Overexpression of Sequestosome-1 as a Protective Measure against Oxidative Stress in the Retinal Pigment Epithelium

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Overexpression of Sequestosome-1 as a Protective Measure against Oxidative Stress in the Retinal Pigment Epithelium
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english
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Liverpool, Danielle L
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Degree:
Master's ( M.S.)
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University of Florida
Degree Disciplines:
Medical Sciences, Medicine
Committee Chair:
Lewin, Alfred S
Committee Members:
Hauswirth, William W
Boulton, Michael Edwin
Kraft, John

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Subjects / Keywords:
epithelium -- liverpool -- oxidative -- pigment -- retinal
Medicine -- Dissertations, Academic -- UF
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Medical Sciences thesis, M.S.
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Abstract:
Dry age-related macular degeneration is a multifactorial disease of the retina that can lead to the severe impairment of or complete loss of vision. Due to the variety of etiological factors involved, this disease has been difficult to treat. It is currently understood that accumulation of damage caused by excess oxidative stress in the eye contributes to pathogenesis of this disease. Our aim in this study is to prevent oxidative stress mediated damage in retinal pigment epithelial cells by increasing the transcription of antioxidant enzymes controlled by the DNA sequence called the Antioxidant Response Element (ARE). We attempt to stimulate this response by increasing the abundance of Sequestosome-1 (SQSTM1), a protein known to mediate the regulation of Nrf2, a transcription factor for several cytoprotective enzymes. We transfected ARPE-19 cells, a cell line of established human retinal pigment epithelial cells, with a plasmid expressing SQSTM1. Overexpression of this protein was verified by immunoblot analysis. Cells were exposed to either hydrogen peroxide or 4-hydroxynonenal to induce oxidative stress. LDH release assay and MTT assays were performed to characterize cytotoxicity and mitochondrial dehydrogenase activity, respectively.  mRNA levels of antioxidant enzymes were quantified by RT-PCR methods. Surprisingly, we discovered that increasing SQSTM1 abundance in ARPE-19 cells did not result in significant protection of cell viability as measured by the MTT assay or an increase in cytoprotective enzyme production. However, cytotoxicity was reduced in SQSTM1- expressing cells. SQSTM1 has several functions in addition to stimulation of the antioxidant response, including stimulation of autophagy. To further understand the mechanism of cytoprotection by gene delivery of SQSTM1, a stable cell line of ARPE- 19 cells that displays constitutive expression of SQSTM1 was established.
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Statement of Responsibility:
by Danielle L Liverpool.
Thesis:
Thesis (M.S.)--University of Florida, 2013.
Local:
Adviser: Lewin, Alfred S.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-08-31

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1 OVEREXPRESSION O F SEQUESTOSOME 1 AS A PROTECTIVE MEASURE AGAINST OXIDATIVE STRESS IN THE RETINA L PIGMENT EPITHELIUM By DANIELLE LORNA LIVERPOOL A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR TH E DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2013

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2 2013 Danielle Lorna Liverpool

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3 To the Gr eat I Am

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4 ACKNOWLEDGMENTS I would like to acknowledge the following people who have been essential to the development of this thesis. Foremost, I would like to thank Dr. Alfred Lewin for his phenomenal support and guidance through out this process. He has been a remarkable example of a mentor and an amazing scientist. I am tremendously grateful for his patience, insight, and encouragement throughout the past two years. I truly could not have asked for a better princi pal investigator. I thank my other committee members, Dr. Michael Boulton and Dr. William Hauswirth for their time patience, and leadership. I thank the members of Dr. Lewin and Dr. Boulton for the knowledge that they have imparted to me and for their wonderful support and friendship From these groups, I would specifically like to mention Dr. Haoyu Mao, Dr. CJ Song, Sayak Mitter, Brian Rossmiller, Hong Li, Dr. Manas Biswal James Thomas, Jr., and Dr. Zhao yang Wang for their recommendations and benevolence. I especially want to thank Dr. Cristhian Ilde fonso for his unceasing assistance and encouragement during the execution of my project. I thank my prior mentors for inspiring me to pursue this degree and allo wing me to learn more about academia and about myself as a researcher: Dr. Mary Kay Schneider Carodine, Dr. Peter Kima, and Dr. Omar Oyarzabal. Finally, I would like to show my deepest grat itude to my family and friends, for they have be en a source of stre ngth and joy during my tenure as a graduate student.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREV IATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 The Mammalian Retina ................................ ................................ ........................... 14 Anatomy of the Retina ................................ ................................ ...................... 14 Retinal Pigment Epithelium ................................ ................................ .............. 16 Age Related Macular Degeneration ................................ ................................ ....... 17 Epidemiology ................................ ................................ ................................ .... 17 Role of Oxidative Stress in AMD ................................ ................................ ...... 18 Role of Mitochondrial Function in AMD ................................ ............................ 19 Nuclear factor erythroid 2 related factor 2 ................................ ........................ 21 Sequestosome 1/p62 ................................ ................................ ................. 22 Treatments for AMD ................................ ................................ ......................... 25 2 MATERIALS AND METHODS ................................ ................................ ................ 28 DNA Techniques ................................ ................................ ................................ ..... 28 Preparation of Plasmid DNA ................................ ................................ ............. 28 Transformation ................................ ................................ ................................ 28 Plasmid Maxi Preparation ................................ ................................ ................ 29 Tissue Culture Techniques ................................ ................................ ..................... 31 Cell Culture ................................ ................................ ................................ ....... 31 Transfections ................................ ................................ ................................ .... 31 Induction of Oxidative Stress ................................ ................................ ............ 32 RNA Techniques ................................ ................................ ................................ ..... 33 RNA Extraction ................................ ................................ ................................ 33 Real Time PCR ................................ ................................ ................................ 33 Protein Techniques ................................ ................................ ................................ 34 Protein Extraction from cells ................................ ................................ ............. 34 Protein Quantitation ................................ ................................ .......................... 35 Protein Extraction and Immunoblot Analysis ................................ .................... 35 Blocking of Membrane and Application of Antibodies ................................ ....... 35 Creation of Lentivirus transduced Stable Cell Lines ................................ ............... 36

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6 Transformation ................................ ................................ ................................ 36 Packaging of Virus ................................ ................................ ........................... 39 Transduction ................................ ................................ ................................ ..... 39 Selection ................................ ................................ ................................ ........... 40 Microscopy ................................ ................................ ................................ ....... 40 Immunoblot Analysi s ................................ ................................ ........................ 40 Functional Assays ................................ ................................ ................................ ... 41 Thiazolyl Blue Tetrazolium Bromide (MTT) Assay ................................ ............ 41 LDH Release Assay ................................ ................................ ......................... 42 Statistical Analysis ................................ ................................ ............................ 42 3 RESULT S ................................ ................................ ................................ ............... 43 Overexpression of SQSTM1 ................................ ................................ ............. 43 Effect of Oxidative Stress on Viability and Proliferation Activity of ARPE 19 cells ................................ ................................ ................................ ............... 43 SQSTM1 Expre ssion Does Not Protect ARPE 19 Cells From External Oxidative Stress ................................ ................................ ............................ 44 Overexpression of SQSTM1 Prevents Oxidative Stress Mediate d Toxicity as Measured by Release of Lactate Dehydrogenase ................................ .... 45 HO 1 mRNA Levels are Increased Upon Exposure to Oxidative Stressors ..... 45 Establishment of SQSTM1 Stable Cell Line ................................ ..................... 46 4 DISCUSSION AND CONCL USIONS ................................ ................................ ...... 65 REFERENCES ................................ ................................ ................................ .............. 69 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 73

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7 LIST OF TABLES Table page 2 1 Oligonucleotide primers used for mRNA level detection in RT PCR .................. 34 2 2 RT PCR conditions ................................ ................................ ............................. 34 2 3 List of antibodies utilized for immunoblot analysis ................................ .............. 36

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8 LIST OF FIGURES Figure page 1 1 Anatomy of the Retina.. ................................ ................................ ...................... 15 1 2 An illustration of the structure, function, and binding interactions of Sequestosome 1 (SQSTM1/p62).. ................................ ................................ ..... 25 2 1 Plasmids used for transfection of ARPE 19 cells. ................................ ............... 30 2 2 Plasmids used for lentiviral vector mediated delivery of SQSTM1 or GFP.. ....... 38 3 1 Immunoblot ana lysis of transfected ARPE 19 cells. ................................ ........... 48 3 2 ARPE 19 response to increasing concentrations of hydrogen peroxide.. ........... 49 3 3 ARPE 19 response to increasing concentrations of 4 hydroxynonenal.. ............ 50 3 4 Transfected ARPE ........................ 51 3 5 Transfected ARPE ....................... 5 2 3 6 Transfected ARPE ........................ 53 3 7 hydroxyn onenal on ARPE 19 cell viability.. ............................ 54 3 8 hydroxynonenal on ARPE 19 cell viability. ............................. 55 3 9 hydroxynonenal on ARPE 19 cell viability.. ............................ 56 3 10 Lactate dehydrogenase (LDH) release as a measure of cytotoxicity in ARPE ................................ .............. 57 3 11 Lactate dehydrogenase (LDH) release as a measure of cytotoxicity in ARPE hydroxynonenal.. ................................ ................. 58 3 12 hydrogen peroxide ................................ ................................ ............................. 59 3 13 peroxide.. ................................ ................................ ................................ ............ 60 3 14 hydroxynonenal. ................................ ................................ ................................ 61 3 15 hydroxynonenal.. ................................ ................................ ................................ 62

