The Role of Annexin A4 in Response to Biomechanical and Ischemic Insult

Nevena Vicic

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Laboratory Medicine and Pathobiology University of Toronto

Nevena Vicic

Master of Science

Laboratory Medicine and Pathobiology University of Toronto

2016

Abstract

Glaucoma is a progressive neurodegenerative disorder characterized by the loss of retinal ganglion cells (RGCs). Elevation of intraocular pressure (IOP) is commonly observed in glaucoma patients and can exert biomechanical and ischemic insults on the optic nerve head

(ONH) resulting in astrocyte reactivity, cytoskeletal dysfunction, and loss of structural support to

RGC axons. Proteomic analysis of human ONH astrocytes revealed an increase of Annexin A4

(ANXA4) following application of a pathologically relevant biomechanical stress. The function of ANXA4 is unknown in the retina but members of this family have been implicated in membrane repair processes that involve interactions with cortical actin. In this study, ANXA4 was upregulated during glaucomatous stresses. Mechanical insult increased membrane permeability, and ANXA4 knockdown increased permeability to this insult. Furthermore,

ANXA4 interacted with F-actin. These studies highlight the importance of membrane dynamics during biomechanical and ischemic insult and the active role ANXA4 has on membrane stability.

Acknowledgments

First and foremost, I would like to express my sincere gratitude to my supervisor, Dr. Jeremy Sivak. I tremendously appreciate you dedicating your time in helping me throughout my project. Thank you for listening to my countless hypotheses and theories, and being incredibly supportive after hearing each one. Your ability to encourage me during times when I felt stuck is greatly appreciated. I would like to express my gratitude to Dr. John Flanagan for being a terrific and very supportive mentor. Your advice and wisdom has helped to shape and better my project. Thank you to my committee members, Dr. Paulo Koeberle and my committee chair Dr. Valerie Wallace. Your invaluable advice and comments have changed the way I look at my experiments for the better and taught me to perceive science in a new perspective.

I would like to give special thanks to everyone in the Sivak, Flanagan, and all surrounding labs at KDT who have helped me out tremendously during my studies. Thank you to Qi, Rachel, Darren, Izzy, Samih, and Ali for being incredibly supportive lab members. Many thanks especially to Cindy. Without your patience and help, my project would not be where it is today.

Finally, a special thank you goes out to my family and friends. Thank you for remaining understanding as you listen to my pain and struggles with science. To my family, you have been my rock and have lovingly remained by my side throughout this entire process. To my friends, you have made this journey that much more enjoyable and without you, I would not have had as much fun as I did.

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Table of Contents Abstract ...... ii

Acknowledgments ...... iii

List of Tables ...... vii

List of Abbreviations ...... viii

List of Figures ...... x

List of Appendices ...... xi

Chapter 1 Introduction ...... 1

1 Introduction ...... 2 1.1 Hypothesis ...... 4 1.2 Specific Aims ...... 4

Chapter 2 Literature Review ...... 5

2 Literature Review ...... 6 2.1 Overview of Glaucoma ...... 6 2.1.1 Risk factors Associated with Glaucoma ...... 7 2.1.2 Current Treatments for Glaucoma ...... 8 2.2 The ONH Structure and Composition ...... 9 2.3 ONH and Biomechanics ...... 11 2.3.1 Finite Element Modeling ...... 12 2.3.2 The Molecular Mechanisms of Biomechanical Insult on RGC Cell Death ...... 13 2.4 ONH and the Vasculature ...... 15 2.4.1 The Molecular Mechanisms of Ischemia on RGC Cell Death ...... 16 2.5 Astrocytes in Response to Biomechanical and Ischemic Insult ...... 17 2.6 Annexins ...... 19 2.7 Annexins and Membrane Dynamics ...... 21 2.8 Annexins and Actin Dynamics ...... 22 2.9 Annexins and Membrane Repair ...... 23 2.10 ANXA4 ...... 24

Chapter 3 Methods ...... 27 iv

3 Methods ...... 28 3.1 Cell Cultures ...... 28 3.1.1 Primary Rat Retinal Astrocyte Isolation and Culture ...... 29 3.2 Induction of Biomechanical and Hypoxic Insults ...... 29 3.2.1 Biomechanical Insult ...... 29 3.2.2 Hypoxic Insult ...... 30 3.3 Elevated IOP Model in vivo ...... 30 3.3.1 Elevated IOP in Rat ...... 30 3.3.2 Elevated IOP in Pig ...... 31 3.4 Gene Expression Analysis through qPCR ...... 31 3.5 Protein Analysis ...... 32 3.5.1 Cell and Tissue Lysate Collection and Protein Quantification ...... 32 3.5.2 SDS-PAGE and Protein Detection ...... 33 3.6 Flow Cytometry and Cell Viability Assay ...... 34 3.7 Pharmacological Treatments ...... 35 3.8 Cell Membrane Integrity Assay ...... 35 3.9 Molecular Biology and ANXA4 Gene Manipulation ...... 36 3.9.1 ANXA4 Overexpression ...... 36 3.9.2 ANXA4 siRNA Knockdown ...... 37 3.9.3 Viral Plasmid ANXA4 shRNA Generation ...... 37 3.9.4 Viral shRNA Expression in vitro and in vivo ...... 38 3.10 Imaging ...... 39 3.10.1 Cell Immunofluorescence ...... 39 3.10.2 Tissue Immunofluorescence ...... 40 3.10.3 Microscopy ...... 40 3.11 Statistical Analysis ...... 40

Chapter 4 Results ...... 41

4 Results ...... 42 4.1 Aim 1: Characterize ANXA4 Regulation in Response to Biomechanical and Hypoxic/Ischemic Stress ...... 42 4.1.1 ANXA4 is Upregulated in Response to Biomechanical and Hypoxic Stress in vitro ...... 42 4.1.2 ANXA4 is Upregulated in Response to Biomechanical and Ischemic Stress in vivo ...... 42

4.1.3 ANXA4 Does Not Influence Cell Death During Biomechanical and Hypoxic Stress in vitro ...... 45 4.2 Aim 2: Elucidate the Functional Role of ANXA4 on Membrane Permeability During Biomechanical Insult ...... 47 4.2.1 Membrane Permeability Increases in a Calcium-Dependent Manner Upon Biomechanical Insult ...... 47 4.2.2 ANXA4 Translocates to Membrane Structures Upon Elevation of Intracellular Calcium .... 47 4.2.3 ANXA4 Knockdown Induces Membrane Permeability During Biomechanical Insult ...... 49 4.3 Aim 3: Determine ANXA4 Interaction With Cytoskeletal Dynamics in vitro and in vivo ...... 51 4.3.1 ANXA4 Co-Localizes With F-actin at the Membrane Cortex and Responds to Actin Disruption in vitro ...... 51 4.3.2 Conserved ANXA4 Expression is Found at the Inner Retina ...... 53 4.3.3 ANXA4 Co-Localizes With F-actin in the Retina ...... 54 4.4 Aim 4: Develop Tools to Generate ANXA4 Knockdown in vivo ...... 56 4.4.1 shRNA Vector Plasmids Generates a Significant Knockdown of ANXA4 ...... 56 4.4.2 ANXA4 shRNA Sequence Was Successfully Cloned into dsAAV Viral Vector Backbone 57 4.4.3 AAV2-ANXA4-shRNA Successfully Infected Primary Rat Retinal Astrocytes in vitro and the Rat Retina in vivo ...... 58

Chapter 5 Discussion ...... 59

5 Discussion ...... 60 5.1 Biomechanical and Hypoxic Stresses Induces ANXA4 Expression and Elevated Protein Levels in vitro and in vivo ...... 61 5.2 ANXA4 Knockdown Contributes to Increased Membrane Permeabilization During Biomechanical Insult ...... 62 5.3 ANXA4 Co-Localizes with F-actin and Responds to Actin Disruptors ...... 65 5.4 Development of Tools to Knockdown ANXA4 in the Retina ...... 68 5.5 Implications in Disease ...... 68

6 References ...... 71

Appendices ...... 89

List of Tables

1. List of Primers Used For quantitative PCR. 2. List of Antibodies and Dilutions For Incubation. 3. List of Primers Used for shRNA PCR Product.

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List of Abbreviations

2D – 2-dimensional

ANXA4 – annexin A4

ATP – adenosine triphosphate

CaCC – calcium activated chloride conductance channels

Calpains – calcium dependent proteases

CaMKII – calcium/calmodulin-dependent protein kinase II

DAPI – 4’6-diamidino-2-phenylinolide

DMSO – dimethyl sulfoxide

F-actin – filamentous actin

FITC – fluorescein isothiocyanate

GAPDH – glyceraldehyde 3-phosphate dehydrogenase

GFAP – glial fibrillary acidic protein

GFP – green fluorescent protein

HBSS – Hank’s Balanced Salt Solution

HEK293 – human embryonic kidney 293 cells

HSP27 – heat shock protein 27

IOP – intraocular pressure

MAPs – microtubule associated proteins

MCAO – middle cerebral artery occlusion

NF-κB – nuclear factor kappa-light-chain-enhancer of activated B cells

NO – nitric oxide

NT – non-targeting

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ON – optic nerve

ONH – optic nerve head

PACG – primary angle closure glaucoma

PFA – paraformaldehyde

PI – propidium iodide

POAG – primary open angle glaucoma

PRRA – primary rat retinal astrocytes

PVDF – polyvinylidene fluoride qPCR – quantitative reverse transcriptase polymerase chain reaction

RGCs – retinal ganglion cells

RIPA – radioimmunoprecipitation assay

ROS – reactive oxygen species

SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis

TBP – TATA-binding protein

TNFα – tumor necrosis factor alpha

TRAIL – TNF-related apoptosis-inducing ligand

List of Figures

1. Lamina Cribrosa Structure. 2. Spatial Distribution of the First Principal Strain on a Finite Element Model Where Donor Eyes Were Exposed to Elevated IOP. 3. The ONH Vasculature. 4. Annexin Properties. 5. ANXA4 is Upregulated in Response to Biomechanical and Hypoxic Stress in vitro. 6. ANXA4 is Upregulated in Response to Ischemic/reperfusion Stress in vivo. 7. ANXA4 Does Not Influence Cell Death During Biomechanical and Hypoxic Stress in vitro. 8. Membrane Permeability Increases in a Calcium Dependent Manner Upon Biomechanical Insult. 9. ANXA4 Translocates to Membrane Structures Upon Elevation of Intracellular Calcium. 10. ANXA4 Knockdown Induces Membrane Permeability During Biomechanical Insult. 11. ANXA4 Co-localizes With F-actin at the Membrane Cortex. 12. ANXA4 Responds to Actin Disruption. 13. Conserved ANXA4 Expression is Found at the Inner Retina. 14. ANXA4 Co-localizes With F-actin in the ON. 15. ANXA4 Co-localizes With F-actin at the ONH and Retina. 16. ANXA4 shRNA Sequences Generates a Significant ANXA4 Expression Knockdown. 17. ANXA4 shRNA Sequence With GFP Reporter Gene was Successfully Ligated into dsAAV Viral Vector Backbone. 18. AAV2-shRNA Successfully Infected PRRA and the Retina.

List of Appendices

1. Confirmation of ANXA4 Knockdown Through Protein Analysis. 2. Confirmation of ANXA4 Overexpression Through Protein and Fluorescence Analysis. 3. FM1-43 dye Fluoresces Upon Membrane Breaks.

Chapter 1

Introduction

1 Introduction

Glaucoma is a progressive neurodegenerative disorder characterized by the gradual loss of retinal ganglion cells (RGCs). Glaucomatous pathology is associated with increased intraocular pressure (IOP) that exerts mechanical insult at the optic nerve head (ONH), the site suggested to be the principle location of RGC axonal damage1–3. IOP-related strain can exert compressive effects on the ONH, resulting in mechanical damage, abnormal vasculature and micro-ischemic/reperfusion conditions4–6. These factors contribute to modified cytoskeletal components, reduced nutrient availability and diffusion, and the para-inflammatory response of astrocyte activation7. Combined, these stresses can influence calcium dynamics within RGCs and astrocytes, and contribute to neuronal pathology. Therefore, studying calcium dependent proteins and their relation to glaucoma is needed to better understand the disease.

We have previously conducted a proteomic analysis of human ONH astrocytes undergoing various magnitudes of a pathologically relevant biomechanical strain10. Astrocytes are crucial for providing structural and homeostatic support to delicate RGC axons at the ONH8.

Biomechanical strain is defined by the change of length divided by the original length of the tissue, which is a pathologically relevant stress at the ONH9. Generating this type of stress in vitro mimics the conditions seen in the ONH environment in glaucoma and provides a model to study the mechanotransduction mechanisms associated with elevated IOP. It is therefore crucial to identify proteins that contribute to pathological mechanisms during biomechanical insult. Of notable interest is Annexin A4 (ANXA4), a calcium-dependent membrane binding protein that was significantly upregulated in this model10.

ANXA4 belongs in a large family of structurally related proteins characterized by their ability to reversibly bind to negatively charged phospholipids in a calcium dependent manner11,12. Upon elevation of intracellular calcium, ANXA4 translocates to membranes and

3 forms lateral assemblies of 2-dimensional trimer structures13. This crystalline arrangement affects membrane properties including rigidity, fluidity, and lipid domain stabilization14,15.

Although a main annexin function is still unclear, members of this ubiquitous family have a role in a variety of essential cellular functions linking calcium, membrane, and cytoskeletal dynamics16,17. In particular, several annexins have been upregulated in neurodegenerative pathologies, including glaucoma18,19 and during ischemic/reperfusion injury20–23. However the functional implication of this is unknown. Annexins have been implicated in calcium dependent membrane repair processes, and are needed to reseal membranes through the formation of trimer structures after membrane disruption24–27. Actin is an important component in membrane repair and involves extensive cytoskeletal reorganization. Annexins have intimate contact with actin and some members help reseal plasma membranes by facilitating polymerization of filamentous actin (F-actin)24.

