Disclosing the Mechanisms of Retina Regeneration

MIAMI UNIVERSITY The Graduate School

Certificate for Approving the Dissertation

We hereby approve the Dissertation

Nancy Paola Echeverri Ruiz

Candidate for the Degree

DOCTOR OF PHILOSOPHY

______Katia Del Rio-Tsonis, Director

______Tracy Haynes, Reader

______Michael Robinson, Reader

______Paul James, Reader

______Peter Stambrook, Reader

______Jonathan Scaffidi, Graduate School Representative

DISCLOSING THE MECHANISMS OF RETINA REGENERATION

Nancy Paola Echeverri Ruiz

The process of regeneration was recognized in ancient Greek mythology, with the story of Prometheus. Also, the empiricist Aristotle described some aspects of regeneration in lizard appendages and snake tails. However, it was not until the seventeenth century that a better understanding of regeneration was obtained with studies in hydra. Nevertheless, this research generated debates about how development occurs and different theories about regeneration (Lenhoff and Lenhoff, 1991).

Regeneration continues to be a very interesting area of research. Luckily, we now have more powerful techniques to elucidate the processes of regeneration. Whereas, regeneration occurs in some organisms more efficiently than others, several questions remain. These include: Why do mammals have such a restricted capacity to regenerate, and how can we improve regenerative capabilities? Although these questions remain unanswered, our studies have shown that factors such as fibroblast growth factor 2 (FGF2), the complement components C3a and C5a, as well as some antioxidants, are able to induce retina regeneration in chicken embryos.

Our lab is focused in understanding the mechanisms that trigger retina regeneration in chicken embryos because the regeneration of the retina in the chick embryos is not spontaneous. Elucidating the mechanisms that trigger regeneration in the chicken embryo may elucidate how to achieve retina regeneration in humans.

In 2014 the world health organization (WHO) declared that close to 285 million people were visually impaired worldwide. Among the causes for blindness is the high production of reactive oxygen species (ROS) in the eye. This is not surprising because the retina produces a large amount of ROS as a consequence of its high metabolic rate (Wong-Riley.2010).

This dissertation presents two novel approaches to induce retina regeneration. The first mechanisms explain how the complement component C3a (Chapter 2) is involved in the process of retina regeneration through the activation of the transcription factor pS727 Stat3. The activation of Stat3 was essential for retinal regeneration. Finally, Chapter 3 discusses the role of the redox status to promote stem/progenitor cell self-renewal and retina regeneration.

The results of this research increase our knowledge of the different mechanisms that are involved in organisms that have the capability to regenerate. Such results could be used in further studies in mammals, including humans to treat retinopathies.

DISCLOSING THE MECHANISMS OF RETINA REGENERATION

A DISSERTATION

Presented to the Faculty of

Miami University in partial

Fulfillment of the requirements

For the degree of

Doctor of Philosophy

Department of Biology

Nancy Paola Echeverri Ruiz

The Graduate School Miami University Oxford, Ohio

2016

Dissertation Director: Katia Del Rio-Tsonis

TABLE OF CONTENT

Chapter 1: Introduction Overview 1 Background and significance 1 References 4 Figure and legends 9

Chapter 2: Complement anaphylatoxin C3a induces retina regeneration through Stat3 activation (Published in Nat Commun. 2013;4:2312. doi: 10.1038/ncomms3312) Abstract 12 Introduction 12 Material and Methods 16 Results 17 Discussion 19 References 19 Figure and legends 26 Tables 31

Chapter 3: The antioxidant N-Acetylcysteine is a powerful retina regeneration inducer (Paper in progress) Abstract 34 Introduction 34 Material and Methods 38 Results 41 Discussion 46 References 49 Figure and legends 56 Tables 72

Chapter 4: Conclusions Final remarks 83 Questions of immediate interest 83 Future directions 85 References 87

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LIST OF TABLES

Chapter 2: Complement anaphylatoxin C3a induces retina regeneration through Stat3 activation Table1 31 Table 2 32 Chapter 3: The antioxidant N-Acetylcysteine is a powerful retina regeneration inducer

Table 1 72

Table 2 73 Table 3 74 Table 4 75 Table 5 77 Table 6 79 Table 7 80

Table 8 80

Table 9 81 Table 10 82

LIST OF FIGURES

Chapter 1: Introduction Figure 1 9 Figure 2 10

Chapter 2: Complement anaphylatoxin C3a induces retina regeneration through Stat3 activation Figure 1 26 Figure 2 27 Figure 3 28 Figure 4 29 Figure 5 30

Chapter 3: The antioxidant N-Acetylcysteine is a powerful retina regeneration inducer Figure 1 56 Figure 2 57 Figure 3 58 Figure 4 59 Figure 5 60 Figure 6 61 Figure 7 63 Figure 8 64 Figure 9 65 Figure 10 66 Figure 11 67 Figure 12 68 Figure 13 69 Figure 14 70 Figure 15 71

DEDICATION

This work is dedicated to my mother Bernarda Ruiz, for all her teachings and advice, and most importantly for encouraging me to follow the path leading to my own happiness. My mentor Margarita Zuleta (R.I.P. 2007) for all her teachings and for injecting me the love and passion for science and teaching.

ACKNOWLEDGMENTS

I thank my mentor P. Stambrook from University of Cincinnati for all his input, advise and support. I could have not made it without his mentoring. To all the lab members, and my advisor Katia Del Rio-Tsonis for all the teachings and support. My soul siblings Gladys Villegas, Aminata Culibaly, Jayanti Shriram, Aswati Subramanian, Jennifer Di Napoli, Matthew Denney and Rafael Herrera for all their support, love and advise. My Russell family, Dave and Jill, they are my biggest and strongest support in live and science. To the members of the Center for Advanced Microscopy & Imaging, Dr. R. Edelmann and Mr. M. Duley; and the Department of Biology for providing services required for the completion of this work; To Drs. L.J. Niedernhofer, P. Wipf, for supplying the antioxidant XJB 5-131, from Pittsburg University. To H. Charles and V. Malinn for technical assistance. This work was supported by Sigma Xi grants #G20130315164134 and #G201503151092235 and an Academic Research Challenge grant/MU to NEPR, and a Faculty Research Challenge Grant/MU and EY023925 grant to KDRT as well as by the Department of Biology/MU.

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CHAPTER 1

INTRODUCTION 1. Overview

According to the World Health Organization (2014), about 285 million people worldwide are visually impaired. The majority of diseases in developed countries are associated with retinopathies, including macular degeneration and diabetic retinopathy (www.who.int). Currently, there are many research efforts directed to establishing the mechanisms of retina regeneration in different animal species. One of the main limitations is the scarce information about the different pathways involved in retina regeneration. The chick embryo has been a useful model to study development, regeneration and pharmaceutical therapies (Vargas et al., 2007; Vergara and Canto-Soler V. 2012; Belecky-Adams et al., 2008). Due to its easy accessibility for surgical procedures, the chick embryo is one of the most versatile and feasible experimental systems (Kotwani, 1998; Vergara and Canto-Soler V. 2012; Belecky-Adams et al., 2008).

The chick embryo has the ability to regenerate the retina following retinectomy. Regeneration depends on mitogens such as fibroblast growth factor 2 (FGF2). Up to day 4-4.5 the retina can regenerate by the activation of stem/progenitor cells present in the ciliary margin (CM) located in the anterior part of the eye and/or by transdifferentiation (TD) of the retinal pigmented epithelium (RPE) (Spence et al., 2004).

2. Background and Significance

2.1 Eye and retinal development in vertebrates

Eye development in vertebrates starts with the formation of the optic vesicle. The optic vesicle evaginates from the diencephalon and after contacting the surface ectoderm, the outer wall of the optic vesicle invaginates to give rise to the optic cup. The proximal layer of the optic cup forms the neural retina, while the distal layer forms the RPE. Successively, retinal histogenesis and lamination occurs (Colozza et al., 2012; Fuhrmann, 2010) (Figure 1).

The vertebrate neural retina consists of eleven major resident cell types: six neuronal and Müller glial (Zhu et al., 2012). Retina neurogenesis is organized and

1 conserved among vertebrates. The order of cell birth is as follows: ganglion cells, horizontal cells, cone photoreceptors, amacrine cells, rod photoreceptors, bipolar cells, and Müller glia (Cepko, 2014). The cone and rod photoreceptors are located in the outer nuclear layer (ONL), the bipolar cells, amacrine and horizontal cells are located in the inner nuclear layer (INL), and ganglion cells are located in the ganglion cell layer (GCL). The synapses between photoreceptors, bipolar and horizontal cells take place in the outer plexiform layer (OPL). The synapses between bipolar, ganglion and amacrine cells take place in the inner plexiform layer (IPL), while the ganglion cell axons make up the nerve fiber (NF) layer (Zhu et al., 2012) (Figure 1).

During early stages of retina development, the mitotic progenitor cells reside in the outer neuroblastic layer (ONL), while newborn neurons such as amacrine and bipolar cells are located in the inner nuclear layer (INL). The location of the mitotic progenitor cells residing in the ONL varies depending upon their progress through the cell cycle. For example, cells in S phase are found on the vitreal side of the ONL, near to the border with the INL. Cells in M phase are found on the sclera side of the ONL, adjacent to the RPE (Doh et al., 2010).

2.2 Retinal regeneration in vertebrates

The retina is a complex multilayered tissue responsible for receiving visual stimuli from the external environment. Approximately, 285 million people worldwide (www.who.int) are visually impaired, and one of the main causes is related with diseases that affect the retina. Once the retina degenerates, the process is irreversible, leading to permanent loss of vision. In the last decade significant advances have been achieved to elucidate the molecular basis of retina regeneration (Colozza et al., 2012; Barbosa-Sabanero et al., 2012). In order to understand better how the retina can regenerate, it is important to work with animals that possess the capability to regenerate. Among the vertebrates that can regenerate, amphibians such as frogs and salamanders as well as fish show a high regenerative potential. That potential is conserved for a great part of their life. Lower vertebrates have the potential to regenerate, while higher vertebrates such as birds and mammals show limited retina regenerative ability upon injury during a brief window of embryogenesis (Karl and Reh, 2010; Barbosa-Sabanero et al., 2012).