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9 3 16 Verification of GFP Expression in GFP stable cell line... ................................ .... 63 3 17 Immunoblot analysis depicting SQSTM1 or GFP levels of stable cell lines.. ...... 64

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10 LIST OF ABBREVIATIONS 4 HNE 4 H ydroxynonenal AD isease AMD Age Related Macular D egeneration AMP A mpicillin ARE Antioxidant Response E lement ATCC American Type Culture C ollection ATP Adenosine T riphosphate BSA Bovine Serum A lbumin CBA C hicken Beta A ct in cDNA C omplementary D NA CEP C arboxyethylpyrrole CMV IE Cytomegalovirus Immediate E arly cPPT Central Polypurine T ract DMEM erum DMSO Dimethyl S ulfoxide ER Endoplasmic R eticulum FDA Food and Drug D dministration FBS Fetal Bovine S erum GCL Ganglion Cell L ayer GFP Green Fluorescent P rotein GSTM1 Glutathione S Transferase M u 1 HEK Human Embryonic K idney HO 1 Heme O xygenase 1 INL Inner Nuclear L ayer

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11 KEAP1 Kelch like ECH Associated P rotein 1 KIR K eap 1 Interacting R egion LDH Lactate D ehydrogenase LV L entivirus MDA M alondialdehyde mtDNA Mitochondrial Deoxyribonucleic A cid MTT 3 (4,5 Dimethylthiazol 2 yl) 2,5 Diphenyltetrazolium B romide NAD(P)H Nicotinamide Adenine Dinucleotide P hosphate NQO1 NAD(P)H Q uino ne O xidoreductase NRF 2 Nuclear Factor E rythro id 2 Related F actor 2 ONL Outer Nuclear L ayer OPL Outer Plexiform L ayer PBS Phosphate Buffered Saline S olution POS Photoreceptor Outer S egments PuroR Puromycin R esistance ROS Reactive O xy gen S pecies RNS Reactive Nitrogen S pecies RRE Rev Resp onsive E lement RPE Retinal Pigment E pithelium RT PCR Real T ime PCR SQSTM1 S equestosome 1 SV 40 Simian V irus 40 TNF Tumor Necrosis F actor A lpha TRIS Tris H ydroxyethylaminomethane VEGF Vascular E ndothel ial Growth F actor

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12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science OVEREXPRESSION OF SEQUESTOSOME 1 AS A PROTECTIVE MEASURE AGAINST OXIDATIVE STRESS IN THE RETINAL PIGMENT EPITHELIUM By Danielle Lorna Liverpool August 2013 Chair: Alfred S. Lewin Major: Medical Sciences Translational Biotechnology Dry a ge related macular degeneration is a multifactorial disease of the retina that can lead to the severe impairment of or complete loss of vision. Due to the variety of etiological factors involved, this disease has been difficult to treat. It is currently understood that accumulation of damage caused by excess oxidative stress in the eye contributes to pathogenesis of this disease. Our aim in thi s study is to prevent oxidative stress mediated damage in retinal pigment epithelial cells by increasing the transcription of antioxidant enzymes controlled by the DNA sequence called the Antioxidant Response Element (ARE) We a ttempt to stimulate this response by increasing the abundance of Sequestosome 1 (SQSTM1) a protein known to mediate the regulation of Nrf2, a transcription factor for several cytoprotective enzymes. We transfected ARPE 19 cells, a cell line of established human retinal pigment epithelial cells, with a plasmid expressing SQSTM1. O verexpression of this protein was verified by immunoblot analysis. Cells were exposed to either hydrogen peroxide or 4 hydroxynonenal to induce oxidative stress. LDH release assay and MTT assays were performed to characterize cytotoxicity and mitochondrial dehydrogenase activity, respectively. mRNA

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13 levels of antioxidant enzymes were quantified by RT PCR methods. Surprisingly, we discovered that increasing SQSTM1 abundance in ARPE 19 cells did not result in significant protection of cell viability as measured by the MTT assay or an increase in cytoprotective enzyme production. However, cy to toxicity was reduced in SQSTM1 expressing cells. SQSTM1 has several functions in addition t o st imulation of the antioxidant response, including stimul ation of autophagy. To further understand the mechanism of cytoprotection by gene delivery of SQSTM1 a stable cell line of ARPE 19 cells that display s constitutive expression of SQSTM1 was established

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14 CHAPTER 1 INTRODUCTION The Mammalian Retina The eye is composed of multiple types of tissue each with its own function impact and anatomical characteristics The human eye is a conscious sense organ that permits the interpretation of motion, shape, contrast, color, and dimensions of objects by perceiving different wavelengths of light. This perception of light is required to initiate phototransduction. Photo transduction is the process by which photoreceptors absorb ph otons of light and transduce this information into an electrical response that can be relayed to the neural layers of the retina and ultimately, to the brain ( Arshavsky 2012). Diseases of the eye can result in skewed or decreased vision or even blindness, and is to describe the efforts to prevent the exacerbation of symptoms of a very common eye disease, dry age related macular degeneration (dry AMD) Anatomy of the Retina A depiction of the structural organization of the retina can be found in Figure 1 1. The retina, composed of two delicate layers of tissue, is the innermost layer of the three layers of tissue that line the wall of the eyeball. From anterior to posterior, these three layers of tissue are the retina, the vascular choroid, and the sclera. The neural retina and the retinal pigment epithelium (RPE) are the two layers that form the retina ( Khandhadia, 2012 )

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15 Figure 1 1. Anatomy of the Retina. A) A cross section of the eye globe. B) A cross section of the retina. The neural retina, which consists of the ONL, INL, GCL, and photoreceptors are located anterior to the RPE. C) Organization of the tissues in the pos terior of the eyecup. D) A light microscopic view of the layers of the retina displayed in (C). This figure is adapted from reference (Bird, 2010).

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16 The process of visual interpretation starts at the photoreceptors, where photons of light are absorbed by visual pigment and converted into an electrical signal that is relayed to bipolar cells in the outer plexiform layer (OPL ) (Kaneda, 2013) The process by which absorbed photons are converted into electrical signals is known as phototransduction. In vertebrates, this mechanism transpires in the photoreceptor outer segment. Briefly, photons of light instigate the isomerization and activated form of rhodopsin, which activates the G protein transducin. Transducin activates an enzyme that hydrolyzes c GMP, which closes cGMP gated channels and leads to hyperpolarization of the cell (Yau, 2009 ). The resulting electrical signal is processed and transmitted to retinal ganglion cells in the ganglion cell layer (GCL). During this step, visual information such as the color, contrast, brightness, and movement are perceived by amacrine cells and transferred to the optic nerve to be processed by the brain. Other cells in the retina such as amacrine cells and horizontal cells support signal modulation (Kaneda, 2013 ). Retinal Pigment Epithelium The RPE is a single monolayer of cuboidal pigmented epithelial cells that attach to each other via tight junctions. The RPE serves multiple functions in order to regulate retinal homeostasis (Barnett, 2012). Although separat ed fro m the membrane, it transports ions, water, and metabolic end products from the subretinal space to the blood. It extracts nutrients such as glucose, retinol, and fatty acids from the blood and delivers them to photoreceptors These cells also secrete growth factors that support the structural integrity of the photorecep tors and the surrounding retina. The RPE is in constant communication with photoreceptors as it re isomerizes molecules

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17 necessary for the visual cycle an d transports it to and from the photoreceptors The outer segments of photoreceptors are structured as a stack of discs that contain pigments necessary for the visual cycle to occur. These discs are shed at the distal end of the segment primarily with the first light of the morning and, importantly, phagocytosed by the RPE (Strauss, 2005). Undegraded material from this process can accumulate within RPE cells with age (Bird, 2010) quality control mechanisms can lead to the accumulation of lipofuscin granules that increases with aging ( Rodrg uez Muela, 2013; Krohne, 2010). Failure of the RPE to perform its innate functions lead to deterioration of other cells in the retin a (Bird, 2010). Age Related Macular Degeneration Compromised function of the eye is a co mmon trait in aging populations. A plethora of ocular diseases such as glaucoma, diabetic retinopathy, and age related macular degeneration threaten the eyesight of the elderly population. Glaucoma a degenerative optic neuropath y is strongly correlated with increased intraocular pressure (Tezel, 2011). In the case of d iabetic retinopathy the structure and function of the retinal vasculature is compromised by persistent high glucose levels (Santos, 2012). Age related macular deg eneration (AMD) is a disease of the retina that leads t o the distortion or loss of central vision AMD is currently the leading cause of irreversible vision loss in the elderly in industrialized countries (Voleti, 2013). Epidemiology There are two related forms of AMD : exudative and non exudative or atrophic. blood vessels that invade the space below the retina or the retina itself. This formation of blood vessels termed choroidal neovasculariz ation, can disrupt the structural