There have been limited studies on the role of annexins, and specifically ANXA4, in the retina and their response to biomechanical and ischemic stresses. Furthermore, literature on membrane dynamics is diminutive in the context of biomechanical insult, yet can have vast implications on the pathological consequences of membrane permeability in astrocytes and

RGC axons of the ONH. Specifically, ANXA4 and the role it has on membrane stability during biomechanical insult have never been studied. Given that ANXA4 has the necessary properties to integrate calcium signaling with a variety of diverse membrane dynamics, it is important to investigate the role ANXA4 has in biomechanical insult in terms of membrane permeability, and to elucidate how it is regulated in ischemic/reperfusion conditions.

1.1 Hypothesis

I hypothesize that ANXA4 plays an important role in biomechanical and ischemic stress and is an important contributor to membrane stability during mechanical insult in the inner retina and ONH.

1.2 Specific Aims

1. Characterize ANXA4 Regulation in Response to Biomechanical and Hypoxic/Ischemic

Stress.

2. Elucidate the Functional Role of ANXA4 on Membrane Permeability During

Biomechanical Insult.

3. Determine ANXA4 Interaction With Cytoskeletal Dynamics in vitro and in vivo.

4. Develop Tools to Generate ANXA4 Knockdown in vivo.

Chapter 2

Literature Review

2 Literature Review

2.1 Overview of Glaucoma

Glaucoma is a progressive neurodegenerative disorder characterized by the gradual loss of retinal ganglion cells (RGCs). It is the leading cause of irreversible blindness worldwide and has been estimated that nearly 80 million people will be affected worldwide by 202028.

Glaucoma was also calculated to be responsible for 15% of all causes of blindness, thus being the second most common cause of blindness in the world after cataracts. There are two main forms of glaucoma. Primary open angle glaucoma (POAG) is the most common form of the disease and is characterized by optic nerve damage associated with gradual increased intraocular pressure (IOP), although not all patients experience elevated pressure29. The second form is primary angle closure glaucoma (PACG), characterized by the closure of the angle between the iris and cornea resulting in obstructed outflow of aqueous humour and consequently elevation of

IOP29.

A challenging aspect of glaucoma is that it is mostly asymptomatic until the late stages of the disease when central visual acuity and temporal visual field deficits arise. Typically, at this stage there is a substantial number of RGCs lost in the inner retina, characterized by the thinning of the retinal nerve fiber layer and substantial loss of axons in the optic nerve29. Optic disc excavation at the optic nerve head (ONH) due to alterations of connective tissue and loss of axons is characteristic of glaucoma and is a predominant feature for diagnosis30–32. Although the cause of this damage is still unknown, there have been many mechanisms described as key contributors to the ultimate death of RGCs. To name a few: astrocyte activation8, ischemia and reduced blood perfusion33, extracellular matrix remodeling34,35, metabolic and oxidative injury36 have all been implicated in the progression of disease and will be discussed in the review.

2.1.1 Risk factors Associated with Glaucoma

Numerous risk factors have been identified that can contribute, or play an essential role, in the development of glaucoma. Examples of these risk factors are age, increased IOP, altered vasculature, family history, and ethnicity. Older age is consistently associated with increased prevalence of POAG37. Older individuals may have also had the disease for a longer period of time, therefore are exposed to other risk factors that contribute to the development of disease.

Elevated IOP is currently the only known modifiable risk factor for the treatment of

POAG. Higher IOP at baseline was found to be a predictive risk factor for glaucoma progression in the Early Manifest Glaucoma Trial38. The higher the IOP, the greater likelihood of developing the disorder and more rapidly it worsens39, although it is not a defining criterion.

Many patients with POAG have normotensive IOP40 and many patients with IOP above the normal range (defined as ocular hypertension), do not develop the disease - although they are at increased risk41. It is also not clear whether it is the elevation or fluctuation of IOP that contributes to disease42.

Factors that can compromise the vascular supply to the optic nerve, leading to ischemic conditions, are closely related to glaucomatous progression43,44. Low systolic perfusion pressure and decreased blood flow velocities in the short posterior ciliary arteries and central retinal arteries were present in patients with severe glaucomatous visual field loss45. Older patients with a history of cardiovascular disease, such as hypertension, were more likely to have POAG than younger patients with hypertension, suggesting a cross-play of risk factors46.

Several genes and biomolecules have been studied over the past few years for their potential as disease markers and predictors of progression. The Rotterdam Glaucoma Study examined all available family members of a population-based group of POAG patients47. It was found that the relative risk for POAG was more than 10 times higher in first-degree relatives.

Furthermore, the Barbados study revealed that 10% of living relatives examined had POAG48.

Several genes have been identified to be related to the development of POAG. Family member genotyping revealed two genes, optineurin and myocilin, are related to early onset POAG, although mutations in these molecules are present in only a small number of POAG patients49. It is difficult to establish a genetic link that can directly influence glaucoma progression. Family members are genetically similar in other factors that are related to POAG, such as disc morphology, IOP level, and low perfusion pressure50. There are also a multitude of environmental factors that are shared by family members that can contribute to glaucoma progression39.

Ethnicity has been also considered as a risk factor associated with glaucoma. Persons who are African-derived are more likely to have POAG than all other ethnicities. They develop the disorder earlier in life and more frequently, increasing the chance for blindness51. PACG is more prevalent in Asian populations due to the tendency for this population to have narrow irido-corneal angles28.

In conclusion, glaucoma is a multifactorial disease, and can be influenced by a multitude of genetic and/or environmental risk factors that eventually lead to the incidence and progression of the disease.

2.1.2 Current Treatments for Glaucoma

Currently, there is no cure or treatment for glaucomatous retinal pathology. For patients diagnosed with POAG optic nerve injury, benefits are seen in IOP lowering drugs, irrespective of whether IOP is high or normal52,53. Existing treatments include the application of daily eye drops, laser treatment, or through surgical means. It is a widely accepted clinical practice to prescribe patients with eye drops that are often prostaglandin analogues or beta-adrenergic antagonists. Through mechanisms still unclear, these eyedrops are effective in lowering IOP,

9 although problems arise when patient compliance is low29,54. This treatment can lower the IOP by about 20-40% and reduce the average rate of progressive field loss by half29. If eye drop treatment proves inadequate, laser delivery to the trabecular meshwork can lead to lowering of

IOP with minimum risk and side effects. Furthermore, a trabeculectomy, which forms a controlled leak area for aqueous humour from the anterior chamber, can also benefit patients55.

If this treatment fails, surgical means by the insertion of an artificial tube reservoir into the anterior chamber could prove to be beneficial56. Other surgical means of lowering IOP are through the destruction of the ciliary body57.

Although reducing IOP is the current main form of treatment, evidence indicates that high IOP is not always a strong risk factor for glaucoma development, and that lowering IOP does not always halt progression in patients with normal or even high IOP52,58,59. There is little known about the link between IOP, risk factors and the development of POAG neuropathology.

Elevated IOP and reduced perfusion pressure in the eye that lead to strenuous and ischemic conditions at the ONH are important risk factors in glaucoma and can initiate a multitude of stress pathways. Yet, as mentioned previously, not all glaucoma patients experience these risk factors, and those that do may not develop glaucoma. It is important to find a “common insult” that is experienced in all glaucoma patients in order to halt disease progression. Experimental glaucoma treatments are currently investigating factors such as ischemic/reperfusion stress, glial cell activation, oxidative stress, apoptosis, calcium deregulation (as a result of aforementioned factors), and inflammation that are associated with biomechanical and vascular insults related to glaucoma pathology36,60–64.

2.2 The ONH Structure and Composition

The ONH tissues make up a dynamic environment wherein 1.2 to 2.0 million RGC axons converge, turn at a 90° angle, and exit through the neural canal to their respective higher

10 visual centers within the brain65. Bundled axons pass from the relatively high-pressure environment of the eye into a lower pressure region in the retrobulbar cerebrospinal space2. To protect the axons from this pressure environment, primates have developed a unique structure called the lamina cribrosa. The lamina cribrosa is a delicate structure that underlies the ONH and consists of a 3-dimensional meshwork of fenestrated connective tissue beams that are lined by an intimate web of astrocytes and capillaries enveloping axons (Figure 1A)66. Astrocytes are a major glial cell type in the non-myelinated ONH in most animals and are arranged in a manner where they form glial columns that run perpendicular to neural bundles in a “honeycomb” appearance, while also interfacing and interacting between connective tissue surfaces and surrounding blood vessels (Figure 1B)67. In the retina, astrocytes are located between fasciculated axons of the nerve fiber layer. In normal ONH conditions, astrocytes are crucial in performing a regulatory role to ensure a homeostatic environment for RGC axons. Disruption of these important cells can result in loss of structural and metabolic support for RGC axons and their cell bodies in the lamina cribrosa and retina, respectively8,68. In addition to astrocytes, the primate ONH exhibit lamina cribrosa cells to provide the structural support in response to biomechanical strain69. Non-primates, due to their lack of lamina cribrosa cells, depend on astrocytes to maintain support within the ONH2.

Within the lamina, RGC survival is dependent on the movement of oxygen and nutrients through the capillaries, to the astrocytes and into axon bundles via cell processes70. This structure is paramount in RGC survival, as they provide structural and metabolic support to these sensitive cells. Any kind of mechanical deformation or displacement on the connective tissue matrix can result in physical damage to optic nerve axons and contribute to decreased blood flow to the region of the ONH, which in turn would decrease nutrition supply and weaken the laminar tissue30,71.

Figure 1. Lamina Cribrosa Structure. The ONH is made up of prelaminar, laminar and retrolaminar regions. RGCs axons collect and bundle together at the ONH and exit through the eye through the lamina cribrosa (A). The lamina cribrosa contains lamina cribrosa cells (primates-only) and astrocytes, which bundle together in to a “honey comb” appearance (B). (A) Adapted from Anderson et al (1969)66. (B) Adapted from Quigley et al. (1990)72.

2.3 ONH and Biomechanics

IOP can play a central role in the physiology and the pathophysiology of the ONH tissue types: the load-bearing connective tissue (lamina cribrosa), the axonal (retinal ganglion cells), and cellular (astrocytes, glial cells, endothelial cells)32. The ONH, and particularly the lamina cribrosa, is a site of interest due to evidence suggesting that it is the principal site of RGC axonal insult in glaucoma1,3,73. Many studies support the importance of the ONH during pathology, describing profound alterations and axonal disruption within the prelaminar, laminar, and retrolaminar tissues of the ONH in not only human patients, but in monkey, rat and mouse models30,71,74–76. Elevated levels of IOP can act on these tissues of the eye and produce stress, deformations and strain, leading to pathologic changes in cellular cascades, and remodeled tissue microarchitecture. This biomechanical injury ultimately results in RGC axonal damage.

Given the concealed location of the ONH, accessibility to this area to study the biomechanical

12 environment during elevation of IOP has proven to be challenging. It is important to study the biological response in this area to better understand RGC pathology.

2.3.1 Finite Element Modeling

A tool used to help elucidate the biomechanical environment at the ONH during elevation of IOP is finite element modeling77. Using this strategy, the type of strain that is exerted on the ONH during elevation of IOP can be examined and quantified through computational modeling. Mechanical strain applied to the eye by the elevation of IOP has determined that the highest magnitude of strain is in the delicate lamina cribrosa region of the

ONH3,78. In a study conducted by Sigal et al.9, computational models were constructed to measure the magnitude of IOP deformation of the ONH, as well as the type of deformation that occurs. The highest levels of all modes of strain were found in the neural tissue region. Strain is a measure of deformation, and is defined as the fractional elongation of a tissue element i.e. the change of length divided by the original length of that tissue line. A positive strain (first principle strain) represents extension, while a negative strain (third principle strain) represents compression. In addition, shearing, defined as the deformation of a tissue line in a direction other than along the axis of the line, was also observed. These strains were observed at the ONH in the pre-laminar neural tissue and lamina cribrosa of eyes that experienced elevation of IOP from 5mmHg to 50mmHg, in the range of 5-15%9,79 (Figure 2). Mechanical insult can have detrimental pathological influences on RGC axons, ONH astrocytes, and optic nerve blood vessels. This stress can cause disruption of axonal transport, ischemia/reperfusion injury, and glial activation, as described below.

Figure 2. Spatial Distribution of the First Principal Strain on a Finite Element Model Where Donor Eyes Were Exposed to Elevated IOP. Strain is defined as the change of tissue length divided by the initial tissue length of that tissue. The lamina cribrosa structure experienced 15% mechanical strain. N = nasal; T = temporal; S = superior; I = inferior. Adapted from Sigal et al. (2007)9.

2.3.2 The Molecular Mechanisms of Biomechanical Insult on RGC Cell

Death

There has been much research conducted to understand RGC degeneration after biomechanical injury at the ONH. Several factors can come in to play such as reduced axonal transport, limited nutrient diffusion, extrinsic apoptosis, and excitotoxicity, to name a few.

Many studies have examined numerous pathways involved that can contribute to the exacerbation of RGC death in glaucoma, although the initiating factor, or the “primary insult” to

RGCs that initiate the neuropathology is relatively unknown.

A current model of glaucomatous pathogenesis suggests that the primary site of insult due to biomechanical insult is on the axons and supporting astrocytes of the ONH. In addition to generating action potentials, axons are important in transporting molecules in the anterograde

14 and retrograde direction in an energy-dependent manner. The axon cytoskeleton is crucial for not only providing structural support, but for forming “tracks” for protein movement.

Neurofilaments, microtubules, microtubule-associated proteins (MAPs) and actin are all important components of the axon cytoskeleton. In many neurodegenerative diseases, axonal pathology precedes cell body loss80. This process includes axonal swelling, microtubule disassembly and the eventual fragmentation of the cytoskeleton81. Numerous experimental glaucoma animal models show that anterograde and retrograde transport of axons is blocked and deficits can arise in the transport of important neurotrophic factors to RGC bodies82,83.

Furthermore, physical disruption of the axon cytoskeleton was reported to occur within 3 hours of acute IOP elevations and precede axon transport abnormalities in the porcine eye84.

Additionally, F-actin at the ONH was observed to reorient early in the response of elevated

IOP85. Elevated IOP can contribute to the blockade of axonal transport and cytoskeletal disassembly and therefore compromise RGC axon longevity83,86,87. Molecular manipulations to delay the degeneration of severed axons can attenuate the progression of animal models of disease such as multiple sclerosis, ischemic stroke, and glaucoma88–90.