Mammals have a limited ability to regenerate, however, they possess cells with stemness traits in the ciliary body located between the retina and the iris. Although, these “stem-like” cells are normally mitotically quiescent, they have the ability to

2 clonally proliferate in vitro and can, upon inductive conditions, express markers of differentiated retina cells (Ahmad et al., 2000; Coles et al., 2004; Tropepe et al., 2000; Ballios et al., 2012; Balenci et al., 2013). Lower vertebrates such as fish and amphibians in turn possess a region adjacent to the ciliary body (CB) called the ciliary marginal zone (CMZ). This region contains actively proliferating retinal progenitor cells that contribute to the perpetual expansion of the retina throughout life. These cells are capable of regenerating all types of retinal neurons and Müller glia cells, except rod photoreceptors in the case of fish (Otteson and Hitchcock, 2003; Wetts and Fraser, 1988; Wetts et al., 1989, Barbosa-Sabanero et al., 2012). After injury, the CMZ cells respond and contribute to the replacement of the lost cells (Locker et al., 2009; Locker et al., 2010; Moshiri et al., 2004).

In amphibians (salamanders and frogs), if the retina is surgically removed, the cells of the RPE respond to injury by re-entering the cell cycle losing the traits of differentiated RPE such as pigmentation, and forming a new layer, giving rise to a complete retina (Mitashov and Maliovanova, 1982; Araki, 2007; Barbosa-Sabanero et al., 2012; Chiba, 2013; Islam et al., 2014). In chick embryos, when the neural retina is surgically removed, no regeneration will occur (Figure 2), unless inducing factors are added into the operated eyes (Del Rio-Tsonis and Tsonis, 2003; Haynes and Del Rio-Tsonis, 2004; Barbosa-Sabanero et al., 2012). Both the CB/CMZ and RPE are capable of regenerating the retina after retinal insults (Figure 2) (Spence et al., 2004). The hatched chicken conserves the CMZ, although with a more limited potential compared with fish and amphibians and does not respond to injury for retina repair (Fischer and Reh, 2000).

Müller glia also exhibit neurogenic capacity and contribute to the regenerative process after injury in different vertebrate retinas (Fischer and Reh, 2001; Fischer and Reh, 2003; Yurco and Cameron, 2005; Das et al 2006; Fausett and Goldman, 2006; Bernardos et al., 2007; Monnin et al., 2007). Müller glial cells need to re- enter the cell cycle for regeneration to take place. Müller glia are the major source of retina regeneration, capable of replacing all retinal cell types in fish (Bernardos et al., 2007; Fausett and Goldman, 2006; Ramachandran et al., 2010; Gorsuch, and Hyde, 2014). Upon directed photoreceptor cell death in transgenic zebrafish expressing nitroreductase (NTR) Müller glial cells are able to re-enter cell cycle and regenerate lost neurons (Montgomery et al., 2010). However, activated Müller glia are somehow limited in their neurogenic potential in mature retina of chicken and mammals, which rarely regenerate after injury (Fischer and Reh, 2001; Ooto et al., 2004). However, treatment with specific soluble factors (such as retinoic acid or

Wnt) or transgenic expression of neurogenic factors such as Ascl1a can promote Müller Glia cells to reprogram to limited types of neurons (Ooto et al., 2004; Osakada et al., 2007; Ueki et al., 2015).

Our lab is testing different factors in order to induce retina regeneration in chick embryos after injury. Chapter 2 and 3 of this dissertation, evaluate two different soluble factors and the mechanisms involved in retina regeneration. The fourth chapter is the conclusions. The second chapter describes the role of complement component C3a, concentrating on the Stat3 signaling pathway regulated by C3a. Our hypothesis was that C3a promotes retina regeneration from the ciliary margin by inducing the phosphorylation of S727 of STAT3. We were able to determine that Stat3 is activated through the phosphorylation at the serine 727 residue (S727) and we found that the activation of pS727 Stat3 is independent of FGF2 signaling. The third chapter describes the role of N-acetyl cysteine (NAC) during the process of chick retina regeneration. Our first hypothesis was that NAC induces retina regeneration through an increase of progenitor cells. We found that NAC was able to enhance the expansion of progenitor cells present in the CM and delay cell differentiation. The second hypothesis was that the regeneration induced by NAC was due to the increase in the levels of glutathione (GSH). Our results showed that the increased levels of GSH were not necessary for the induction of regeneration. Finally, we hypothesized that NAC activates the MEK/Erk1/2 pathway for the induction of regeneration. And in fact, NAC induced regeneration through the activation of Erk1/2. This activation was mediated through the thiol disulfide exchange activity of NAC.

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4. Figure and figure legends Figure 1.

Eye and retina development in vertebrates. (A) The neural ectoderm evaginates, followed by the flattening of its distal portion. A two-walled optic cup is formed following invagination of the optic vesicle (Figure A from Colozza, et al., 2012 with permission of Baishideng Publishing Group Inc.). Once the neuroepithelium forms it differentiates into all the retinal cell types. (B-C) A schematic representation of the neural retinal organization (B) and glia cell organization (C). (Figure B and C from Zhu et al, with permission of John Wiley & Sons, Inc.).

Figure 2.

Retina regeneration in the chick embryo. (A) Chick embryo at day 4 (E4) of development. (B) Histology section of an E4 chick eye. (C) Histological section after mechanical removal (retinectomy) of the neuroepithelium (NE). (D) Histological section of a retinectomized eye after 7day post-retinectomy (dPR) in presence of fibroblast growth factor 2 (FGF2). Two types of regeneration occur: regeneration from the CM and RPE TD. (E) In absence of any growth factor, regeneration does not occur. NE: Neuroepithelium, CM: Ciliary margin, (CM), RPE: Retinal pigmented epithelium, TD: Transdifferentiation, CR: Ciliary regeneration, L: Lens.

CHAPTER 2

COMPLEMENT ANAPHYLATOXIN C3A INDUCES RETINA REGENERATION THROUGH Stat3 ACTIVATION

Abstract

Complement is an ancient immunological system, which has its origins in some of the earliest metazoans (Leslie and Mayor, 2013). The function of complement components is not only associated with immunity, but also it has been related with regenerative properties (Mastellos et al., 2001; 2013). We demonstrate that the carboxyl terminal (C-Ter) domain of C3a (C3a-p) signals through its own receptor and activates Stat3 in an FGF-independent manner, to induce retinal regeneration in chick embryos. The activation of Stat3 was observed mainly through the phosphorylation at the residue serine 727 (S727), while the phosphorylation at residue Y705 STAT3 did not show changes during the regeneration induced with C3a-p. We also show that the activation of the MAPK (a group of S/T kinases) is important for the phosphorylation at the residue S727 Stat3. These results highlight the relevance of the activation of this residue during retina regeneration, and are important to elucidate the mechanisms that may induce retina regeneration in mammals.

1. Introduction

In order to understand the process of regeneration, we need to understand the signaling pathways and activators that play a role during injury and inflammation. The inflammation process has two faces; it can be beneficial or detrimental for the tissue. It is well known that some pro-inflammatory molecules, such as some cytokines and the complement components, are important during development (Symonds et al., 2001; Mourik et al., 2009, Rutkowski et al., 2010,). The complement components in addition to their role as immune effectors are also involved in a wide variety of physiological and pathological processes. Studies in different organ systems demonstrate that complement components play a direct role in cellular turnover, maintenance, healing, proliferation and regeneration, including regeneration after injury of the limb, eye and liver (Del Rio-Tsonis, 1998; Mastellos et al., 2001; Kimura et al., 2003; Strey et al., 2003; Markiewski et al., 2009).

Three different pathways are involved during complement activation: the classical, lectin, and alternative pathways. Eventually, all three pathways lead to the formation of C3 convertases (C4b2b represents the classical/lectin pathway C3 convertase, and the C3bBb the alternative pathway C3 convertase) that activate the central component C3. These C3 convertases cleave C3 into C3a and C3b fragments (Figure 1). C3a is an anaphylatoxin that mediates inflammatory processes. C3b is a larger fragment that can covalently bind to adjacent molecules and surfaces via its thioester group. The C3b fragment, can bind to specific complement receptors (Sarma and Ward. 2011; Mihá ly, 2011).

1.2 Complement proteins mediate tissue regeneration in vertebrates

In urodeles, after limb amputation, there are mature cell types present at the injured site. These cells suffer different changes. They undergo dedifferentiation, proliferation forming blastema cells. Blastema cells are important for repopulating and reforming old structures through re-differentiation into mature tissue, such as muscle, bone and connective tissue. Both C3 transcripts and protein are specifically expressed in urodele blastema cells but not in intact limb tissues, suggesting that C3 plays an important role mediating regeneration (Del Rio-Tsonis et al., 1998).

Complement components are essential for liver regeneration as they induce a network of signaling pathways that promote hepatocyte proliferation. Deficiencies in C3 or C5 in mice result in high mortality, parenchymal damage, and impaired regeneration after partial hepatectomy. Dual C3 and C5 deficiency creates a more severe phenotype. These phenotypes could be reversed by combined reconstitution of C3a and C5a. Inhibition of C5a receptor signaling after hepatectomy resulted in a reduced activation of NF-ĸB/Stat3. These results suggest a requirement of C3a and C5a for the early priming stages of hepatocyte regeneration (Strey et al., 2003).