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18 integrity of the retina, choroid, and B The majority of the many of the complications associa ted with choroidal neovasculariz ation. There are currently 8 million people in the United States (Klein, 2011) and more than 50 million people worldwide that are afflicted with age related macular degeneration (Barnett, 2012). It is expected that ap proximately two thirds of the population over the age of 80 will have some signs of AMD (Khandadi a, 2012). Although the majority of the diagnoses are patients with dry AMD, 90% of severe vision loss can be attributed to patients with wet AMD. In wet AMD, blood vessels can grow from beneath the retina toward the macula. The combination of inflammation, angiogenesis, and tissue invasion within the eye globe lead to rapid onset of disease and loss of vision in wet AMD patients (Campa, 2010). Both environmen tal and genetic factors contribute to the pathogenesis of this disease. Age and smoking are two of th e highest risk factors for AMD. The cell types most affected include photoreceptors, the RPE, Bru Genetic studies have concl uded that misregulation of the immune system can serve as a contributing factor in AMD (Barnett 2012). Role of Oxidative Stress in AMD Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in several processes that are vital for proper cellular function. Cellular function can be disrupted in the event that there is a surplus or deficit of ROS in the cell. Oxidative stress is the state in wh ich the amount of ROS present in the cell is greater than what the cell can effici ently detoxify (Halliwell, 2006). ROS are relatively short lived within the cellular environment but damage caused by these species can

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19 accumulate. All ROS in the ce ll are not eliminated because ROS perform important functions. In the event that the amoun t of ROS is greater than what the cell is equipped to keep at bay, the cell will mount defenses such as antioxidants to eliminate these species and minimize damage (Halliwell, 2006). Enzymes such as superoxide dismutases and catalases in addition to other antioxidant and cytoprotective factors demonstrate the ability to reduce or neutralize ROS levels in the cell. Glutathione (GSH) neutralizes lipid peroxides and H 2 O 2 Melanin is a pigment molecule that offers tocopherol and ascorbate also contribute to the maintenance of cellular redox homeostasis (Plafker, 2012). Oxidative stress can originate from either exogenous or endogenous sources. Exogenous stressors, such as cigarette smoke, can instigate the production of chemical oxidants that will modify existing proteins, DNA, and lipids in the retina (Barnett, 2012) These oxidation events result in the formation of reactive molecules and advanced glycation end products that can cause damage to the sensitive tissue. Some of the molecules that have been found as a result of these incidences are 4 hydroxynonenal (4 HNE), mal ondialdehyde (MDA), and carboxy ethylpyrrole (CEP ). Proper regulation of the environmental balance of the retina is achieved by crosstalk between various element s of the eye. The retina has the richest oxygen demand by weight of any organ in the body. The RPE is situated in a location in the eye that makes it very suscep tible to oxidative stress (Barnett, 2012). Role of Mitochondrial Function in AMD The mitochond rion is an organelle necessary for proper cellular function and oxidative phosphorylation is the process by which mitochondria oxidize nutrients to form

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20 adenosine tripho sphate (ATP), a molecule that suppl ies energy for metabolic events Mitochondria function in fatty acid oxidation, amino acid and heme biosynthesis, antioxidant regulation, apoptosis, and cytoprotection ( Olszewska 2013). Mitochondrial decay is a normal result of cellular aging. Increased chronological age results in increased damage to mtDNA, reduced activity of the enzymes that perform in the respiratory chain, ROS imbalance, and decreased ability to defend against oxidat ive stress (Wenzel, 2008). There is growing evidence that confirms that mitochondrial impairment and the absence of effective mtDNA repair can contribute to pathogenesis in degenerative retinal diseases and diseases related to aging (Jarrett, 2010). Mutati ons in mitochondrial DNA may lead to severe congenital defects affecting the endocrine system the central nervous system, the heart and the musculoskeletal system (Koopman, 2007). More commonly, however, mitochondrial defects have been associated with neu rodegenerative diseases such as Parkinson disease and Alzheimer dise ase (AD) Specifically, in AD, dysfunction of the ETC, increased oxidative stress, subcellular fractionation, misregulation of Ca 2+ content, mtDNA mutations, and an impair ed balance of fusion and fission are some of the factors that contribute to pathogenesis. Redistribution of mitochondria is also noted in the AD afflicted hippocampus and neurons (DuBoff, 2013). The uniformity and integrity of the mitochondria shape, size and functio n tivity and state of replication Alterations in lipid peroxidation, cytoplasmid superoxide dismutases, and oxidative stress marker s have been observed in aging cells (He, 2010). Importantly, cellular ROS content can serve as a determinant of mitochondrial shape and function (Koopman, 2007).

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21 Cellular stress may serve as the impetus for changes in mitochondrial functions. Endoplasmic reticulum (ER) stress leads to misregulation of respiratory activities and activates apoptotic pathways in the mitochondria (Kwong, 2007). Excess oxidative stress inhibits proper regulation of oxidative phosphorylation and causes mitochondrial fragmentation (Koopman, 2007). Due to the close proximity of mitochondrial DNA (mtDNA) to the oxy gen radical producing respiratory chain, mtDNA is more vulnerable to oxidative damage than nuclear DNA ( Olszewska 2012). In the event that the homeostatic state of mitochondria is disrupted, ATP synthesis is disturbed, which can lead to the production o f excess ROS and proteins that participate in cell death pathways, such as apoptosis, autophagy, or mitotic catastrophe (Blasiak, 2013). Cells are enabled with a defense mechanism that sequesters and degrades dysfunctional mitochondria before it causes cel l death. The process by which mitochondria are selectively sequestered and targeted for destruction is known as mitophagy. It is possible for mitochondria to activate b oth cell death and mitophagy, two different processes, in response to th e same stimulus. (Kubli, 2012). Overall, the state of mitochondrial health can greatly affect overall cellular viability. Nuclear factor erythroid 2 related factor 2 Normal aerobic metabolic activity results in the production of ROS in unstressed conditions. In order to control the redox status of the cell and to eliminate ROS that are not needed for proper cellular function, a cis acting element termed the Antioxidant Response Element (ARE) is transcribed to produce cytoprotective enzymes that detect and eliminate excess ROS (Nguyen 2009). of phase II detoxification enzymes (Liu, 2007). The ARE is activated in response to reactive oxygen species and other electrophilic compounds that have the ability to

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22 undergo redox cycling (Ng uyen, 2009), a series of chemical reactions that result in the production of the reac tive species (Gutierrez, 2000). The prominence of the ARE in maintaining endogenous redox has been demonstrated in several studies that conclude that nrf2 / mice lack the ability to mount a response to oxidative stress via ARE controlled genes (Nguyen, 2009). Glutathione S transferases (GST ), heme oxygenase 1 (HO 1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) are some genes that are activated by the ARE (Li u, 2007) The transcription factor nuclear factor erythroid 2 related factor 2 (Nrf2) is a leucine zipper transcription factor that regulates the expression of a number of genes that are transcribed in response to electrophiles, pro oxidants, ROS, or RNS Nrf2 is regulated in part by the cytosolic metallo protein Kelch Li ke ECH associated protein 1 (Keap 1) This interaction is depicted in Figure 1 2. The Neh2 domain of Nrf2 interacts with the Kelch repeat domain of Keap 1. This interaction is disrupted in r esponse to r e active species in the cell. This occurrence is the impetus for Nrf2 translocation to the nucle u s, where it binds to the ARE and promotes transcription of the antioxidant regulators mentioned previously. The activation of the Nrf2 antioxidant a nd cellular stress (Tew, 2011). Sequestosome 1/p62 Sequestosome 1 (SQSTM1), or p62 is a 65 kDa protein located in both the cytoplasm and the nucleus in order to participa te in protein trafficking and ubiquitination events. SQSTM1 participates in the mediation of several signaling pathways In addition to its role in the nucleus as a transcriptional co activator, SQSTM1 serves as a modifier of the K + 8 pathways, and a molecular bridge in the

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23 process of selectively activating NF The UBA domain in the C terminus of SQSTM1 has an affinity for polyubiquitinated proteins and serves to recruit and bind ubiquitinated prot eins (Fig. 1 2) (Geetha, 2002) 1 interacting region (KIR) motif binds to the Kelch repeat domain of KEAP1, the location of Nrf2/Keap1 binding. SQSTM1 serves as a competitor of Nrf2 binding, which promotes free cytosolic Nrf2 (Ne zis, 2012). In addition to having multiple binding partners that participate in different cellular pathways, SQSTM1 has the unique ability to homodimerize and polymerize itself This conformation, however, does not prevent the binding of SQSTM1 to Nrf2 or KEAP1 (Nezis, 2012). Interestingly, SQSTM1 has an ARE DNA element in its promoter, permitting positive induction of SQSTM1 transcription via Nrf2. Therefore, Nrf2 and SQSTM1 are in a positive feedback loop, where Nrf2 increases SQSTM1 production and SQSTM 1 encourages nuclear translocation of Nrf2 ( Jain, 2010 ). SQSTM1 plays a crucial role as a cargo receptor in the regulation of autophagy. Autophagy is a process that is obligatory for cell survival, maintenance, and differentiation. This process involves t he collection of cytosolic contents into a double membrane vacuole to form an autophagosome. The autophagosome then fuses with and releases its contents into lysosomes, where acid hydrolases degrade the cellular material (Fan, 2010). In the event that prot eins for potentially harmful aggregates or are misfolded, ubiquitin molecules are added to the structures to target them for degradation. SQTSM1 recognizes mono and polyubiquitin ated structures via its UBA domain and assists in delivery of the targeted as semblies to autophagic machinery Inhibition of autophagy results in the accumulation of ubiquitinated protein aggregates,