The primary mechanism of RGC damage in glaucoma is not known, but much evidence suggests that neuronal loss occurs largely by apoptosis7. Apoptotic processes can be triggered by a variety of stimuli, leading to the activation of the extrinsic or intrinsic apoptotic cascade.

Extrinsic apoptotic signals include a wide array of death-receptor ligands such as tumor necrosis factor (TNF) alpha, FasL, and TNF-related apoptosis-inducing ligands (TRAIL). These ligands initiate activation of downstream initiator caspases, leading to the activation of executioner caspases. In the intrinsic pathway, which may be triggered by neurotrophic factor deprivation, a variety of kinases can activate downstream factors and leads to mitochondrial membrane

15 instability and dysfunction by the continuous release of cytochrome C and loss of ATP productions91.

2.4 ONH and the Vasculature

The superficial nerve fiber layer, which consists primarily of neurons, receives blood supply mainly from the central retinal artery92. Posterior to this, the prelaminar region receives its blood supply from the branches of the peripapillary choroid and from the short posterior ciliary arteries. The lamina cribrosa is nourished by the short posterior ciliary arteries and the circle of Zinn-Haller, which penetrate into the peripapillary sclera and feed into capillaries contained within the laminar beams93 (Figure 3). Under normal conditions, blood flow is autoregulated. Autoregulation is defined as the ability of a tissue to maintain blood flow at a constant level despite changes in perfusion pressure94. The retina lacks autonomoic innervations and thus blood flow is regulated by muscle-driven negative feedback mechanisms. Arterioles are thought to be the major site for the regulation of perfusion and can be controlled by metabolic, myogenic, and hormonal mechanisms95. Altered autoregulation and changes in perfusion pressure have been shown in patients with normotensive glaucoma and in POAG in response to elevation of IOP96.

Along with the biomechanical theory, the vasogenic theory has been proposed to also contribute to glaucomatous optic neuropathy. Several epidemiological studies have shown that low ocular perfusion pressure is a risk factor for the prevalence, incidence and progression of glaucoma97. IOP-related strain within the peripapillary sclera exerts a compressive effect on volume of blood flow within the branches of the posterior ciliary arteries and reduces vascular perfusion at the level of the lamina cribrosa65. This can induce micro-ischemic conditions that reduce flow of nutrients and oxygen to surrounding axons and force mitochondria to function at reduced energy levels43. In these conditions, the increased metabolic demands are not met and

16 can ultimately lead to the death of RGCs. It is likely that the biomechanical and vascular theories of glaucomatous neuropathology are not two separate entities of glaucoma, but act in concert with one another to contribute to disease.

Figure 3. The ONH Vasculature. The inner retina receives most blood supply from the central retinal artery (CRA), while the short posterior ciliary arteries (PCA) and the circle of Zinn- Haller (Z-H) nourish the lamina cribrosa (LC). NFL = nerve fiber layer; PLC = prelaminar region; RLC = retrolaminar region; ON = optic nerve. Adapted from Cioffi et al. (1996)98.

2.4.1 The Molecular Mechanisms of Ischemia on RGC Cell Death

There is evidence to suggest that fluctuations in IOP could cause changes in blood flow to the ONH and would theoretically cause ischemia and reperfusion injury43. As a result of ischemia, intracellular calcium levels can increase by several mechanisms. These mechanisms are through the activation of ionotropic glutamate receptors (as seen during excitotoxic conditions), by reversal of sodium-calcium exchange channels, through voltage sensitive calcium channels or by stimulation of metabotropic neurotransmitter receptors which can consequently release calcium from intracellular stores4. Increased calcium levels can activate numerous enzymes dependent on calcium such as protein kinase C, calcium dependent preoteases (calpains), and nitric oxide synthase99,100. The activation of these proteins can lead to breakdown of cellular homeostatic mechanisms and reduce the cytoskeletal integrity of the cell.

Ischemic conditions can lead to deprivation of oxygen, metabolic substrates and inefficient waste product removal4. Loss of these responses will lower homeostatic responses and will subsequently induce injury to tissue. The tissue damage and functional deficits that follow ischemic injury can result in drastic changes in ion movements, neutrotransmitter release and an increase in free radical production. Oxidative stress induced by glucose/oxygen deprivation can result in an increase in toxic reactive oxygen species (ROS) generation such as free radicals, hydrogen peroxide and singlet oxygen101. ROS-induced alterations of macromolecules can subsequently result in damage to cells. During ischemia, free radicals accumulate and ATP is degraded, which leads to the formation of hypoxanthine and increases in intracellular calcium levels in neurons102. This increase in calcium triggers calpains, which can activate enzymes for further free radical production during reperfusion. Free radical bursts can overwhelm normal cellular antioxidant defense mechanisms and cause additional oxidative stress102. Furthermore, mitochondria become overloaded with high calcium levels and release excess cytochrome C103.

In conclusion, there are a variety of mechanisms leading to RGC death in glaucoma that are dependent on biomechanical and ischemic insults. It is likely that all of these pathways are not mutually exclusive and are in fact synergistic to one another.

2.5 Astrocytes in Response to Biomechanical and Ischemic Insult

Glial cells have received considerable attention due to their vast changes seen during glaucomatous damage8. In normal conditions, astrocytes are “quiescent” and perform many regulatory roles to ensure a homeostatic environment to RGCs. Astrocytes supply energy substrates to axons and maintain extracellular pH, ion homeostasis and are efficient at buffering extra synaptic glutamate104,105. Furthermore, astrocytes regulate water exchange between brain and vascular space through expression of water channel aquaporin 4106. Astrocytes can become

“reactive” in the sense that they respond to injury and disease and can participate in a variety of pathological consequences to RGCs. Reactive astrocytes display enlarged cell bodies

(hypertrophy) and a thick network of processes with increased glial fibrillary acidic protein

(GFAP), vimentin, and HSP27107. Cardinal features of reactive laminar astrocytes in experimental and human glaucoma are astrocytic proliferation, migration and altered gene expression8,18. Moreover, reactive astrocytes participate in the formation of a glial scar and participate in remodeling of the ONH by the expression of many extracellular matrix proteins such as matrix metalloproteinases laminin, collagen, tenascin C and proteoglycans108–111. Not only can astrocytes contribute to remodeling of the ONH environment, they produce and/or respond to neurotoxic molecules such as NO, TNFα, interleukin cytokines and endothelins leading to a multitude of dysfunctions in the surrounding axons and blood vessels112–115.

Calcium can play a very important role in glial activation. Calcium waves are propagated intracellularly through the diffusion of second messengers through gap junctions and by extracellular release of ATP116. Astrocyte injury and mechanical disturbance can induce an astrocytic calcium wave and induce GFAP expression via the JNK/c-Jun/AP-1 pathway117.

Blocking calcium induction with intracellular chelators can reduce GFAP and glial scar formation. Moreover, calcium increases in astrocytes have been accompanied by the release of glutamate118,119, which can lead to activation of glutamate receptors and even more calcium influx into astrocytes, ultimately leading to an autocrine oxidative and excitotoxic environment120,121.

In summary, astrocytes have an important role in maintaining homeostasis within the

ONH and lamina cribrosa. These regulatory roles can be lost during disease progression and can activate numerous pathways and mechanisms to abrogate healthy conditions for RGCs.

There is a lack of knowledge in integrating the aforementioned pathological factors such as biomechanics, ischemia/reperfusion, and glial activation associated with glaucoma. A common relation between these factors is the dysfunction of calcium dynamics. A thorough understanding of calcium dependent proteins and its relation to glaucoma is needed to better understand the disease.

2.6 Annexins

The annexins are a family of structurally related proteins characterized by their ability to reversibly bind to negatively charged phospholipid membranes in a calcium dependent manner16. The name annexin was derived from the Greek word “annex” meaning to “bring/hold together”. This name describes a principal property of nearly all annexins, which is to bind to certain biological structures, particularly membranes. All annexins consist of a typical conserved COOH-terminal core domain, which is comprised of four annexin repeats (in annexin

A6, there are eight) tightly packed into a highly alpha-helical disk with a slight curvatures and two principal sides122. The convex side of the core domain harbors the calcium and membrane binding sites and is responsible for mediating the canonical membrane binding properties of annexins. The concave side faces the cytoplasm and contains the NH2-terminal domain. In contrast to the COOH-terminal domain, the NH2-terminal domain is variable in length and can regulate specific annexin functions by interacting with cytoplasmic binding partners12,16 (Figure

4A).

In the presence of phospholipids, the calcium affinity of the binding sites located in the core domain is in the low micromolar range, although this can vary between different annexins123–125. Furthermore, certain annexins have different affinities for acidic phospholipids123. For example, annexin A5 needs a large free calcium concentration of around

20uM to bind to phosphatidylserine-containing liposomes while annexin A1 and A2 need

20 submicromolar calcium concentrations of around 100nM to bind to phosphatidylserine- containing membranes. This indicates that annexins can have diverse functions within the cell that can be induced by a variety of calcium concentrations. Upon calcium binding, annexins can undergo conformational changes where the NH2-terminal domain can be exposed from the core of the molecule. Many cytosolic protein ligands can interact with specific members of the annexin family. Examples of these are the EF-hand-containing calcium binding S100 proteins, which are known to interact with annexin A1, A2, A7 and A1116. Proteolytic cleavage of annexin A1 and A2 can also occur by calpains, plasmins and chymotrypsin123.

Although a main function for the annexin family as a whole remains to be assigned, members of this ubiquitous family have a role in a variety of essential cellular functions such as in calcium, membrane, and cytoskeletal dynamics, which will be discussed later in the review.

Annexins are present in many eukaryotic organisms, including plants and fungus, and are present in a wide variety of cell types. It is interesting to note that no single annexin is present in all cells, suggesting tightly controlled gene expression12. Annexins are regulated through changes in their expression, properties or localization, which may contribute to the pathophysiology in many disease phenotypes12. No human diseases have been linked to alterations or mutations to an annexin gene as its primary cause, although the involvement of annexins has been linked to many diseases. Annexins are upregulated in a number of pathologies, including those related to neurodegeneration19 and glaucoma126. Furthermore, they have been linked to inflammation where annexin antibodies have been found in models of ischemia/reperfusion, and in patients with autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus22,23,127–129.

2.7 Annexins and Membrane Dynamics

Annexins are involved in a multitude of membrane-related roles such as membrane trafficking and organization, membrane domain stabilization, vesicle aggregation, and ion channel activity12. Annexins can form lateral assemblies of 2D crystals on the lipid bilayer to form trimer structures, as first seen using cryo-electron and atomic-force microscopy of annexin

A5130,131 (Figure 4B-4C). Trimer formation depends on calcium and membrane binding and seems to be mainly mediated by the core domain. Crystalline structure arrangement can affect membrane properties including rigidity, fluidity, and lipid segregation and can therefore affect the regulation or stabilization of various membrane domains14,16.

Not only do annexins bind to membrane structures within the cell, some members are able to aggregate membrane vesicles in a calcium dependent manner. Some of these include segregation of membrane lipids to establish certain phospholipid domains in cells, as seen with annexin A2 and A4, or membrane vesicle aggregation, as seen with annexins A1, A2, A4, A6, and A712. The mechanisms of aggregation activity differ between members of the family.

Annexin core domains that are facing adjacent membranes interact to promote contact between membranes, defined as ‘bivalent’ annexin activity11. Furthermore, some annexins are able to participate in exocytotic events by promoting close attachment of membranes to one another to help facilitate membrane fusion during calcium regulated exocytosis132,133.

Figure 4. Annexin Properties. Annexins have a conserved C-terminal domain that contain 4 annexin repeats and harbor the calcium and membrane binding domains (A). The N-terminal domain faces the cytoplasm. Noise-filtered electron microscopy images show crystalline trimer formation of ANXA4 at the plasma membrane (B). Schematic representation of calcium induced ANXA4 trimer formation into 2D crystal arrays on the plasma membrane (C). (A) Adapted from Gerke et al. (2005)16. (B) Adapted from Newman et al. (1991)13. (C) Adapted from Piljic et al. (2006)14.

2.8 Annexins and Actin Dynamics

In addition to membrane-related events, annexins can also interact with components of the actin cytoskeleton17. The role of annexins in actin dynamics are not well understood, but have been implicated in mediating, stabilizing, and/or regulating membrane-actin interactions.

Annexin-actin interactions are highly dynamic in nature and occur only in close proximity to cellular membranes. The C-terminal sequence required for filamentous actin (F- actin) binding is conserved in a number of annexins123. Many members are found primarily at the highly dynamic actin structures, in particular during phagocytosis, vesicle transport, endocytosis and exocytosis17. Annexin A2 was the first annexin described to have F-actin binding properties in a calcium dependent manner134,135. Annexin A2 can participate as a scaffold protein in the stabilization and organization of membrane rafts to the cortical actin

23 cytoskeleton. Annexin A2 can associate with Ahnak (also known as desmoyokin)136. Ahnak is found in the nucleus and at desmosomes, where it associates with G and F-actin and accumulates following cell-cell contact. A functional role of Ahnak is the maintenance of the structural and functional organization of the subsarcolemmal cytoarchitecture137. Ahnak has also been found to interact with “enlargosomes”, which are redistributed to the external surface of the plasma membrane in response to large increases in calcium for membrane repair138.

Downregulation of annexin A2 inhibits re-loclaization of Ahnak, and therefore can potentially participate as a scaffold protein linking plasma membrane domains with actin136.

2.9 Annexins and Membrane Repair

Many cells are prone to destruction and repair as a result of mechanical strain. Control measures need to be in place to ensure that the membrane remains intact and is not compromised, especially in cells that are irreplaceable such as neurons. Rapid resealing mechanisms prevent consequent calcium entry, cytoskeletal breakdown and rapid cell death139,140. There have been many models suggested for membrane resealing, such as the patch or membrane shedding model141. In the patch model, calcium entry at the wound site triggers recruitment of certain liposomes, such as endosomes, enlargosomes, or lysosomes, which then fuse with the plasma membrane to close the wound142,143. Lesion removal can also occur through membrane shedding at sites of calcium entry, where membranes can be folded to form an outward curvature followed by the release of microparticles into the extracellular space144.

