical neurons were observed under 100X objective lens, with the aim to locate individual mitochondria in the axons, and evaluate the distribution and localization of zinc and its relation to mitochondria. As we can see in Figure 8, single axons were imaged under higher magnification. Red fluorescence signal was emitted from mitochondrial indicator, it showed what mitochondria aligned linearly in the axons. These mitochondria differed in size, some resembled squiggly lines, while others were dot-like, reflects the morphological heterogeneity of mitochondria, which is a phenomenon comes from the constant division, fusion and elongation of mitochondria. Interestingly, the green 52 fluorescence signals come from the zinc indicator was highly overlapped with the red fluorescence signals emitted from the mitochondria indicator, which indicated the co- localization of zinc and mitochondria.

Figure 8. The observation of zinc and mitochondria co-localization in single mitochondrion. Rat primary cortical neurons were dual-stained with 10 µM Zinpyr-1 and 250 nM MitoFluor Red 589. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. The cells were observed and recorded under 100X objective lens, respectively. Scale bars indicate 10 µm.

1.9 Co-localization of Zinc and ER in HeLa Cells

To determine whether ER was one of the storage sites for zinc in HeLa cells, fresh cells were dual-labeled with Zinpyr-1 and ER tracker Red, an ER specific fluorescent probe. Images captured by fluorescence microscopy showed significant colocalization of zinc indicator and the ER Tracker Red, indicating that free zinc were located within the 53 lumen of the ER (Figure 9). These results were consistent with our previous study in which I showed that zinc was released from thapsigargin-sensitive and IP3R-mediated stores of the ER (Stork and Li 2009).

Figure 9. Co-localization of zinc and ER in HeLa cells. Rat primary cortical neurons were dual-stained with 10 µM Zinpyr-1 and 250 nM MitoFluor Red 589. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. The cells were observed and recorded under 100X objective lens, respectively. Scale bars indicate 10 µm.

1.10 Co-localization of Zinc and ER in Pancreatic Beta Cells HIT-T15

Considering the critical role of zinc in insulin biosynthesis and storage in pancreatic beta-cells, here I evaluated whether ER could be a possible zinc storage site in pancreatic beta cells HIT-T15. I stained the freshly cultured HIT-T15 cells with the specific fluorescent indicators for ER (red fluorescence signal) and zinpyr-1 (green fluorescence signal), the co-localization analysis showed that the overlap of the two 54 fluorescence signals is significant, it suggests that endoplasmic reticulum serves as one of the storage sites of intracellular zinc in pancreatic HIT-T15 cells.

Figure 10. Zinc is localized to the ER in HIT T15 cells. Images of HIT T15 cells co-labeled with Zinpyr-1 and a live cell marker for the Endoplasmic Reticulum, ER Tracker Red. HIT T15 cells were stained with 2.5 µM ER Tracker Red and 2.5 mM Zinpyr-1 for 30 min at 37°C. Two representative cells were showed. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. Scale bars indicate 10 µm.

1.11 Co-localization of Zinc and Golgi in HeLa Cells and HIT-T15 Cells

The Golgi apparatus is usually located near the nucleus. Here, I probed the Golgi apparatus using BODIPY TR ceramide. In HeLa cells, dual-staining with Zinpyr-1 and

BODIPY TR ceramide showed that the signals from the Golgi dye co-localized with the perinuclear fluorescence of zinc dye, indicating that free zinc is localized to the Golgi apparatus, as shown in Figure 11A. As the main function of the Golgi apparatus is to process and package macromolecules such as proteins and lipids after their synthesis, it is 55 especially prominent in cells involved in secretion, such as pancreatic cells. I used the pancreatic ß cell line, HIT-T15 to evaluate further the presence of zinc in the Golgi apparatus as a case of positive control (Figure 11B). The Golgi apparatus in pancreatic β- cells is one of the zinc sources, because the insulin packaging and secretion requires a high amount of free zinc. Here the HIT-T15 cells were double stained with BODIPY TR ceramide and Zinpyr-1. Bright Zinpyr-1 fluorescence emanating from the perinuclear zones, indicating the presence of high concentration of free zinc in the regions. BODIPY

TR ceramide stained the same perinuclear zones, which proved that the co-localization of zinc with the Golgi apparatus.

Figure 11. Zinc is localized to the Golgi apparatus in HeLa cells and HIT T15 cells. Images of HeLa cells or HIT T15 cells co-labeled with Zinpyr-1 and a live cell marker for the Golgi apparatus. A. Zinpyr-1 co-localizes with BODIPY TR ceramide, the marker for 56 the Golgi apparatus, in HeLa cells. B. Zinpyr-1 co-localizes with BODIPY TR ceramide in pancreatic ß cells HIT T15. HeLa cells or HIT T15 cells were stained with 5 mM TR ceramide for 30 min at 4°C, then stained with 2.5 mM Zinpyr-1 for 30 min at 37°C. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. Scale bars indicate 10 µm.

1.12 Zinc was not Detectable in the Nucleus of Viable HeLa cells

Syto Red 64 was used to identify nucleus in HeLa cells because it reliably labeled nucleus in live cells. As shown in Figure 12, Syto Red 64 labeled HeLa cell nuclei with red fluorescence. The same cells were also stained with Zinpyr-1 but zinc fluorescence was not observed in the nucleus of HeLa cells. Nucleus is known for its packaging of many zinc binding proteins or zinc binding motif for hundreds transcription factors.

However, it didn’t surprise that zinc was not detectable within the nucleus using a fluorescence indicator. This was probably because most of the nuclear zinc was bound to nuclear proteins in a healthy cell and the concentration of free chelatable zinc was negligible.

Figure 12. Absence of zinc staining in the nucleus. Images of HeLa co-labeled with Zinpyr-1 and Syto Red 64, a live cell marker for the nucleus. HeLa cells were stained with 10 mM Zinpyr-1 for 10 min at 37°C, and then stained with 2.5 mM SYTO Red 64 for 10 min at 37°C. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. Scale bars indicate 10 µm. 58

2. To Investigate the Effect of Zinc on Mitophagy during Hypoxia by Modulating the

Zinc Levels

2.1 Mitophagy Observation

Damaged or excessive mitochondria are cleared by a process called mitochondrial autophagy, or mitophagy. During mitophagy, the targeted mitochondria are engulfed by autophagosomes and delivered to lysosomes. The fusion of a autophagosome and a lysosome will generate an autolysosome. Therefore, a milestone of mitophagy is the existence of autolysosome (Zuo et al. 2014). Autolysosome can be identified by co- localization of mitochondria and lysosome. Here, LysoTracker Green and MitoTracker

Red were used to label lysosome and mitochondria in freshly cultured HeLa cells, respectively. As shown in Figure 13 top panel, the co-localization of green fluorescence and red fluorescence was not observable, which states that, at physiological condition, the level of mitophagy was low and barely detectable by live-cell imaging.

To induce mitophagy, I treated HeLa cells with a mitochondrial uncoupler,

Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). FCCP disrupts ATP synthesis by transporting protons across the mitochondrial inner membrane, interfering with the proton gradient. Therefore, the mitochondrial membrane potential is compromised, which makes the mitochondria dysfunctional, then triggers mitophagy.

Freshly cultured HeLa cells were co-labeled with lysosome indicator and mitochondria indicator. Then, the stained cells were treated by 5 µM FCCP. Upon treatment, the cells were observed and recorded, as shown in Figure 13 bottom panel.

Comparing with the control group, FCCP treatment did not affect the overall appearance 59 of the green fluorescence in HeLa cells. It indicates that the lysosome has a relative consistent level and morphology with or without FCCP treatment. However, a dramatic change was observed in the red fluorescence signal with FCCP administration. As the red fluorescence signal was from MitoTracker Red, the mitochondria indicator, it proved that the mitochondria were heavily affected by FCCP. Morphologically, the most apparent change was the increase of fragmented mitochondria. Furthermore, the merged image showed that the fragmented mitochondria were highly overlapped with lysosomes. The co-localization of mitochondria and lysosomes observed upon FCCP administration, was a direct proof of mitophagy. The meaning of this preliminary result is at two folds, first, it showed the different mitophagy levels at physiological condition and FCCP-treated HeLa cells, second, it proved the feasibility and reliability of assessing mitophagy by co- localization of lysosomes and mitochondria.

Figure 13. Mitophagy observation. Freshly cultured HeLa cells were pre-stained with lysosome indicator, LysoTracker Green, and mitochondria indicator, MitoTracker Red. Then the cells were treated with either control solution or FCCP. Upon treatment, the cells were observed and recorded by Nikon A1R Confocal Microscope and analyzed using Nikon imaging software. 60

2.2 Hypoxia Induced Mitophagy Can be Alleviated by Zinc Chelator TPEN

Zinc is an important element in physiology which plays critical roles functionally and structurally. Therefore, intracellular level of zinc must be strictly controlled and monitored. Intracellular zinc accumulation is cytotoxic and reported to be a great causal factor for the development of neuronal death after hypoxic injury (Stork and Li 2009).

The cytotoxicity can be reduced by removing zinc with a chelator (Frederickson, Koh, and Bush 2005).

The changes of intracellular zinc level during hypoxia was examined. A published study from us showed that hypoxia exposure induced a rapid zinc transient (Slepchenko,

Lu, and Li 2017). Here, I further investigated the roles of zinc during hypoxia.

To induce a hypoxia-like condition in live cells, sodium dithionite was used.

Sodium dithionite, also named sodium hydrosulfite, was an oxygen scavenger and reducing agent. This method of “chemical” hypoxia has been widely used to rapidly induce a low-oxygen environment with reliability.

To evaluate mitophagy under hypoxia condition in HeLa cells, first, freshly cultured HeLa cells were pre-stained with lysosome indicator and mitochondria indicator; next, hypoxia condition was established in the pre-treated HeLa cells with hypoxic buffer by using 4mM final concentration of sodium dithionite solution.

As shown see in Figure 14, upon hypoxia treatment, the morphology of mitochondria changed, showing the appearance of mitochondrial fragments. The merged image of green fluorescence and red fluorescence in the hypoxia group, showing some

Figure 14. Hypoxia induced mitophagy was alleviated by zinc chelation. HeLa cells were co-stained with LysoTracker Green (1uM) and MitoTracker Red (100nM) for 30min at 37oC. The overlap of mitochondria and lysosomes indicated mitophagy which was demonstrated by yellow fluorescence. The stained HeLa cells were treated with FCCP, dithionite, or the combination of dithionite and TPEN. FCCP, an activator of mitophagy. Dithionite (DT), an inducer of hypoxia. TPEN, a zinc chelator. The cells were observed and recorded by Nikon A1R Confocal Microscope and analyzed using Nikon imaging software. 62 degree of overlap. The breakdown of filament mitochondria into fragments, along with their co-localization with lysosomes, served as the hallmarks for mitophagy.

I have shown that intracellular zinc concentration would increase during hypoxia, moreover, the rise in zinc contributed to mitochondrial reactive oxygen species (ROS) accumulation (Slepchenko, Lu, and Li 2016), mitochondrial ROS is a major contributor to mitochondrial damage. On the other hand, FCCP-treated, damaged mitochondria, would induce zinc release and elevation (Slepchenko, Lu, and Li 2017). Therefore, it is reasonable to explore whether zinc cross talked with mitophagy during hypoxia.

Since intracellular zinc level rises during hypoxia, I managed to reduce the intracellular zinc level by applying TPEN, the zinc chelator. When TPEN was applied to the hypoxic HeLa cells, as shown in the group of Hypoxia/TPEN in Figure 14, the mitophagy activity was reduced. The overall mitochondrial morphology and quality was improved when comparing to the Hypoxia group. It showed that, if the zinc accumulation was abolished, the injured mitochondria would be rescued, therefore, there was less mitophagy happening. The alleviation of mitophagy activity and improvement in mitochondrial morphology observed after zinc chelation, was possibly through reducing the zinc-induced toxic or stress to mitochondria. The observation in Figure 14 was further quantitatively measured, as the mitophagy activity can be measured from the overlap of the lysosome-specific green fluorescence signal and the mitochondrial-specific red fluorescence signal. The average level of mitophagy activity in control, FCCP, hypoxia, and hypoxia/TPEN groups were showed in Figure 15. Pearson’s correlation coefficient for co-localization analysis revealed that FCCP treatment, also serves as a positive 63 control in its ability to uncouple mitochondrial membrane potential to trigger the occurrence of mitophagy, induced dramatic mitophagy. Under hypoxia condition, the mitophagy was statistically significant with a p <0.01. Moreover, the hypoxia induced mitophagy was effectively reduced upon zinc chelation through TPEN administration.