1.3 Stat3 is activated after complement activation and plays an important role during tissue regeneration

Stat3 is a transcription factor whose signaling is involved in proliferation, stem cell self-renewal and cell survival in response to cytokines and growth factors (Zhang et al., 2005). The activity of Stat3 depends on several post-translational modifications (i.e, phosphorylation and acetylation). Depending on the post- translational modification, Stat3 will have differential gene transcriptional activity.

Stat3 can be phosphorylated on Tyrosine (Y) 705 by the Janus Kinase (Jak), and on Serine (S) 727 via mitogen-activated protein kinase 1 (MAPK1), as a result of growth factor signaling like FGF2 and Epidermal Growth factor (EGF). Phosphorylation at the S727 residue gives a maximum transcriptional effect (Zhang et al., 2005; Chung et al., 1997). Phosphorylation of Stat3 facilitates its homo or hetero-dimerization with other Stat proteins, and modulates its translocation to the nucleus via importins, where Stat3 can induce gene transcription (Garcia et al., 2001). Phosphorylation of each residue can work independently or together to regulate different Stat3 transcriptional outcomes (Togi et al., 2009).

Stat3 can interact with other transcription factors such as: Islet1 (Isl 1), HepG2, c- Jun, Sp1 and NF-κβ (Hao et al., 2005). Additionally, Stat3 and cMyc can share downstream targets. cMyc is well known as one of the four important factors (cMyc, Oct4, Sox2, and Klf4) involved in Embryonic Stem cell (ESC) self-renewal, independent of LIF signaling (Cartwright et al., 2005). cMyc is mainly associated with gene activation, while Stat3 has been associated with activation and repression (Kidder et al., 2008). Gata2, Gata3, Gata6, Lhx1, Pax5, Sox15, and Sox21 are among the genes targeted by Stat3 and expressed in mice. Additionally, HOX genes such a HOXA3, HOXA10, HOXA11, HOXB4, HOXB6, HOXB8, HOXB9, HOXB13, HOXC6, HOXC8, HOXC12, HOXC13, HOXD9, HOXD10, and HOXD13 are repressed in ESCs by Stat3 (Kidder et al., 2008). Stat3 plays an important role in the G1 to S cell-cycle progression through the up-regulation of cyclins D2, D3 and A, and cdc25A with the concomitant down regulation of p21 and p27 (Fukada et al., 1998).

During development, Stat3 is important for survival. It has been shown that Stat3 knockout mice die at early embryonic stages (Takeda et al., 1997). The role of Stat3 is essential for proliferation and/or survival through promoting mitogen factors and inhibiting apoptosis by inducing anti-apoptotic genes of the Bcl family (Regisa et al., 2008). Additionally, Stat3 activity is important to keep mouse embryonic stem cells (ESCs) in an undifferentiated state (Matsuda et al., 1999) by controlling the expression of Dax1, a protein involved in self-renewal and inhibition of cell differentiation (Sun et al., 2009).

During regeneration or tissue repair, Stat3 is an important factor in different tissues. It has been reported that Stat3 is a very important factor in regrowth of damaged axons in the adult central nervous system (Qin et al., 2013). Also, the induction of Stat3 appears to be essential in the initial response of liver regeneration after hepatectomy (Cressman et al., 1995). In the eye, Stat3 is

13 activated in the retina during stress induced by sub-toxic bright light, mechanical trauma, systemic administration of α (2)-adrenergic agonist xylazine and ocular hypertension (Ji et al., 2004). In zebrafish, Stat3 is essential for maximal proliferation status of Müller glia cells during regeneration of the damaged retina (Nelson et al., 2012).

1.4 Role of Stat3 in the eye

Stat3 plays a central role in specification of glia cell fate in a dissociated cell culture system, and in mature retinal cells as a response to stress (Bonni et al., 1997; Rajan and McKay, 1998; Peterson et al., 2000). During mouse retina development, Stat3 is present in the inner nuclear layer (INL), ganglion cell layer (GCL), and in the retinal pigmented epithelium (RPE) (Zhang et al., 2003), and it can be activated as a response to ciliary neurotrophic factor (CNTF), leukemia inhibitor factor (LIF) and Fibroblast Growth Factor 1 and 2 (FGF1 and 2) (Zhang et al., 2005).

In adult mouse retina, Stat3 is expressed in the INL and the pigmented epithelium (PE) of the ciliary margin (CM), and less significantly in the GCL, inner limiting membrane (ILM) and RPE (Peterson et al., 2000; Zhang et al., 2003). In-ovo electroporation of Stat3 in chicken embryos demonstrated that it regulated specific cis-elements and initiated expression of a reporter gene construct in the CNS and the eye. This demonstrates that endogenous Stat3 protein is active during development in both the eye and the CNS (Yan et al., 2004). Stat3 activity in different cell types in the eye is not only important during development, but also it is important during tissue repair and regeneration.

In the eye, there are different cells that can be activated to replace dead/damaged or missing cells. For example, in some animal species, the CM contains neural stem and progenitor cells that are mitotically active and could serve as a potential source to replace damaged retina (Morris et al., 1976; Fischer et al., 2000; Fischer et al. 2013). However, not much is known about the role of Stat3 in the CM after damage or during regeneration. Müller glia cells are another good source for retina repair/regeneration. Studies performed in post-natal chick eyes showed that Müller glia cells are potential sources of neural progenitor cells. These cells can re-enter the cell cycle and dedifferentiate into Pax6, Chx10 and CASH 1 positive cells after local damage (Fischer et al., 2001; Reh et al., 2001; Fischer et al., 2002). Recently, it has been shown in zebrafish, that after light-induced retinal damage, Müller glia require CNTF to proliferate via a Stat3 dependent but AKT and MAPK independent pathway (Kassen et al., 2009). Studies performed in both cats and rabbits, showed

14 that Stat3 can be phosphorylated at day 1 and 3 after retinal detachment in the INL cells. At these times there is a temporal correlation with Müller glia cell proliferation and expression of intermediate filaments. After 3 days of retinal detachment, CREB and NF-κβ activity also increased in the INL (Lewis et al., 1989; Geller et al., 2001). All these proteins respond to growth factors (such as FGF and CNTF) (Lewis et al., 1994; Kahn et al., 1997).

1.5 Retina Regeneration and inflammatory molecules

We were interested in studying the role of some molecules involved in the immune system regulation during retinal regeneration in chick embryos. Since complement components are expressed during newt limb and lens regeneration (Del Rio-Tsonis et al., 1998; Kimura et al., 2003), and because C3a can induce liver regeneration as well as brain neurogenesis (Strey et al., 2003; Rahpeymai et al., 2006; Shinjyo et al., 2009; Järlestedt; 2013), we decided to explore complement component C3a during chick retina regeneration.

Lower vertebrates possess a high ability to regenerate most of their tissues (Baird et al., 1996; Mitashov, et al., 1996), whereas, mammals have very limited capacity for regeneration after birth (Roberson and Rubel, 1994, Stocum, 2005). Recently, efforts have been directed to induce regeneration by using cell potency (multipotecy, pluripotency and totipotency) both in vivo and in vitro. The use of mitogens is necessary to enhance regeneration. Important molecules that have been previously tested in tissue regeneration include: growth factors such as CNTF, FGF, BMP, PDGF, VDGF, and EGF (Tabata, 2003, Koria, 2012, Doan et al., 2013), cytokines such as TNFα and IL-6 (Fausto et al., 1995), and complement components from the adaptive immune system (Rutkowski et al., 2010).

Complement components are important players during healing, cellular turnover, proliferation and regeneration following injury. They maintain the integrity of the tissue in the absence of inflammation, through the disposal of cellular debris and waste, an important process to prevent autoimmune disease. This characteristic contributes to their title of “tissue housekeepers” (Rutkowski et al., 2010). Complement components can activate different cell signal responses, including Stat3 signaling. Stat3 signaling plays an important role in cell growth, differentiation and survival. In developing mice, Stat3 is localized to specific regions of the brain, neural tube and eye (Yan et al., 2004). Recent studies showed that Stat3 is an important factor during skin (Zhu et al., 2008), liver (Riehle et al., 2008), fin (Schebesta et al., 2006) and axon regeneration (Qin et al., 2010). We recently

15 demonstrated that the C3a activates Stat3 via its receptor C3aR to induce retinal regeneration in chick embryos (Haynes et al., 2013).

2. Materials and methods

2.1 Chick embryos

Fertilized Specific Pathogen Free (SPF) chicken eggs were purchased from Charles River Laboratories Inc. (Wilmington, MA, USA) and incubated in a humidified incubator at 38°C until they reached the developmental stage HH 22-24 (E4) for surgery (Hamburger and Hamilton, 1951).

2.2 Surgical Procedure

A window was created in the egg shell using forceps, and microsurgical removal of the retina was carried out at E4 as previously described (Spence et al., 2004). The retina was removed, taking care not to damage the RPE, CM or other parts of the eye. After retina removal, eyes were incubated with either with 50 µg of C3a-p (an active C3a peptide), specific inhibitors (FGFR inhibitor: 1 µM PD173074, MAPK inhibitor: 1 µM PD98059, Stat3 dimerization inhibitor: 2 µM STATIC, and a blocking antibody against C3aR that recognizes the ligand binding site for C3a: 6 µg of C3aRAb). C3a-p was added along with DMSO or IgG as a control for inhibitors or C3aRAb respectively.

Tissue fixation and sectioning

Embryos were collected at specified times after retinectomy and fixed according to the procedure. For histology, the tissue was fixed in 10% buffered formalin (Richard-Allan Scientific. Ref: 5701) at 4°C for at least 24h, and then transferred to 70% ethanol for dehydration. Finally, the tissues were placed in a tissue processor (Leica TP 1020) and embedded (Thermo electron corporation, Shandon histocentre 3) in paraffin wax. The tissues were sectioned at 10µm thickness using a Microm HM 355s microtome, and subsequently stained using hematoxylin and eosin (H&E).