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24 which can be a source of cellular toxicity (Schreiber, 2013). Through polymerization of its PB1 domain, SQSTM1 is recruited to mitoch ondrial substrates by the E3 ubiquitin ligase Parkin, which ubiquitinates specific protein substrates on damaged mitochondria. SQSTM1 then serves to mediate aggregation of ubiquitinated mitochondria for targeted destruction, similar to its role in the aggr ega tion of ubiquitinated proteins. (Narendra, 2010). Recruitment of SQSTM1 alone is not sufficient for induction of mitophagy, but it has been reported that knockdown of SQSTM1 substantially inhibits mitophagy (Kubli, 2012). SQSTM1 (here also referred to as p62) forms protein bodies or p62 bodies, that consist of accumulations of SQSTM1 and ubiquitinated proteins. p62 bodies either reside in the cytosol, nucleus or are present within autophag osomes and lysosomal structures. In ord er for formation of p 62 bodies, the UBA domain of SQSTM1 must be present and unbound to other proteins ( Bjrky 2005). The PB1 domain of SQSTM1 is necessary for SQSTM1 to interact with itself. These protein bodies are found both as membrane free protein a ggregates (sequestosomes) and as membrane confined autophagosomal and lysosomal structures ( Bjrky 2005) SQSTM1, as a protein, is involved in numero us pathways that arbit rate regulation of cellular function Although it is primarily regarded as a cyto solic protein, it contains both a nuclear localization signal (NLS) and nuclear export signal to facilitate transport between the two compartments (Pankiv, 2010). The interplay between the various pathways in both homeostatic and perturbed conditions is of great interest in relation to RPE function in AMD.

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25 Figure 1 2. An illustration of the structure, function, and binding interactions of Sequestosome 1 (SQSTM1/p62). A) Sequestosome 1 acts as a scaffolding protein that serves in many pathways. The diff erent domains of SQSTM1 are responsible for mediating its activity in different pathways associated with cell survival, protein trafficking, and proliferation. B) The Keap 1 Interacting region (KIR) in the C terminus of SQSTM1 binds to the Kelch repeat dom ain of KEAP1. This same domain regulates abundance of cytosolic Nrf2 by binding to its Neh2 domain in the N terminus. This figure was adapted from reference (Nezis, 2012). Treatments for AMD Although treatments approved by the U.S. Food and Drug Admini stration (FDA) have been identified and are currently being utilized for exudative AMD, no FDA

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26 approved treatments or preventative measures for non exudative AMD exist (Voleti, 2013). Vascular endothelial growth factor (VEGF) is a circulating serum cytoki ne that promotes angiogenesis and vascular permeability. Inhibition of VEGF activity has been the foremost method for treatment of choroidal neovascularization This is being achieved through use of antibody fragments that bind to all VEGF isoforms ( Ranibi zumab ), RNA aptamers that inhibit VEGF from binding to its receptor ( Pegaptanib ), and receptor decoys that trap VEGF ( Aflibercept ) ( Cheung, 2013). A more thorough understanding of the pathogenesis of t he disease is needed before eff ective therapeutic treat ments can be developed. Specific Background and Objectives: After exposure to oxidative or ine residues undergo oxidation, leading to a conformational change in the protein. This change inhibits KEAP1 from serving as a substrate ubiquitin ligase adaptor, therefore discontinuing the targeted destruction of Nrf2. This sequence of events leads to t he increased translocation of Nrf2 from the cytosol to the nucleus, where it can then increase transcription of ARE enzymes (Jain, 2010). Several studies have demonstrated that the activation of the ARE by manipulation of the Nrf2/Keap1 pathway have facil itated in vitro protection from oxidative stress ( Reuland, 2012; Ha 2006 ). A 48 hour transfection of SQSTM1 encoding cDNA into primary cortical neurons doubled the amount of nuclear Nrf2 protein levels and increased ARE cDNA levels (Liu, 2007). With the r esults of these experiments in mind, the goal of this project is to determine if overexpression of SQSTM1 can protect retinal pigment epithelial cells from oxidative stress. To

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27 accomplish this, I proposed the following aims: I) To achieve increased express ion of SQSTM1 in ARPE 19 cells using transfection based methods II) To determine the impact of varying concentrations of hydrogen peroxide or 4 hydroxynonenal on cell survival III) To assess the ability of SQSTM1 to mediate protection of ARPE 19 cells ex posed to oxidative stress. IV) To measure the effect of SQSTM1 overexpression on regulation of ARE antioxidant enzymes.

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28 CHAPTER 2 MATERIALS AND METHODS DNA Techniques Preparation of Pla smid DNA The pTR SB smCBA SQSTM1 plasmid DNA ( Fig. 2 1 ) was a gift from Dr. Diego Fajardo The pTR smCBA h GFP plasmid DNA ( Fig. 2 1 ) was graciously supplied by Dr. Cristhian Ildefonso Both plasmids contain a cytomegalovirus immediate early enhancer (CMV IE) coupled to a stro ng and cons t itutively active chicken beta actin promoter to maximize gene expression. The ampicillin resistance gene (AmpR) was used as a marker for selective propagation of bacteria. The vector includes a chimeric intron to increase transgene expression and a bacteriophage f1(+) origin of replication To ensure fidelity of the plasmid DNA, sequencing of the DNA was executed by the DNA Sequencing Core Facility, Interdisciplinary Center for Biotechnology R esearch (ICBR) at the University of Florida. Transformation E. c oli SURE (Invitrogen) cells were used for the transformation of the plasmids used in downstream experiments. Electroporation was performed at 1.5 volts for 5 msec in chilled 1mm pla stic medium ( Life Technologies) by incubating at 37 C for 1 hour in a shaking incubator. Recovered cultures were spread on Luria Bertani Broth (LB) agar plates containing 100 ampicillin (amp) to select for transformed cells. Plates were incubated overnight (12 16 hours) at 30 C.

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29 Plasmid Maxi Preparation Colonies containing a single clone were selected after the overnight incubation. Individual clones were chosen fr om the agar plates and 50 mL flasks containing 5 mL LB and ampicillin were inoculated with a single colony of transformed bacteria. Samples were incubated in a shaking water bath at 37 C for approximately 8 hours. The samples were then added to 145 mL LB containing ampicillin and incubated 37 C overnight in a shaking water bath. Plasmid DNA was isolated using the Plasmid Maxi Kit (Omega Bio Tek). Purif i ed plasmid DNA was eluted in sterile, double disti lled water and sto r e d at 20C.

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30 A B Figure 2 1 Plasmids used for transfection of ARPE 19 cells. A) Plasmid map of pTR SB smCBA SQSMT1 B) Plasmid map of control vector pTR SB smCBA hGFP. Plasmids were introdu ced into ARPE 19 cells using NovaFector transfection reagent (Venn Nova). The Cytomegalovirus immediate early (CMV IE) enhancer and the chicken beta actin promoter were included to increase gene expression. TR: terminal repeat, SV40: Simian virus 40 poly (A) tail; AmpR: Ampicillin resistance gene; SB: simple basic.

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31 Tissue Culture Techniques This section describes the general maintenance and specific techniques employed while using an established human cell line of retinal pigment epithelial cells. The cell line, ARPE 19, is a spontaneously arising human RPE cell li ne with normal karyology that forms a polarized monolayer in vitro (Dunn, 1996). Cell Culture ARPE 19 cells were obtained from American Type Culture Collection ( ATCC ) at ampule passage number 19 and immedia tely reconstituted according to the supplier protocol. 12 (DMEM/F 12), supplemented with 10% fetal bovine serum ( G IBCO) (FBS) 100 glutamine. This mixture will b e referr Cells were incubated at 37 C with 5% CO 2 until 85% confluence. All experiments were performed with cells between passages 21 and 41 Transfections ARPE 19 cells were counted by trypan blue exclusion using a hemacytometer. De pending on the assay that was going to be performed, cells were seeded in tissue culture treated plates ( Genesee Scientific ) with the appropriate number of wells and incubated overnight for 12 16 hours in complete DMEM/F 12 to allow them to attach to the p late. After the overnight incubation, the plasmid DNA was diluted in Opti MEM reduced serum medium (Invitrogen). NovaFECTOR (Venn Nova), the transfection reagent, was added to the plasmid DNA at 4 of plasmid DNA. Solutions were prepare d according to the number of cells that were seeded. Dilutions of

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32 The DNA and reagent were incubated in Opti MEM (Invitrogen) at room temperature for 30 minutes to allow the reagent to form a complex with the DNA. Cells were washed with phosphate buffered saline ( PBS ) (GIBCO) (1.06 mM KH 2 PO 4 155.17mM NaCl, 2.97 mM Na 2 HPO 4 7H 2 O ) to remove a ny residual serum from the medium that may lower transfection efficiency. The cells were incubated with the DNA and transfection reagent mixture for 6 hours at 37 C. The mixture was removed and the cells were incubated in DMEM/F 12 for an additional 42 hours to accomplish a 48 hour transfectio n. Induction of Oxidative Stress Hydrogen p eroxide ( Sigma) (H 2 O 2 ) was used t o induce oxidative stress to the cells. The solution was always prepared fresh by diluting stock H 2 O 2 in Opti MEM to At the end of the 48 hour transfection, cells were washed once wi th PBS to remove lingering medium Samples were incubated with the freshly prepared solution containing H 2 O 2 for six hours at 37 C with 5% CO 2 Stoc k H 2 O 2 was st ored at 4C and in the dark to p revent decomposition of the solution. In a similar manner, 4 Hydroxynonenal (Calbiochem) (4HNE) was diluted in PBS to final concentrations of 5 prepared during the last hour of the 48 hour transfection an d added to the cells immediately following the end of the transfection period Samples were incubated with the diluted 4HN E solutions for six hours at 37 C with 5% CO 2 Stock solutions of 4HNE were stored at 80C to preserve chemical activity.