Figure 15. Quantification of mitophagy levels in control, FCCP, hypoxia, hypoxia/TPEN groups. Histogram showing the Pearson’s correlation coefficient for co-localization in Control, FCCP, Hypoxia, Hypoxia/TPEN groups. For the quantification of colocalization, Pearson's correlation coefficient (PCC) calculated using JACoP (Just Another Colocalization Plugin) for each group was normalized to the control group and the average of the normalized PCC is represented as a graph. The statistical significance assessed using one-tailed t-test. Data are shown as mean ± SEM of three independent experiments. ** p<0.01, *** p<0.001. (n>=8) 64

2.3 Zinc Induced Mitophagy is Alleviated by TPEN

I examined the effect of zinc influx on mitophagy by applying exogenous zinc. To induce the elevation of intracellular zinc, fresh HeLa cells were exposed to 50 µM ZnCl2 solution supplemented with 50 µM pyrithione which facilitates the influx of zinc into cells. The mitophagy activity of the zinc-treated cells were evaluated by co-staining the cells with lysosome and mitochondria fluorescent indicators. The occurrence of mitophagy was intensive upon exogenous zinc administration, as observed in Figure 16.

This observation can be explained by our previous findings that zinc elevation or zinc overload triggers the generation of oxidative stress, especially reactive oxygen species, which in turn damages mitochondria to induce mitophagy. If the zinc chelator TPEN was applied to zinc-treated HeLa cells, the mitophagy occurrence reduced, and the overall appearance was more resemble to the control group. It suggests that zinc removal by chelation could rescue mitochondria from the zinc induced damages.

The observations were further evaluated by quantification analysis of the mitophagy levels. The mitophagy activity can be measured from the co-localization of the lysosome-specific green fluorescence signal and the mitochondrial-specific red fluorescence signal. The average level of mitophagy activity in control, zinc, and zinc/TPEN groups were showed in Figure 17. Pearson’s correlation coefficient for co- localization analysis revealed that exogenous zinc induced significant mitophagy, and the mitophagy level was effectively reduced by zinc chelation.

Figure 16. Zinc induced mitophagy was alleviated by zinc chelation HeLa cells were co-stained with LysoTracker Green (1uM) and MitoTracker Red (100nM) for 30min at 37oC. The overlap of mitochondria and lysosomes indicated mitophagy which was demonstrated by yellow fluorescence. The stained HeLa cells were treated with ZnCl2, or the combination of ZnCl2 and TPEN. The cells were observed and recorded by Nikon A1R Confocal Microscope and analyzed using Nikon imaging software.

Figure 17. Quantitative analysis of the mitophagy level in zinc and zinc/TPEN groups For the quantification of colocalization, Pearson's correlation coefficient (PCC) calculated using JACoP (Just Another Colocalization Plugin) for each group was normalized to the control group and the average of the normalized PCC is represented as a graph. The statistical significance assessed using one-tailed t-test. Data are shown as mean ± SEM of five independent experiments. ** p<0.01, *** p<0.001.

2.4 Mitochondrial Morphology as an Indicator of Mitochondrial Wellness

Mitochondria are very dynamic organelles that can change shape, size and subcellular distribution. They are constantly undergoing fusion and fission to reach mitochondrial homeostasis. Mitochondria are strictly regulated within a cell to maintain the number and morphology and perform their biological functions. However, when challenged with stresses, mitochondria change morphologies.

Figure 18. Representative images showing one of the typical morphologies of stressed mitochondria HeLa cells were stained with mitochondria specific dye, MitoTracker Red. Stress in mitochondria was induced by 200 µM ZnCl2 solution. Images were randomly captured using a customized fluorescence microscopy and analyzed using ImagePro.

Under physiological conditions, mitochondria are in tubular shapes, interconnected and present a network-like behavior, as revealed by live cell imaging in the bottom image of Figure 18. The interconnected networks of mitochondria may suggest an efficient system to deliver energy or perform physiological roles. Under stress conditions or when the mitochondrial function is compromised, a decrease in connectivity was observed, along with the formation of fragmented, short, and round mitochondria. A representative image of the stressed mitochondria was shown in the top image of Figure 18. Moreover, the changes in mitochondrial morphology are qualitative and quantitative. Therefore, the changes in mitochondrial morphology can serve as a hallmark to indicate the wellness of mitochondria. 68

2.5 The Analysis of Mitochondrial Morphology under the Stresses of Hypoxia and

Zinc Influx

I observed that both hypoxia stress and zinc influx will induce mitophagy activity; moreover, hypoxia induced mitophagy can be alleviated by TPEN. It suggested a role of zinc in the hypoxia induced mitophagy. I aimed to analyze the role zinc played in the occurring of mitophagy under hypoxic condition. As mitophagy is initiated by mitochondrial damage, here I evaluated the effect of hypoxia or zinc influx on mitochondrial quality, as indicated by mitochondrial morphology.

Freshly cultured HeLa cells were pre-stained with mitochondrial specific indicator, MitoTracker Red to enable the detection of mitochondria. The HeLa cells were then treated to induce the following stresses: hypoxia induced by dithionite, zinc influx induced by exogenous zinc administration. Here two different concentrations were employed: 10 µM ZnCl2 and 50 µM ZnCl2. The stained and treated HeLa cells were observed under 1000X resolution to ensure the visualization of subtle mitochondrial morphology.

Representative images were shown in Figure 19. In control group, the mitochondria were robust, in tubular shape and interconnected to form a network-like structure. Under hypoxic condition, mitochondria were heavily damaged that they were fragmented, much shorter, or even show a dot shape; the changes did not only happen in the mitochondrial shape, but also in mitochondrial quantity, evidenced by the decreasing in the dense of mitochondria in hypoxia group. The mitochondrial morphologies in 10

µM ZnCl2 or 50 µM ZnCl2 groups were changed as well; compare to the mitochondrial 69 morphology in control group, after zinc treatment, the mitochondria were fragmented into slimmer and shorter pieces, which was possibility due to an increase in the fission/division of individual mitochondria. If compare the mitochondrial morphologies between 10 µM ZnCl2 treatment and 50 µM ZnCl2 treatment, the damages to mitochondria were more severe with 50 µM ZnCl2 treatment. While the zinc influx with both 10 µM ZnCl2 and 50 µM ZnCl2 induced the stressed conditions in HeLa cells, the low dose of 10 µM ZnCl2 is enough to induce mitochondrial damage and trigger mitochondrial fragmentation. The changes in mitochondrial dense was not observed in 10

µM ZnCl2 or 50 µM ZnCl2 treated HeLa cells.

Figure 19. Mitochondrial morphology under the stress of hypoxia and exogenous zinc HeLa cells was incubated with mitochondria specific dye, MitoTracker Red (100nM) for 30min at 37oC. The pre-stained HeLa cells were treated with the hypoxia inducer, dithionite, 10 µM ZnCl2 or 50 µM ZnCl2. The images were captured by a customized fluorescence microscope and analyzed by ImagePro. Representative images were showed here.

2.6 Qualitative and Quantitative Analysis of Mitochondrial Morphology by Evaluating

Mitochondrial Roundness

The morphology of mitochondria can be qualitatively and quantitatively described by parameters including mitochondrial roundness, mitochondrial circularity, major axis, and aspect ratio. Mitochondrial roundness shows how much the mitochondria represents a perfect circle compared to an elongation or uneven shape. The smallest value of 71 mitochondrial roundness is 0.0, the highest value is 1.0, shows the mitochondria is a perfectly round circle. Mitochondrial roundness was measured in individual mitochondria to determine the changes in mitochondrial morphology between control HeLa cells and hypoxia or zinc treated HeLa cells.

The statistical analysis was shown in Figure 20. Three stress types were analyzed: hypoxia, 10µM zinc influx, 50µM zinc influx. There are no significant differences between mitochondrial roundness induced by oxygen limitation (hypoxia) or low does rapid exogenous zinc influx, comparing with the control group. Mitochondria were fragmented and shortened in these cells, but mitochondrial roundness analysis did not distinguish a significant difference. But when the zinc concentration increased to 50µM, the mitochondrial roundness was significantly increased.

n.s. * n.s.

Figure 20. Changes in mitochondrial roundness induced by hypoxia and exogenous zinc influx Mitochondrial roundness was observed in HeLa cells after hypoxia or exogenous zinc treatment. Hypoxia was induced by dithionite, two concentrations of ZnCl2 (10 µM, 50 µM) were used for exogenous zinc treatments. For each group, mitochondria from 4 independent experiments were analyzed. Data are shown as mean ± SEM. *p<0.05, **p<0.01

2.7 Qualitative and Quantitative Analysis of Mitochondrial Morphology by Evaluating

Mitochondrial Circularity

Mitochondrial circularity describes how closely the measured mitochondria represent a circle, the value of mitochondrial circularity ranges from 0.0 to 1.0.

Mitochondria with circularity value 1.0 indicates it is a perfect circle, the smaller the value, the closer it is representing an elongated shape. Mitochondrial circularity is 73 determined by the balance between mitochondrial fission and fusion, high fission activity generate a large value of mitochondrial circularity, while active fusion between mitochondria result in a small mitochondrial circularity.

Mitochondrial circularity was measured in individual mitochondria for each group to determine the changes in mitochondrial morphology. The result was shown in Figure

21. After hypoxia or zinc influx treatment, significant increases in mitochondrial circularity were observed in all treatment groups. It suggests that mitochondrial circulation happens intensively after the stresses of oxygen limitation and rapid zinc influx. Exogenous zinc influx showed a dose effect as evidenced by the result that 50 µM

ZnCl2 administration had a greater mitochondrial circulation than 10 µM ZnCl2 administration. The mitochondrial circulation induced by hypoxia was greater than by 10

µM ZnCl2. Mitochondrial circulation as a phenomenon directly showing mitochondrial damage, was induced the stress or toxic conditions, here I analyzed the degrees of mitochondrial circulation under the conditions of oxygen limitation and rapid zinc influx.

The relation between mitochondrial circulation and mitochondrial elongation could be further analyzed to evaluate whether there is a balance between mitochondrial circulation and mitochondrial elongation. Moreover, it will be interesting to further understand whether circulated mitochondria can be reversed to the elongated shape, and what is the critical value that once mitochondrial circularity reached this value, the circulated mitochondria are non-reversible.

* ** *

Figure 21. Changes in mitochondrial circularity induced by hypoxia and exogenous zinc influx Mitochondrial circularity was observed in HeLa cells after hypoxia or exogenous zinc treatment. Hypoxia was induced by dithionite, two concentrations of ZnCl2 (10 µM, 50 µM) were used for exogenous zinc treatments. For each group, mitochondria from 4 independent experiments were analyzed. *p<0.05, **p<0.01

2.8 Qualitative and Quantitative Analysis of Mitochondrial Morphology by Evaluating

Major Axis and Aspect Ratio

Mitochondrial morphology can also be qualitatively and quantitatively described by the following two parameters: mitochondrial major axis and aspect ratio.

Mitochondrial major axis is the length of the mitochondria; aspect ratio is the ratio of major axis over minor axis. 75

Mitochondrial major axis and aspect ratio were measured in individual mitochondria for each group to determine the changes in mitochondrial morphology. The result was shown in Figure 22. A decrease was observed in all groups after hypoxia or zinc influx treatment; however, the changes were not statistically significant. It suggests that even though the stresses from hypoxia or zinc influx reduced the mitochondrial lengths, but the degree of change was not significant and can’t be used as a strong proof.

A possible reason is that the base length of mitochondria in HeLa cells is different among cells and cell-dependent, therefore, it would be better to observe the changes in mitochondrial major axis and aspect ratio before and after treatment in a cell, rather than observe the changes in mitochondrial major axis and aspect ratio among different groups of cells. But the method is verified to be feasible, because mitochondrial morphology can be quantitatively and qualitatively measured by the parameters including major axis and aspect ratio.