Plasma membrane repair is an active process that depends on membrane rearrangement, membrane fusion, and extensive cytoskeletal reorganization139. Annexins are known to promote membrane aggregation and fusion, as well as have interaction with actin dynamics. This makes them well suited for a role in membrane repair processes. Annexins are implicated in sensing and repairing plasma membrane lesions triggered by a variety of stimuli including smaller

24 injuries induced by pore forming toxins to larger injuries induced by laser irradiation24,25,27.

Furthermore, a study by Lennon et al. found that annexin A1 and A2 were found to associate with dysferlin, a protein linked to skeletal muscle repair, in a calcium and injury-dependent manner145. Dysferlin mouse mutants exhibited disrupted annexin distribution and it was proposed that annexins could facilitate plasma membrane repair by the aggregation and fusion of intracellular vesicles at the site of injury. As previously discussed, members of the annexin family are able to form 2D trimer structures at the membrane in a calcium dependent manner. In response to a large influx of extracellular calcium by laser irradiation, annexin A5 was shown to be recruited to the wound site, followed by the formation of 2D arrays25. This prevented the wounded membrane from expanding and contained any further elevation of intracellular calcium.

Cortical actin cytoskeleton is in intimate contact with the plasma membrane and is a fundamental regulator of plasma membrane repair146,147. Tight coupling of cortical actin to the membrane can cause membrane tension during many cellular processes, which can inhibit passive resealing after injury148. Following injury, it is important for actin to quickly depolymerize at the wound site to release membrane tension so the wounded edges can subsequently be brought together146. This is then followed by quick actin re-synthesis. Calcium influx is able to trigger recruitment and accumulation of annexin A2 and S100A11 at site of laser irradiation, which is then followed by rapid buildup of F-actin at site of repair. This buildup of actin was annexin A2 and S100A11 dependent, as the absence of either these proteins prevent F-actin buildup and plasma membrane repair following injury.

2.10 ANXA4

Annexin A4 (ANXA4) was first identified as lipid binding protein II during experiments in attempt to search for F-actin-binding proteins that depend on calcium in porcine intestinal

25 cells135. Similarly to other annexins, ANXA4 is able to self-associate on selective membrane surfaces and aggregate into 2D arrays on the plasma membrane13,149. Limited research has been conducted to elucidate the phospholipid binding function of ANXA4. Membrane-bound

ANXA4 can reduce the mobility of transmembrane and plasma membrane associated proteins14.

Furthermore, ANXA4 2D array formation can inactivate calcium activated chloride conductance channels (CaCC) by preventing the channel phosphorylation by the calcium/calmodulin- dependent protein kinase II (CaMKII)150,151. ANXA4 has been reported to have a role in the regulation of vesicle trafficking by self-associating on membrane surfaces and aggregating phospholipid membranes152 dependent on phosphorylation by protein kinase C153, and also by participating in synaptic exocytosis154.

Limited research has been conducted on ANXA4 knockout mice to elucidate function. A gene trap was inserted into the first intron of ANXA4, although not all expression was eliminated in some tissues155. Further investigation revealed that ANXA4 gives rise to three transcripts, ANXA4 a/b/c which all produce the same identical ANXA4 protein. Studies have been conducted that demonstrated ANXA4 membrane binding in the presence of calcium resulted in liposome aggregation and reduced membrane water permeability due to ANXA4- dependent rigidity of the outer membrane leaflet of these liposomes156. Although, ANXA4a knockout mice were unable to show defective barrier function (transepithelial resistances, water permeabilities and urea permeabilities) after application of hydrostatic pressure in mouse bladders157. In response to stress, ANXA4 is upregulated during ischemia/reperfusion in a middle cerebral artery occlusion (MCAO) and intestinal ischemic injury in mice21,20.

Interestingly, the application of tetrandrine, a calcium channel blocker and anti-inflammatory mediator, reduced the elevation of ANXA4.

Work from our laboratory has identified ANXA4 to be upregulated in a model of biomechanical insult, a stress that mimics the conditions observed at the glaucomatous ONH during elevation of IOP, in human primary astrocytes10. The function of ANXA4 has been unexplored in the retina or in models that relate to glaucoma injury, but has the potential to be a critical factor in integrating calcium and membrane dynamics during biomechanical and ischemic stresses in ONH tissue such as RGC axons and astrocytes.

Chapter 3

Methods

3 Methods

3.1 Cell Cultures

The astroglial A7 cell line derived from the optic nerve of a neonatal rat was used for

158 silencing experiments . Cells were maintained at 37°C at 5% CO2 with Dulbecco’s media H21

(Gibco, Ref#12100) supplemented with 10% fetal bovine serum (Multicell, Ref#080105) and

1% penicillin/streptomycin (GIBCO, Ref#15-140-122). Media replacement occurred 2-3 times a week and passaged when reached 100% confluency. Cells were plated onto new 100mm polystyrene tissue culture dishes (Sarstedt, Ref#83.1802) at least twice a week using TrypLE

(GIBCO, Ref#12604-013) for detachment, followed by inactivation in complete media. Cells were pelleted by centrifugation at 200g for 5 minutes, followed by resuspension in fresh media.

A7 cells were quantified using a hemocytometer and replated at appropriate densities.

The human embryonic kidney (HEK293) cell line was used for plasmid overexpression experiments and shRNA optimization due to its ease of transfection. HEK293 cells were maintained at 37°C at 5% CO2 with Dulbecco’s Modified Eagle Medium (GIBCO, Ref#11995-

065) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Media was changed every other day and passaged using TrypLE for cell detachment three times a week.

HEK293 cell quantification was implemented as stated above.

The PC12 cell line was used for shRNA optimization due to its ease of transfection and overexpression of ANXA4. PC12 cells were maintained at 37°C at 5% CO2 with RPMI media

(GIBCO, Ref#11-875-093) supplemented with 10% horse serum (Invitrogen, Ref#16050-122),

5% fetal bovine serum and 1% penicillin/streptomycin. Cells were passaged once a week using

TrypLE for cell detachment and quantified as described above.

3.1.1 Primary Rat Retinal Astrocyte Isolation and Culture

Our laboratory has previously established an effective protocol for the isolation and culturing of primary adult rat retinal astrocytes159. Briefly, P21 Wistar rats were sacrificed by

CO2 asphyxiation with a protocol approved by the Animal Care and Use Committee of the

University Health Network. Eyes were rapidly enucleated and immediately placed in ice-cold

MEM-H17 media (Gibco, Ref#61100) supplemented with 5% horse serum and 5% fetal bovine serum. Retinas were dissected out and placed in serum-free MEM-H17 supplemented with

TrypLE and 5µg/mL DNase I (Roche, Ref#10104159001) for 20 minutes. Cells were resuspended in specialized astrocyte growth media (Lonza, Ref#CC-4123) and seeded onto T-75 flasks (Corning, Ref#430641). On the following day, media was replaced with complete MEM-

H17 to stimulate growth and to remove debris and other cell types. Media replacement was carried out three times per week. Upon satisfactory growth, cells were shaken at 100 rpm for five hours, after which media was replaced to remove non-astrocytic cells. One day after shaking, astrocyte cultures were detached from the T75 flasks using TrypLE and plated accordingly.

3.2 Induction of Biomechanical and Hypoxic Insults

3.2.1 Biomechanical Insult

Mechanical insult was induced using the FlexerCell® Tension Plus FX-4000T system using a vacuum pump connected to a steel base plate with 4 6-well Bioflex plates slots as previously described10. The pre-coated collagen IV Bioflex wells are composed of a flexible silicone bottom. Prior to plating, wells were coated with additional rat-tail collagen I (Corning,

Ref#354236) to maximize adherence. After one hour incubation at room temperature, collagen I was removed and wells were thoroughly rinsed with PBS three times. Cells were then plated

30 according to the confluency needed for the experiment. Once cells have reached about 70-80% confluency, media was removed and replaced with appropriate media. Cells that were being stretched for long periods of time were serum-deprived for 1 hour prior to stretch. Plates were then loaded onto the baseplate and 12% equiaxial strain at 1Hz was applied in a humidified incubator at 37°C and 5% CO2 for the indicated times.

3.2.2 Hypoxic Insult

In some instances, hypoxic conditions were induced simultaneously with mechanical insult. Prior to hypoxia, cells were plated and serum deprived as described above. The stretch base plate was then placed in a plastic chamber connected to a nitrogen tank and a calibrated oxygen sensor. Nitrogen gas was flushed into the chamber until the oxygen reading was at 1%.

The oxygen levels were kept between 1% and 2% throughout the time course, as previously

36 described . CO2 was maintained constantly at 5%.

3.3 Elevated IOP Model in vivo

3.3.1 Elevated IOP in Rat

To study the acute effects of elevated IOP in rat retinas, P21-30 Wistar rats were used.

All animal experiments were conducted under a protocol approved by the University Health

Network (UHN) animal care committee. Rats were anesthetized with an intraperitoneal injection of 80-100mg/kg ketamine (Bioniche) and 10mg/kg xylazine (Bayer). A drop of topical anaesthetic, 0.5% proparacaine (Alcon), was also applied on the eyes. Body temperature was maintained at 37°C using a heating pad. Once the animal was fully anaesthetized, the anterior chamber at the limbus was cannulated with a 30-gauge needle. The needle was connected to a reservoir of sterile saline water, which was elevated to successfully raise the IOP to 100mmHg for 1 hour. IOP recordings were taken before and after elevation of IOP and throughout the

31 experiment using a TonoLab (iCare) to ensure accurate IOP elevation. Furthermore, proper elevation of IOP was monitored by the complete cessation of retinal and choroidal blood flow resulting in blanching and therefore producing ischemic conditions within the eye. After one hour, the saline reservoir was slowly lowered, blood flow was observed to return to the eye, and the needle was removed. A drop of antibiotics was applied on the cannulated eye and the rat was returned to its cage for recovery. The corresponding eye of the same rat was used as a control.

Control eyes were cannulated with a needle, but no elevation of IOP occurred. After various time points, animals were sacrificed by CO2 asphyxiation. Eyes were then rapidly enucleated and retinas dissected and processed for analyses.

3.3.2 Elevated IOP in Pig

To study the acute effects of elevated IOP in pig eyes, previous members of our laboratory used 17 adult Yorkshire pigs. Animals were anaesthetized with 2.5% isoflurane. Eyes were then cannulated with two 27-gauge needles inserted into the anterior chamber of the eye.

One needle was attached to a saline reservoir and used to elevate IOP. The other needle was connected to a pressure inducer to measure IOP. Pressure was slowly increased at increments of

20mmHg up to 60mmHg then back down to baseline. This was completed four times over a 1 hour period. Eye pressure measurements were additionally confirmed with a tonometer (Mentor

Tonopen XL). Control eyes were cannulated with the needles but had no manipulation of IOP.

After the elevations of IOP, eyes were removed and the retina was dissected out. The ONH was collected and snap frozen using liquid nitrogen and stored at -80°C. ONHs were analyzed for gene expression using quantitative real time polymerase chain reaction (qPCR).

3.4 Gene Expression Analysis through qPCR

Total RNA was extracted from cultured cells using TRIzol® reagent (Life Technologies,

Ref#15596-026). Rodent retinal tissue was collected using RNeasy spin columns (Qiagen,

Ref#74034). RNA was then resuspended in nuclease-free water and incubated at 60°C for 10 minutes. To ensure no DNA contamination, resuspended RNA was digested using RQ1 RNase- free DNase I (Promega, Ref#M6101). RNA was then reverse transcribed using Superscript III

Reverse Transcriptase kit (Invitrogen, Ref#18080-093). After ensuring appropriate cDNA concentrations using NanoDrop 2000 Spectrophotometer, qPCR was performed using the SYBR

Green PCR Master Mix (Applied Biosystems, Ref#4367659) with 200nM of primers (Table 1). qPCR samples were loaded into a Mastercycler Eppendorf Realplex for the running of 40 cycles and annealing temperature of 60°C. This was followed by a melting curve analysis to confirm the purity and specificity of the product. Gene expression values were determined using the comparative Ct method, and normalized to the housekeeping gene TATA-binding protein (TBP) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Table 1. List of Primers Used For quantitative PCR.

Target Protein Forward Reverse ANXA4 – rat 5’-ACCAGCAGCAATATGGAAGG-3’ 5’-GTCCCCCATCTCTTCTCTCC-3’ TBP - rat 5’-ACAGGTGGCAGCATGAAGTG-3’ 5’-GCAGGGTGATTTCAGTGCAGA-3’ ANXA4 - pig 5’-ACAAGAGTACCATCGGCAGG-3’ 5’-GTCTGGTTTATGCGCCGAAT-3’ GAPDH - pig 5’-TTTGACGTTGTAGCCAGCAG-3’ 5’-AGCTTGACGAAGTGGTCGTT-3’

3.5 Protein Analysis

3.5.1 Cell and Tissue Lysate Collection and Protein Quantification

For the preparation of total protein extraction, plated cells had media removed and were washed twice with PBS. Total protein was extracted from cultured cells by applying appropriate amounts of ice-cold RIPA buffer (Cell Signaling, Ref#9806) supplemented with protease inhibitors (Roche, Ref#11836170001) and phosphatase inhibitors (Roche, Ref#04906845001).

A cell scraper was used to remove cultured cells off wells. Protein lysate was then placed in

1.5mL tubes, vortexed and incubated on ice for 10 minutes. Retinal tissue samples were rinsed in PBS two times following dissection and placed in 2xRIPA buffer supplemented with protease and phosphatase inhibitors. The lysate was then incubated on ice for 1 hour and vortexed periodically. To fully digest tissue clumps, a 25-gauge needle was used to passage the lysate several times until no tissue clumps were observed. The lysate was vortexed again and incubated on ice for another 15 minutes. All protein lysates were pelleted by centrifugation at

12000g for 10 minutes at 4°C to remove lipids and debris. The supernatant was removed and transferred to a new tube. Protein lysates were stored at -80°C until needed for SDS-PAGE.