2.3 Protein collection and Western Blot

The CM was collected after 2h of incubation on cold RIPA plus protease inhibitors, keeping all the samples on ice during the entire process of collection to avoid any

16 degradation. The protein was quantified using the BSA method and 100µg of total protein was loaded in a denatured gel (10%, SDS PAGE). The transfer was performed for 2h at 100V in the cold room. The antibodies used to detect pS727- Stat3 were obtained from Cell Signaling (#9134). The immune reactive band corresponding to pS727 Stat3 in the Western blot was normalized to ß-actin and the results were analyzed with Image-Quant.

3. Results

3.1 C3a-p induces retinal regeneration primarily from the ciliary margin

Based on previous studies where C3a was able to induce liver regeneration, we were wondering if C3a has the potential to induce retina regeneration as well. Immediately after retinal removal at E4, we added 50 µg of a C-terminal peptide of C3a (C3a-p) or full length C3a to the chick eyecup followed by incubation for 3 or 7 days post-retinectomy (dPR). We used the C3a (full length) and the C-terminal domain of C3a (C3a-p) that includes the domain important for binding with the C3a G-coupled receptor, and as a negative control a scrambled C3a-p sequence (Scramble). Histological analysis revealed that both C3a and C3a-p were able to induce regeneration after 3d PR from the CM. C3a-p induced a stronger regeneration response than C3a (Figure 2 A, B). After 7d PR both C3a-p and our comparative control FGF2 induced a similar amount of regeneration from the CM (Figure 2 A, C). Regeneration from RPE transdifferentiation was significantly less in eyes treated with C3a and C3a-p vs FGF2, demonstrating that the main effect of C3a is in the CM. Since the regeneration from the CM was more robust than the one from RPE transdifferentiation, we decided to study further the effect of the C3a-p on the CM.

3.2 C3a-p induces the MAPK pathway and phosphorylation at residue S727 of Stat3 independently of FGFR signaling to activate retina regeneration in chick embryos

After observing that C3a-p was able to induce regeneration, we evaluated the different pathways that were involved in retinal regeneration. MAPK has previously been demonstrated to be essential for FGF2-induced retina regeneration (Spence et al., 2004). Therefore, we used the specific inhibitor PD98059 for Mitogen- activated protein kinase kinase (MEK) in retinectomized eyes that were posteriorly treated with C3a-p, and found significantly reduced retina regeneration (Figure 3). Knowing that the MEK was induced and that FGF2 through its own receptor can

17 activate the MEK/Erk1/2 (Spence et al., 2004; Ku et al., 1995), we inhibited FGFR with a specific inhibitor (PD173074) followed by the addition of C3a-p in retinectomized eyes. We were able to see regeneration in the absence of FGFR signaling (Figure 3). In order to confirm that C3a was activating its own receptor, we used an antibody (Ab) which bocks the binding site of C3a to the C3a receptor (C3aRAb). Once the receptor was blocked with C3aRAb in the presence of C3a-p, no regeneration was observed, highlighting the importance of this pathway to induce regeneration (Figure 3).

As part of this project, my role was to evaluate the role of Stat3 during the C3a- induced regeneration. Stat3 was chosen because it is present in the eye during development and during healing processes after insults (Peterson et al., 2000; Zhang et al., 2003). In addition, it was previously shown that complement proteins are able to activate Stat3 through the MEK/Erk1/2 pathway by the phosphorylation of the residue S727 (Gao et al., 2004). Phosphorylation at this residue confers Stat3 with higher transcriptional activity (Zhang et al., 2005). We analyzed pS727 Stat3 after 2 hour (h) PR with and without the different treatments because it was previously reported that Stat3 can be activated as early as 10 minutes after receiving stimuli through the MEK/Erk1/2 pathway (Chung et al. 1997).

We tested different times of activation from 10 minutes to 2h, where 2h showed the highest activation after the treatment with C3a-p. Then we collected the CM of retinectomized eyes and extracted protein to be analyzed by Western blotting for pS727 Stat3. Our study demonstrated that the phosphorylation levels of S727 Stat3 were significantly higher in C3a-p treated CM samples vs samples from intact CM of E4 eyes or E4 retinectomized eyes (Figure 4). Using an anti C3aR antibody (C3aRAb) that inhibits the binding with the ligand (C3a-p), we demonstrated that C3a-p activates Stat3 in the CM through its receptor (C3aR) (Figure 4). In order to confirm the relevance of pS727 Stat3 activation, we used Stattic, which is able to inhibit Stat3 dimerization and activation. We found that Stattic significantly reduced the amount of regeneration (Figure 3). Likewise, a reduction in the levels of phosphorylation was also noted (Figure 4). In order to confirm the relevance of Stat3 activation, we added the same inhibitors used for the histological studies (Figure 3) such as Stattic, PD98059 and AbαC3aR in retinectomized eyes and observed a significantly reduced phosphorylation at residue S727 of Stat3 when the MAPK inhibitor (PD98059) was used, as well as the C3aRAb (Figure 4). This implies that the phosphorylation at S727 Stat3 takes place through the C3a/C3aR pathway mediated by the MAPK (a member of a family of S/T kinases) (Figure 4).

Some reports on human neutrophils have shown that C5a can initiate the phosphorylation of S727 Stat3 through a MAPK pathway (Kuroki and O’Flaherty, 1999), but very little is known about the activation of the pS727 Stat3 through C3a. Our study is the first to show that C3a/C3aR can induce such phosphorylation of Stat3 during regeneration. In Figure 5 a model of retina regeneration induced by C3a is shown.

4. Discussion

The immune response after injury has been shown to be essential for the process of healing and regeneration (Strey et al., 2003; Markiewski et al., 2009; He et al., 2009; Kyritsis et al., 2012). Among the proteins responsible for the activation of proliferation and wound healing are the STAT proteins. STAT proteins have essential roles for regeneration and function of the immune system (Leonard and O’Shea, 1998). Stat3 proteins co-evolved along with the innate and adaptive immune system from low to higher organisms (Darnell, 1997). Disruptions in most of the Stat proteins in mouse had different effects in malfunction of cellular and hormonal immune response. However, STAT3 knockouts are lethal (Leonard and O’Shea, 1998. Takeda et al., 1997).

Our results revealed an important role in the activation of the pS727 Stat3 through the C3a/C3aR pathway in the induction of retina regeneration. We noticed that the CM responded more robustly to the treatment. and therefore we further elucidated the C3a/C3aR signaling pathway during CM regeneration using different inhibitors and antibodies. We found that C3a binds to its receptor and activates the MEK/Erk1/2 pathway in an FGF-independent manner. This kinase induces the phosphorylation of Stat3 at the S727 residue. The phosphorylation at this residue was indispensable to induce chick retina regeneration.

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6. Figure and figure legends

Figure 1

The complement cascade through the Lectin, Classical and Alternative pathways. Whereas the activation and amplification of each pathway is exclusive, they all converge with the cleavage of C3 into its main products: C3a and C3b which are important for the formation of the membrane attack complex (MAC). The complement cascade has been well known for its role in innate immunity, though recent research has demonstrated that complement components play a very important role in proliferation and regeneration of different tissues including: bone, marrow, liver, limbs, lens and connective tissues. This figure is used with permission of Springer. Rutkowski et al., 2010 (License # 3885480440077).

Figure 2

C3a induces retina regeneration in chick embryos. A. Histological (H&E staining) analysis after 3 and 7 days post-retinectomy (dPR). (B and C) with the respective quantification of the regenerated area from the ciliary margin (cr) and transdifferentiation of the RPE (td). Scale bar in Scramble and FGF2 treated is 100µm and applies to all. The quantification of the regenerated area was performed with image-pro 6.3 and the data was analyzed with the two-tailed permutation test at 3 and 7 dPR (Haynes et al., 2013). Statistical analysis of the quantification of regeneration comparing the regeneration from RPE transdifferentation as a response to C3a-p and FGF2. Transdifferentiation in C3a-p- treated eyes was significantly less than in FGF2-treated eyes at both 3d (P=0.0012) and 7d PR (P=0.0182) using a two-tailed permutation test. *P˂0.05 and **P˂0.01. Standard deviation (Stdev) and averages for each treatment are provided in table 1 (Haynes et al., 2013).

Figure 3

C3a activates Stat3 through the MAPK, independent of the FGF2 pathway. Histological analysis at 3dPR with a Stat3 dimerization inhibitor (Stattic), the MAPK inhibitor (PD98059), the FGFR inhibitor (PD173074), C3aRAb, and respective controls: DMSO (vehicle for the inhibitors) and IgG as a control for the antibody (ab). Scale bar in C3a-p+Stattic, FGF2+C3aR ab and FGF2+PD98059 is 100µm and applies to each group (Haynes et al., 2013). Standard deviation (Stdev) and averages for each treatment are provided in table 2 (Haynes et al., 2013).

Figure 4

C3a-p induces S727 Stat3 phosphorylation in the CM. (A) Western Blot showing the phosphorylation status of Stat3 in the CM after different treatments (Lane 1: Development, Lane 2: 2h Retinectomy, Lane 3: C3a-p. Lane 4: C3a-p scramble. Lane 5: Control for the antibody with IgG. Lane 5: Inhibition of the C3aR with a blocking antibody (C3aRAb). Lane 6: Positive control, DF-1 cells treated with interferon gamma (INFγ). (B) Graphical representation of the western blot analysis from (A) shows the level of phosphorylation of S727 Stat3 at 2 h PR. In E4 developmental eyes (Dev), retinectomized eyes with no treatment (Ret), C3a-p-treated eyes and eyes treated with Scrambled C3a (Scr). *P=0.03125 using the exact binomial test comparing C3a-p treatment and Scr treatment (n=5). (C) Graphical representation of the western analysis shows the level of phosphorylation of S727 Stat3 (pS727 Stat3) at 2 h PR in eyes treated with C3a- pþIgG and eyes treated with C3a-pþC3aR-Ab. *P=0.03125 using the exact binomial test; n=5 biological samples. Error bars represent Stdev. The mean ratio of pS727 STAT3 to actin, Sdev and range are provided in table 1. (D) Western blot using the CM samples treated with C3a-p and inhibitors: Lane 1: C3a-p+ pS727 dimerization inhibitor (Stattic). Lane 2: C3a-p + MAPK inhibitor (PD98059). Lane 3: C3a-p + DMSO. Lane 4: C3a-p. (E) Quantification of the amount of regeneration at 3d PR shows that C3a- p-induced regeneration is significantly reduced in the presence of Stattic (**P=0.0054; n=10) and PD98059 (**P=0.0081; n=9) when compared with C3a-p+DMSO (n=9). A two- tailed permutation test was used for statistical analysis. Error bars represent standard deviation (Stdev) and averages for each treatment are provided in table 1 (Haynes et al., 2013).