Figure 22. The effects of hypoxia and exogenous zinc on mitochondrial major axis and aspect ratio Mitochondrial major axis and aspect ratio were observed in HeLa cells after hypoxia or exogenous zinc treatment. Hypoxia was induced by dithionite, two concentrations of ZnCl2 (10 µM, 50 µM) were used for exogenous zinc treatments. For each group, mitochondria from 4 independent experiments were analyzed. *p<0.05, **p<0.01 77

3. Investigating the Genes Involved in Zinc-Mitophagy Interplay during Hypoxia by

Large-scale Screening and Fine-scale Validation

3.1 Gene Expression Omnibus Analysis to Reveal the Genes of Interest

The aim is to investigate how zinc and mitophagy crosstalk by screening for the candidate genes involved in the hypoxia triggered zinc-mitophagy signaling pathway, and eventually draft a possible signaling pathways to explain the zinc-mitophagy regulation during hypoxia.

First, I performed large-scale screening using the Gene Expression Omnibus

(GEO) database. GEO was created in 2000 as a worldwide resource for gene expression studies. It is an international public repository that archives and freely distributes high- throughput gene expression and other functional genomics data sets (Clough and Barrett

2016). From the NCBI GEO database, I filtered and selected the studies which employed

HeLa cells to study the gene expression profiles under hypoxic condition (GSE79069,

Bourseau-Guilmain et al. 2016), zinc addition (GSE49657, Homma et al. 2013), or FCCP

(GSE84631, (Matilainen, Quirós, and Auwerx 2017).

3.2 Identifying the Candidate Genes of in Hypoxic HeLa Cells by Comparing with Their

Overlapping with Mitochondria Genes and Zinc Genes

The purpose of experimental GSE79069 was to analyze the gene expression in hypoxic and normoxic HeLa cells. Hypoxic HeLa cells were achieved by 1% oxygen at

6h. The total RNA was obtained for microarray (Bourseau-Guilmain E, Lidfeldt J, 2016).

The GEO record was retrieved from NCBI GEO database. I analyzed the GEO data with 78

GEO2R, and identified 1415 genes that were differentially expressed (DEG) between

Hypoxia and Normoxia. To evaluate how many genes were related with mitochondria, I tracked the mitochondrial related genes from GO website, the genes were filtered for homo sapiens and retrieved, the total number of mitochondrial related genes retrieved was 1286.

Figure 23 The comparison of DEG with mitochondria related genes revealed 154 overlapped genes. Blue solid circle: Genes differentially expressed between Hypoxia and Normoxia. P<0.01 Pink solid circle: Mitochondria related genes retrieved from GO website, filtered by eukaryote -> mammalia ->Homo Sapiens.

The DEG between hypoxia and normoxia treatments, was compared with the mitochondrial related genes in homo sapiens. As we can see in Figure 22, there were 154 overlapped genes between the two groups. 79

I also tracked zinc related genes from GO website, the genes were filtered for homo sapiens and retrieved, the total number of zinc related genes identified was 1718.

The DEG between hypoxia and normoxia treatments, was compared with the zinc related genes in homo sapiens. As we can see in Figure 23, 97 overlapped genes between the two groups were identified.

Figure 24. The comparison of DEG (Hypoxia v.s. Normoxia) with zinc related genes revealed 97 overlapped genes Blue solid circle: Genes differentially expressed between Hypoxia and Normoxia. P<0.01 Pink solid circle: Zinc related genes retrieved from GO website, filtered by eukaryote -> mammalia ->Homo Sapiens.

I did a further comparison among the three groups: differentially expressed genes in response to hypoxia, mitochondrial related genes, zinc related genes. The comparison revealed that 7 genes were overlapped among the three groups (Figure 24). A list of the 7 overlapping genes is shown in table 3. 80

Figure 25. The comparison of DEG from hypoxic HeLa cells with mitochondrial genes and zinc genes revealed 7 overlapped genes Blue solid circle: Genes differentially expressed between Hypoxia and Normoxia. P<0.01 Pink solid circle: mitochondrial related genes retrieved from GO website, filtered by eukaryote -> Mammalia ->Homo Sapiens. Green solid circle: zinc related genes retrieved from GO website, filtered by eukaryote -> Mammalia -> Homo Sapiens

Table 3: List of 7 overlapped genes

3.3 Identifying the Candidate Genes of in FCCP HeLa Cells by Analyzing their

Overlapping with Mitochondrial Genes and Zinc Genes

GSE84631 was the GEO record from NCBI GEO database. The purpose of the

GSE84631 study was to assess the gene expression profiling in HeLa cells upon treatment with mitochondrial stressors. FCCP was used in this study to introduce mitochondrial stress (Auwerx J, Quiros PM, 2016). I retrieved the GSE84631 data from

NCBI. After analyzing the GEO data with GEO2R and identified 1149 genes that were differentially expressed (DEG) between FCCP treatment and control (p<0.01). To evaluate how many genes were related with mitochondria, I searched the mitochondrial related genes from GO website, the genes were filtered for homo sapiens and retrieved, the total number of mitochondrial related genes retrieved was 1149.

The DEGs in FCCP treated HeLa cells were compared with the mitochondrial related genes in homo sapiens. As we can see in Figure 25, there were 100 overlapped genes between the two groups.

I also tracked zinc related genes from GO website, the genes were filtered for homo sapiens and retrieved, the total number of zinc related genes identified was 1719.

The DEG between hypoxia and normoxia treatments, was compared with the zinc related genes in homo sapiens. As we can see in Figure 26, 74 overlapped genes between the two groups were identified.

I further analyzed and compared the differentially expressed genes in FCCP treated HeLa cells with the total mitochondrial related genes and total zinc related genes. 82

The comparison revealed that 8 genes were overlapped among the three groups (Figure

27). A list of the 8 overlapping genes is described in table 4.

Figure 26. The comparison of DEG in FCCP treated HeLa cells with mitochondria related genes revealed 100 overlapped genes. Blue solid circle: Genes differentially expressed between FCCP and control. P<0.01 Pink solid circle: Mitochondria related genes retrieved from GO website, filtered by eukaryote -> Mammalia ->Homo Sapiens.

Figure 27. The comparison of DEG in FCCP treated HeLa cells with zinc related genes revealed 74 overlapped genes. Blue solid circle: Genes differentially expressed between FCCP and control. P<0.01 Pink solid circle: Zinc related genes retrieved from GO website, filtered by eukaryote -> Mammalia ->Homo Sapiens. 83

Figure 28. The comparison of DEG from FCCP treated HeLa cells with mitochondrial genes and zinc genes revealed 8 overlapped genes Blue solid circle: Genes differentially expressed between FCCP and control. P<0.01 Pink solid circle: mitochondrial related genes retrieved from GO website, filtered by eukaryote -> Mammalia ->Homo Sapiens. Green solid circle: zinc related genes retrieved from GO website, filtered by eukaryote -> Mammalia -> Homo Sapiens

Table 4. List of 8 overlapped genes

3.4 Candidate Genes Identified by Large-scale Screening and Literature Research

Through GEO analysis, I identified 14 different genes that were differentially expressed under the stresses of hypoxia or FCCP, also were categorized as mitochondrial related genes and zinc related genes. After a careful evaluation of the 14 genes, I decided to move on with SOD1, LACTB2, TIMM8B, SPG7, TSPO and TRIM14. Along the several other candidate genes that were identified through literature research. A list of the finalized candidate genes with description was listed in table 5.

Table 5. List of finalized candidate genes identified by large-scale screening and literature research

3.5 The Superoxide Dismutase Multigene Family CuZn-SOD (SOD1) and Mn-SOD

(SOD2) Act Differently in Response to FCCP, Hypoxia or Zinc Addition

I examined the relative gene expression levels of the three superoxide dismutase family genes: CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) under FCCP administration, hypoxia treatment, or exogenous zinc addition. CuZn-SOD (SOD1), Mn-

SOD (SOD2), EC-SOD (SOD3) are three members of the superoxide dismutase multigene family, they are the first and most important line of antioxidant enzyme defense systems against ROS and particularly superoxide anion radicals (Zelko, Mariani, and Folz 2002). Interestingly, the three genes were acting differently in response to the stresses from different sources.

CuZn-SOD (SOD1) gene expression level did not alter notably upon FCCP administration but increased dramatically under the stress of hypoxia or exogenous zinc, and increased to the similar degree, which was above 2 folds compare to control group or

FCCP group (Figure 25 A). In contrast, the relative gene expression level of Mn-SOD

(SOD2) increased significantly in response to FCCP; even though increases of Mn-SOD

(SOD2) were also observed in response to Hypoxia and ZnCl2, but the changes were not statistically significant (Figure 25 B). The results suggested that, under the stress of hypoxia and exogenous zinc, CuZn-SOD (SOD1) is actively transcribed and takes its role as the antioxidant to defend against hypoxia and exogenous zinc induced ROS. In contrast, Mn-SOD (SOD2) is more sensitive to ROS induced by FCCP, as evidenced by the robust increase in the transcription level upon FCCP administration.

Figure 29. Hypoxia and ZnCl2 induces the expression of SOD1, FCCP induces the expression of SOD2 qPCR analysis of the relative gene expression levels of CuZn-SOD (SOD1) and Mn-SOD (SOD2) under three different stresses: FCCP, Hypoxia, and ZnCl2. RPS18 was used for normalization. Data are the mean ± SEM from three independent experiments. Each experiment includes three technical repeats. *p<0.05, **p<0.01, ***p<0.001

EC-SOD (SOD3) is the most recently characterized SOD, it is structurally close to CuZn-SOD (SOD1), and both proteins containing copper and zinc. While CuZn-SOD

(SOD1) is almost exclusively in intracellular cytoplasmic spaces, EC-SOD (SOD3) exclusively locates in extracellular spaces (Zelko, Mariani, and Folz 2002). I evaluated the relative gene expression levels of EC-SOD (SOD3) under stresses of FCCP, hypoxia or ZnCl2, the results are as shown in Figure 26. The results suggested that EC-SOD

(SOD3) is not involved in the antioxidant defense against ROS during the stress conditions induced by FCCP, Hypoxia, and exogenous ZnCl2.

The results comprehensively compared the involvement of the three superoxide dismutase genes under the stresses of FCCP, Hypoxia, and ZnCl2. The results from

Figure 25 and Figure 26 revealed that even though all these SODs are able to defend against ROS, but they are responding to different stimuli. I introduced FCCP stress, hypoxic condition, and exogenous zinc to fresh HeLa cells. During hypoxia and ZnCl2 treatment, SOD1 is highly expressed to lead the role as an antioxidant, SOD2 expression has a slight increase but not significant, SOD3 expression is not affected. Under the stress of FCCP, SOD2 is highly expressed, while SOD1 and SOD3 expression remain the same.

The differences in SODs expression levels in regarding to different stimuli may suggest that, even though all three stimuli (FCCP, Hypoxia, ZnCl2) induce ROS, but the sources and locations of the ROS are different, therefore, only SOD in the same space was stimulated to increase expression. Considering CuZn-SOD (SOD1) is exclusively in intracellular cytoplasmic spaces, Mn-SOD (SOD2) exclusively locates in mitochondrial spaces, and EC-SOD (SOD3) goes exclusively to extracellular spaces; it is reasonable to 88 state that hypoxia and exogenous zinc induce-ROS generation happen mostly in the intracellular space (some ROS generation in mitochondria), while FCCP triggers the ROS generation in mitochondria.

Figure 30. EC-SOD (SOD3) is not responsive to FCCP, Hypoxia or ZnCl2 stimuli qPCR analysis of the relative gene expression levels of EC-SOD (SOD3) under three different stresses: FCCP, Hypoxia, and ZnCl2. RPS18 was used for normalization. Data are the mean ± SEM from three independent experiments. Each experiment includes three technical repeats. *p<0.05, **p<0.01, ***p<0.001

3.6 HIF-1a Gene Expression Level was not Altered by FCCP, Hypoxia or ZnCl2

Administrations

HIF-1 is the mediator of physiological and pathophysiological responses to hypoxia. HIF-1 is a heterodimer composed of an alpha subunit and a beta subunit. The biological activity of HIF-1 is determined by the expression and activity of the HIF-1a subunit (Semenza 2000). Under normal conditions, HIF-1 alpha is hydroxylated by prolyl-hydroxylases (PHD) and degraded; under hypoxic condition, HIF-1 alpha is stabilized and translocated to the nucleus, where it binds to the subunit HIF-1 beta and regulates the expression of multiple genes (Semenza 2000; Manalo et al. 2005).