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33 RNA Techniques RNA Extraction RNA was extracted from samples after transfection and induction of oxidative protocol. RNA was eluted in 50 L sterile double distilled water. S amples were quantified by spectrophotometry by reading the OD 260 The purity of the RNA was determined by analyzing the OD 260 /OD 280 ratio. Real Time PCR Real Time PCR ( RT PCR ) was a technique used to quantify the production of mRNA for different antioxidant enzymes after stimulation of ARPE 19 cells by oxidative stressors. From the purified RNA, first strand cDNA synthesis sample was executed using the iScript cDNA synthesis kit (Bio Rad). Reactions were completed in a fi sample. Primer sequences are listed in T able 2 1. The total volume for each reaction loaded in a 96 well plate sealed, and centrifuged at 7,600 x g for 2 minutes. The reaction mixture was then subjected to optimized reaction conditions listed in T able 2 2 using the CFX96 Touch RT PCR Detection system (Bio Rad). Gene expression was extrapolated from the data after analyzing with CFX Manager Software ( Bio Rad).

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34 Table 2 1 Oligonucleotide p rimers used for mRNA level detection in RT PCR Primer Orientation Sequence GAPDH GAPDH Sense GAG TCA ACG GAT TTG GTC GT Antisense TTG ATT TTG GAG GGA TCT CG GSTM1 GSTM1 Sense ATG CCC ATG ATA CTG GGG TA 3 Antisense GTG AGC CCC ATC AAT CAA GT HO 1 HO 1 Sense ACA TCT ATG TGG CCC TGG AG Antisense CGC TTC ACA TAG TGC TGC AT NQO1 NQO1 Sense AAA GGA CCC TTC CGG AGT AA Antisense Table 2 2 RT PCR conditions Step Temperature Duration 1 95.0 C 3:00 2 95.0 C 0:10 3 60.0 C 0:20 4 Go To Step 2 39 more times N/A 5 95.0 C 0:30 6 55.0 C 0:30 7 Melt curve 60 C to 95 C increment 0.5 C 0:50 8 4.0 C Indefinite 9 END N/A Protein Techniques Protein Extraction from cells ARPE 19 cel ls were rinsed with sterile PBS to remove any residual medium The cells were trypsinized and collected in a 1.5 mL microcentrifuge tube. The cells were centrifuged at 16,00 x g for 1 minute at room t emperature in an E ppendorf 5415 C centrifuge. The trypsin was discarded. Pe llets were washed once with PBS to remove remaining trypsin and centrifuged for 5 minutes at 16,000 x g at room temperature. The wash buffer was discarded. Pel lets were stored at 80C until they were ready to be utilized in later experiments. Cell pellets were removed from 80C, placed on ice, and resuspended in 2 L RIPA buffer containing 1% Protease Inhibitor ( Sigma Aldrich ) and 1% 0.5 M EDTA. Samples were mixed by vortexing every 10 minutes for 30 minutes to complete cell

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35 lysis. Samples w ere centrifuged at 1400 x g for 15 minutes at 4C to pellet extracellular material. Supernatant was collected and transferred t o a 1.5 mL microfuge tu be. 120 of the supernatant freshly prepared Laemmli sample buffer (Laemmli, 1970) and heate d at 97 C for 5 minutes to denature an d dissociate proteins Protein Quantitation Protein concentratio n was determined using a DC Protein Color imetric Assay (Bio Rad). Samples of cell supernatant were loaded in trip licate onto an optically clear 96 well plate. A standard curve using known concentrations of b ovine serum albumin (BSA) (Promega) was generated to determine the sample protein concentr ation. The standard curve ranged from 0.30 mg/mL to 1.5 mg/mL of BSA diluted in RIPA buffer Absorbance value s were read at 750 nm after the required incubation period. Protein Extraction and Immunoblot Analysis Immunoblot analysis was performed using Tri s HC l ready gels containing 10% polyacrylamide (Bio Rad). 20 run at approximately 250 Volts for 2 hours. Prot m p olyvinylidene fluoride membrane using an iBlot gel transfer device (Invitrogen). The protein transfer was performed for 7 minutes. Blocking of Membrane and Application of Antibodies Membranes were incubated in methanol for 5 minutes at room temperature prior to blocking. Membr anes were then blocked in blocking buffer (Odyssey) for 30 minutes, shaking, at room temperature. Primary antibodies were diluted in fresh blocking buffer using the dilution scheme displayed in Table 2 3 Primary antibodies were applied overnight by incub ating the membranes at 4C, shaking, for 12 16 hours. Mem branes were then washed with P BS + 0.1% Tween 20 (Fisher Scientific ) (PBS/Tween) three

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36 times for 5 minutes. Secondary antibodies were diluted in PBS/Tween as described in Table 3 3 Membranes were incubated with infrared labeled secondar y antibodies for 1 hour, shaking, at room temperature. Imaging was performed using a quantitative fluorescence imaging system at the appropriate wavelengths. Image s of the bands were digitized and de n sitometric analy sis was performed using ImageJ. Table 2 3 List of antibodies utilized for immunoblot analysis Antibody Vendor Dilution Mouse Anti p62 Lck ligand BD Biosciences 1:4000 Rabbit Anti GFP Invitrogen 1:5000 Rabbit Anti Actin Odyssey 1:5000 Goat Anti Mouse Odyssey 1:5000 Goat Anti Rabbit Odyssey 1:5000 Creation of Lentivirus transduced Stable Cell Lines Transformation DH5 ith the plasmids shown in Fig. 3 3. Expression of SQSTM1 and GFP were under the contr ol of the elongation factor 1 alpha (EF1) promoter The HIV 1 l ong terminal repeat (LTR) served as part of the prom oter but also allowed the lentivirus vector to integrate into the genome of both dividing a nd non divid i ng cells (Moldt, 2011). A T2A reporter peptide gene was included to track cells that were actively expressing the gene of interest. The puromycin resistance gene allowed for selection of transduced cells that were expressing the gene of intere st. The ampicillin resistance (AmpR) gene and pUC origin

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37 of replication allow for selection and propagation in bacteria. DH5 aliquots were subjected to heat shock at 42C for 1.5 minutes and immediately placed on ice for 2 Life Technologies) by incubating at 37 C for 1 hour in a shaking incubator. Recovered cultures were spread on Luria Bertani Broth (LB) agar pl ates containing ampicillin (amp) to select for transformed cells. Plates were incubated overnight (12 16 hours) at 30 C. 10 mL LB broth containing ampicillin were inoculated with one transformed colony and incubated overnight at 37 C. Plasmid DNA extraction from bacterial cells was performed using the PureLink Quick Plasmid Miniprep Kit (Invitrogen) according to

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38 Figure 2 2 Plasmids used for lentiviral vector mediated delivery of SQSTM1 or GFP. The Puromycin resistance ( PuroR ) sequence allows for selection of ARPE 19 cells that have incorporated the plasmid of interest. RSV: Rous sarcoma virus promoter; LTR: long terminal repeat; RRE : rev responsive element; WPRE: woodchuck hepatitis posttranscriptional regulatory element; cPPT: central polypurine tract; EF1: elongation factor 1 alpha promoter

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39 Packaging of Virus Human Embryonic Kidney 293T (HEK293T ) cells were seeded at 3x10 6 cells per well in 10 cm plates containi ng complete medium and incubated overnight at 37 C with 5% CO 2 After the overni ght incubation, the growth medium was replaced with 9 mL of transfection medium (DMEM/F 12 supplemented with 10% FBS) In a microcentrifuge tu EF1 hGFP T2A Puro plasmid DNA or pCDH EF1 SQSTM1 T2A Puro plasmid DNA was Mem. Twenty microliters of pPACK HI packaging plasmid mix (Systems Biosciences) was added to each mixture and incubated at room t emperature for 15 minutes. In a separate microcentrifuge tube, Mem. The diluted Lipofectamine solution was added dropwise to the diluted DNA mixture and mixed by gentle pipetting. The transfection mixture of Lipofectamine and DNA was incubated at room temperature for 15 minutes to allow the reagent to form a complex with the DNA. The complexed DNA was then added dropwise to the HEK293T cells. The HEK293T cells were incubated with t he DNA/Lipofectamine complex at 37 C with 5% CO 2 for 48 hours. After the 48 hour transfection period, the cell supernatant was harvested and centrifuged at 1,811 x g at room temperature for 5 minutes to pellet cellular debris that still remained in the m edium The supernatant containing the viral particles was nylon syringe filter (Fisher Scientific) and stored in 1 mL aliquots at 80C. Transduction 1 x 10 6 ARPE 19 cells were seeded in T 25 flasks in DMEM/F 12 ( supplemented with 10% FBS, 1 00 U/mL penicill and incubated