Figure 5

Retina regeneration induced by C3a/C3aR Model. The model describes the mechanism by which C3a induces chick retina regeneration. C3a binds to its receptor, C3aR, this G- coupled protein receptor activates the Ser/Thr kinase MAPK, which in turn phosphorylates the transcription factor Stat3 in the residue Ser727, enhancing its homodimerization and translocation into the nucleus, promoting gene transcription. Among the up-regulated genes by pS727 Stat3 are IL-6, TNFα and IL-8 that respond to injury and Six3 and Sox2 that are important for retinal progenitor cells. Wnt2b transcripts were down-regulated (Haynes et al., 2013).

7. Tables

Table 1 Mean ratio of C3 to actin or pS727 STAT3 to actin with std dev. and range. Each set of biological samples were run on separate gels but all comparisons were done within the same gel. C3a-p + IgG (1) was compared to C3a-p + C3aR-Ab and C3a-p + IgG (2) was compared to C3a + IL-6 Ab.

Treatment Ratio Mean Std dev Range

E4 No Treatment C3/actin 0.97 0.02 0.93-1.00

E4 Retinectomy 2 h PR C3/actin 1.22 0.30 0.77-1.79

E4 No Treatment C3/actin 1.43 0.46 0.94-2.35

E4 Retinectomy 6 h PR C3/actin 1.59 0.51 0.84-2.55

E5 No Treatment C3/actin 0.93 0.12 0.81-1.05

E4 Retinectomy 24 h PR C3/actin 1.01 0.22 0.79-1.23

E4 Development pS727 0.11 0.06 0.05-.17 STAT3/actin

E4 Retinectomy pS727 0.16 0.10 0.05-0.23 STAT3/actin

C3a-p pS727 0.35 0.17 0.1-0.80 STAT3/actin

Scr pS727 0.22 0.14 0.06-0.50 STAT3/actin

C3a-p + IgG (1) pS727 0.25 0.07 0.06-0.50 STAT3/actin

C3a-p + C3aR-Ab pS727 0.17 0.06 0.05-0.40 STAT3/actin

C3a-p + IgG (2) pS727 0.40 0.13 0.1-0.86 STAT3/actin

C3a-p + IL-6 Ab pS727 0.18 0.07 0.04-0.38 STAT3/actin

Table 2

Mean measurement of regeneration with std dev. and range for each treatment Stage/Treatment Mean Std dev Range (eyes with Measurement regeneration/n)

3d PR Scrambled C3a (Scr) 0 0 0 (0/8)

3d PR FGF2 1054 144 231-1418 (10/10)

3d PR C3a 307 93 245-620 (5/7)

3d PR C3a-p 754 170 407-1904 (9/11)

3d PR C5a-p 1028 313 137-2213 (7/8)

7d PR FGF2 1929 372 1089-3793 (9/10)

7d PR C3a-p 1417 329 1155-3237 (8/12)

3d PR FGF2 258 51.6 231-804 (9/10) Transdifferentiation

3d PR C3a-p 13 8.2 68 and 73 (2/11) Transdifferentiation

7d PR FGF2 938 242.6 354-2423 (9/10) Transdifferentiation

7d PR C3a-p 51 39.4 305 (1/12) Transdifferentiation

3d PR C3a-p + DMSO 746 187 369-1691 (7/9)

3d PR C3a-p + PD173074 790 332 543-1534 (6/8)

3d PR FGF2 + PD173074 0 0 0 (0/5)

3d PR FGF2 + C3aR-Ab 954 170 428-2045 (12/14)

3d PR C3a-p + IgG 974 212 755-1671 (5/7)

3d PR C3a-p + C3aR- Ab 49 16 439 (1/9)

3d PR C3a-p + PD98059 94 94 852 (1/9)

3d PR FGF2 + PD98059 56 56 279 (1/5)

3d PR C3a-p + Stattic 140 80 278-688 (4/9)

3d PR IL-6 508 109 143-1006 (8/10)

3d PR DPBS 0 0 0 (0/6)

3d PR C3a-p + IL-6 Ab 215 146 651 and 1070 (2/8)

3d PR C3a-p + CHIR 99021 98 36 148-420 (6/16)

3d PR C3a-p + DMSO (CHIR 547 163 167-1966 (8/11) 99021 equivalent)

CHAPTER 3

THE ANTIOXIDANT N-ACETYL CYSTEINE IS A POWERFUL RETINA REGENERATION INDUCER

Abstract

Stem cells are important for the maintenance and repair of many tissues. Their fate is regulated by the redox status present intracellularly and in the stem cell niche, which in turn controls their self-renewal, differentiation or survival, and ultimately their ability to regenerate tissue after damage. The use of antioxidants in tissue regeneration has been studied, but their mechanism of action is not well understood. Here, we analyze the role of the antioxidant N-acetylcysteine (NAC) in retina regeneration. The embryonic chick is able to regenerate its retina after its complete removal from retinal stem/progenitor cells present in the ciliary margin (CM) of the eye. The regeneration is dependent on fibroblast growth factor 2 (FGF2), however, this study shows that NAC, unaided by FGF2, modifies the redox status of the CM, enhances self-renewal of stem/progenitor cells present in the CM and induces regeneration. NAC works as an antioxidant by scavenging free radicals either independently or through the synthesis of glutathione (GSH), and/or by reducing oxidized proteins through a thiol disulfide exchange activity. We dissected the mechanism used by NAC to induce retina regeneration through the use of inhibitors of GSH synthesis and the use of other antioxidants with varying modes of action, and found that NAC induces retina regeneration through its thiol disulfide exchange activity. Thus, our results provide, for the first time, a biochemical basis for induction of retina regeneration. Furthermore, NAC induction was independent of FGF receptor signaling, but dependent on the MAPK (pErk1/2) pathway. Other observations during regeneration induced with NAC were the increase in the size of the optic tectum (OT), due to increased cell proliferation.

1. Introduction

1.1 Role of redox status in cell differentiation in mammals

During embryogenesis, before the morula stage, each individual blastomere is a totipotent stem cell (TSC) and retains the capacity to generate a complete organism as well as its placenta. TSCs undergo only few self-renewing divisions and rounds of DNA replication, but there is no net growth (Leese, 1995). The

TSC’s metabolism is controlled by limiting the glycolytic enzymes hexokinase (HK) and phosphofructokinase 1 (PK1) that are responsible for reducing the glycolytic rate (Barbehenn et al., 1978). Therefore, in TSCs, the energy and carbon sources come from pyruvate analogs. At the Morula stage, glucose is taken up by the blastomeres and it is oxidized to pyruvate, which in turn enters the Krebs cycle in the mitochondria, generating carbon intermediates and fueling oxidative phosphorylation (OxPhos). Despite the preference for glucose, the oxygen (O2) consumption is very low in TSCs (Brinster and Troike, 1979). The low O2 consumption is consistent with structurally immature mitochondria in the TSCs (Van Blerkom, 2009).

During compaction, blastomeres undergo the first round of cell differentiation, to segregate into precursors of trophoectoderm (which will become the placenta) and the inner cell mass (ICM), which are pluripotent cells that will become the embryo proper (Pantaleon and Kaye, 1998). The mitochondria in the trophoectoderm of the blastula are very well developed, and perform OxPhos at high rates. On the other hand, mitochondria from the ICM are not well developed and therefore glycolytic pathways remain the main source of energy (Van Blerkom, 2009; Shyh-Chang N, 2013). Glycolysis is not necessary for pluripotency, but it represents the preferred metabolic state for rapidly proliferating cells (Hanna et al., 2009). This condition is essential during development, cancer and induced pluripotent stem cells (iPSc) (Cooper 2000; DeBerardinis et al., 2008; Espejel et al., 2010).

Importantly, during the process of embryonic stem (ESC) differentiation, the cells change the use of their metabolic pathways, switching from glycolysis to OxPhos. During this process the mitochondria mature and start producing ROS as a byproduct. ROS are involved in the decrease in transcription or activity of the stemness factors Oct4, Tra 1-60, Nanog, and Sox2, and in the up-regulation of mesodermal and endodermal markers (Facucho-Oliveira et al., 2007; Lonergan et al., 2007; Ji et al., 2010).

In addition to the metabolic traits, ESCs have different ways to keep low levels of ROS. For example, ESCs are located in a hypoxic environment, and have few developed mitochondria. Additionally, the percentage of unsaturated lipids (reduced lipids according to their high index of hydrogen deficiency) is higher in ESCs in comparison with differentiated neurons and cardiomyocytes. Interestingly, the inhibition of enzymes necessary for lipid oxidation such as cyclooxygenase (COX) and lipooxygenase (LOX) enhances the undifferentiated state of ESCs (Yanes et al., 2010).