Considering the significance of HIF-1a in the hypoxia induced signaling pathways, I evaluated the mRNA levels of HIF-1a under the three different stresses.

However, the relative gene expression level of HIF-1a remain relatively stable under the stresses of FCCP, Hypoxia, and ZnCl2, as shown in Figure 27. It was summarized that the regulation of HIF-1a expression and activity in vivo occurs at multiple levels, mRNA expression, protein expression, nuclear localization and transactivation (Semenza 2000).

The result in Figure 27 suggested that, in response to the acute stresses such as FCCP, hypoxia, and exogenous zinc, due to the short time during, the gene expression of HIF-1a is not altered yet; instead, the regulation of HIF-1a expression and activity principally occur on the preexisting proteins. Our observation and hypothesis are in consistence with the role of HIF-1a under hypoxia: instead of being degraded, the HIF-1 stabilizes and mobilizes to nucleus and functions as transcriptional factor to induce hypoxia-related 90 genes. It requires further study to distinguish whether regulation of HIF-1a activity occurred post-translationally under FCCP and exogenous zinc.

Figure 31. mRNA level of HIF-1a is not altered by FCCP, Hypoxia or ZnCl2 stimuli qPCR analysis of the relative gene expression levels of HIF-1a under three different stresses: FCCP, Hypoxia, and ZnCl2. RPS18 was used for normalization. Data are the mean ± SEM from three independent experiments. Each experiment includes three technical repeats. *p<0.05, **p<0.01, ***p<0.001

3.7 The Expression of SPG7 is Highly Increased under Hypoxia and Exogenous Zinc

Administration

SPG7 is one of the candidate genes screened from GEO analysis. It encodes a mitochondrial metalloprotease protein. SPG7 is best known as the spastic paraplegia gene, the mutations in SPG7 are responsible for an autosomal recessive form of 91 hereditary spastic paraplegia (HSP) (Settasatian et al. 1999). A recent work showed that

SPG7 serves as a core component in the formation and regulation of the mitochondrial permeability transition pore (PTP), and promotes Ca2+- and ROS-induced PTP opening

(Shanmughapriya et al. 2015).

The gene expression level of SPG7 is highly increased in response to hypoxia and exogenous zinc, as shown in Figure 28, the relative mRNA level of SPG7 in hypoxia is three folds higher than in control group and FCCP, the relative SPG7 mRNA level in zinc treatment group is two folds higher, both are statistically significant. The mitochondrial permeability transition pore was known as a Ca2+ activated pore, which functions as a mitochondrial calcium release channel (Bernardi and Petronilli 1996). Opening of the mitochondrial PTP causes massive swelling of mitochondria, rupture of the outer membrane and release of intermembrane components that induce apoptosis (Halestrap,

McStay, and Clarke 2002). It was reported that during hypoxic stress, an increase in permeability of the mitochondrial membranes due to the opening of the mitochondrial

PTP is prominent in the hypoxia induced cell death and tissue injury (Assaly et al. 2012).

I hypothesize that the up regulation of SPG7 mRNA level under hypoxia stress indicate that SPG7 may play a role in the hypoxia induced mPTP opening. Moreover, I observed that exogenous zinc treatment increased the SPG7 expression level, considering the role of SPG7 in the calcium activated mPTP, it may suggest that, in addition to the well- known calcium, zinc also contribute to the opening of mPTP, possibly through the role of

SPG7. 92

Figure 32. The expression of SPG7 is highly increased under hypoxia and exogenous zinc administration qPCR analysis of the relative gene expression levels of SPG7 under three different stresses: FCCP, Hypoxia, and ZnCl2. RPS18 was used for normalization. Data are the mean ± SEM from three independent experiments. Each experiment includes three technical repeats. *p<0.05, **p<0.01, ***p<0.001

3.8 The Gene Expression of TRIM 14 is Elevated during Hypoxia, and Remains Stable

during FCCP and Exogenous Zinc Stresses

Tripartite motif-containing 14 (TRIM14) is a candidate gene identified by the previous GEO analysis. So far there is very limited study on the function of TRIM14. It is reported that TRIM14 is abnormally expressed in several human cancers, but the function 93 and expression of TRIM 14 are still largely unknown (Hai et al. 2017). A recent report showed that TRIM14 may act as an oncogene in human breast cancer by promoting cancer cell proliferation and inhibiting apoptosis (Hu, Pen, and Wang 2019).

Figure 33. Hypoxia elevated the gene expression of TRIM14. qPCR analysis of the relative gene expression levels of TRIM14 under three different stresses: FCCP, Hypoxia, and ZnCl2. RPS18 was used for normalization. Data are the mean ± SEM from three independent experiments. Each experiment includes three technical repeats. *p<0.05, **p<0.01, ***p<0.001

I evaluated the gene expression levels of TRIM14 under the following three stresses: FCCP treatment, hypoxia, exogenous zinc administration. As shown in Figure

29, the mRNA level of TRIM14 was not affected by either FCCP treatment or zinc treatment, comparing to the control group. But an elevation of TRIM14 mRNA was observed during hypoxia, it suggests that TRIM14 is involved in hypoxia response. 94

3.9 The Expression of Parkin was Elevated upon Mitochondrial Stresses, while PINK1

Expression Level Remained Unaffected

Parkin is central to mitochondrial quality control. PINK1 and Parkin mediates mitophagy in the same pathway (Jin and Youle 2012), PINK1 activate Parkin to damaged mitochondria (Nguyen, Padman, and Lazarou 2016). I evaluated the gene expression levels of Parkin and PINK1 in the HeLa cells treated by FCCP, hypoxia, or exogenous zinc. As shown in Figure 33, the relative mRNA level of Parkin was increased upon

FCCP treatment, which serves as a positive control that induce mitophagy. Both hypoxia and exogenous zinc induced Parkin expression. It suggests that hypoxia and exogenous zinc induced mitophagy activity, employs the same signaling pathway as the mitophagy induced by FCCP, in which Parkin is the key regulator. The gene expression level of

PINK1 remain relatively stable, after stresses of hypoxia or exogenous zinc. The transcription of PINK1 was not altered in the FCCP treatment as well.

PINK1 was synthesized in cytosol and transported into mitochondria (Kawajiri et al. 2010:1) , and the degraded in the mitochondrial matrix (Kato et al. 2013) under physiological conditions. PINK1 is selectively stabilized on the damaged mitochondria

(Narendra et al. 2010:1). Combines with our observations, the result suggests that PINK1 functions in the mitophagy signaling pathway through the regulation of PINK1 protein level, PINK1 transcription level was not affected during mitophagy.

Figure 34. The mRNA level of Parkin (PRNK) was elevated upon mitochondrial stresses. Quantitative RT-PCR was used to measure relative PRNK and PINK1 mRNA expression in HeLa cells treated with FCCP, hypoxia, or ZnCl2. The graph represents the results from three independent experiments. PRNK and PINK1 mRNA expression levels were normalized to the housekeeping gene RSP18. Data are the mean ± SEM from three independent experiments. Each experiment includes three technical repeats. *p<0.05.

3.10 PGC1A is Up-regulated in Response to Hypoxia and Zinc Influx

Peroxisome proliferator-activated receptor gamma coactivator-1 (PGC1A) is a transcription factor that regulates cellular energy metabolism. PGC1A stimulates mitochondrial biogenesis (Liang and Ward, 2006). It was stated that mitochondrial biogenesis contributes to ischemic neuroprotection by contributing to ischemic tolerance

(Stetler and Chen, 2012).

I evaluated the gene expression level of PGC1A in HeLa cells that subjected to

FCCP treatment, hypoxia, or exogenous zinc influx. As shown in Figure 34, the PGC1A mRNA level was not affected by the mitochondrial uncoupler FCCP, but under the stresses of hypoxia and exogenous zinc influx, PGC1A was up-regulated to express. It suggests that the mitochondrial biogenesis activity was increased in response to the rapid hypoxia and zinc influx.

The results suggested that, the increasing of mitochondrial biogenesis activity through PGC1A up-regulation, serves to improve the tolerance to hypoxia and zinc influx. Under stresses, the damaged mitochondria were cleared through mitophagy, which disturbs the mitochondrial homeostasis by reducing the mitochondrial content/quantity. The loss in mitochondrial content/quantity could be replenished by the process of mitochondrial biogenesis.

Figure 35. PGC1A was up-regulated in response to hypoxia and zinc influx. Quantitative RT-PCR was used to measure relative PGC1A mRNA expression in HeLa cells treated with FCCP, hypoxia, or ZnCl2. The graph represents the results from three independent experiments. PGC1A mRNA expression levels were normalized to the housekeeping gene RSP18. Data are the mean ± SEM from three independent experiments. Each experiment includes three technical repeats. *p<0.05, **p<0.01, ***p<0.001

3.11 Mitophagy Regulators NIX1 and FUNDC1 were Not Associated with the FCCP,

Hypoxia or Zinc Influx Induced Mitophagy

In addition to Parkin-PINK1 mediated mitophagy, mitochondria can also be cleared through a Parkin-independent pathway, in which an autophagy receptor protein on the outer membrane of mitochondria interact directly with LC3 on the autophagosome to form autolysosome and degrade the engulfed mitochondria. In HeLa cells, NIX1 protein serves as an autophagy receptor for the selective clearance of mitochondria. The gene expression of NIX1 was examined in HeLa cells treated with FCCP, hypoxia, or 98 exogenous zinc. However, I did not observe changes on the relative mRNA level of NIX1 in the above conditions, as shown in Figure 35A.

Figure 36. Mitophagy regulators NIX1 and FUNDC1 were not involved. Quantitative RT-PCR was used to measure relative NIX1 and FUNDC1 mRNA expression in HeLa cells treated with FCCP, hypoxia, or ZnCl2. The graph represents the results from three independent experiments. NIX1 and FUNDC1 mRNA expression levels were normalized to the housekeeping gene RSP18. Data are the mean ± SEM from three independent experiments. Each experiment includes three technical repeats. n.s. not significant.

FUNDC1 is another protein located on mitochondrial outer membrane that has been reported to involve in the mitochondrial clearance in response to hypoxic condition

(We et al, 2014). It was stated that FUNDC1 could act as a receptor protein to mediate mitophagy (Liu et al. 2012; Wu et al 2014). Here the gene expression level of FUNDC1 was tested in the stressed HeLa cells, even though decreases in FUNDC1 mRNA level in hypoxic HeLa cells and zinc-influxed HeLa cells were observed, as recorded in Figure

35B, the changes were not identified as statistically significant.

100

DISCUSSION

Ischemic stroke is a devastating disease with a complex pathophysiology.

Previous study from our lab had shown that zinc is a major factor in neuronal cell death during ischemic stroke. But it is still unclear how alterations of zinc concentration contribute to neuronal cell death during ischemic stroke. Here I further demonstrated that zinc plays a critical role in ischemic stroke through my study in the in vitro stroke model, hypoxic HeLa cells.

First, I comprehensively explored the intracellular distribution of free zinc ions.

Zinc specific fluorescent indicator, Zinpyr-1, was used to monitor the intracellular zinc distribution. The concentration of zincpyr-1 used in cells was optimized to obtain the most reliable zinc monitoring results (Figure 1). The specificity of zinpyr-1 was verified by exogenous zinc addition or intracellular zinc removal, as shown in Figure 2, along with the increasing or decreasing intracellular zinc levels, the fluorescence signals of zinpyr-1 change accordingly. It proved zinpyr-1 was sensitive and specific to intracellular zinc ions and serves as a feasible tool to monitor and investigate the activity of intracellular zinc.