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40 overnight to permit attachment to the flask. Five hundred microliters of v iral supernatant was recovered from 80C and added to the cells following the overnight incubation. The cells were incubated with the sterilized supernatant for 48 hours at 37 C with 5% CO 2 Selection In order to ensure that cells expressed the plasmid of interest cells were incubated with puromycin to exterminate cells that did not have the DNA incorpor ated into the cellular genome. Transduced ARPE 19 c ells were rinsed with PBS. Cells were then inc ubated in complete medium Life Technologies ) for 72 hours. Wild type ARPE 19 cells of the same passage were included as a negative control. The negative control was in cubated with this complete medium supplemented with puromycin to ensure destruction of all cells that did not contain the plasmid DNA. Microscopy Fluorescence microscopy was performed to characterize expressio n of GFP in the GFP stable cell line. A Keyence BZ 9000 inverted fluorescent microscope was used to obtain i mages of the cells Images of live cells in complete m edium were obtained using a 1 0X objective and analyzed using BZ II Analyzer software (Keyence) Immunoblot Analysis While the stable cells were trypsinized during routine passaging, a liquots of 1 x 10 5 stable cells were collected in microcentrifuge tubes and pell eted by centrifugation at 7,200 x g for 2 minutes. Ce ll pellets were rinsed with P BS and centrifuged again at the same conditions. buffer containing 1% Protease Inhibitor ( Sigma Aldrich) and 1% 0.5 M EDTA. Samples

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41 were vortexed every 10 minutes for 30 minutes to complete cell lysis. Samples w ere centrifuged at 1400 G for 15 minutes at 4C to pellet extracellular material. Supernatant was collected and transferred to a 1.5 mL microfuge tube. 120 heated at 97C for 5 minutes to denature and dissociate proteins. Protein concentration was determined using a DC Protein Colorimetric Assay (Bio Rad). Functional Assays Thiazolyl Blue Tetra zolium Bromide ( MTT ) Assay In order to determine cellular metabo lic activity, a functional assay that utilizes (3 (4,5 Dimethylthiazol 2 yl) 2,5 diphenyltetrazolium bromide (MTT) (Sigma) was performed. MTT is a tetrazolium dye that is reduced to formazan by mitochondrial dehydrogenases. ARPE 19 cells were seeded at 30, 000 cells/well in a 96 well tissue culture treated plate (Olympus) and transfected according to the steps previously described. The cells were subjected to oxidative stress by adding either H 2 O 2 or 4HNE diluted in the appropriate solvent for 6 hours. MTT p owder was diluted in RPMI 1640 (Invitrogen) medium without phenol red to a final concentration of 5 mg/mL to create the stock solution. MTT stock solution was filter (Fisher Scientific ) and added to RPMI 1640 to a final volume of one tenth of the culture dimethyl sulfoxide (DMSO) ( Fisher Scient ific ). Absorbances were read at 750 nm using a spectrophotometer.

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42 LDH Release Assay Plasma membrane damage was assessed by quantifying the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) from cells after insult (Kozeniewski and Callewaert, 1983) An LDH release cytotoxicity kit II (Abcam) was used according medium. Statistical Analysis Statistical analysis was performed by analysis of variance and Bonferroni post test. P <0.05 was considered statistically significant.

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43 CHAPTER 3 RESULTS Overexpression of SQSTM1 E xpression of SQSTM1 and GFP in ARPE 19 cells was achieved using transfection techniques. Immunoblot analysis displayed increase of protein expression 3 1). Similarly, increasing transfecting DNA from 1.5 to 2.5 g did not appear to lead to increased production of GFP. Expression levels of the respective proteins varied between three biological replicates (data not shown), displaying inconsistency in transfection efficiency. It is unknown whether th e source of the incons istency wa s the transfection reagent or a physiologic characte ristic of the ARPE 19 cell line. Western blots were sensitive enough to detect basal levels of SQSTM1 (see no DNA control lane and pTR smCBA GFP transfection lanes) Effect of Oxidative Stress on Viability and Proliferation Activity of ARPE 19 cells To c onstruct a dose response curve, untransfected ARPE 19 cells were exposed to increasing concentrations of hydrogen peroxide or 4 hydroxynonenal for 6 hours. The v iability of ARPE 19 cells in response to increasing amounts of oxid ative stress was measured us ing the MTT assay, which reflects the activity of NADH or NADPH redox enzymes inside cells. Samples of ARPE 19 cells without stressor were incl uded as a negative control. Abso rbance values of treated samples were compared to the untreated, untransfected co ntrol cells. ARPE 19 cells appeared to be resistant to treatment with lo wer levels of hydrogen peroxide. There is no significant difference in 2 O 2 compared to the control (Fig. 3 2) Only 60% of the cells were still viable after 6 hour s of

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44 peroxide Increasing hydrogen peroxide levels further led to an additional loss of cell viability by this measure. Treatment with 4 hydroxynonenal (4 HNE) led to a steeper threshold in viability ARPE 19 cells were insensitive to 5 M, 10 M, and 20 M, but MTT reduction was reduced by 75% at 40 M and by 80 M only 10% of the reduci ng activi ty was detected (Fig. 3 3). Fig 3 2 and 3 3 display the mean of three independent experiments, each inclu ding samples performed in triplicate. SQSTM1 Express ion Does Not Protect ARPE 19 Cells From External Oxidative Stress To determine if increased expression of SQSTM1 could protect ARPE 19 cell from oxidative stres s, cells were transfected with 0.5 to 2.5 g of pTR SB smCBA SQSMT1 or pTR SB smCBA GFP, and 48 hours later they were exposed to increasing levels of H 2 O 2 ranging from 200 to 600 M for 6 hours. Following this, cell viability was measured using the MTT as say as described (Fig. 3 4 3 6 ). No sign ificant difference was seen in viability of cells expressing SQTSM1 in comparison to untransfected cells or cells expressing GFP. Similarly, to determine if increased expression of SQSTM1 could protect ARPE 19 cell from oxidative stress arising from 4 HN E, cells were transfected with 0.5 to 2.5 g of pTR SB smCBA SQSMT1 or pTR SB smCBA GFP and 48 hours later they were exposed to 4 HNE levels ranging from 20 60 M for 6 hours Then cell viability was measured with the MTT assay (Fig. 3 7 3 9 ) As for oxidat ive stress induced by hydrogen peroxide, increased expression of SQSTM1 did not lead to increased MTT reduction relative to untransfected cells or cells transfected with the GFP plasmid.

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45 Overexpression of SQSTM1 Prevents Oxidative Stress Mediated Toxicity as Measured by Release of Lactate Dehydrogenase Because the MTT assay depends on the activity of NADH or NADPH linked dehydrogenase enzymes, we also used an independent assessment of cytotoxicity of hydrogen peroxide and 4 HNE: release of lactate dehydrog enase ( LDH ) Lact ate dehydrogenase is a cytoplas mic enzyme that interconverts lactate and pyruvate in the cells. Its release from the cytoplasm is widely used as a measure of cell integrity. Assessment of cell membrane in tegrity after 6 hours oxidative stress with H 2 O 2 was achieved by measuring levels of lactate dehydrogenase released into the c ulture medium (Fig. 3 10 ). A s i g nificant difference was seen between GFP and SQSTM1 hydrogen peroxide. This result suggests t hat overexpression of SQSTM1 may b e able to prevent cytotoxicity related to oxidative stress in human RPE cells. Similarly, a significant increase in cytotoxicity was prevented in cells expressing SQSTM1 in comparison to the untransfected cells that were e xposed to 4 hy droxynonenal for 6 hours (Fig. 3 11 ). However, there was no significant difference in cells that were expressing SQSTM1 in comparison to the GFP expressing control cells. HO 1 mRNA Levels are Increased Upon Exposure to Oxidative Stressors Re al time polymerase chain reaction ( RT PCR ) experiments were performed to determine if when exposed to oxidative stress, induction of ARE enzymes in ARPE 19 cells is increased in cells overexpressing SQSTM1 (Fig 3 12 3 15 ). ARPE 19 cells were transfected with 0.5 g of pTR SB smCBA SQSMT1 or pTR SB smCBA GFP for

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46 4 hydroxynonenal for 6 hours. Cells treated with the corresponding stressor and u nstr essed ARPE 19 cells and were included as controls. RT PCR was used to determine mRNA levels of NQO1, HO 1, and GSTM1. GAPDH mRNA levels were analyzed and used an internal control. Results displayed represent either relative fold expression or expression af ter normalization to the GAPDH internal control. Results are the mean of three independent experiments, with samples read in duplicate for each experiment. I hypothesized that increasing SQSTM1 expression would decrease cytosolic Keap1 availability, makin g cytosolic Nrf2 more available to be imported into the nucleus and upregulate ARE enzymes. HO 1 expression was upregulated i n response to either hydroxynonena l. Approximately a thirteen fold and eighteen fold increase i n HO 1 response was observed in all samples after treatment with hydrogen peroxide and 4 hydroxynonenal, respectively in comparison to untreated cells (Fig 3 13 3 15 ). Hydrogen peroxide instigated no increase in GSTM1 levels, but a two f old increase in NQO1 levels was observed. 4 hydroxynonenal did not instigate an increase in NQO1 levels. However, a two fold increase of GSTM1 mRNA levels was observ ed in control cells treated with the stressor and cells transfected with pTR SB smCBA GFP. A slight decreas e in GSTM1 express ion was measure d in cells transfected with pTR SB smCBA SQSMT1. Because of the variability between replicates in this assay, the difference between cells transfected with pTR SB smCBA SQSMT1 and pTR SB smCBA GFP was not statistically sign ificant. Establishment of SQSTM1 Stable Cell Line As an alternative to p lasmid transfection, Lentiviral (LV) mediated gene transfer was used to produce a cell line of human retinal pigment epithelial cells that