It is clear that the redox status is tightly related with cell differentiation as it was previously discussed. In order to neutralize the effects of ROS, various endogenous antioxidant strategies have evolved, which employ both enzymatic and non-enzymatic mechanisms. Therefore, antioxidants have been used to delay or inhibit ESCs differentiation. Studies have demonstrated that the use of vitamin C (Vc) in presence of a well-known ROS producer buthionine sulfoximine (BSO) inhibits the expression of differentiation factors in neurons (Ji et al., 2010). Other studies with curcumin, an antioxidant and inhibitor of the eicosanoid pathway, increased the levels of Nanog and Oct4 to promote the pluripotent state of ESCs (Yanes et al., 2010). Additionally, antioxidants play very important roles enhancing somatic cell reprogramming to induce pluripotent stem cells (iPSCs). Antioxidants such as Vitamin C (Vc) can increase the in vitro efficiency of cell reprogramming both in human and mouse somatic cells. Vc accelerates gene expression changes and promotes the transition of pre-iPSC colonies to a fully reprogrammed state (Esteban et al., 2010). Another antioxidant, NAC, has been showed to diminish differentiation of hematopoietic stem and progenitor cells in bone marrow by enhancing the expression of CD34+ cells due to elimination of ROS, and inhibited erythroid differentiation through the inhibition of gene expression and reduced the number of cells expressing the erythroid-specific antigen TER119 (Fan et al., 2007, Nagata et al., 2007).

1.2 Antioxidant NAC and its role in tissue regeneration

NAC is an aminothiol and synthetic precursor of GSH, used in some disorders related with oxidative stress and acetaminophen intoxication (Lauterburg et al., 1983; Cotgreave., 1997; Zafarullah et al., 2003). The chemical properties of NAC rely on the cysteinyl thiol group that is important for its nucleophilicity and redox reactions essential for the antioxidant properties through thiol disulfide exchange (Kim et al., 2001). NAC has the ability to reduce free radicals or cellular proteins. Specifically, NAC has been reported to directly interact with target proteins that contain cysteine residues or thiol groups such as Raf-1, Mek, and Erk (Cotgreave, 1997; Kim et al., 2001).

NAC is a well-known antioxidant that has been found to play a role in stem cell renewal. NAC is a precursor for cysteine, which is necessary for the production of glutathione (GSH), a free radical scavenger that decreases the level of ROS (Barford, 2004). In addition, NAC also has the ability to reduce free radicals or cellular proteins through its thiol-disulfide exchange activity. Specifically, NAC has

36 been reported to directly interact with target proteins that contain cysteine residues or thiol groups such as Raf-1, Mek, and Erk, further supporting the role for ROS as second messengers (Cotgreave,1997; Kim et al., 2001; Zafarullah et al., 2003). Among other roles, NAC has been shown to promote or enhance tissue repair or regeneration such as liver regeneration in rat after partial hepatectomy (Uzun et al., 2009), neuroprotection and enhanced growth of sensory neurons in adult rats with injured peripheral nerves (Welin et al., 2009), and induced bone regeneration and osteogenesis in rats by improving tissue formation and survival of gingival fibroblasts and dental pulp cell using biomaterials (Yamada et al., 2013). Treatment of muscle-derived stem cells (MDSCs) with NAC previous to implantation improved cardiac function over non-treated MDSCs after myocardial infarction in a murine model (Drowley et al., 2010). Several studies have demonstrated that the use of antioxidants such as vitamin C, α-tocopherol, carotene and NAC, inhibit cellular differentiation and enhance the induction of pluripotent stem cells (Allen and Venkatraj, 1992; Neroev et al., 2008; Bernal et al., 2013; Yamada et al., 2013).

1.3 Redox status in the retina

The retina is one of the tissues with highest risk of ROS-induced damage for the following reasons: (i) retina contains high content of polyunsaturated fatty acids, which are targets for ROS attack, (ii) retina is exposed to light producing different kinds of ROS as singlet oxygen, (iii) oxygen concentration in the retina increases as a consequence of the contact with the hyaloid artery, and (iv) retina is a tissue with high respiratory activity, which means that ROS production by the mitochondria is high (Barnett and Handa, 2013).

Excessive light exposure in the eye may enhance the progression and severity of human age-related macular degeneration (AMD) and some forms of retinitis pigmentosa. Intense light exposure induces lipid peroxidation of the disc membranes of photoreceptor outer segments. This peroxidation is caused by free radicals such as ROS (Organisciak and Vaughan, 2010). ROS are also involved in loss of RPE cells. This loss represents an early event in AMD. RPE degradation is primarily attributed to oxidative stress, as a consequence of attenuated antioxidant defense. Different antioxidants have been used to protect the retina, and antioxidants with an endogenous thiol system such as GSH and thioredoxin (TRX) are considered regulators of endogenous redox in the retina (Tanito et al., 2002).

Different studies involving plastoquinone 1 (SkQ1), an antioxidant used in different animal models animals such as rabbits, dogs, cats, and horses, has been shown to

37 improve vision in very low concentrations. SkQ1 helped prevent glaucoma, uveitis, conjunctivitis, and cornea diseases. Also, in animals with cataracts or retinopathies, SkQ1 helped to recover vision (Neroev et al., 2008). All these eye pathologies are related to oxidative stress resulting from the deleterious effects of ROS. The antioxidant SkQ1 not only prevented loss of vision, but also helped recover vision after oxidative events (Neroev et al., 2008).

Although it is known that NAC plays a role in regeneration, the specific inductive mechanisms are not clear. Here, we analyze the antioxidant properties of NAC to determine its role in retina regeneration using the embryonic chick as a model. Chick embryos cannot regenerate their retina spontaneously, however, retina regeneration can be induced using ectopic factors such as fibroblast growth factor 2 (FGF2). FGF2-induced retina regeneration occurs by the activation of retinal stem/progenitor cells present in the CM of the eye and via the transdifferentiation of the retinal pigmented epithelium (RPE) (Spence et al., 2004). We have found that NAC is able to induce retina regeneration in the absence of any exogenous growth factors. NAC decreases the level of ROS induced by injury leading to the activation of the retinal stem/progenitor cells and ultimately regeneration. The regenerative potential of NAC is not dependent on its free radical scavenging ability as our results support a model in which NAC activates the MAPK pathway, independent of the FGF receptor signaling, through its thiol-disulfide exchange activity.

2. Methods

2.1 Chick embryos

Fertilized chicken eggs were purchased from Michigan State University, poultry farm. The eggs were incubated at 38°C in approximately 40-70% humidity until they reached the developmental stage HH 22-24 (embryonic day 4- E4) (Hamburger and Hamilton, 1951).

2.2 Surgical Procedure

Microsurgical removal of the retina was carried out at E4 as previously described (Spence et al., 2004; Haynes et al., 2007; Haynes et al., 2013). After retina removal, 4µl of the different treatments were added in the eye cup; 100mM NAC (Sigma A9165), 100mM NAG (Sigma-Aldrich A16300), 100mM NAS (Sigma- Aldrich A2638), 10 mg/ml vitamin C (Sigma A4544), 5 mM XJB 5-131 (obtained

38 from Dr. Wipf from University of Pittsburg), 200µM Hydrogen peroxide or 1ug/ul of FGF2 (R&D Systems) was added inside the eyecup. Controls for NAC and FGF2 included retinectomy plus vehicle (1% glycerol in 1X sterile PBS).

2.3 Tissue fixation and sectioning

Embryos were collected at specified times after retinectomy and fixed according to the procedure. For histology, the tissue was fixed in 10% buffered formalin (Richard-Allan Scientific. Ref: 5701) at 4°C for at least 24h, and then transferred to 70% ethanol for dehydration. Finally, the tissues were placed in a tissue processor (Leica TP 1020) and embedded (Thermo Electron corporation, Shandon histocentre 3) in paraffin wax. The tissues were sectioned at 10µm thickness using a Microm HM 355s microtome, and subsequently stained using hematoxylin and eosin (H&E).

For immunohistochemistry and Click iT Edu analysis, tissues were fixed in 4% paraformaldehyde overnight (O/N) at 4°C, rinsed in 1X PBS three times for 5 minutes, and cryopreserved in 30% sucrose for 2 days at 4°C. The tissues were embedded in optimal cutting temperature media (O.C.T, from Tissue tek. Ref: 4583) and sectioned at 10µm thickness using a Microm HM 505N cryotome. The slides are kept at -20°C until use.

2.4 Immunohistochemistry

Immunohistochemistry was performed on frozen sections. Three different biological samples were used in triplicate for each experiment. The tissues were permeabilized with 1% saponin for 5 mins, blocked for 30 mins in 10% goat or donkey serum followed by an incubation with the primary antibody O/N at 4°C. The secondary antibody was incubated for 2h in the dark. The primary antibodies used for immunohistochemistry include: Vsx2 (1:50; Exalpha biological, X1180P), DMPO (1:1500; Cayman chemical #10006170), phospho histone 3 (Ser 10) (1:500; Cell signaling # 06-570), phospho-P44/42 Erk1/2 (1:500; Cell signaling # 4370), Brn3a (1:50; Chemicon International) and Sox2 (1:1000; Santa Cruz, sc 17319). SSEA1, Vimentin, Visinin, Ap2, Pax6, and Napa (1:100) were obtained from the Hybridoma Bank. Secondary antibodies include: donkey anti-sheep IgG 488 (Invitrogen A11015), Goat anti-mouse IgG 546 (Invitrogen A11003), Goat anti-mouse IgM 488 (Invitrogen A21042), Goat anti-rabbit IgG 488 (Invitrogen A11008), Goat anti-rabbit IgG 546 (Invitrogen A11010), Goat anti-mouse IgG (Invitrogen A11001).

Fluorescence was evaluated using a laser scanning confocal microscope (Olympus FV500).

2.5 Cell proliferation

The Click iT EdU Alexa fluor 488 image kit (Invitrogen # C10337) was used. One hour before collecting the embryo, 30µl of 5mM EdU (Component A) was added on the top of the eye (making sure there were no membranes on top). We performed the click reaction as per manufacturer instructions.