The zinc distribution was first observed in HeLa cells in my study (Figure 3), further studies were performed in cortical neurons and pancreatic HIT-T15 cells. I found out that mitochondria (Figure 4, 5, 7, 8), along with ER (Figure 9, 10), the Golgi apparatus (Figure 11), serves as major storage sites of intercellular zinc, these organelles sequester or release zinc in responses to different cellular conditions. Even nucleus was reported to contain high amount of zinc binding proteins and have a high amount of zinc 101 binding with the proteins, but the free zinc in nucleus was not detectable (Figure 12).

Moreover, the absence of zinc fluorescence in nucleus supports that zinc fluorescence detected in mitochondria, ER and the Golgi apparatus represent the free chelatable zinc.

Next, the effect of zinc on mitophagy during hypoxia was investigated by modulating the zinc levels. Mitophagy was monitored by live cell fluorescent imaging of mitochondria and lysosome simultaneously. MitoTracker Red, the mitochondrial specific dye and LysoTracker Green, the lysosome specific dye, were used to label fresh HeLa cells. The co-localization was tested as the overlay of green fluorescence and red fluorescence.

The mitophagy level was low and barely detectable at physiological condition; when HeLa cells treated by FCCP, the mitochondrial uncoupler, the mitophagy activity was highly elevated (Figure 13). This result shows that mitophagy was not observable at basal level, once the mitochondria were damaged, mitophagy occurs intensively to clear the damaged mitochondria. It also proved that the method of assessing mitophagy by co- localization of evaluating lysosomes and mitochondria was feasible and reliable.

The same methodology was used to evaluate the mitophagy level in hypoxic

HeLa cells, the result was shown in Figure 14. The hypoxic condition was achieved by chemical hypoxia inducer, sodium dithionite, which removes oxygen as a reducing agent.

Sodium dithionite induced hypoxia was widely used to rapidly induce the hypoxic condition with reliability. Moreover, when TPEN was applied to the hypoxic HeLa cells, the mitophagy activity level was alleviated (Figure 14, Figure 15). This observation was consistent with our previous report that intracellular zinc accumulates. 102

I also reported that zinc elevation contributes to ROS generation (Slepchenko, Lu, and Li 2016). The rapidly generated ROS damages mitochondria and triggers mitophagy.

When the zinc accumulation was abolished by zinc chelator TPEN, the mitochondria can be saved from the zinc toxicity and as a result of reduced mitophagy and rescued mitochondrial overall wellness. The effect of zinc influx was tested by bringing up the intracellular zinc level. Zinc level up-regulation was achieved by applying exogenous zinc solution to the fresh HeLa cells, pyrithione was supplemented in the solution to facilitate the uptake of zinc ion by HeLa cells. Pyrithiones are chelated zinc via oxygen and sulfer center. Two pyrithiones coordinated with one zinc to form a centrosymmetric dimer. Pyrithione works as zinc ionophore, facilitating zinc transport across membranes

(Reeder et al. 2011). Pyrithione was tested in HeLa cells to verify the effect of pyrithione alone, the result showed that intracellular free zinc was not changed when HeLa cells were treated with pyrithione solution but without exogenous zinc (data not shown). The mitophagy activity level was increased in zinc treated HeLa cells, and zinc removal by

TPEN alleviated the intensive mitophagy occurrence (Figure 16, Figure 17).

In this study, mitophagy was observed by the co-localization of mitochondria and lysosome, this is the most feasible method for my research. But there are other methods, probably can fulfill my research purpose better, but could not be utilized due to the limitation of resources. One of the methods is mitophagy observation and analysis by electron microscopy. Images from electron microscopy are able to show the content in the cells with sufficient details, including mitochondrial number and mitochondrial morphology. The number of normal mitochondria and damaged mitophagy is countable, 103 therefore, the percentage of mitochondria undergoing mitophagy can be calculated. It provides a better understanding on the occurrence of mitophagy and to what degree the mitophagy occurred.

To further demonstrate that the treatments do increase mitophagy, mitophagy inhibitors could be applied. Mitochondrial division/mitophagy inhibitor (Mdivi) is one of the commonly used mitophagy inhibitors, it functions by inhibiting mitochondrial fission during mitophagy (Givvimani et al. 2012). Cells incubated with dithionite, ZnCl2 or

FCCP, should then further treated with Mdivi. I would expect to see decreasing in mitophagy level upon Mdivi administration. Mdivi experiment is a good supplemental experiment, but not necessary, because both positive control and negative control were tested, both controls have demonstrated that mitophagy activity was induced by both hypoxia and zinc overload.

Fluorescence microscopy for mitochondria-lysosome colocalization is a well- recognized method to monitor mitophagy for live-cell imaging microscopy. This approach is an indirect measurement of mitophagy compared with electron microscopy

(EM). EM is one of the best approaches to provide direct evidence for mitophagy, the morphological hallmark of mitophagy could be faithfully identified and recorded.

However, each EM image presents limited number of cells, and it uses fixed samples only. To assess a large number of cells, or to monitor the dynamic process of mitophagy in a live cell setting, would require the use of co-localization method in live-cell imaging microscopy. Gene expression study was performed to target the genes involved in the process, however, gene expression is not a feasible tool to monitor the occurrence of 104 mitophagy. The major reason is that mitophagy is a dynamic process that consists of a series of morphological hallmarks, including mitochondrial fission, formation of autophagosomes, engulfing of mitochondrial by autophagosomes, and fusion of autophagosomes with lysosomes. The morphological changes could not be timely reflected by the changes in gene expression.

Throughout the process of mitophagy, mitochondria are different in their structures. Therefore, the different stages of mitophagy can be identified. For example, the early stage of mitophagy and late stage of mitophagy are distinguishable in electron microscopy images, different outcomes happen at different mitophagy stages. The cells with more late-stage mitophagy are more likely in the process of cell death; cells with more early-stage mitophagy probably can still be rescued, the TPEN chelation is expected to have a better protective effect on these cells.

In addition to mitophagy activity level, I also examined the mitochondrial wellness under the stress of hypoxia and zinc influx. Mitochondrial wellness was indicated by the mitochondrial morphology. As dynamic organelles that constantly changes shape, size and quantity, mitochondrial overall quantity/quality were strictly regulated to maintain mitochondrial homeostasis, or a relatively stable number and morphology. Stresses induce changes to mitochondrial morphology. Figure 18 showed the morphology of normal mitochondria and stressed mitochondria. The shape of mitochondria changes from tubular and interconnected form to fragmented, even dotted discrete form. The changes in mitochondrial morphology are qualitatively and 105 quantitatively measurable, therefore, it can be used as an indicator to show the mitochondrial wellness.

Mitochondrial roundness, circularity, aspect ratio and major axis were evaluated in HeLa cells under the following stresses: hypoxia, 10 µM ZnCl2 and 50 µM ZnCl2.

High resolution fluorescence microscopy enables the visualization of individual mitochondrion. Quantitatively analysis was performed and confirmed the observations

(Figure 19, Figure 20). After hypoxic treatment or zinc treatment, the mitochondria were fragmented into smaller pieces with less elongation and larger circularity, comparing with un-treated HeLa cells. Moreover, differences in mitochondrial roundness and circularity were not observed between hypoxia treatment and zinc treatment. But higher dose of exogenous zinc (50 µM) have a notable but not statistically significant greater effect than lower dose of exogenous zinc (10 µM).

To investigate how zinc and mitophagy interplay during hypoxia, large-scale screening for the genes associated with this process was performed by Gene Expression

Omnibus (GEO) analysis. Totally three GEO records were retrieved from the database to identify the genes of interest in my study. The three GEO records were generated from studies to reveal the gene expression profiles under hypoxic condition (GSE79069,

Bourseau-Guilmain et al. 2016), zinc addition (GSE49657, Homma et al. 2013), or FCCP

(GSE84631, Matilainen, Quirós, and Auwerx 2017). I also retrieved a list of mitochondrial related genes and a list of zinc related genes in Homo Sapiens from Gene

Ontology (GO) database. I first took the top differentially expressed genes (DEG) at a cuf-off of p<0.01. The DEG genes were compared with the mitochondrial related genes 106 and zinc related genes to identify the overlapping genes. In total, there were 14 identified genes that were differentially expressed and associated with zinc and mitochondria under the stresses of hypoxia or FCCP from the large-scale screening (Figure 21 – Figure 27).

After further evaluation, the SOD1, LACTB2, TIMM8B, TRIM14 and TSPO were shortlisted, along with DNM1, SOD2, SOD3, FUNDC1, HIF-1a, NIX1, PRKN, PGC1-a,

PINK1, SPG7 were validated by quantitative real-time PCR.

The HeLa cells were treated by dithionite, FCCP and 50 µM zinc solution for one hour, then total RNA were extracted and genes of interest were analyzed by quantitative real-time PCR.

SOD1, SOD2, and SOD3 are three superoxide dismutase, they all function in the oxidative stress defense system, but they were acting differently in response to different stresses (Figure 28, Figure 29). SOD1 was relatively unresponsive to FCCP, but the gene expression level of SOD1 was dramatically increased with the treatment of hypoxia or exogenous zinc. In contrast, SOD2 is more sensitive to FCCP, as evidenced by the observation that FCCP induced a significant up-regulation of SOD2 mRNA level. SOD3 is structurally similar with SOD1, both harbor copper and zinc. However, the gene expression of SOD3 is not altered by any of the above treatments.

Through a comprehensive evaluating of the three members in the oxidative stress defend system, I suggest that the sources and locations of the ROS generated by FCCP, hypoxia, and exogenous zinc are different, and only the SOD in the same space was stimulated to increase the transcription level. SOD1 is exclusively located in intracellular cytoplasm, SOD2 locates exclusively in mitochondria, SOD3 locates exclusively to 107 extracellular matrix. The results (Figure 28, Figure 29) indicated that the ROS generated under the stress of hypoxia and exogenous zinc majorly is in the intracellular space, while the ROS generated by FCCP is in the mitochondria.

The gene expression level of HIF-1a was studies to reveal whether the stressors will change the transcription of HIF-1a in HeLa cells. The quantitative RT-PCR result showed that HIF-1a mRNA level was not altered under the stresses of FCCP, hypoxia, and exogenous zinc influx (Figure 30). HIF-1a is the critical mediator protein in the physiological and pathophysiological responses to hypoxia, under physiological condition, HIF-1a is hydroxylated and degraded, under hypoxic condition, HIF-1a, as a transcription factor, stabilize and translocate to nucleus and regulate the expression of hypoxia related genes. The result suggested that the regulation of HIF-1a is at the protein level, instead the mRNA level. Further study can be performed the verify the changes of

HIF-1a protein level under the stresses of FCCP, hypoxia and exogenous zinc influx.

SPG7 was significantly up-regulated in the hypoxic HeLa cells and zinc-treated

HeLa cells (Figure 31). SPG7 is one of the genes identified by GEO analysis, it is best known as the spastic paraplegia gene, because the mutation is SPG7 is the major cause of spastic paraplegia (Settasatian et al. 1999:7). SPG7 is also reported to promote calcium and ROS induced mitochondrial PTP opening, as a key component in the formation and regulation of the mPTP (Shanmughapriya et al. 2015:7). It is very possible that zinc also contributes to the opening of mPTP, and the accumulation of zinc and generation of ROS promotes the opening of mPTP in hypoxic HeLa cells and zinc-treated HeLa cells. 108

TRIM14 is also one of the candidate genes identified from GEO analysis, quantitative RT-PCR revealed that the expression level of TRIM14 is highly elevated in the hypoxic HeLa cells, but not in FCCP treated or zinc treated HeLa cells. The functions of TRIM14 is largely unknown, one of the clues is that TRIM14 promotes cancer cell proliferation by inhibiting apoptosis.

Proteins involved in the mitophagy signaling pathways were also evaluated.

Currently mechanisms for damaged mitochondrial clearance can be classified into two categories: Parkin-dependent mitophagy pathway and Parkin-independent pathway. In the Parkin-dependent pathway, Parkin was recruited by damaged mitochondria by

PINK1. PINK1 is transported into the mitochondrial matrix and degraded under physiological conditions, the damaged mitochondria failed to transport PINK1 and result in a stabilization and accumulation of PINK1 on the outer membrane of damaged mitochondria, which gives a signal to Parkin and initiates the process of mitochondrial clearance (Kato et al. 2013:1). The mRNA level of Parkin was elevated under the stress of FCCP, hypoxia and exogenous zinc, however, PINK1 gene expression level remained relatively stable under the stresses (Figure 33). It suggests that the regulation of PINK1 activity during mitophagy was not at transcription level, instead, PINK1 is regulated post- transcriptionally at the protein level.