Protein concentrations were quantified using the Bio-Rad DC Protein Assay Kit using the Lowry method (Bio-Rad, Ref#500-0116). Standard bovine serum albumin (Bio-Rad,

Ref#500-0007) was used to generate standard curve values.

3.5.2 SDS-PAGE and Protein Detection

Protein samples were diluted in RIPA buffer and 4x Laemmelli Loading Buffer (Bio-

Rad, Ref#161-0747) containing 10% β-mercaptoethanol (Sigma, Ref#M3148) to achieve equal volumes for SDS-PAGE loading. Samples were boiled for 5 minutes at 95°C and loaded onto a

12% SDS-polyacrylamide gel made from a 30% acrylamide stock (Biorad, Ref#1610158). The gel was run at 55V for 30 minutes, or until the loading buffer passed the stacking gel, followed by 120V for 90 minutes, or until the loading buffer reached the end of the separating gel in Tris-

Glycine-SDS running buffer. The gel was then transferred onto PVDF membrane (Millipore,

Ref#IPFL00010) by either using the Bio-Rad wet transfer (tissue protein) for 1 hour at 0.28mA or by the Pierce Fast Semi-Dry Transfer System buffer (Thermo Scientific, Ref#PI-88217) for 1 hour at 13V. Subsequently, membranes were rinsed in water and Tris-buffered saline containing

0.1% Tween 20 (TBS-T) and then blocked in 5% BSA (FisherScientific, Ref#BP1600-100) diluted in TBS-T. Primary antibodies were diluted in 1% BSA in TBS-T and were shook on

34 transferred membranes overnight at 4°C. Following primary antibody incubation, membranes were rinsed in TBS-T three times for 10 minutes. Secondary antibodies were then incubated on membranes for 1 hour at room temperature. After TBS-T rinsing, membranes were scanned using the LI-COR Odyssey Infrared Quantitative Imaging System. Protein densitometry was analyzed using the LI-COR Odyssey software.

Table 2. List of Antibodies and Dilutions For Incubation. (WB = western blot, IF = immunofluorescence).

Target Protein WB IF Supplier, Ref# ANXA4 1:2000 1:400 Prestige Antibodies, HPA007393

GAPDH 1:10,000 Calbiochem, CB1001

Donkey Anti-Rabbit IgG IRDye 1:10,000 Mandel Scientific, 926-68023

Donkey Anti-Mouse IgG IRDye 1:10,000 Mandel Scientific, 926-32212

GFAP 1:200 Sigma, G3893

β3 tubulin 1:200 Abcam, ab78078

AlexaFluor 647-Phalloidin 1:50 Life Technologies, A22287

AlexaFluor 488 Goat Anti- 1:200 Life Technologies, A11034 Rabbit IgG

3.6 Flow Cytometry and Cell Viability Assay

After removal of media and wash with PBS, cells were detached using TrypLE. TrypLE was neutralized with complete media and cells were collected into 1.5 mL tubes. Tubes were spun down at 300g for 5 minutes at 4°C to pellet cells. Media was aspirated from the tubes and cells were washed with cold PBS, followed by centrifugation. Cells were re-suspended in cold

PBS and filtered through a cell strainer into a flow cytometry tube (Falcon, Ref#352235). Cells were maintained on ice and were analyzed by the BD FACS Canto™ Flow Cytometry Machine.

To measure cell viability, the cells were re-suspended in Annexin V binding buffer (Life

Technologies, Ref#V13246) containing Annexin-V conjugated to Alexa Fluor®488 (Life

Technologies, A13201) and 10µg/mL of propidium iodide (PI) (Sigma, Ref#4170). Annexin V conjugate was excited by the 488nm laser line and emitted at 520nm. PI was excited by the

PercP-Cy5.5 fluorochrome by the 488nm laser line and emitted at a wavelength greater than

650nm.

3.7 Pharmacological Treatments

To stimulate elevation of intracellular calcium, ionomycin was used. Ionomycin is a calcium ionophore that can induce an influx of calcium ions from extracellular medium or release of free calcium from intracellular calcium stores160,161. Ionomycin was reconstituted in

DMSO at a concentration of 1.3mM. To manipulate actin dynamics, jasplakinolide and cytochalasin D were used162,163. Jasplakinolide, an F-actin stabilizer, was reconstituted in DMSO at a concentration of 190uM. Cytochalasin D, a potent inhibitor of actin polymerization, was diluted in DMSO at a concentration of 986uM. All drugs were diluted at appropriate concentrations in complete media and applied on cells for various time points.

3.8 Cell Membrane Integrity Assay

To measure membrane integrity, cells were incubated with either FM1-43FX dye

(Molecular Probes, Ref#F-35355) or impermeant DRAQ7™ (Cell Signaling, Ref#7406) dye.

FM1-43 is a well-established dye used to measure cell membrane integrity. FM dyes are lipophilic styryl compounds used in studies involving the plasma membrane. The water-soluble dye is virtually non-fluorescent in aqueous media but increases its fluorescence immensely when bound in lipid environments (Appendix 3)164. They are believed to anchor onto the outer leaflet of the surface membrane with its lipophilic tail, while the ammonium head prevents the

36 molecule from permeating the cell165. DRAQ7 is a new generation dye that is compatible with fixation and permeable in cells only during conditions of compromised membranes where it then binds to nuclear DNA166. Fluorescence is observed at the nucleus in the Cy5 channel during membrane disruption while FM1-43 fluoresces in the FITC channel. FM1-43 was diluted in

HBSS with (GIBCO, Ref#14025-092) or without (GIBCO, Ref#14175-095) calcium at a concentration of 5µg/mL and applied on cells that were stretched for 10 minutes at 25°C.

Following stretch, dye was removed and 4% PFA was placed on cells on ice for 10 minutes.

DRAQ7™ was diluted at a concentration of 3µM in HBSS with calcium. The dye was placed on cells for 2 hours during stretch. After stretch, cells were washed with PBS and fixed with 4%

PFA.

3.9 Molecular Biology and ANXA4 Gene Manipulation

3.9.1 ANXA4 Overexpression

Bacteria colonies containing full sequence human ANXA4 (GenBank ID: BC011659) tagged to GFP (pcDNA-DEST47) or V5 (pcDNA3.2-DEST) at the C-terminal were purchased at the SPARC BioCenter at the Hospital for Sick Children. A bacterial single colony was isolated and grown in 5mL LB Broth supplemented with 0.1% ampicillin in a bacterial shaker overnight at 200rpm in 37°C. The next day, bacteria was isolated using centrifugation and plasmid DNA was isolated using Qiagen Spin Miniprep Kit (Qiagen, Ref#27106).

Subsequently, DNA plasmid underwent Sanger sequencing at the Center of Applied Genomics at the Hospital for Sick Children to confirm proper sequence. Furthermore, plasmids underwent restriction enzyme digestion and then run on a 1% agarose DNA gel to confirm proper size of plasmid DNA fragments. Lipofectamine 3000 (Thermo Fisher, Ref#L3000008) reagent was used to transfect plasmids into cells overnight. ANXA4 overexpression was evaluated by GFP fluorescence imaging and western blot (Appendix 2).

3.9.2 ANXA4 siRNA Knockdown

siRNAs were used to generate ANXA4 knockdown in cells. siRNAs are short double stranded RNAs that target complementary mRNAs for degradation via the RISC complex167.

ON-TARGETplus SMARTpool rat ANXA4 siRNAs (Dharmacon, Ref#L-097163-02-0005) were purchased and transfected into cells using Dharmafect 1 Transfection Reagent

(Dharmacon, Ref#D001630-01) in media containing no antibiotics for 24 hours. After 24 hours, media was changed and cells were incubated in complete media with 1% fetal bovine serum for an additional 24 hours. ANXA4 knockdown was assessed by western blot. ANXA4 knockdown was evaluated by qPCR and western blot (Appendix 1).

3.9.3 Viral Plasmid ANXA4 shRNA Generation

4 shRNA sequences targeting ANXA4 (Genecopoeia, Ref#RSH050780) and 1 non- targeting (NT) control (Genecopoeia, Ref#CSHCTR001-CU6) was purchased. A7 and PC12 cells were transfected with 5 different ANXA4 shRNA sequences containing GFP reporter gene using Lipofectamine 3000. 48 hours post transfection, mRNA was isolated using TriZol®, followed by cDNA synthesis. qPCR analysis was conducted to assess mRNA knockdown. After validation of sufficient knockdown, 2 shRNA sequences with the best knockdown were chosen.

RT-PCR with Platinum Taq Polymerase (Invitrogen, Ref#10966-018) was used with appropriate primers (Table 1) to generate the GFP-U6-shRNA cassette sequence from the Genecopoeia vectors. The PCR products were then run on a DNA gel and purified. In order to engineer appropriate sticky ends on the PCR products for efficient ligation into the viral vector backbone, the PCR fragments underwent an adenosine-tailing procedure using Taq DNA Polymerase

(Promega, Ref#M8291) and dATP (Invitrogen, Ref#10216-018) for 30 minutes at 70°C and then ligated into a pGEM-T Easy vector (Promega, Ref#A1360) using T4 DNA ligase (NEB,

Ref#M0202S) at 4°C overnight. Ligated vectors were then transformed into SURE2 competent

38 cells (Agilent, Ref#200152) and then grown onto LB Agar plates containing ampicillin overnight at 37°C. The next day, 6 colonies were picked and amplified in LB Broth containing ampicillin overnight. Bacteria were then collected and plasmids were purified using Qiagen

Spin Miniprep Kit. To confirm correct orientation of the insert in the pGEM-T easy vector, plasmids were digested with restriction enzyme NcoI-HF (NEB, Ref#R3193S) at 37°C for 1 hour and then run on a DNA gel. pGEM –T easy purified vectors were digested with NcoI-HF and SacI-HF (NEB, Ref#R3156S) for 1 hour at 37°C followed by DNA gel purification. scAAVeGFP backbone vector was also cut at the NcoI and SacI sites followed by gel purification to release its GFP sequences and to produce 2 sticky ends. The backbone vector and

PCR product was then ligated using T4 DNA Ligase at 4°C overnight. The plasmids were transformed into SURE2 competent cells. NcoI and SacI digestions were then completed to ensure the insertion of the PCR shRNA product into the scAAVeGFP backbone after plasmid purification. Each time the scAAVeGFP vector was amplified or transformed, several colonies were picked and SmaI (NEB, Ref#R0141S) digestion was completed to choose plasmids without recombination artifacts. Purified plasmids were sequenced and validated for the correct insert sequence. Furthermore, new ANXA4 shRNA and NT shRNA sequence vectors were transfected using Lipofectamine 3000 into A7 and HEK293 cells to validate GFP expression.

Table 3. List of Primers Used For shRNA PCR Product.

Target Gene Forward Reverse GFP-U6- 5’-AGGCCCAGAAGATCGAGTG-3’ 5’-CCTTCACAAAGATCCCAAGC-3’ shRNA Cassette

3.9.4 Viral shRNA Expression in vitro and in vivo

Primary rat retinal astrocytes were infected with the virus for 2 weeks at various MOIs to evaluate GFP expression and knockdown. For intravitreal AAV2 injections, P20 Wistar rats

39 were anesthetized by intraperitoneal injection as described above. AAV2-ANXA4shRNA or

AAV2-NTshRNA was injected in both eyes of the animal using a Hamilton syringe (~1.5-4uL =

3.0x1010 PFU). To see infection efficiency, rat eyes were harvested at 3 weeks and flatmount was conducted to evaluate GFP expression.

3.10 Imaging

3.10.1 Cell Immunofluorescence

For immunofluorescence staining, cells were plated on Bioflex silicone membrane 6- well plates s mentioned above. Following treatment, cells were fixed with 4% paraformaldehyde

(PFA) for 30 minutes on ice. Cells were then either processed for immunofluorescence imaging or maintained at 1% PFA at 4°C. If processed for immunofluorescence imaging, the silicone bottom was removed from the plate using a sharp scalpel, followed by cutting of the silicone membrane into wedges. The cells on the wedges were washed with PBS and treated with 0.2%

Triton-X diluted in PBS for 30 minutes. Cells were then blocked with 5% goat serum diluted in

0.1% Triton-X PBS for 1 hour. Primary antibodies were diluted in PBS and applied on the wedges overnight at 4°C. The next day, primary antibody was washed off with PBS, followed by application of secondary antibody incubation for 1 hour at room temperature. Secondary antibody was then washed off cells with PBS. To stain for F-actin, phalloidin conjugated to Cy5 fluorophore (Molecular Probes, Ref#A22287) was applied to cells for 1 hour at room temperature, followed by washing with PBS. Finally, wedges were mounted on slides using

MOWOIL® mounting media (Sigma, Ref#81381) supplied with DAPI (Sigma, Ref#D9564) and allowed to dry overnight at room temperature.

3.10.2 Tissue Immunofluorescence

Eyes were harvested from rats and washed in PBS after appropriate treatment. Eyes were then placed in 4% PFA and allowed to fix at 4°C for 4 hours. Scissors were then used to cut around the limbus to remove the anterior portion of the eye, including the lens. Eyes were placed back in 4% PFA for an additional 2 hours. Following fixation, eyes were dehydrated in a

30% sucrose solution overnight. Retinas were dissected and prepared for immunofluorescence staining in a similar method as described above, followed by flatmounting on slides, or embedded in TissueTek® Optimum Cutting Temperature Compound (VWR, Ref#25608-930) for cryostat sectioning.

3.10.3 Microscopy

Cells and tissue sections were imaged on Zeiss Confocal Microscope LSM 780 or using the Nikon Eclipse Ti epifluorescence microscope. Image analysis was performed on Zen or NIS

Elements software.

3.11 Statistical Analysis

All statistical analysis was performed using a student’s T-test or ANOVA for multiple groups. Graphs represent ±SEM values that were obtained from independent experiments.

Significance was determined by a P value of less than 0.05 (*p). Results denoted by 2 asterisks

(**p) represented a P value of less than 0.01 and 3 asterisks (***p) denoted a P value of less than 0.001. The number of biological replicates for each experiment is described in the figures.