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47 c onstitutively expresses SQSTM1 A cell line of ARPE 19 cells that constitutively expresses GFP was established concurrently as a control. LV mediated gene transfer is a method that is more comparable to a gene therapy treatment in that a transgene coding for SQSTM1 will be delivered to c ells and the gene of interest will be integrated into the host genome ( Moldt, 2011 ) This method will abolish inconsistencies in results due to varying transfection efficiencies between biological replicates and cells transfected with different plasmids Formation of this cell line will also allow us to examine the physiological effects of persistent expression of SQSTM1 in ARPE 19 cells. Selection o f cells that produce the gene of interest was achieved by including a puromycin resistant gene downstream o f the transgene. GFP production in the control cell line was effectively confirmed by fluorescence microscopy (Figure 3 16 ) Quantification of protein expression was accomplished by immunoblotting methods (Figure 3 17 )

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48 Figure 3 1 Im munoblot analysis of transfected ARPE 19 cells. Cells were transfected with increasing amount s of pTR SB smCBA SQSTM1 or pTR SB smCBA hGFP for 48 hours and lysat e was run on a 10% Tris HCl gel Transfection with 0.5 g plasmid DNA resulted in an increase i n the quantity of the protein s of interest. Expression seem ed to plateau when cells we re transfect ed with 1.5 g of DNA. Untransfected cells were included as a negative control. Actin was included as a loading control. Data presented is representative of one western blot Three independent experiments were performed (data not shown).

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49 Figure 3 2 ARPE 19 response to increasing concentrations of hydrogen peroxide. Untransfected ARPE 19 cells were exposed to increasing concentrations of hydrogen peroxide for six hours in order to establish a dose response curve. Cell viability was assessed using an MTT Assay with samples analyzed in triplicate Cells not exposed to oxidative stress were included as a negative control. Resu lts are displayed as residual cellular viability relative to the negative control after induction of oxidative stress. Cells show no significant change in viability until they are exposed to 600 M of hydrogen peroxide. Data are mean SD of three independent experime nts.

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50 Figure 3 3 ARPE 19 response to increasing concentrations of 4 hydroxynonenal Untransfected ARPE 10 cells were exposed to increasing concentrations of 4 hydroxynonenal f or six hours in order to establish a dose response curve. Cell viability was assessed using an MTT Assay with samples analyzed in triplicate. Cells not exposed to oxidative stress were included as a negative control. Results are displayed as residual cellu lar viability relative to the negative control after induction of oxidative stress. Cells show no significant change in viability until they are exposed to 40 M of 4 hydroxynonenal. Cell via bility is severely affected at higher concentrations. Data are me an SD of three independent experiments.

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51 Figure 3 4 Transfected ARPE Cells were transfected with the indicated amount of plasmid DNA for 48 hours Samples viability was determined using an MTT assay with samples analyzed in triplicate. Untransfected ARPE 19 cells exposed to hydrogen peroxide a re displayed on the graph as a negativ e control. A second negative control (not displayed) of ARPE 19 cells transfected with the indicated amount of plasmid DNA but not exposed to oxidative stress was included Data is shown as residual cellular viability of each sample relative to the corresp onding second negative control. Results are representative of the mean SD of three separate experiments

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52 Figure 3 5 Transfected ARPE transfected with the indicated amount of plasmid DNA for 48 hours. Samples viability was determined using an MTT assa y with samples analyzed in triplicate. Untransfected ARPE 19 cells exposed to hydrogen peroxide are displayed on the graph as a negative control. A second negative control (not displayed) of ARPE 19 cells transfected with the indicated amount of plasmid DN A but not exposed to oxidative stress was included. Data is shown as residual cellular viability of each sample relative to the corresponding second negative control. Results are representative of the mean SD of three separate experiments

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53 Figure 3 6 Transfected ARPE transfected with the indicated amount of plasmid DNA for 48 hours. Samples viability was determined using an MTT assa y with samples analyzed in triplicate. Untransfected ARPE 19 cells exposed to hydrogen peroxide are displayed on the graph as a negative control. A second negative control (not displayed) of ARPE 19 cells transfected with the indicated amount of plasmid DN A but not exposed to oxidative stress was included. Data is shown as residual cellular viability of each sample relative to the corresponding second negative control. Results are representative of the mean SD of three separate experiments

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54 Figu re 3 7 hydroxynonenal on ARPE 19 cell viability. Cells were transfected with the indicated amount of plasmid DNA for 48 hours. Samples hydroxynonenal for six hours. Cellular viability w as determined using an MTT assay with samples analyzed in triplicate. A slight reduction in cell viability was detected amongst all samples. Untransfected ARPE 19 cells exposed to 4 hydroxynonenal are displayed on the graph as a negative control. A second negative control (not displayed) of ARPE 19 cells transfected with the indicated amount of plasmid DNA but not exposed to oxidative stress was included. Data is shown as residual cellular viability of each sample relative to the corresponding second negati ve control. Results are representative of the mean SD of three separate experiments.

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55 Figure 3 8 hydroxynonenal on ARPE 19 cell viability. Cells were transfected with the indicated amount of plasmid D NA for 48 hours. Samples hydroxynonenal for six hours. Cellular viability was determined using an MTT assay with samples analyzed in triplicate. Untransfected ARPE 19 cells exposed to 4 hydroxynonenal are displayed on the graph as a negative control. A second negative control (not displayed) of ARPE 19 cells transfected with the indicated amount of plasmid DNA but not exposed to oxidative stress was included. Data is shown as residual cellular viability of each sample relative t o the corresponding second negative control. Results are representative of the mean SD of three separate experiments.

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56 Figure 3 9 hydroxynonenal on ARPE 19 cell viability. Cells were transfected with the indicated amount of plasmid DNA for 48 hours. Samples hydroxynonenal for six hours. Cellular viability was determined using an MTT assa y with samples analyzed in triplicate. Untransfected ARPE 19 cells exposed to 4 hydroxynonenal are displayed on the graph as a negative control. A second negative control (not displayed) of ARPE 19 cells transfected with the indicated amount of plasmid DNA but not exposed to oxidative stress was included. Data is shown as residual cellular viability of each sample relative to the corresponding second negative control. Results are representative of the mean SD of three separate experiments.

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57 Figure 3 10 L actate dehydrogenase (LDH) release as a measure of cytotoxic i ty in ARPE ARPE 19 cells were transfected with 0.5 SB smCBA SQSTM1 or pTR SB smCBA GFP for 48 hours. Cells were then treated with hours. An LDH release assay was performed, with samples analyzed in duplicate, to quantify the amount of lactate dehyd rogenase released into the medium of each sample after exposure to stress. A negative control of untransf ected ARPE 19 cells treated with hydrogen peroxide is defined as NC (+). Likewise, untransfected cells that were not exposed to oxidat i ve stress were included as another control and defined as NC ( ). A reduction in cytot o xicity of cells expressing SQSTM1 was observed. Statistical significance was measured by analysis of variance, P < 0.05. Results shown represent the mean of three separate experiments SD.

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58 Figure 3 11 Lactate dehydrogenase (LDH) release as a measure of cytotoxic i ty in ARPE 19 cells hydroxynonenal. ARPE 19 cells were transfected with 0.5 SB smCBA SQSTM1 or pTR SB smCBA GFP hours. An LDH release assay was performed, with samples analyzed in duplicate, to quantify the amount of lactate dehyd rogenase released into the medium of each sample after exposure to stress. A negative control of untransfected ARPE 19 cells treated with hydrogen peroxide is defined as NC (+). Likewise, untran sfected cells that were not exposed to oxidat i ve stress were included as another control and defined as NC ( ). A reduction in cytot o xicity of cells expressing SQSTM1 compared to NC (+) was observed. Statistical significance was measured by analysis of var iance, P < 0.05. Results shown represent the mean of three separate experiments SD.

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59 Figure 3 12 Relative Fold Expression of ARE antioxidant genes after insul t with 800 1. D) GAPDH. ARPE 19 cells were transfected with 0.5 SB smCBA SQSTM1 or pTR SB smCBA GFP for 48 hours. Cells were then treated with H 2 O 2 for six hours RT PCR was then performed to analyze change in mRNA levels of antioxidant genes. GAPDH levels were measured as an internal control. A negative control of untransfected ARPE 19 cells treated with hydrogen peroxide is defined as NC (+). Likewise, untransfected cells that were not exposed to ox idat i ve stress were included as another control and defined as NC ( ). Results are displayed as mRNA levels relative to NC ( ). Data shown represent the mean of three separate experiments SD.