In addition to Parkin-dependent mitophagy pathway, the mitochondrial clearance can also be mediated by mitochondrial receptor proteins such as NIX1 and FUNDC1, both of they are located on mitochondrial outer membrane and interact directly with LC3 109 on autophagosome to form autolysosome, the hallmark structure of mitophagy. However, either NIX1 or FUNDC1 was altered at the transcription level under the stresses.

GEO data analysis is a feasible tool to screen for genes of interest in a specific study. However, it is not likely to find the gene expression profiles of the exactly same study I planned to investigate, such as 10 µM ZnCl2 and 50 µM ZnCl2 treatment to HeLa cells. But similar studies were retrieved, and the gene expression profiles from the studies can be used as a reference, but solid analysis and consideration are required to screen the candidate genes from the gene expression profiles.

The gene expression studies were done to the HeLa cells with 2-hour treatment.

One of the reasons to set the administration period at 2-hour is that our published data showed that rapid hypoxia induces a burst generation of ROS and suggested early response of superoxide dismutase genes (Slepchenko, Lu, and Li 2017). Moreover, a time course study was done to check multiple time points: 30 min, 60 min, 120 min, 180 min

(data not shown). Changes in SODs mRNA levels were observed from 60 min of treatment. However, a time range from 30 min to 180 min is still short. A short time range brings several limitations, the biggest limitation is that I won’t be able to catch the genes that take longer time to response. To solve this problem, a longer time period could have been tested, for example, have the cells treated for 3 hours, 6 hours, 9 hours, 15 hours and 24 hours. With longer treatment, I would expect to see the changes in the current “non-responsive” genes, especially PINK1, NIX1, FUNDC1.

In this study I searched for genes of candidate in the crosstalk of zinc and mitophagy during hypoxia. But pathway analysis should be a better choice to reveal the 110 zinc-mitophagy crosstalk during hypoxia, however, to analyze pathway, I need to obtain the gene expression profiles from my own research, instead of retrieving data from GEO database. Because the selected pathways are hard to be verified and validated after screening. Due to the limitation of resources, and without access to microarray for RNA sequencing, the comprehensive gene expression profiles in HeLa cells with the hypoxia and exogenous zinc treatments can’t be obtained in this study.

Another pitfall in this study is that the protein level or post-translational levels of candidate genes were not determined. Protein expression level and post-translational modification should be analyzed for the candidate genes. In addition to candidate genes, protein expression levels of marker proteins for hypoxia effect and mitophagy should also be evaluated, and be used as proofs to show the effect of chemical hypoxia treatment and

FCCP treatment. However, even though a direct proof of the hypoxia effect was not available, indirect proofs were provided, as evidenced by the increase in ROS gene expression level. 111

Figure 37．Zinc and mitophagy interplay during hypoxia There pathways were identified in the crosstalk between zinc and mitophagy during hypoxia: Parkin-dependent mitophagy pathway, PGC1a-mediated mitochondrial biogenesis, and oxidative defense by SOD2.

As concluded in Figure 37, under the stress of stroke/hypoxia, zinc overload was caused by the rapid accumulation of zinc from different sources. The possible sources include zinc preserving organelles, zinc binding proteins, and extracellular environment.

In this study I confirmed that mitochondria, ER, and the Golgi apparatus are the major zinc storage sites in cells.

Zinc binding proteins include enzymes, storage proteins, zinc figure transcription factors etc. The release of zinc from the binding proteins is triggered under hypoxic condition, it increases the burden of zinc accumulation. 112

Zinc level can be modulated by membrane transport proteins ZnT and ZIP. ZnT is zinc exporter which controls the efflux of zinc from the cytoplasm out of the cell and from the cytoplasm into organelles. ZIP is zinc importer which controls the influx of zinc into the cytoplasm from outside the cell and from organelles.

Mitochondria are the major target of zinc overload during hypoxia. The oxidative stress, as a result of hypoxia, increases ROS while decreasing the activity of the normal cellular antioxidant system. ROS causes damages to mitochondria, and the damaged mitochondria undergoes mitophagy. Moreover, the hypoxia induced zinc accumulation, also contributes to the generation of ROS. The occurrence and process of mitophagy were observed, and the pathways involved in the process were evaluated. Collectively, three pathways were identified: Parkin-dependent mitophagy pathway, PGC-1α mediated mitochondrial biogenesis, and oxidative defense by SOD2.

The findings revealed that under the hypoxic condition, in response to the stresses, mitophagy was ongoing through the Parkin-dependent pathway to remove the damaged mitochondria. To compensate the loss of mitochondria and the reduce of mitochondrial number in the cells, the generation of new mitochondria were modulated by PGC-1α, the master regulator of mitochondrial biogenesis. At the same time, to defend the oxidative stress, SOD2, the superoxide dismutase which located in mitochondria, was highly expressed. I also found that TRIM14 and SPG7 were involved in the zinc-mitophagy crosstalk during hypoxia. The functions of the two genes are very elusive. The very limited reports showed that TRIM14 is an oncogene that promotes breast cancer cell proliferation by inhibiting apoptosis (Hu, Pen, and Wang 2019:14) and 113 a mitochondrial adaptor that facilitates retinoic acid-inducible innate immune response

(Zhou et al. 2014:14), while SPG7 is an essential and conserved component of the mitochondrial permeability transition pore (Shanmughapriya et al. 2015:7). It suggested that during hypoxia, the opening of mitochondrial permeability transition pore occurred, likely triggered by zinc accumulation.

Through my study, it is demonstrated that zinc and mitophagy crosstalk with each other during hypoxia. Moreover, zinc and mitochondria can be the target for therapeutic interventions in stroke. The possible strategies include remove excessive zinc by zinc chelators, and reduce mitophagy by applying mitophagy inhibitors. Because of the interplay between zinc and mitophagy during hypoxia, it is promising to identify the regulator or reagent that modulates both zinc and mitophagy.

In conclusion, even though the functions of zinc and mitophagy during stroke/hypoxia are very intricate, our study revealed that the roles of zinc and mitophagy are overlapping and even synergistic. Recognizing the interplay between zinc and mitophagy during hypoxia and understanding their multimodal impact they have on stroke is a very promising direction for the therapeutic interventions.

114

REFERENCES

Akira, Toshiaki, Didier Henry, Roger A. Baldwin, and Claude G. Wasterlain 1994 Nitric Oxide Participates in Excitotoxic Mechanisms Induced by Chemical Hypoxia. Brain Research 645(1): 285–290.

Alker, Wiebke, Tanja Schwerdtle, Lutz Schomburg, and Hajo Haase 2019 A Zinpyr-1-Based Fluorimetric Microassay for Free Zinc in Human Serum. International Journal of Molecular Sciences 20(16): 4006.

Assaly, Rana, Alexandra d’Anglemont de Tassigny, Stéphanie Paradis, et al. 2012 Oxidative Stress, Mitochondrial Permeability Transition Pore Opening and Cell Death during Hypoxia–Reoxygenation in Adult Cardiomyocytes. European Journal of Pharmacology 675(1–3): 6–14.

Baesler, Jessica, Johannes F. Kopp, Gabriele Pohl, et al. 2019 Zn Homeostasis in Genetic Models of Parkinson’s Disease in Caenorhabditis Elegans. Journal of Trace Elements in Medicine and Biology 55: 44–49.

Baker, Brooke M., and Cole M. Haynes 2011 Mitochondrial Protein Quality Control during Biogenesis and Aging. Trends in Biochemical Sciences 36(5): 254–261.

Balaban, Robert S., Shino Nemoto, and Toren Finkel 2005 Mitochondria, Oxidants, and Aging. Cell 120(4): 483–495.

Bernardi, Paolo, and Valeria Petronilli 1996 The Permeability Transition Pore as a Mitochondrial Calcium Release Channel: A Critical Appraisal. Journal of Bioenergetics and Biomembranes 28(2): 131–138.

Bliek, Alexander M. van der, Qinfang Shen, and Sumihiro Kawajiri 2013 Mechanisms of Mitochondrial Fission and Fusion. Cold Spring Harbor Perspectives in Biology 5(6): a011072.

Bolaños, Juan P, and Angeles Almeida 1999 Roles of Nitric Oxide in Brain Hypoxia-Ischemia. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1411(2–3): 415–436.

Bourseau-Guilmain, E., J. A. Menard, E. Lindqvist, et al. 2016 Hypoxia Regulates Global Membrane Protein Endocytosis through Caveolin-1 in Cancer Cells. Nature Communications 7: 11371.

Bright, C. M., and D. Ellis 115

1992 Intracellular PH Changes Induced by Hypoxia and Anoxia in Isolated Sheep Heart Purkinje Fibres. Experimental Physiology 77(1): 165–175.

Brose, Stephen A., Svetlana A. Golovko, and Mikhail Y. Golovko 2016 Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia. Frontiers in Neuroscience 10. https://www.frontiersin.org/articles/10.3389/fnins.2016.00546/full, accessed November 17, 2019.

Budde, T., A. Minta, J. A. White, and A. R. Kay 1997 Imaging Free Zinc in Synaptic Terminals in Live Hippocampal Slices. Neuroscience 79(2): 347–358.

Calderone, Agata, Teresa Jover, Toshihiro Mashiko, et al. 2004 Late Calcium EDTA Rescues Hippocampal CA1 Neurons from Global Ischemia- Induced Death. Journal of Neuroscience 24(44): 9903–9913.

Chan, David C. 2006 Mitochondria: Dynamic Organelles in Disease, Aging, and Development. Cell 125(7): 1241–1252.

Chan, Pak H. 2005 Mitochondrial Dysfunction and Oxidative Stress as Determinants of Cell Death/Survival in Stroke. Annals of the New York Academy of Sciences 1042(1): 203– 209.

Chen, Zhaogui, Yanhua Lu, Zhengtao Wang, Xinyi Tao, and Dongzhi Wei 2007 Protective Effects of Salidroside on Hypoxia/Reoxygenation Injury by Sodium Hydrosulfite in PC12 Cells. Pharmaceutical Biology 45(8): 604–612.

Chen, Zhou-qing, Ru-tao Mou, Dong-xia Feng, Zhong Wang, and Gang Chen 2017 The Role of Nitric Oxide in Stroke. Medical Gas Research 7(3): 194–203.

Clough, Emily, and Tanya Barrett 2016 The Gene Expression Omnibus Database. Methods in Molecular Biology (Clifton, N.J.) 1418: 93–110.

Cousins, Robert J., Juan P. Liuzzi, and Louis A. Lichten 2006 Mammalian Zinc Transport, Trafficking, and Signals. Journal of Biological Chemistry 281(34): 24085–24089.

Dagda, Ruben K., Salvatore J. Cherra, Scott M. Kulich, et al. 2009 Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission. The Journal of Biological Chemistry 284(20): 13843–13855. 116

Dineley, Kirk E., Tatyana V. Votyakova, and Ian J. Reynolds 2003 Zinc Inhibition of Cellular Energy Production: Implications for Mitochondria and Neurodegeneration. Journal of Neurochemistry 85(3): 563–570.

Dong, Wen, Zhifeng Qi, Jia Liang, et al. 2015 Reduction of Zinc Accumulation in Mitochondria Contributes to Decreased Cerebral Ischemic Injury by Normobaric Hyperoxia Treatment in an Experimental Stroke Model. Experimental Neurology 272. Pangu Stroke Conference: 181–189.

Eide, David J. 2006 Zinc Transporters and the Cellular Trafficking of Zinc. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1763(7). Cell Biology of Metals: 711–722.

Eliasson, Mikael J. L., Zhihong Huang, Robert J. Ferrante, et al. 1999 Neuronal Nitric Oxide Synthase Activation and Peroxynitrite Formation in Ischemic Stroke Linked to Neural Damage. Journal of Neuroscience 19(14): 5910–5918.