Chapter 4

Results

4 Results

4.1 Aim 1: Characterize ANXA4 Regulation in Response to Biomechanical and Hypoxic/Ischemic Stress

4.1.1 ANXA4 is Upregulated in Response to Biomechanical and Hypoxic Stress in vitro

In order to corroborate previous findings of increased ANXA4 protein levels after mechanical insult10, A7 cells were stretched for 2, 6, and 24 hours using the Flexercell® Tension

Plus FX-4000T system. Although a trend of elevated ANXA4 protein was seen after 24 hours of stretch, it was not significant (Figure 5A). Literature has suggested that hypoxic conditions can induce elevation of intracellular calcium levels168. To see if this stress is relevant in ANXA4 response, primary rat retinal astrocytes (PRRA) were stretched for 24 hours in a 1% oxygen chamber. There was no difference of ANXA4 levels with hypoxia or stretch alone, although combining the stresses together resulted in a 1.5-2-fold increase in ANXA4 levels (Figure 5B).

4.1.2 ANXA4 is Upregulated in Response to Biomechanical and Ischemic Stress in vivo

During elevation of IOP, biomechanical and ischemic stress can occur in the retina and the ONH4,65. Rat eyes were subject to an acute 100mmHg increase of IOP and retinas were collected after various time points. To determine if ANXA4 expression is regulated during these conditions, qPCR, western blot, and immunofluorescence analysis was conducted. 6 hours after elevation of IOP, there was no significant rise in ANXA4 mRNA levels, but after 18 hours, there was a significant 6-7-fold increase in ANXA4 mRNA expression (Figure 6A). Western blot analysis revealed an increase of protein 1 day after elevation of IOP, and a significant 3- fold elevation of ANXA4 protein levels 3 days after elevation of IOP (Figure 6B). Confocal immunofluorescence microscopy was used to confirm the increase of ANXA4 expression in the

retina and to determine its localization. ANXA4 staining was visibly strongly induced at the

Müller glia as soon as 1 day after elevation of IOP and throughout the entire retina 2 days after

IOP (Figure 6C).

To further corroborate these results, pig ONH samples were analyzed that had been

previously obtained after exposure to 60mmHg IOP elevation for 1 hour35. Pig eyes are

excellent candidates for the study of IOP elevation due to their similarity to human eyes169.

After IOP elevation, pig eyes were immediately collected, and the ONH was dissected out for

mRNA collection. qPCR was performed and revealed a 6-fold increase of ANXA4 mRNA

expression after 1 hour of elevation of IOP (Figure 6D).

A B

2 ** 2 Control 1.5 1.5 Stretch 1 Control 1 Stretch ANXA4 Hypoxia 0.5 0.5 Change of Control) Change of Control)

(Fold Levels Protein Hypoxia and ANXA4 Protein Levels Levels ANXA4 Protein

(Fold Change of Control) Change(Fold of Control) 0 0 Stretch 2h 6h 24h 24h

Figure 5. ANXA4 is Upregulated in Response to Biomechanical and Hypoxic Stress in vitro. A7 cells that underwent 12% stretch at 2, 6, and 24 hour time points showed no significant elevation of ANXA4 protein levels (A, n=4). PRRA that underwent 12% stretch for 24 hours also did not exhibit elevated ANXA4 protein levels (B, n=3). When coupled with 1% hypoxia, ANXA4 protein levels increased (B, n=3, **p<0.01).

A B 10 4 **

) 8 * 3 6 Control 2 Control 4 High IOP High IOP 1

Change of Control 2 ANXA4 mRNA (Fold (Fold ANXA4 mRNA ANXA4 Protein Levels Levels ANXA4 Protein

Change(Fold of Control) 0 0 6hr 18hr 1 Day 3 Day

C DAPI ANXA4 Merge Control D Control 9 * 8

7 Δ Ct)

Δ 6 1 Day 5 Post-IOP 4 3 2 1

( TBP to Normalized 2 Day 0 Post-IOP Expression ANXA4 mRNA Relative Control High IOP

Figure 6. ANXA4 is Upregulated in Response to Ischemic/reperfusion Stress in vivo. Rats underwent elevation of IOP to 100mmHg. ANXA4 mRNA expression was elevated in the retina 18 hours after elevation of IOP to 100mmHg in rat eyes (A, n=4, p<0.05). Elevated ANXA4 protein levels were seen 3 days after elevation of IOP (B, n=3, p<0.01). ANXA4 staining was seen in Müller glia (white arrowhead) as soon as 1 day after injury, followed by ANXA4 staining throughout the retina 3 days after injury (C, n=2, scale bar=50µm). Blue staining represents DAPI. Elevated ANXA4 mRNA expression was seen 1 hour after elevation of IOP to 60mmHg in pig eyes (D, n=17, p<0.05).

4.1.3 ANXA4 Does Not Influence Cell Death During Biomechanical and Hypoxic Stress in vitro

To evaluate cell death, PRRA were stretched for 24 hours in a 1% oxygen chamber.

Annexin V and PI staining measured through flow cytometry revealed elevated apoptotic and

cell death counts after 24 hours (Figure 7A-7B). To assess whether ANXA4 is involved in cell

death associated with stretch and hypoxia, ANXA4 siRNA was transfected into A7 cells and

used to knockdown protein levels (Appendix 1). Although elevated PI staining was seen

between control and combined 12% stretch and 1% hypoxia conditions, there was no significant

change between the NT siRNA and ANXA4 siRNA group (Figure 7C). Subsequently, due to

ease of plasmid transfection, HEK293 cells were transfected with either pcDNA empty vector or

ANXA4 tagged to V5, followed by 12% stretch and 1% hypoxia for 24 hours (Appendix 2).

Again, PI staining revealed an increase between control and combined stretch and hypoxia

conditions, but there was no significant change compared to the pcDNA and ANXA4-V5 group

(Figure 7D). Annexins have been documented to translocate to membranes upon elevation of

calcium. To examine ANXA4 translocation under conditions of stress, PRRA were stretched in

hypoxic conditions for 24 hours. Immunofluorescence staining of ANXA4 revealed that cells

that were smaller or with condensed nuclei had concentrated ANXA4 staining at the nuclear and

cellular membrane (Figure 7E).

A B 46 2 2 Control Control 1.5 1.5

V (Fold (Fold V Hypoxia Only Hypoxia Only 1 1 Stretch Only Stretch Only Control) Control) Annexin

Change Control) Change Control) 0.5

% 0.5 Stretch and Stretch and Hypoxia Change% PI (Fold Hypoxia 0 0 24h 24h

C D * 1.5 ** * 1.5 *

1 Control Control 1

Hypoxia and Control) Control) 0.5 Stretch and Control) 0.5 Stretch Hypoxia %PI (Fold Change%PI (Fold %PI (Fold Change%PI (Fold 0 0 NT ANXA4 siRNA pcDNA ANXA4-V5

ANXA4 Merge+DAPI E

Control

12% Stretch and 1% Hypoxia

Figure 7. ANXA4 Does Not Influence Cell Death During Biomechanical and Hypoxic Stress in vitro. A trend of elevated cell death was seen in PRRA, as measured by Annexin-V and PI after 24 hours of 12% stretch and 1% hypoxia (A-B, n=3). Increased membrane permeability, measured by PI, was seen in A7 (C, n=4, NT **p<0.01, ANXA4 siRNA *p<0.05) and HEK293 (D, n=4, pcDNA *p<0.05, ANXA4-V5 *p<0.05) 24 hours after stretch and hypoxia. Knocking down ANXA4 in A7 cells did not influence membrane permeability when compared to non-targeting control after 24 hours of stretch and hypoxia (C, n=4). Overexpressing ANXA4-V5 in HEK293 cells did not influence membrane permeability, as well (D, n=4). ANXA4 staining of PRRA revealed a cytoplasmic distribution in control conditions (E). After stretch and hypoxia, cells that were shrinking exhibited increased ANXA4 staining at the nuclear and cytoplasmic membrane (E, white arrows). Blue staining represents DAPI.

4.2 Aim 2: Elucidate the Functional Role of ANXA4 on Membrane Permeability During Biomechanical Insult

4.2.1 Membrane Permeability Increases in a Calcium-Dependent Manner Upon Biomechanical Insult

Annexins have been shown to contribute to membrane repair processes during membrane breaks by laser irradiation24–27. To study this mechanism in a pathologically relevant biomechanical insult model, membrane permeability was first determined during stretch conditions. A7 cells were incubated with FM1-43, a dye that fluoresces in a lipid environment, in HBSS with or without calcium and stretched at 12% for 10 minutes. After stretch, FM1-43 dye was quickly removed and cells were fixed in 4% PFA. FM1-43 fluorescence intensity was increased during stretch with extracellular calcium compared to the non-stretch control (Figure

8A-8B). In conditions without extracellular calcium, FM1-43 fluorescence intensity was increased even further compared to its non-stretch control and to cells that were stretched with extracellular calcium. This suggests that extracellular calcium is important for inhibiting plasma membrane permeability. To further validate these results and to highlight the importance of extracellular calcium in stretch conditions, A7 cells were stretched in HBSS with or without calcium and collected for flow cytometry. Cells that were stretched with calcium had no difference in PI staining while cells that were stretched without calcium had significantly increased PI incorporation compared to its non-stretch control (Figure 8C).

4.2.2 ANXA4 Translocates to Membrane Structures Upon Elevation of Intracellular Calcium

To visualize ANXA4 localization during elevation of intracellular calcium, A7 cells were transfected with ANXA4-GFP plasmids (Appendix 2) and subsequently treated with 10µM of the calcium ionophore, ionomycin, for 5 minutes. Live imaging was conducted to visualize

ANXA4 membrane translocation, as well as microscopy after PFA cell fixation in A7 and

HEK293 cells, respectively. After treatment of ionomycin, ANXA4 rapidly translocates to

membrane structures within the cell, including cellular and nuclear membrane (Figure 9A-9B).

ANXA4-GFP also localized to punctate areas within the cytoplasm, so it is likely that it interacts +Ca2+ A with other membrane structures within the cell as well.

Control

2+ 2+ A +Ca -Ca B 3 * ** Control

2 * Control

12% Stretch Control) 1 12% Stretch

Intensity a.u.Change (Fold Fluorescence Mean FM1-43 0 + Ca2+ - Ca2+

C ** 10 **

6 Control

% PI 4 12% Stretch

2 0 +Ca2+ -Ca2+

Figure 8. Membrane Permeability Increases in a Calcium-Dependent Manner Upon Biomechanical Insult. A7 cells incubated with calcium showed an increase in FM1-43 fluorescence intensity after 10 minutes of stretch (A-B, n=3, *p<0.05). A7 cells incubated without calcium showed an even higher increase in FM1-43 fluorescence intensity (A-B, n=3, **p<0.01, scale bar=50µm). A7 cells that were stretched for 10 minutes without calcium had increased membrane permeability as measured by PI (C, n=3, **p<0.01).

A +10µM ionomycin

B +10µM ionomycin

Figure 9. ANXA4 Translocates to Membrane Structures Upon Elevation of Intracellular Calcium. HEK293 (A) and A7 (B) cells transfected with ANXA4-GFP were treated with 10µM ionomycin for 5 minutes. HEK293 cells were fixed with 4% PFA and showed clear ANXA4 translocation to membrane structures after ionomycin treatment (A, white arrowhead). A7 cells also showed clear ANXA4 translocation after ionomycin treatment using live imaging (B, white arrowhead).

4.2.3 ANXA4 Knockdown Induces Membrane Permeability During Biomechanical Insult

Annexins have been known to translocate to membrane structures upon apoptosis and during elevation of intracellular calcium, yet little is known on the physiological importance of this contrivance. To see if ANXA4, a calcium-dependent membrane binding protein, is important in membrane dynamics during stretch, ANXA4 siRNA was used to knock down the protein (Appendix 1). A7 cells were then stretched at 12% for 2 hours. DRAQ7 was incubated in HBSS with calcium and applied on the cells during stretch. After stretch, cells were washed in cold HBSS and fixed in 4% PFA on ice. Each biological replicate is an image that has 20 cells imaged quantified for each condition. ANXA4 knockdown cells had increased DRAQ7 intensity at the nucleus in non-stretch conditions when compared to NT siRNA control (Figure

10A-10B). Increased DRAQ7 fluorescence intensity was also seen in stretched cells that had no

manipulations of ANXA4 levels. Furthermore, ANXA4 knockdown increased DRAQ7

fluorescence intensity during stretch, suggesting that ANXA4 contributes to membrane stability

and/or repair.

A NT siRNA ANXA4 siRNA B

Control 120 **

100 ***

80 *** 60 40 12% Stretch

Control siRNA 20 *

0 Intensity Compared to NT to NT Intensity Compared in DRAQ7 Mean Difference NT ANXA4 NT ANXA4 -20 Control siRNA Stretch siRNA Control Stretch Figure 10. ANXA4 Knockdown Induces Membrane Permeability During Biomechanical Insult. A7 cells with ANXA4 knockdown showed increased DRAQ7 fluorescence intensity after 2 hours of stretch compared to NT siRNA stretch (**p<0.01) and NT siRNA control (A-B, n=9, ***p<0.001). Increased DRAQ7 fluorescence intensity was seen in ANXA4 siRNA Control (*p<0.05) and NT siRNA stretch (***p<0.001) when compared to NT siRNA control (A-B, n=9).

4.3 Aim 3: Determine ANXA4 Interaction With Cytoskeletal Dynamics in vitro and in vivo

4.3.1 ANXA4 Co-Localizes With F-actin at the Membrane Cortex and Responds to Actin Disruption in vitro

Previous literature has suggested interactions with annexins and F-actin, although there

is limited research in vivo and no literature has reported ANXA4 interaction with F-actin.

Furthermore, annexin-actin interactions are important in terms of membrane repair24,147,170,171.

A7 cells were used to observe endogenous ANXA4 cellular distribution. ANXA4 staining was

distributed fairly evenly in the cytoplasm and nucleus, but had strong staining at the plasma

membrane in areas of cell-cell contact (Figure 11A), and in areas where cortical F-actin present

in control conditions (Figure 11B-11D).