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60 Figure 3 13 mRNA levels of ARE peroxide. Expression of ARE hydrogen peroxide normalized to GAPDH mRNA levels A) GSTM1. B) HO 1. C) NQO1. ARPE 19 cells were transfected with 0.5 SB smCBA SQSTM1 or pTR SB smCBA GFP for 48 hours. Cells were then treated with H 2 O 2 for six hours. RT PCR was then performed to analyze change in mRNA levels of antioxidant genes. A negative control of untransfected ARPE 19 cells treated with hydrogen peroxid e is defined as NC (+). Likewise, untransfected cells that were not exposed to oxidat i ve stress were included as another control and defined as NC ( ). Results are displayed as mRNA levels relative to NC ( ). Data shown represent the mean of three separate experiments SD.

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61 Figure 3 14 Relative Fold Expression of ARE antioxidant genes after insult with 4 hydroxynonenal. A) GSTM1. B) NQO1. C) HO 1. D) GAPDH. ARPE 19 cells were transfected with 0.5 SB smCBA SQSTM1 or pTR SB smCBA GFP for 48 hours. Cells were then treated with 4 HNE for six hours. RT PCR was then performed to analyze change in mRNA levels of antioxidant genes. GAPDH levels were measured as an internal control. A negative con trol of untransfected ARPE 19 cells treated with hydrogen peroxide is defined as NC (+). Likewise, untransfected cells that were not exposed to oxidat i ve stress were included as another control and defined as NC ( ). Results are displayed as mRNA levels re lative to NC ( ). Data shown represent the mean of three separate experiments SD.

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62 Figure 3 15 4 hydroxynonenal normalized to GAPDH mRNA levels. A) HO 1. B) GSTM1. C) NQO1. ARPE 19 cells were transfected with 0.5 SB smCBA SQSTM1 or pTR SB smCBA GFP for 48 hours. Cells were then treated with 4 HNE for six hours. RT PCR was then performed to analyze change in mRNA levels of antioxidant genes. A negative control of untransfected ARPE 19 cells treated with h ydrogen peroxide is defined as NC (+). Likewise, untransfected cells that were not exposed to oxidat i ve stress were included as another control and defined as NC ( ). Results are displayed as mRNA levels relative to NC ( ). Data shown represent the mean of three separate experiments SD.

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63 Figure 3 16 Verification of GFP Expression in GFP stable cell line Fluorescence microscopy was used to assess if the cells were expressing the transgene of interest A) GFP stable ARPE 19 cells displayed expression of the GFP transgene in ARPE 19 cells that persisted after selection. B) SQSTM1 stable ARPE 19 cells lacked expression of GFP. C) An overlay of the images in (A) and (B).

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64 Figure 3 17 Immunoblot analysis depicting SQSTM1 or GF P levels of stable cell lines. Cell lysates were extracted and run on a 10% Tris HCl gel. Expression of SQSTM1 or GFP can be seen in the sample extract Densitometry was performed u sing ImageJ. actin was included as a loading control. This e xperiment was performed once, with all samples displayed representative of one western blot.

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65 CHAPTER 4 DISCUSSION AND CONCLUSION S Th e results of these experiments confirm that we were able to successfully overexpress SQSTM1 in A RPE 19 cells without seeing a drastic change in cell viability in compar ison to wild type ARPE 19 cells However, the ability of SQSTM1 to activate the Nrf2/ARE cascade and aid in protection against oxidative stress is not clear. It is evident that mitochondrial respiratory activity is sensitive to oxidative stress. It is important to note that hydrogen peroxide and 4 hydroxynonenal differed in their ability to instigate dysfunc tion of cellular capabilities. ARPE 19 cells were able to maintain cell viability at higher concentrati ons of hydrogen peroxide than 4 hydroxynonenal. In accordance with other studies, both hydrogen peroxide and 4 hydro xynonena l induced HO 1 expression in RPE cells (Pilat, 2013; Huang, 2012 ) Unfortunately, SQSTM1 overexpression did not instigate a more robust HO 1 response than in the other controls. Future studies may include delivery of SQSTM1 to the RPE in a mouse model of dry AMD. This would allow us to determine if SQSTM1 overexpression would protect mitochondrial function in the context of the disease state. Previously published studies have es tablished that transfection of SQSTM1 cDNA protects primary cortical neuron cells from oxidative st ress via the Nrf2/ KEAP1/ ARE pathway Upon overexpression of SQSTM1 cDNA, NQO1 protein expression was increased 2.6 fold, as verified by western blot (Liu, 20 07). Although our results confirm a slight increase in NQO1 transcript levels (2.0 fold) after exposure to hydrogen peroxide the increase was the same in ARPE 19 cells overexpressing SQSTM1 as it was in both transfected and untransfected controls. In fact untransfected ARPE 19 cells exposed to hydrogen peroxide displayed greater NQO1 expression than

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66 in GFP or SQSTM1 expressing cells. This may indicate that transient t ransfection methods instigate the induction of ARE enzymes and m ight serve as a source of cellular stress. The conflict between our results and those of the Liu group may b e due to cell type specificity or to the relative quantity of SQSTM1 overexpression. Over e xpression of SQ STM1 may modulate the KEAP1/Nrf2/ARE pathway in the same manner bu t to a different extent in primary cortical culture. Necrosis and apoptosis are two distinct mechanisms of cell death. Necrosis is characterized by injury to cytoplasmic organelles, cell swelling, membrane lysis, and release of cytoplasmic contents. Apopt osis is characterized by membrane blebbing, shrinkage of the cell, chromatin condensation, and cross linking of membrane proteins (Bonfoco, 1995) These processes differ in many ways, but importantly here, in the release of cellular content into the neighb oring environment. The results of the LDH assays suggest that SQSTM1 expressing cells released significantly less lactate dehydrogenase than in untreated ARPE 19 cells that were exposed to stress. In order to make further conclusions about overall cellular health from these results, it is necessary to determine if the apoptotic pathway was initiated in SQSTM1 expressing cells. It is possible that apoptosis but not primary or secondary necrosis was initiated in SQSTM1 expressing cells, preventing the release of LDH into the medium but not offering protection to the mitochondria in the MTT assays. It is also possible that SQSTM1 overexpression mediated protection from cell death, but not mitochondrial dysfunction, by a pathway other than the ARE. The additio nal roles that SQSTM1 plays in the cell must be taken into consideration when determining if overexpression of this protein will offer attenuation of

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67 oxidative stress induced damage. Further studies may include identifying the location of overexpressed SQSTM1 using immunocytochemistry. Importantly it has been demonstrated that SQSTM1 is subject to nucleocytoplasmic shuffling in order to participate in multiple pathways (Pankiv, 2010). It is also known that accumulation of SQSTM1 can result in its inclusion in p62 or PML bodies in the nucleus (Pankiv, 2010). If overexpressed SQSTM1 is confined within these bodies or homodimerizes in the nucleus suffici ent sequestrat ion of KEAP1 may not be occu r ring In this case, t he Nrf2/ KEAP1/ ARE pathway may not be responsible for the protection displayed in Figs. 3 6 and 3 7. This proposal, of course, is dependent on the rate of SQSTM1 nuclear import and export, whet her nuclear export of SQSTM1 is a saturable process, and the percentage of overexpressed or endogenous SQSTM1 that is appropriated into protein aggregates. A greater understanding of these events could shed light on the results that we report. Lastly, the positi ve feedback loop created by Nrf2 and SQSTM1 should not be ignored. In correlation with its role in Nrf2 induction, SQSTM1 levels increase after oxidative stress (Ishii, 1997). This interaction must be consid ered. If overexpressing SQ STM1 by transfec of plasmid DNA leads to toxic levels of either Nrf2 or SQSTM1, then our meth ods may be injurious to cellular function Importantly, this may not interfere with plasmid membrane tenacity resulting from cell death explaining the conflicting results in the MTT and LDH release assays. However, there may be an optimal degree of SQSTM1 overexpression for our desired purposes that can lead to nontoxic levels of nuclear Nrf2 to induce ARE expression.

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68 In order to further investigate the role of SQ STM1 in protection from oxidative stress without the interfering effects of a transfection reagent, ARPE 19 cell lines that constitutively express SQSTM1 or GFP were established. Further studies using these cell lines will need to be performed in order to assess the feasibility of a gene therapy treatment geared toward overexpressing SQSTM1 in the RPE. This cell line will be able to demonstrate the effects of viral vector based delivery of SQSTM1 to RPE cells. In conclusion, it is still possible that overex pression of SQSTM1 may mediate protection of retinal pigment epithel ial cells from oxidati ve stress, although its ability to protect certain features of the cell may differ. Altering the Nrf2/ARE pathway by increasing SQSTM1 availability will need to be in vestigated in vivo in order to fully asse s s the potential of this alteration as a gene therapy treatment for dry AMD patients.

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73 BIOGRAPHICAL SKETCH Danielle Lorna Liv erpool was born in Danbury, CT to Joseph and Kathleen Liverpool. She was raised in Brandon, FL by a loving family tha t included her parents and her o lder brother, Stephen Liverpool Although she was diagnosed with terminal cancer at the age of 13, she continues to relentlessly pursue life with stron g faith and a joyful attitude. As a grandchild of four immigrants from the small island of St. Vincent, having the opportunity to pursue higher education has made a tremendous impact in her life. She is the second member of her family to attend the University of Florida Her research endeavors and thought provoking classes inspired her to pursue a career in the biotechnology indu stry. In May 2011, she obtained her Bachelor of Science degree in b i ology with a specialization in b iotechnology. After completing t he requirements for her master of science degree, she will c ontinue to pursue a career in biotechnology