2.6 ROS quantification

Two different methods were used to measure ROS: the chemiluminescent probe (6)-carboxy-2’, 7’dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and the immuno-spin trapping 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The CM- H2DCFDA (Invitrogen Molecular Probes # C6827) was used as described by Owusu-Ansah (2008). The CM was collected after 6h, washed in 1X PBS at room temperature (RT)/5min. In a glass bottom microwell dish (MatTek Part No-P35G- 1.5-14-C) the tissue was incubated for 15min with a final concentration of 1µM CM- H2DCFDA followed by 3 washes in 1X PBS, and immediately photographed using confocal microscopy. The immuno-spin trapping DMPO (Cayman chemical Item No: 10006170) was performed using immunohistochemistry as per manufacturer instructions. The intensity of the signal was quantified using Image Pro 7.4 and reported as a ratio between the intensity/area selected.

2.7 Inhibitors

For inhibition studies, inhibitors were added after retinectomy 30 minutes before the treatments with either FGF2 or NAC. The following inhibitors were added in a volume of 4µl: 1 μM FGFR inhibitor PD173074 (Selleckchem Cat No S1264); 1 μM MEK inhibitor PD98059 (Cell Signaling Cat No 9900); and the GSH inhibitors: 0.9 mM conjugating agent Diethyl Maleate (DEM) (Sigma-Aldrich Cat No D97703) and 10mM L-Buthionine sulphoximine (BSO) (Cayman chemical Item No: 14484). All inhibitors were prepared in 1XPBS, 20% glycerol and 5% DMSO, except for BSO which was prepared in 1XPBS and 20% glycerol.

2.8 Glutathione quantification (GSH)

The embryos were collected after 6h and 24h PR in 1X PBS at RT. The CM was collected and processed immediately for GSH quantification. GSH quantification was determined followed the instructions from Rahman et al., 2006. The samples’ absorbance was read in a spectrophotometer at 412nm at 0, 1 and 2 min. Data was analyzed using the following formulas: y=AX+B. [GSH]TOTAL=2*((∆OD412/min- B)/A)*Sample dilution, [GSSH]=((∆OD412/min-B)/A)*Sample dilution. [GSH/GSSH]= ([GSH]TOTAL-2[GSSH])/[GSSH]. Were the GSH is the reduced form and the GSSH is the oxidized form of GSH.

2.9 Statistical analysis

The statistical analysis was performed using SAS/STAT software, Version 9.4 of the SAS System for Windows. Copyright © 2016 SAS Institute Inc. SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc., Cary, NC, USA. Mixed model analyses of variance (ANOVA) were performed for examining treatment comparisons (Figures 4, 5) with Dunnett-adjusted multiple comparisons to control Type I error in treatment comparisons against a control performed of analyses in Figures 1, 3, 6, and 7. All model assumptions of normality and homoscedasticity were checked and verified, with occasional implementation of log transformations to stabilize error variance. Mann-Whitney-Wilcoxon rank-based tests were used for analyses of skewed response data in Figure 2.

3. RESULTS

3.1 Redox status changes in the ciliary margin in response to injury

Since the redox status is important for stem cell self-renewal, we wanted to determine if this was critical for the induction of retina regeneration. To determine this, we first investigated the redox status in the CM, the retinal stem/progenitor cell niche of the embryonic chick, following retinectomy. Previous work in our laboratory determined that activation of transcription factors necessary for induction of regeneration occurs in response to injury by 6 hours (h) post- retinectomy (PR) (Luz-Madrigal et al., 2014). Therefore, we investigated changes in the redox status starting at 6h PR and up to 24h PR in the CM, by measuring levels of immunofluorescence when using an antibody against 5,5-dimethyl-1- pyrroline-N-oxide nitrone adduct (DMPO) which covalently binds to oxidized adducts in proteins. When NAC was introduced, the levels of oxidized protein were

41 diminished significantly at both 6h and 24h PR compared to eyes receiving retinectomy only (Figure 1A and B). Interestingly, FGF2, which can induce retina regeneration, also decreased the level of oxidized proteins at 6 and 24h PR compared to eyes receiving retinectomy only (Figure 1A and B) suggesting that the redox status is important for induction of regeneration. In order to determine the antioxidant scavenging capacity of NAC, the level of immunofluorescence with DMPO was also determined in retinectomized embryos treated with NAC in the presence or absence of hydrogen peroxide (H2O2). H2O2 is a known precursor of free radicals, which in turn will induce a more oxidized state. Eyes treated with H2O2 showed an increase in the levels of oxidized proteins compared to treatment with NAC alone and this increase was significantly diminished by NAC by 24 h PR (Figure 1A and B). However, NAC was not able to reduce the level of oxidized proteins to the low level seen when H2O2 is not added, demonstrating the ability of H2O2 to maintain the oxidized state longer (Figure 1A and B). These results were corroborated with the fluorescent probe, (6)-carboxy-2’,7’dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), which oxidizes in the presence of ROS (Figure 2A-D).

3.2 NAC is able to induce retina regeneration

To determine if a reduction in ROS is indeed critical for induction of retina regeneration in the embryonic chick, NAC was added after retinectomy into eyecups at embryonic day 4 (E4), and eyes were collected 7d PR. We found that NAC in the absence of FGF2 was able to induce regeneration (Figure 3 A) from the CM. As a matter of fact, NAC was able to induce regeneration from the CM comparable to FGF2 (Figure 3 B). No significant difference was seen in the level of regeneration induced by both treatments (Figure 3 F). Coincidently, transdifferentiation (TD) of the RPE was also induced, however it was significantly less in the NAC treated eyes when compared to FGF2 treated eyes (Figure 3 F). Because of these observations, we focused on retina regeneration induced from the CM. NAC was even able to induce regeneration from the CM in the presence of a more oxidized state induced by H2O2 as shown by the significant regeneration observed 3d PR in eyes treated with NAC + H2O2 (Figure 3 D, E, G) suggesting that the free radical scavenger activity of NAC is not needed for induction of regeneration.

3.3 NAC induces proliferation

Low levels of ROS create/maintain an optimal redox status conducive for stem cell self-renewal (Urao and Ushio-Fukai, 2013). To test if NAC is indeed necessary for stem cell proliferation and maintenance, we compared the number of proliferating cells present in the CM after retinectomy in response to treatments with NAC or FGF2 (Figure 4A). EdU was added to eyes one hour before collection to detect cells in S phase. Then, double immunohistochemistry was performed using antibodies against EdU as well as phospho-histone 3 (PH3), which detects cells in G2/M phase (Figure 4A). We found a significantly higher level of Edu+ and PH3+ cells at 24h and 3dPR in the CM exposed to NAC compared to eyes receiving no treatment (retinectomy only), showing that in the presence of NAC the retinal stem/progenitor cells do proliferate in a similar level as eyes exposed to FGF2 (Figure 4B and C). Additionally, we noted there was no increase in apoptosis during NAC-induced regeneration as no observable TUNEL positive cells were seen in eyes treated with either NAC or FGF2 (Figure 5).

3.4 Cell differentiation is delayed in NAC-induced regeneration

Next, we examined if NAC induced neuroepithelium differentiation. During retina development, the retinal neurons are formed in a specific order. Amacrine cells are formed first followed by horizontal, rods, bipolar and lastly Müller glia (Cepko, 2014) We evaluated the differentiation of each retinal cell type by immunohistochemistry using antibodies to detect Brn3a (ganglion cells), Napa (ganglion cell axons), Visinin (photoreceptors), Ap2 (amacrine cells), Vimentin (Müller glia), Vsx2 (bipolar cells) and Lim 1/2 (horizontal cells). In response to FGF2 induction, all major retinal cell types were detected by 7d PR (Figure 6 A, E, I, M, Q, U) while NAC treated eyes at 7d PR had significantly less number of each of the cell types (Figure 6 B, F, J, N, R, V). However, by 11d PR, NAC did promote differentiation of most of the cell types (Figure 6 D, H, L, P, T, X).

We hypothesized that NAC treatment results in a delay in differentiation because lower levels of ROS can favor stem cell renewal and maintenance instead of differentiation (Xiao et al., 2014). Therefore, we performed immunohistochemistry against the stage-specific embryonic antigen-1 (SSEA-1), a marker for immature retinal progenitor cells in mice (Koso et al., 2006). In NAC treated eyes, SSEA-1 was present in the anterior retina at higher levels than in FGF2- treated eyes 7 dPR (Figure 7 A and C). Co-expression of Vsx2/Pax6 is also indicative of neural stem/progenitor cells and these markers were also significantly increased in the

NAC treated eyes at 7dPR in the anterior and posterior regions of the regenerated retina when compared to FGF2 treated eyes (Figure 7 B and D). This expansion of Vsx2/Pax6 positive cells beyond the CM in the NAC treated eyes compared to FGF2 treated eyes supports the role of low ROS in stem/progenitor cell maintenance. Taken together these results show that NAC enhances self-renewal of progenitor cells during chick retina regeneration resulting in a delay in their differentiation. Other progenitor markers used were the ribonucleoprotein Lin28 (Figure 8A) and the transcription factor Sox2 (Figure 8B). We did not see significant difference between NAC and FGF2 for these markers. Whereas, Lin28 was expected to be localized in the cytoplasm, Lin28 was found in the nucleus in the eyes treated with NAC 3 d PR. Previous studies have found that Musashi 1 is involved in Lin28 translocation into the nucleus in order to control transcription (Kawahara et al., 2010).