Literature has also suggested that ANXA2 localization can be altered following

treatment of actin disruptors such as jasplakinolide and cytochalasin D24. Treatment with these

pharmacological actin disrupters revealed remarkably altered actin staining when compared to

DMSO control (Figure 12A-12C). ANXA4 staining became more punctate upon actin

disruption, especially around the perimeter of the cell and in areas where disrupted cortical F-

actin is present.

ANXA4

52 ANXA4 F-actin Merge+DAPI A

Figure 11. ANXA4 Co-Localizes with F-actin at the Membrane Cortex. ANXA4 (green) staining was concentrated in areas of membrane-membrane contact between adjacent A7 cells (A, white arrowhead, scale bar=20µm). ANXA4 staining co-localized with the actin cortex (red) in A7 cells (B, scale bar=20µm). Blue represents DAPI. Zoom-in subsets of (B) reveal co- localization with cortical F-actin (C-D, white arrowheads, scale bar=5µm).

ANXA4 F-actin Merge+DAPI A DMSO Control

+1µM cytochalasin D

+0.5µM jasplakinolide

Figure 12. ANXA4 Responds to Actin Disruption. ANXA4 (green) staining becomes punctate around the perimeter of the cell after treatment with actin disruptors cytochalasin D (B) and jasplakinolide (C) for 1 hour in A7 cells compared to DMSO control (A). ANXA4 staining is punctate at areas of disrupted cortical actin (white arrowheads). Blue represents DAPI. Scale bar=20µm.

4.3.2 Conserved ANXA4 Expression is Located at the Inner Retina

Since there has been very limited published research on ANXA4 in the retina, human, mouse, and rat retinal sections were stained with ANXA4. Interestingly, a prominent conserved staining pattern was observed at the inner retina, particularly at the nerve fiber layer, in astrocytes and in vascular endothelial cells in all of the species mentioned above (Figure 13).

Additionally, there was staining in the outer plexiform layer of the rat, but not in the mouse or human.

ANXA4 Merge+DAPI A

Human

Mouse

-ve staining control C

Rat

Figure 13. Conserved ANXA4 Expression is Found at the Inner Retina. ANXA4 staining is prominent at the nerve fiber layer of human (A), mouse (B), and rat retina (C) (white arrows). ANXA4 staining is also seen in vascular endothelial tissue (white arrowhead) and at the outer plexiform layer of the rat. Blue represents DAPI. Scale bar=50µm.

4.3.3 ANXA4 Co-Localizes With F-actin in the Retina

Strong co-localization was seen with ANXA4 and F-actin, but not with tubulin (stained

with β3 tubulin) or intermediate filaments (stained with GFAP) in normal rat retina and ONH

(Figure 14, 15). Cross section co-localization coefficient measurements throughout the whole

ONH revealed strong co-localization coefficients with ANXA4 to F-actin, and low co-

localization coefficients to GFAP or β3 tubulin (Figure 14D). Cross sections of the ON and

ONH reveal strong expression of ANXA4 with F-actin in striated perpendicular astrocyte fibers

in respect to RGC axons (β3 tubulin). This suggests high expression of ANXA4 with F-actin in

supporting astrocytes85. Interestingly, ANXA4 co-localized with actin only at the nerve fiber

layer and outer plexiform layer of the retina (Figure 15D).

A ANXA4 β3 tubulin Merge+DAPI

B GFAP D 0.8

0.6

0.4

Coeficient 0.2 Co-localization F-actin C 0

Figure 14. ANXA4 Co-Localizes with F-actin in the ON. ANXA4 (green) staining of ON cross-sections revealed high co-localization with F-actin (A, phalloidin) but not with intermediate filaments (B, GFAP) or tubulin (C, β3 tubulin) (A-D). Co-localization coefficients were generated and show high co-localization with ANXA4 (green) to F-actin (red) (D, n=2). Blue represents DAPI. Scale bar=50µm.

β3 tubulin Merge+DAPI A ANXA4

B GFAP

F-actin C

D ANXA4 F-actin Merge+DAPI

Figure 15. ANXA4 Co-Localizes with F-actin at the ONH and Retina. ANXA4 (green) staining highly co-localized with F-actin (C, phalloidin) but not with tubulin (A, β3 tubulin) or intermediate filaments (B, GFAP) in the normal rat ONH (scale bar=100µm). ANXA4 co- localized with F-actin in the retina but only in the inner retina and outer plexiform layer (D, white arrowhead, scale bar=50µm)

4.4 Aim 4: Develop Tools to Generate ANXA4 Knockdown in vivo

4.4.1 shRNA Vector Plasmids Generates a Significant Knockdown of

ANXA4

4 shRNA vectors targeting rat ANXA4 with GFP reporter gene were purchased. GFP

expression was strongly induced in the A7 and PC12 rat cell line 48 hours post transfection

(Figure 16A). qPCR analysis revealed a knockdown of ANXA4 in A7 and PC12 cells in all of

the shRNA sequences tested (Figure 16B-16C). 2 of the 4 shRNA sequences (shRNA 1 and 3)

were selected for AAV2 viral vector cloning.

1 shRNA 2 shRNA 3 shRNA 4 shRNA C shRNA A

PC12

B C ** ** * 1 1

Non-targeting Non-targeting Control Control 0.5 0.5 shRNA shRNA

Anxa4 mRNA Anxa4 mRNA Anxa4 mRNA 0 0 shRNA) Control (Fold (Fold Control shRNA) Control (Fold

Figure 16. ANXA4 shRNA Sequences Generates A Significant ANXA4 Expression Knockdown. ANXA4 shRNA transfection produces a significant knockdown in A7 cells (A, B, n=2-3, *p<0.05, **p<0.01) and PC12 cells (A, C, n=1) 48 hours post-transfection as validated by GFP reporter gene expression and qPCR.

4.4.2 ANXA4 shRNA Sequence Was Successfully Cloned into dsAAV Viral Vector Backbone

ANXA4 shRNA sequences were cloned into a scAAVeGFP backbone as described

above. Briefly, appropriate PCR sequences were inserted into the digested viral scAAVeGFP

backbone vector. NcoI and SacI digestion was used to confirm correct insertion of shRNA PCR

product to viral plasmid backbone. This confirmed insertion of the GFP sequence, U6 promoter

and shRNA sequence from purchased shRNA plasmids. SmaI digestion revealed low

recombination of the plasmid, as seen with complete or near complete digestion. To confirm

proper expression of constructed viral vectors with respective shRNA sequences, vectors were

transfected into A7 or HEK293 cells and revealed strong GFP expression (Figure 17).

Packaging of viral vectors by Vector Biolabs produced high titer virus. AAV2-ANXA4-

1shRNA, AAV2-ANXA4-3shRNA, AAV2-ANXA4-NTshRNA produced 1.8x1013, 8.9x1012,

7.8x1012 GC/mL, respectively, as measured by bGH qPCR.

scAAVeGFP A 1 shRNA 3 shRNA C shRNA Only

HEK293

Figure 17. ANXA4 shRNA Sequence with GFP Reporter Gene Was Successfully Ligated into dsAAV Viral Vector Backbone. shRNA sequences with GFP reporter gene were cloned into dsAAV vectors and then transfected into A7 and HEK293 cells. Clear GFP expression was seen in the cells.

4.4.3 AAV2-ANXA4-shRNA Successfully Infected Primary Rat Retinal Astrocytes in vitro and the Rat Retina in vivo

PRRA were infected at an MOI of 50 000 by diluting the virus in complete media. 2 weeks after infection, GFP fluorescence was observed (Figure 18A). AAV2-3shRNA infected

PRRA exhibited the highest amount of cells expressing GFP (preliminary observations). No cytotoxicity was observed in all virus groups. In a pilot study, rat retinas were collected 3 weeks after intravitreal injection of 3.0x1010 PFU and prepared for flatmounting. All viruses were able to successfully cells at the inner retina (Figure 18B). Again, AAV2-3shRNA exhibited the highest amount of cells expressing GFP (preliminary observations).

1 shRNA 3 shRNA C shRNA A

PRRA

B 1 shRNA 3 shRNA C shRNA

Retina

Figure 18. AAV2-ANXA4shRNA successfully infected PRRA and the retina. AAV2 viruses were infected into PRRA at an MOI of 50 000 (A, scale bar=50µm) and the rat retina at 3.0x1010 PFU (B, scale bar=100µm). GFP expression was seen 2 weeks after infection in the PRRA and 3 weeks after infection in the rat retina.

Chapter 5

Discussion

5 Discussion

Glaucoma is a disease characterized by a multitude of factors that lead to the degeneration of RGCs, ultimately leading to blindness. Many glaucoma patients experience biomechanical insult at the ONH due to the elevated IOP, along with other disruptions involving the vasculature2. This can cause mechanical and physiological stress on the RGC axons and retinal astrocytes at the ONH, leading to cytoskeletal disruption, and the eventual death of delicate RGCs65,86. This project examines the regulation of the calcium-dependent membrane binding protein, ANXA4, during biomechanical and ischemic/reperfuson stress, as well as its important role in membrane stabilization during biomechanical insult.

In my experiments, ANXA4 was upregulated in retinal astrocytes following biomechanical and hypoxic stress. Furthermore, ANXA4 was strongly upregulated in an elevated IOP model of ischemia/reperfusion in the rat. Knockdown or overexpression of

ANXA4 in cells undergoing biomechanical and ischemic insult did not affect cell death as measured by PI, but it was observed that cells and nuclei that were condensed had ANXA4 staining at the nuclear and cytoplasmic membrane, suggesting important implications at the membrane. To study the role ANXA4 may play in relation to membrane dynamics, biomechanical stretch on cells was applied to cultured cells. Cells incubated with extracellular calcium had more permeable membranes during mechanical insult. Interestingly, cells that were incubated without extracellular calcium had even more permeable membranes suggesting the importance of calcium-dependent proteins relevant in membrane repair. ANXA4 knockdown increased membrane permeability in cells undergoing biomechanical insult. Given that membrane stability/repair is dependent on cortical actin dynamics, ANXA4 was stained in vitro and in vivo and shown to co-localize closely with cortical actin and F-actin, respectively.

Furthermore, disruption of F-actin by pharmacological agents drastically altered ANXA4

61 localization. ANXA4 staining became more punctate at areas around the perimeter of the cell where F-actin was abundant, suggesting close ANXA4-actin interactions.

5.1 Biomechanical and Hypoxic Stresses Induces ANXA4 Expression and Elevated Protein Levels in vitro and in vivo

In the present study, ANXA4 protein levels were upregulated during biomechanical insult coupled with hypoxia. Furthermore, ANXA4 expression was increased in a model of elevated IOP in rat eyes (which lead to ischemic/reperfusion conditions) and in pig eyes. This result is consistent with previous publications reporting that other members of the annexin family were upregulated in animal models of elevated IOP18. Furthermore, several members of the annexin family have been upregulated in ischemia/reperfusion models20,21,23. ANXA4 protein levels have been reported to be upregulated in a transient reversible MCAO model and during intestinal ischemia/reperfusion injury, although the functional role and the significance of this increase has not yet been clearly established20,21. In a mouse model of MCAO, brains treated with tetrandrine resulted in reduced ANXA4, mitigated cerebral neurological deficits and decreased infarct volume. Tetrandrine is an analgesic medicine that can reduce inflammation and act as a potent inhibitor of elevation of intracellular calcium by blocking calcium channels172–174. Ischemia/reperfusion can result in the elevation of intracellular calcium via excitotoxic mechanisms175. Considering that ANXA4 responds in a calcium dependent manner, it would be interesting to see if the calcium levels within a cell (and/or the subsequent ischemia/reperfusion damage) can regulate its expression. Future experiments can be conducted to closely elucidate the relationship between intracellular calcium concentrations with ANXA4 expression.

Limited studies have been conducted so far on the functional role of ANXA4 in response to ischemia/reperfusion. A study that induced intestinal ischemia/reperfusion injury identified a

62 novel natural IgM antibody that was able to react on the surface of intestinal epithelial cells20.

This antibody was found to recognize ANXA4 and was able to subsequently cause complement activation, neutrophil recruitment, and exacerbate intestinal injury. Furthermore, “inhibiting”

ANXA4 reactive natural antibody repertoire by pre-injection of recombinant ANXA4 protein blocked the inflammatory process associated with intestinal reperfusion injury. The pre- injection of other annexin proteins before ischemia/reperfusion was able to inhibit inflammatory responses in other studies, as well22,23. Annexins, particularly ANXA5, have been widely used as a tool to measure early apoptosis through their upregulation and/or translocation to the membrane in dying cells176,177. In the current study, elevated cell death was seen in biomechanical insult and hypoxic conditions, although there was no change in cell death when

ANXA4 levels were manipulated. These results suggest that ANXA4 may not be directly involved in regulating apoptosis or cell death. However, it would be interesting to see if annexins can participate in the final stages of apoptosis by serving as a membrane epitope for antibodies to bind and attract phagocytic immune cells.

5.2 ANXA4 Knockdown Contributes to Increased Membrane Permeabilization During Biomechanical Insult

Annexin upregulation and/or translocation to the membrane has been associated with dying cells, although it is not understood why functionally annexins bind to membranes in a calcium dependent manner. Some members of the annexin family have been suggested to play a role in membrane repair processes, although no research on this topic has been reported for

ANXA4. Given that ANXA4 is able to translocate to membranes and form trimer structures to stabilize membranes, it is a good candidate for a role in calcium dependent membrane repair13.

In the context of biomechanical stress, the membrane is an important cellular structure to focus on, as it is the critical barrier between the extracellular and intracellular environment. It is

63 therefore crucial to understand the stresses that are applied to the membrane and the consequent implications it has within the cell. In the current study, I have shown that cells became permeable to FM1-43 fluorescent dyes as soon as 10 minutes after pathologically relevant mechanical stretching. This is consistent with previous findings that measured membrane permeability in neurons exposed to mechanical stretch178. Embryonic cortical neurons exposed to high magnitudes of stretch were permeable to large fluorescent molecules and remained permeable to smaller molecules up to 5 minutes after stretch. Altered membrane permeability can result in massive ionic disturbances179,180, cytoskeletal breakdown175,181, loss of action potential conduction182,183 and potentially expose the cell to extracellular factors, which can ultimately lead to cellular dysfunction or death. Therefore, this research brings forth the question of the implications membrane permeability has in the context of biomechanical insult environments.