Frederickson, Christopher J., Jae-Young Koh, and Ashley I. Bush 2005 The Neurobiology of Zinc in Health and Disease. Nature Reviews Neuroscience 6(6): 449–462.

Frederickson, Christopher J., Sang Won Suh, David Silva, Cathy J. Frederickson, and Richard B. Thompson 2000 Importance of Zinc in the Central Nervous System: The Zinc-Containing Neuron. The Journal of Nutrition 130(5): 1471S-1483S.

Gegg, Matthew E., J. Mark Cooper, Kai-Yin Chau, et al. 2010 Mitofusin 1 and Mitofusin 2 Are Ubiquitinated in a PINK1/Parkin-Dependent Manner upon Induction of Mitophagy. Human Molecular Genetics 19(24): 4861–4870.

Givvimani, Srikanth, Charu Munjal, Neetu Tyagi, et al. 2012 Mitochondrial Division/Mitophagy Inhibitor (Mdivi) Ameliorates Pressure Overload Induced Heart Failure. PLOS ONE 7(3): e32388.

Grabrucker, Stefanie, and Andreas M. Grabrucker 2017 Chapter 9 - Zinc in Autism. In Biometals in Neurodegenerative Diseases. Anthony R. White, Michael Aschner, Lucio G. Costa, and Ashley I. Bush, eds. Pp. 153–173. Academic Press. http://www.sciencedirect.com/science/article/pii/B9780128045626000099, accessed November 17, 2019.

Greene, Jessica C., Alexander J. Whitworth, Isabella Kuo, et al. 2003 Mitochondrial Pathology and Apoptotic Muscle Degeneration in Drosophila Parkin 117

Mutants. Proceedings of the National Academy of Sciences of the United States of America 100(7): 4078–4083.

Hai, Josephine, Chang-Qi Zhu, Tao Wang, et al. 2017 TRIM14 Is a Putative Tumor Suppressor and Regulator of Innate Immune Response in Non-Small Cell Lung Cancer. Scientific Reports 7(1): 1–13.

Halestrap, Andrew P, Gavin P McStay, and Samantha J Clarke 2002 The Permeability Transition Pore Complex: Another View. Biochimie 84(2): 153– 166.

Hanna, Rita A., Melissa N. Quinsay, Amabel M. Orogo, et al. 2012 Microtubule-Associated Protein 1 Light Chain 3 (LC3) Interacts with Bnip3 Protein to Selectively Remove Endoplasmic Reticulum and Mitochondria via Autophagy. Journal of Biological Chemistry 287(23): 19094–19104.

Hayashi, Teruo, Rosario Rizzuto, Gyorgy Hajnoczky, and Tsung-Ping Su 2009 MAM: More than Just a Housekeeper. Trends in Cell Biology 19(2): 81–88.

Higashi, Y., T. Aratake, S. Shimizu, T. Shimizu, and M. Saito 2019 [Brain zinc dyshomeostasis and glial cells in ischemic stroke]. Nihon yakurigaku zasshi. Folia pharmacologica Japonica 154(3): 138–142.

Homma, Kengo, Takao Fujisawa, Naomi Tsuburaya, et al. 2013 SOD1 as a Molecular Switch for Initiating the Homeostatic ER Stress Response under Zinc Deficiency. Molecular Cell 52(1): 75–86.

Hu, Gaowu, Wei Pen, and Ming Wang 2019 TRIM14 Promotes Breast Cancer Cell Proliferation by Inhibiting Apoptosis. Text. https://www.ingentaconnect.com/content/cog/or/2019/00000027/00000004/art00006, accessed December 4, 2019.

Ji, Sung G., Yuliya V. Medvedeva, Hwai-Lee Wang, Hong Z. Yin, and John H. Weiss 2019 Mitochondrial Zn2+ Accumulation: A Potential Trigger of Hippocampal Ischemic Injury. The Neuroscientist 25(2): 126–138.

Jiang, Pengju, and Zijian Guo 2004 Fluorescent Detection of Zinc in Biological Systems: Recent Development on the Design of Chemosensors and Biosensors. Coordination Chemistry Reviews 248(1–2): 205–229.

Jin, Seok Min, and Richard J. Youle 2012 PINK1- and Parkin-Mediated Mitophagy at a Glance. J Cell Sci 125(4): 795–799. 118

Kambe, Taiho, Tokuji Tsuji, Ayako Hashimoto, and Naoya Itsumura 2015 The Physiological, Biochemical, and Molecular Roles of Zinc Transporters in Zinc Homeostasis and Metabolism. Physiological Reviews 95(3): 749–784.

Karbowski, Mariusz, and Richard J Youle 2011 Regulating Mitochondrial Outer Membrane Proteins by Ubiquitination and Proteasomal Degradation. Current Opinion in Cell Biology 23(4). Membranes and Organelles: 476–482.

Kato, Hiroki, Qiping Lu, Doron Rapaport, and Vera Kozjak-Pavlovic 2013 Tom70 Is Essential for PINK1 Import into Mitochondria. PLoS ONE 8(3): e58435.

Kawajiri, Sumihiro, Shinji Saiki, Shigeto Sato, et al. 2010 PINK1 Is Recruited to Mitochondria with Parkin and Associates with LC3 in Mitophagy. FEBS Letters 584(6): 1073–1079.

Kawashima, Yu, Yui Someya, Shogo Sato, et al. 2011 Dietary Zinc-Deficiency and Its Recovery Responses in Rat Liver Cytosolic Alcohol Dehydrogenase Activities. The Journal of Toxicological Sciences 36(1): 101– 108.

Kiang, Juliann G., Vishal G. Warke, and George C. Tsokos 2003 NaCN-Induced Chemical Hypoxia Is Associated with Altered Gene Expression. Molecular and Cellular Biochemistry 254(1–2): 211–216.

Kim, Jung-whan, Irina Tchernyshyov, Gregg L. Semenza, and Chi V. Dang 2006 HIF-1-Mediated Expression of Pyruvate Dehydrogenase Kinase: A Metabolic Switch Required for Cellular Adaptation to Hypoxia. Cell Metabolism 3(3): 177–185.

Kimura, Eiichi, and Tohru Koike 1998 Recent Development of Zinc-Fluorophores. Chemical Society Reviews 27(3): 179–184.

Kornmann, Benoît, Erin Currie, Sean R. Collins, et al. 2009 An ER-Mitochondria Tethering Complex Revealed by a Synthetic Biology Screen. Science 325(5939): 477–481.

Krohn, Kenneth A., Jeanne M. Link, and Ralph P. Mason 2008 Molecular Imaging of Hypoxia. Journal of Nuclear Medicine 49(Suppl 2): 129S- 148S.

Lai, Ted Weita, Shu Zhang, and Yu Tian Wang 2014 Excitotoxicity and Stroke: Identifying Novel Targets for Neuroprotection. Progress 119 in Neurobiology 115. 2013 Pangu Meeting on Neurobiology of Stroke and CNS Injury: Progresses and Perspectives of Future: 157–188.

Lazarou, Michael, Seok Min Jin, Lesley A. Kane, and Richard J. Youle 2012 Role of PINK1 Binding to the TOM Complex and Alternate Intracellular Membranes in Recruitment and Activation of the E3 Ligase Parkin. Developmental Cell 22(2): 320–333.

Lee, Jae W., Junsuk Ko, Cynthia Ju, and Holger K. Eltzschig 2019 Hypoxia Signaling in Human Diseases and Therapeutic Targets. Experimental & Molecular Medicine 51(6): 1–13.

Lee, Youngil, Hwa-Youn Lee, Rita A. Hanna, and Åsa B. Gustafsson 2011 Mitochondrial Autophagy by Bnip3 Involves Drp1-Mediated Mitochondrial Fission and Recruitment of Parkin in Cardiac Myocytes. American Journal of Physiology - Heart and Circulatory Physiology 301(5): H1924–H1931.

Li, Peiying, R. Anne Stetler, Rehana K. Leak, et al. 2018 Oxidative Stress and DNA Damage after Cerebral Ischemia: Potential Therapeutic Targets to Repair the Genome and Improve Stroke Recovery. Neuropharmacology 134. Cerebral Ischemia: 208–217.

Lill, Roland, and Ulrich Mühlenhoff 2008 Maturation of Iron-Sulfur Proteins in Eukaryotes: Mechanisms, Connected Processes, and Diseases. Annual Review of Biochemistry 77(1): 669–700.

Lin, Yu-Chun, Pei-Feng Tsai, and James Swi-Bea Wu 2014 Protective Effect of Anthocyanidins against Sodium Dithionite-Induced Hypoxia Injury in C6 Glial Cells. Journal of Agricultural and Food Chemistry 62(24): 5603–5608. liu, Guoxiang, seong-hoon Park, Xianghui Zou, et al. 2016 Loss of Sirt2 Alters Mitochondrial Protein Acetylation, Morphology, and Dysregulates Mitophagy. Antioxidants & Redox Signaling. http://online.liebertpub.com/doi/10.1089/ars.2016.6662, accessed August 2, 2016.

Liu, Lei, Du Feng, Guo Chen, et al. 2012 Mitochondrial Outer-Membrane Protein FUNDC1 Mediates Hypoxia-Induced Mitophagy in Mammalian Cells. Nature Cell Biology 14(2): 177–185.

Lu, Qiping, Hariprakash Haragopal, Kira G Slepchenko, Christian Stork, and Yang V Li 2016 Intracellular Zinc Distribution in Mitochondria, ER and the Golgi Apparatus. International Journal of Physiology, Pathophysiology and Pharmacology 8(1): 35–43.

MacDonald, Ruth S. 120

2000 The Role of Zinc in Growth and Cell Proliferation. The Journal of Nutrition 130(5): 1500S-1508S.

Manalo, Dominador J., Ashley Rowan, Tera Lavoie, et al. 2005 Transcriptional Regulation of Vascular Endothelial Cell Responses to Hypoxia by HIF-1. Blood 105(2): 659–669.

Mandemakers, Wim, Vanessa A. Morais, and Bart De Strooper 2007 A Cell Biological Perspective on Mitochondrial Dysfunction in Parkinson Disease and Other Neurodegenerative Diseases. Journal of Cell Science 120(10): 1707–1716.

Maret, Wolfgang 2008 Metallothionein Redox Biology in the Cytoprotective and Cytotoxic Functions of Zinc. Experimental Gerontology 43(5). Zinc and Ageing (ZINCAGE Project): 363–369.

Maret, Wolfgang, and Harold H. Sandstead 2006 Zinc Requirements and the Risks and Benefits of Zinc Supplementation. Journal of Trace Elements in Medicine and Biology 20(1): 3–18.

Matilainen, Olli, Pedro M. Quirós, and Johan Auwerx 2017 Mitochondria and Epigenetics – Crosstalk in Homeostasis and Stress. Trends in Cell Biology 27(6): 453–463.

Matsushima, Yuichi, and Laurie S. Kaguni 2012 Matrix Proteases in Mitochondrial DNA Function. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 1819(9–10). Mitochondrial Gene Expression: 1080–1087.

Maxwell, Patrick H., Michael S. Wiesener, Gin-Wen Chang, et al. 1999 The Tumour Suppressor Protein VHL Targets Hypoxia-Inducible Factors for Oxygen-Dependent Proteolysis. Nature 399(6733): 271–275.

Medvedeva, Yuliya V., Bin Lin, C. William Shuttleworth, and John H. Weiss 2009 Intracellular Zn2+ Accumulation Contributes to Synaptic Failure, Mitochondrial Depolarization, and Cell Death in an Acute Slice Oxygen–Glucose Deprivation Model of Ischemia. Journal of Neuroscience 29(4): 1105–1114.

Michiels, Carine 2004 Physiological and Pathological Responses to Hypoxia. The American Journal of Pathology 164(6): 1875–1882.

Mortiboys, Heather, Kelly Jean Thomas, Werner J. H. Koopman, et al. 2008 Mitochondrial Function and Morphology Are Impaired in Parkin-Mutant Fibroblasts. Annals of Neurology 64(5): 555–565. 121

Murakami, Masaaki, and Toshio Hirano 2008 Intracellular Zinc Homeostasis and Zinc Signaling. Cancer Science 99(8): 1515– 1522.