ANXA4 may respond to localized calcium influxes and bind to the membrane as a

“band-aid” or membrane stabilizer through their trimer structure formation. This activity would ensure efficient repair and stabilization of small membrane breaks and subsequently prevent the further influx of calcium and cellular dysfunction. In the current study, increased fluorescent staining of the membrane impermeable dye DRAQ7 was observed after 2 hours of biomechanical stretch. Interestingly, significantly increased fluorescence intensity was seen after ANXA4 knockdown compared to the NT siRNA stretch conditions. This function of

ANXA4 is consistent with previous work showing the importance of ANXA5 in membrane repair25. ANXA5 is an immediate responder to membrane damage by laser irradiation, and is able to contain the mixing of intracellular and extracellular environments in a calcium dependent manner. ANXA5 binds to plasma membranes exposed to the edges of torn membranes and assembles itself into 2D arrays to subsequently strengthen the membrane and

64 prevent the expansion of the tear. However, ANXA5 trimers only composed of about ~65% of the crystal surface. It is likely that other annexins or proteins are integrated into the 2D arrays.

Given that ANXA5 can form heterogenous assemblies with other annexins such as ANXA1,

ANXA2, and ANXA4 on plasma membranes, it is likely that ANXA4 is an active participant in membrane repair as well15. Further evidence suggesting a role of annexins in plasma membrane repair was seen for ANXA1184. ANXA1 becomes concentrated at disruption sites and promotes membrane resealing. Blocking ANXA1 function by using an antibody, a small peptide competitor, or a dominant-negative ANXA1 mutant protein incapable of calcium binding all inhibited membrane resealing.

Along with forming trimer structures for membrane repair, annexins can also participate in isolating areas of plasma membrane breaks27. Microparticles that contain annexins are shed into the blood stream from blood cells and platelets in response to calcium influx, yet the molecular mechanisms of this process is unknown185,186. Following a streptolysin O-attack,

ANXA1 translocated to perforated “hot-spots” of calcium entry, where the pore-containing region was subsequently quarantined and eliminated by shedding in the form of an extracellular microparticle144. Further studies show that formation of blebs protected cells from the damaged regions of the plasma membrane187,188. Blebbing induced an elevation of intracellular calcium and the translocation of ANXA1 to the plasma membrane of the bleb inhibited further calcium elevation. There was also an accumulation of ANXA1 observed at the perforated bleb base to pinch the membrane and block communication from the bleb and the rest of the cell. Once the perforation in the plasma membrane was resolved, the calcium concentration within the cell returned to normal levels, which dissociated ANXA1 from the plasma membrane followed by bleb retraction.

The characteristic of annexins to contain intracellular or extracellular compartments in their respective environment can be related back to apoptosis. Elevation of intracellular calcium that occurs during the apoptotic cascade can breakdown the cytoarchitecture of the cell, leading to increased internal pressure resulting in blebbing and cell shrinkage189,190. The release of intracellular components into the extracellular space during apoptosis can be detrimental to surrounding cells, as molecules such as ATP can activate nearby receptors leading to an exacerbated inflammatory response191. It would be favourable for the cell to have a mechanism to preserve the plasma membrane until immune cells are recruited to safely recognize, lyse and remove the dying cell189. As mentioned above, circulating antibodies are able to identify cells that have exposed ANXA4 on the cell surface and recruit the complement system20.

Furthermore, it is a well-known characteristic of ANXA5, to translocate to the plasma membrane during apoptosis25,177. Future experiments can address the importance of plasma membrane integrity during apoptosis and the role annexins have in this process; a field relatively unexplored, yet can have major contributions to autoimmune disorders or diseases associated with biomechanical insult.

5.3 ANXA4 Co-Localizes with F-actin and Responds to Actin Disruptors

Extensive actin remodeling needs to occur to facilitate membrane repair146,147. Under physiological conditions, cortical actin helps maintain plasma membrane integrity. Studies have suggested that actin dynamics play an important role in membrane repair, as the inhibition of actin polymerization or demolymerization can inhibit plasma membrane repair processes192. The presence of the cortical F-actin can create tension that would inhibit passive resealing after injury. Depolymerization of actin after injury is therefore needed to prevent the membrane from further damage by decreasing tension, and also facilitate membrane fusion to close the

66 wound148. Some annexins have been shown to interact with actin, although the functional importance of this is still unknown17. Given the evidence suggesting annexins involvement in membrane repair, it is important to elucidate how they interact with actin during the membrane repair process.

A recent study by Jaiswal et al. has highlighted the importance of annexin-actin interaction and proposed a model of the interaction during membrane repair24. As a result of plasma membrane damage by laser irradiation, local calcium entry into the cell triggers ANXA1 recruitment to the injured membrane and subsequent depolymerization of cortical F-actin to release tension. Concomitantly, an S100A11-ANXA2 complex forms proximal to the injury site and initiates cortical F-actin polymerization, aiding in wound closure and the re-establishment of membrane tension. Actin depolymerization of F-actin at the injury site can cause the injured membrane to collapse on itself and be excised from the rest of the cell. Another actin connection is previous work has shown that annexins, particularly ANXA1 and ANXA4, are substrates for calpain cleavage123. Calpains are responsible for cleaving cortical actin associated proteins, such as spectrins, and can target annexins for the excision process of damaged membranes193.

Other studies have confirmed annexin interaction with cortical F-actin. Membrane- bound ANXA6 reduced calcium entry in the cell when treated with thapsigargin. Thapsigargin raises cytosolic intracellular calcium by blocking the ability for the cell to pump calcium into

ER stores, leading to depletion and the opening of plasma membrane calcium channels170.

ANXA6 localization to the plasma membrane resulted in the accumulation of cortical F-actin and inhibited calcium influx. Actin destabilization using latrunculin A abolished this effect.

Therefore, these results implicate ANXA6 in actin-dependent regulation of calcium entry.

In spite of this work, there has been very limited research on ANXA4 specifically and its relation with actin in vitro and in vivo. Results from the present study are the first to show the

67 close interaction between ANXA4 and actin through immunofluorescence microscopy. ANXA4 staining was very evident in areas of cell membrane contact, and was concentrated at sites of cortical F-actin in vitro. This close interaction was seen in the ONH and the retina in vivo as well, as ANXA4 co-localized with F-actin, but not with other cytoskeletal markers β3 tubulin or

GFAP. Astrocytes are positioned perpendicular to RGC axons. F-actin orientation was seen to be perpendicular to β3 tubulin, a marker of RGC axons85. This suggests that ANXA4 is abundant in astrocytes at the ONH. Not only are membrane repairing mechanisms important to

RGCs, but in astrocytes where support is needed to maintain a structural environment for delicate axons. Interestingly, ANXA4 only co-localized with F-actin at the nerve fiber layer.

Since this layer contains RGC axons and astrocytes and is directly exposed to the vitreous, the presence of ANXA4 may provide membrane stability or ensure membrane repair is proficient during mechanical damage. Future experiments using co-immunoprecipitation will reveal if there is a direct interaction with ANXA4 and actin.

As further evidence of an ANXA4 and actin interaction, disrupting actin dynamics resulted in a stark difference in endogenous ANXA4 distribution within the cell. ANXA4 staining became very punctate in cell membrane areas, especially where fragment F-actin is present. This is in agreement with work that shows ANXA1 membrane accumulation in cells treated with cytochalasin D24. Furthermore, altering actin dynamics inhibited annexin-dependent plasma membrane repair. This suggests a close relationship between ANXA4, and actin in terms of repair during biomechanical insult. Future experiments can be conducted to disrupt actin to see whether membrane permeability increases during biomechanical insult.

In summary, these findings suggest a close interaction of ANXA4 with cortical F-actin.

The role of this interaction during biomechanical insult in regards to calcium influx and membrane repair will be addressed in future experiments.

5.4 Development of Tools to Knockdown ANXA4 in the Retina

There is much research to be done in regards to the mechanisms of annexin mediated membrane repair. In the current study, rat ANXA4 siRNA sequences were optimized in vitro and then used to make AAV2 vectors that were successfully tested in vivo. After 3 weeks of viral injection, GFP expression was observed in vitro and in vivo. Future studies will optimize infections with a more thorough time course, followed by qPCR and immunofluorescence microscopy to assess knockdown. To assess membrane integrity during biomechanical elevation of IOP, eyes will be loaded with impermeable fluorescent dextrans. Fluorescent dye incorporation into the retinal cells following mild IOP increase will reveal the biomechanical effect of elevated IOP on cell membrane permeability. It would be interesting to see if ANXA4 knockdown results in increased membrane permeability in this in vivo model. As an alternative strategy, given that injection of recombinant ANXA4 protein was able to block antibody recognition to the ANXA4 epitope after intestinal ischemia/reperfusion injury20, ANXA4 recombinant protein can also be injected into the eye before elevation of IOP and cell death and inflammation can be measured thereafter.

5.5 Implications in Disease

During glaucoma, the retina is exposed to a multitude of stresses, including biomechanical, and ischemic/reperfusion insult. These stresses can induce elevation of intracellular calcium, which can subsequently activate a multitude of apoptotic, necrotic and calcium-dependent death pathways4,194. There are many ways elevation of intracellular calcium can contribute to glaucomatous neuropathy including astrocytic reactivity, excitotoxic, oxidative, and many other stresses117,194. Previous in vitro studies have examined the intracellular calcium concentrations in fura-2/AM-loaded human lamina cribrosa cells

69 undergoing 15% mechanical insult195. Intracellular calcium was immediately elevated at the initiation of stretch and gradually increases over time. This gradual increase was observed even in the presence of L-type calcium channel blocker verapamil suggesting different ways of calcium entry that may not include calcium channels. Furthermore, lamina cribrosa cells from glaucoma donors showed elevated cytosolic calcium levels, although this study linked dysfunctional calcium homeostasis to increased ROS production, compromised anti-oxidant capacity, and decreased mitochondrial membrane potential in glaucomatous lamina cribrosa cells compared to normal controls196. Astrocyte activation has been linked with influxes of calcium116,117,119,197. Previous work suggests that elevated intracellular calcium through scratch injury can activate GFAP expression, and lead to astrogliosis117. Future studies examining the effect of membrane permeability during biomechanical insult, and the subsequent elevation of calcium levels on astrocyte reactivity can have large implications on astrocyte-dependent response to injury.

Much research has been centered on the detrimental effect elevated IOP has on the disruption of cytoskeletal axonal transport. This disturbance can lead to reduced transport of survival factors and a subsequent rise in intra-axonal calcium via activation of calcium channels and leaks from intracellular calcium stores198. There has been no research on how chronic biomechanical insult caused by the elevation of IOP can contribute to increased membrane permeability of RGC axons and the supporting astrocytes. Increased permeability of cells at the

ONH can have detrimental effects and lead to abnormal intracellular calcium accumulation.

More research needs to be conducted to validate the importance of membrane stability and/or repair during biomechanical insult, and the effect this has on delicate RGCs and the astrcoytic environment, which are crucial in maintaining the necessary support for the axons at the ONH.

The dynamic actin cortex is an important component of plasma membrane stability and its participation is crucial in membrane repair processes. Abnormal intracellular calcium accumulation has been described in muscle fibres from patients afflicted with X-linked muscular dystrophy – a disease related to the weakening of the muscoskeletal system from mutations in the dystrophin protein199. Dystrophin is found in muscle fiber membranes and links the cytoskeleton to the surrounding extracellular matrix200. Since dystrinopathies are diseases of the membrane associated cytoskeleton, it is important to consider the effect that disruption of the cytoskeletal network can have on calcium influxes. Destabilization of the cortical actin cytoskeleton can render tissue more susceptible to micro-ruptures and influxes of calcium180.

Similarly in glaucoma, where there is a fluctuating biomechanical component of insult, membrane micro-ruptures at ONH tissue could initiate calcium influxes and therefore over time can activate many injury pathways previously mentioned. It is important to study calcium regulatory proteins that might lead to better understanding of molecular pathways involved in membrane repair and/or stability.

Chronic elevation of IOP in glaucoma may cause membrane breaks and are dependent on annexins to be present to mediate repair processes. Annexins are upregulated in models of disease that experience biomechanical insult, such as glaucoma, muscular dystrophy and cardiovascular diseases18,201–204. Elucidating the pathologic outcome of biomechanical and ischemic stress on cell membranes at the ONH during elevation of IOP is a novel and exciting avenue of research to explore in future studies205.

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Appendices

A B

1 1

Non-targeting Non-targeting control Control 0.5 0.5 ANXA4 Anxa4 siRNA Control) Control) siRNA (Fold Change of (Fold Change of ANXA4 Protein Level ANXA4 Protein Level 0 0

Appendix 1. Confirmation of ANXA4 knockdown through protein analysis. Protein levels of ANXA4 48 hours of transfection with non-targeting siRNA or ANXA4 siRNA in A7 cells. ANXA4 siRNA transfection reagents were incubated for 24 hours, followed by replacement of complete media with 10% FBS for an additional 24 hours (A, n=2). This protocol was followed in Section 4.1.3. ANXA4 siRNA was applied as described above, but replaced by complete media with 1% FBS for an additional 24 hours (B, n=3). This protocol was followed in Section

4.2.3. GFP-only ANXA4-GFP A B

Appendix 2. Confirmation of ANXA4 overexpression through protein and fluorescence analysis. A7 cells were transfected with either GFP-only or ANXA4-GFP for 24 hours. Western blot revealed an endogenous ANXA4 band and an ANXA4-GFP band (A). HEK293 cells were transfected with GFP-only or ANXA4-GFP for 24 hours (B). HEK293 were transfected with either pcDNA or ANXA4-V5 for 24 hours (C).

Appendix 3. FM1-43 dye fluoresces upon membrane breaks. Plated A7 cells were incubated in FM1-43 dye. A 30-gauge needle was then used to scratch the A7 cells. The dye was removed and the cells were fixed in 4% PFA. Cells that came in contact with the scratch showed a clear increase in fluorescence intensity. Scale bar = 50µm.