Narendra, Derek P., Seok Min Jin, Atsushi Tanaka, et al. 2010 PINK1 Is Selectively Stabilized on Impaired Mitochondria to Activate Parkin. PLOS Biology 8(1): e1000298.

Narendra, Derek, Atsushi Tanaka, Der-Fen Suen, and Richard J. Youle 2008 Parkin Is Recruited Selectively to Impaired Mitochondria and Promotes Their Autophagy. The Journal of Cell Biology 183(5): 795–803.

Nguyen, Thanh N., Benjamin S. Padman, and Michael Lazarou 2016 Deciphering the Molecular Signals of PINK1/Parkin Mitophagy. Trends in Cell Biology 26(10): 733–744.

Northington, Frances J., Donna M. Ferriero, Ernest M. Graham, Richard J. Traystman, and Lee J. Martin 2001 Early Neurodegeneration after Hypoxia-Ischemia in Neonatal Rat Is Necrosis While Delayed Neuronal Death Is Apoptosis. Neurobiology of Disease 8(2): 207–219.

Novak, Ivana, Vladimir Kirkin, David G. McEwan, et al. 2010 Nix Is a Selective Autophagy Receptor for Mitochondrial Clearance. EMBO Reports 11(1): 45–51.

Patten, David A., Marc Germain, Melissa A. Kelly, and Ruth S. Slack 2010 Reactive Oxygen Species: Stuck in the Middle of Neurodegeneration. Journal of Alzheimer’s Disease 20(s2): S357–S367.

Qi, Zhifeng, Wenjuan Shi, Yongmei Zhao, Xunming Ji, and Ke Jian Liu 2019 Zinc Accumulation in Mitochondria Promotes Ischemia-Induced BBB Disruption through Drp1-Dependent Mitochondria Fission. Toxicology and Applied Pharmacology 377: 114601.

Quirós, Pedro M., Miguel A. Prado, Nicola Zamboni, et al. 2017 Multi-Omics Analysis Identifies ATF4 as a Key Regulator of the Mitochondrial Stress Response in Mammals. J Cell Biol: jcb.201702058.

Reeder, N. L., J. Xu, R. S. Youngquist, et al. 2011 The Antifungal Mechanism of Action of Zinc Pyrithione. British Journal of Dermatology 165(s2): 9–12.

Samaja, Michele, and Giuseppina Milano 122

2015 Editorial – Hypoxia and Reoxygenation: From Basic Science to Bedside. Frontiers in Pediatrics 3. http://journal.frontiersin.org/article/10.3389/fped.2015.00086/full, accessed August 3, 2017.

Sandoval, Hector, Perumal Thiagarajan, Swapan K. Dasgupta, et al. 2008 Essential Role for Nix in Autophagic Maturation of Erythroid Cells. Nature 454(7201): 232–235.

Santel, A., and M. T. Fuller 2001 Control of Mitochondrial Morphology by a Human Mitofusin. Journal of Cell Science 114(5): 867–874.

Schapira, Anthony HV 2006 Mitochondrial Disease. The Lancet 368(9529): 70–82.

Semenza, Gregg L. 2000 HIF-1: Mediator of Physiological and Pathophysiological Responses to Hypoxia. Journal of Applied Physiology 88(4): 1474–1480.

Sensi, Stefano L., Pierre Paoletti, Jae-Young Koh, et al. 2011 The Neurophysiology and Pathology of Brain Zinc. Journal of Neuroscience 31(45): 16076–16085.

Settasatian, C., S.A. Whitmore, J. Crawford, et al. 1999 Genomic Structure and Expression Analysis of the Spastic Paraplegia Gene, SPG7. Human Genetics 105(1): 139–144.

Shanmughapriya, Santhanam, Sudarsan Rajan, Nicholas E. Hoffman, et al. 2015 SPG7 Is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore. Molecular Cell 60(1): 47–62.

Shuttleworth, C. William, and John H. Weiss 2011 Zinc: New Clues to Diverse Roles in Brain Ischemia 答辩必读. Trends in Pharmacological Sciences 32(8): 480–486.

Sims, Neil R., and Michelle F. Anderson 2002 Mitochondrial Contributions to Tissue Damage in Stroke. Neurochemistry International 40(6). Role of Mitochondria in Neurological Disease: 511–526.

Sinclair, Scott A., Sarah M. Sherson, Renée Jarvis, James Camakaris, and Christopher S. Cobbett 2007 The Use of the Zinc-Fluorophore, Zinpyr-1, in the Study of Zinc Homeostasis in Arabidopsis Roots. New Phytologist 174(1): 39–45. 123

Slepchenko, Kira G., Calvin B. L. James, and Yang V. Li 2013 Inhibitory Effect of Zinc on Glucose-Stimulated Zinc/Insulin Secretion in an Insulin-Secreting β-Cell Line. Experimental Physiology 98(8): 1301–1311.

Slepchenko, Kira G, Qiping Lu, and Yang V Li 2016 Zinc Wave during the Treatment of Hypoxia Is Required for Initial Reactive Oxygen Species Activation in Mitochondria. International Journal of Physiology, Pathophysiology and Pharmacology 8(1): 44–51.

Slepchenko, Kira G., Qiping Lu, and Yang V. Li 2017 Crosstalk between Intracellular Zinc Rises and Reactive Oxygen Species Accumulation in Chemical-Ischemia. American Journal of Physiology - Cell Physiology: ajpcell.00048.2017.

Soubannier, Vincent, Gian-Luca McLelland, Rodolfo Zunino, et al. 2012 A Vesicular Transport Pathway Shuttles Cargo from Mitochondria to Lysosomes. Current Biology 22(2): 135–141.

Stork, Christian J., and Yang V. Li 2006 Intracellular Zinc Elevation Measured with a “Calcium-Specific” Indicator during Ischemia and Reperfusion in Rat Hippocampus: A Question on Calcium Overload. The Journal of Neuroscience 26(41): 10430–10437. 2009 Rising Zinc: A Significant Cause of Ischemic Neuronal Death in the CA1 Region of Rat Hippocampus. Journal of Cerebral Blood Flow & Metabolism 29(8): 1399–1408.

Tang, Yan-Cheng, Hong-Xia Tian, Tao Yi, and Hu-Biao Chen 2016 The Critical Roles of Mitophagy in Cerebral Ischemia. Protein & Cell: 1–15.

Tapiero, Haim, and Kenneth D Tew 2003 Trace Elements in Human Physiology and Pathology: Zinc and Metallothioneins. Biomedicine & Pharmacotherapy 57(9): 399–411.

Thompson, Richard B 2005 Studying Zinc Biology with Fluorescence: Ain’t We Got Fun? Current Opinion in Chemical Biology 9(5): 526–532.

Tønder, N., F. F. Johansen, C. J. Frederickson, J. Zimmer, and N. H. Diemer 1990 Possible Role of Zinc in the Selective Degeneration of Dentate Hilar Neurons after Cerebral Ischemia in the Adult Rat. Neuroscience Letters 109(3): 247–252.

Toyokuni, S. 1999 Reactive Oxygen Species-Induced Molecular Damage and Its Application in Pathology. Pathology International 49(2): 91–102. 124

Truong-Tran, Ai Q., Joanne Carter, Richard E. Ruffin, and Peter D. Zalewski 2001 The Role of Zinc in Caspase Activation and Apoptotic Cell Death. Biometals 14(3–4): 315–330.

Tuboly, Eszter, Andrea Szabó, Dénes Garab, et al. 2013 Methane Biogenesis during Sodium Azide-Induced Chemical Hypoxia in Rats. American Journal of Physiology. Cell Physiology 304(2): C207-214.

Valente, Enza Maria, Patrick M. Abou-Sleiman, Viviana Caputo, et al. 2004 Hereditary Early-Onset Parkinson9s Disease Caused by Mutations in PINK1. Science 304(5674): 1158–1160.

Walkup, Grant K., Shawn C. Burdette, Stephen J. Lippard, and Roger Y. Tsien 2000 A New Cell-Permeable Fluorescent Probe for Zn2+. Journal of the American Chemical Society 122(23): 5644–5645.

Weiss, John H, Stefano L Sensi, and Jae Y Koh 2000 Zn2+: A Novel Ionic Mediator of Neural Injury in Brain Disease. Trends in Pharmacological Sciences 21(10): 395–401.

Wiemerslage, Lyle, and Daewoo Lee 2016 Quantification of Mitochondrial Morphology in Neurites of Dopaminergic Neurons Using Multiple Parameters. Journal of Neuroscience Methods 262: 56–65.

Woodroofe, Carolyn C., Rafik Masalha, Katie R. Barnes, Christopher J. Frederickson, and Stephen J. Lippard 2004 Membrane-Permeable and -Impermeable Sensors of the Zinpyr Family and Their Application to Imaging of Hippocampal Zinc In Vivo. Chemistry & Biology 11(12): 1659–1666.

Wu, Wenxian, Weili Tian, Zhe Hu, et al. 2014 ULK1 Translocates to Mitochondria and Phosphorylates FUNDC1 to Regulate Mitophagy. EMBO Reports 15(5): 566–575.

Yoshii, Saori R., Chieko Kishi, Naotada Ishihara, and Noboru Mizushima 2011 Parkin Mediates Proteasome-Dependent Protein Degradation and Rupture of the Outer Mitochondrial Membrane. Journal of Biological Chemistry 286(22): 19630–19640.

Youle, Richard J., and Derek P. Narendra 2011 Mechanisms of Mitophagy. Nature Reviews. Molecular Cell Biology 12(1): 9–14.

Zelko, Igor N, Thomas J Mariani, and Rodney J Folz 2002 Superoxide Dismutase Multigene Family: A Comparison of the CuZn-SOD 125

(SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) Gene Structures, Evolution, and Expression. Free Radical Biology and Medicine 33(3): 337–349.

Zhang, Huafeng, Marta Bosch-Marce, Larissa A. Shimoda, et al. 2008 Mitochondrial Autophagy Is an HIF-1-Dependent Adaptive Metabolic Response to Hypoxia. The Journal of Biological Chemistry 283(16): 10892–10903.

Zhao, Haiping, Ziping Han, Xunming Ji, and Yumin Luo 2016 Epigenetic Regulation of Oxidative Stress in Ischemic Stroke. Aging and Disease 7(3): 295–306.

Zhao Yongmei, Yan Feng, Yin Jie, et al. 2018 Synergistic Interaction Between Zinc and Reactive Oxygen Species Amplifies Ischemic Brain Injury in Rats. Stroke 49(9): 2200–2210.

Zhou, Zhuo, Xue Jia, Qinghua Xue, et al. 2014 TRIM14 Is a Mitochondrial Adaptor That Facilitates Retinoic Acid-Inducible Gene-I–like Receptor-Mediated Innate Immune Response. Proceedings of the National Academy of Sciences 111(2): E245–E254.

Zuo, Wei, Shuai Zhang, Cong-Yuan Xia, et al. 2014 Mitochondria Autophagy Is Induced after Hypoxic/Ischemic Stress in a Drp1 Dependent Manner: The Role of Inhibition of Drp1 in Ischemic Brain Damage. Neuropharmacology 86: 103–115.

126

PERMISSIONS OF REUSE

Some of the works stated in this dissertation have been published in the following journals, and permissions were requested and granted for reuse in this dissertation. Other works were appropriately referenced.

1. Lu, Qiping, Hariprakash Haragopal, Kira G Slepchenko, Christian Stork, and

Yang V Li

2016 Intracellular Zinc Distribution in Mitochondria, ER and the Golgi

Apparatus. International Journal of Physiology, Pathophysiology and

Pharmacology 8(1): 35–43.

2. Slepchenko, Kira G, Qiping Lu, and Yang V Li

2016 Zinc Wave during the Treatment of Hypoxia Is Required for Initial

Reactive Oxygen Species Activation in Mitochondria. International Journal of

Physiology, Pathophysiology and Pharmacology 8(1): 44–51.

3. Slepchenko, Kira G., Qiping Lu, and Yang V. Li

2017 Crosstalk between Intracellular Zinc Rises and Reactive Oxygen Species

Accumulation in Chemical-Ischemia. American Journal of Physiology - Cell

Physiology: ajpcell.00048.2017.

! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !

Thesis and Dissertation Services ! !