3.5 NAC-induced regeneration is not dependent on GSH-ROS scavenger pathway

We have shown that NAC is able to induce retinal stem/progenitor cell proliferation and subsequent regeneration and we next sought to identify the mechanism of NAC induction. One of the mechanisms cells use to lower the levels of ROS is the synthesis of the small molecule, GSH. GSH can be reduced to GSSH as H2O2 is simultaneously converted to H2O thereby decreasing ROS in the cell. Therefore, the ratio of GSH/GSSH is the main indicator of redox status in the cell. Increased GSH will lead to a reduced state and low ROS, while high levels of GSSH are indicative of high levels of ROS (Forman et al., 2004). NAC is a precursor of GSH, so it is possible that NAC diminishes the levels of ROS by increasing the level of GSH (Figure 9A). We measured the levels of GSH in the CM in the presence and absence of NAC and found that NAC did increase the levels of GSH (Figure 9B) (after 6h PR: GSH/GSSH= 29:1, after 24h PR: GSH/GSSH=9/1) compared to untreated controls (after 6h PR GSH/GSSH=1:4, after 24h PR GSH/GSSH=0.2/8) (Figure 9B). In order to evaluate the role of GSH during NAC-induced regeneration, two inhibitors of the GSH synthesis pathway were used: L-Buthionine sulphoximine (BSO) which reversibly inhibits γ- glutamyl-cysteine synthase, and dietyl maleate (DEM) which conjugates with GSH inhibiting its binding with antioxidant enzymes (Figure 9A). While these inhibitors significantly diminished the ratio of GHS/GSSH in the presence of NAC (at 6hPR BSO+NAC: GSH/GSSH=1:2.33, DEM+NAC: GSH/GSSH=1:8, at 24hPR BSO+NAC: GSH/GSSH=2.8:2, DEM+NAC: GSH/GSSH=1:4) (Figure 9B). Histological analysis performed with eyes collected 3d PR showed that the same level of regeneration was induced with NAC as with

NAC + BSO or NAC + DEM (Figure 9C). These results indicate that NAC’s regenerative ability is not dependent on GSH synthesis (Figure 9D).

3.6 MAPK pathway is necessary for NAC induced regeneration

In addition to GSH synthesis, NAC is also able to reduce proteins at cysteine residues through its thiol sulfate group. Interestingly, MAPK is one of the proteins documented to be reduced by NAC (Kim et al., 2001) and MAPK activation is also necessary for chick retina regeneration by FGF2, BMP, and C3a (Spence et al., 2007; Haynes et al., 2007; Haynes et al., 2013).

Immunohistochemistry results showed that pErk, a MAPK, is transiently activated 6hPR in response to injury and sustained activation of pERK only occurs in the presence of NAC or FGF2 (Figure 10A). NAC is able to activate pErk to a comparable level as that of FGF2 (Figure 10B). These results suggest NAC is inducing regeneration through activation of the MAPK pathway. To test this, we added the MAPK inhibitor PD98059 along with NAC in retinectomized eyes and collected 3dPR. Histological analysis of these eyes showed there was a significant decrease in regeneration in the embryos treated with the PD98059 and NAC (Figure 11A and B). However, the addition of the FGFR inhibitor, PD173074, along with NAC, did not show a significant decrease in regeneration suggesting that NAC-induced regeneration depends on the activation of the MAPK pathway in an FGF-independent manner (Figure 11B). Since NAC has been shown to reduce MAPK, presumably through its thiol disulfate exchange activity (Sun, 2010), we tested the importance of this activity next. We used N-acetylglycine (NAG) which is able to induce GSH synthesis but lacks a thiol group and N-acetylserine (NAS) which is structurally identical to NAC except lacking the thiol group, and found that neither NAG nor NAS were able to induce regeneration (Figure 12A and C) supporting the importance of the thiol sulfate exchange activity of NAC in the induction of retina regeneration. Additionally, two different antioxidants, Vitamin C (Vc) and XJB 5-131, that have been shown to decrease the levels of ROS by scavenging and do not contain a thiol group (Niki, 1991; Wipf et al., 2005), were unable to induce regeneration (Figure 12D and C). Thus, our results support a model in which the thiol disulfide exchange activity of NAC, and not GSH synthesis or ROS scavenging, is critical for the induction of retina regeneration (Figure 13).

3.7 Other effects of NAC during retinal regeneration

After collecting the embryos, we noticed an increase in the volume of the optic tectum in the embryos treated with NAC (Figure 14), histological analysis showed an increase in the size of this area (Figure 14). Immunohistochemistry against two markers Pax7 and Meis2 were performed and we found an increase in positive cells for both markers (Figure 15). Pax7 is initially present in the proliferative neuroepithelial layer that establishes the tectal polarity. Later Pax7 is expressed in neurons of the retino-recipient precursor stratum griseum et fibrosum superficiale laminae (Thomas et al., 2006). On the other hand, Meis2 is essential for tectal development (Agoston et al., 2012).

4. Discussion

Our results indicate that the antioxidant NAC is able to induce retina regeneration in the embryonic chick primarily from retinal stem/progenitor cells in the CM. NAC has been shown to promote or enhance tissue repair or regeneration in other systems. For example, in rats, NAC enhanced liver regeneration after partial hepatectomy (Uzun et al., 2009). NAC enhances peripheral sensory neuron growth after injury (Welin et al., 2009), and induces bone regeneration and osteogenesis (Yamada et al., 2013). In these reports, the antioxidant property of NAC is thought to be critical for its role in regeneration but specific mechanisms were not defined. Here, we separately examined the GSH synthesis and free radical scavenger activity and the thiol disulfide exchange activities of NAC during chick retina regeneration, and concluded that the thiol disulfide exchange activity is critical for the induction of retina regeneration and we suggest that this activity is responsible for activating the MAPK pathway. We show that NAC does increase the level of GSH and modifies the redox status by decreasing the level of ROS after retinectomy. However, inhibition of GSH synthesis does not affect the induction of regeneration by NAC. Also other antioxidants such as Vc and XJB 5-131, are unable to induce regeneration supporting that low levels of ROS are not sufficient for induction of regeneration. Furthermore, the levels of ROS induced by hydrogen peroxide did not inhibit the ability of NAC to induce regeneration further supporting NAC’s ability to induce regeneration independent of its role as cysteine donor for the GSH synthesis and as a ROS scavenger. Instead, we show that induction of regeneration requires the thiol-disulfide exchange activity of NAC since NAS and NAG, two structurally similar molecules to NAC that lack the thiol-sulfate group, were unable to induce regeneration. Although NAG does not have the thiol-sulfate group, it has been

46 reported to have the ability to increase GSH levels (Bloch, 1949), further highlighting the importance of the thiol-disulfide group.

Oxidation of the thiol group in cysteine residues of proteins occurs when levels of ROS increase and consequently change the redox status thereby affecting different signaling pathways and changing gene expression. A large variety of proteins including transcription factors, chaperones, protein tyrosine phosphatases and protein kinases have been shown to be tightly regulated via redox processes (Barford., 2004; Leonard and Carroll., 2011; Gupta and Carroll, 2014). MAPK family, most notably Erk1/2, is one protein that has been shown to be sensitive to the redox status (Murray et al., 2015). The MAPK pathway has been identified as being critical for retina regeneration induced by other signaling molecules including FGF2, BMP, and C3a (Spence et al., 2007; Haynes et al., 2007; Haynes et al., 2013) and now we show that NAC-induced regeneration is also dependent on the MAPK pathway but independent of FGFR activation. Therefore, activation of MAPK by the thiol disulfide exchange activity is a likely possibility. A cysteine residue at position166 of Erk1/2 lies within an important domain for ATP binding, and modifications of this domain with an irreversible inhibitor at this site decreases its interactions with other kinases (i.e. MEK1/2) as well as its translocation to the nucleus (Ohori et al., 2007; Galli et al., 2008; Corcoran and Cotter., 2013), so it is possible that the reduction of this particular residue by NAC can enhance Erk activation. NAC has been reported to enhance activation of Erk1/2 in cultured bovine and human chondrocytes (Zafarullah et al., 2003) as well as in sympathetic neurons and PC-12 cells further supporting our results (Yan and Greene, 1998). In fact, Yan and Greene suggested that the thiol-disulfide exchange activity of NAC was necessary for the in vitro activation of Erk 1/2.

Activation of Erk mediates basal and stimulus-activated gene expression and controls different cellular processes including proliferation, differentiation, and cell death (Ohori et al., 2007; Mebratu and Tesfaigzi. 2009) that are critical mechanisms for regeneration. Tissue regeneration in vertebrates relies mainly on the activation and self-renewal of stem/progenitor cells (Lane et al., 2014) as we see in the embryonic chick. However, regeneration is limited in higher vertebrates. It is possible that stem cells in higher vertebrates are particularly sensitive to changes in redox status. After injury, the production of ROS increases as a consequence of the inflammatory response, and this increase in ROS production modifies the redox status, affecting the process of regeneration by diminishing stem/progenitor cell self-renewal as well as the presence of stemness factors dictating cell differentiation, and inducing apoptosis and replicative senescence (Kobayashi and Suda, 2012).

Our work shows that NAC modifies the redox status as well as induces proliferation and subsequently the number of retinal progenitor cells in the CM as shown by the increased expression of Vsx2/Pax6 and SSEA. NAC is known to increase proliferation and reduce apoptosis and necrosis in adipose-derived stem cells from human subcutaneous adipose tissue (Xiong et al., 2012) as well as to enhance the ability of muscle-derived stem cells used in implantation to improve cardiac function in a myocardial infarction murine model (Young et al., 2003) supporting our results. Previous studies have also shown that NAC delays cell differentiation in rat bone marrow by multipotent adult progenitor cells (Xiao et al., 2014). These results agree with our observations since the NAC-induced retina had delayed cell differentiation compared with FGF2 treated eyes. However, by 11d PR, the retina induced by NAC had increased differentiated photoreceptors, amacrine, and bipolar cells when compared to 7d PR, suggesting that differentiation was not inhibited but delayed by the presence of NAC, as expansion of retinal stem/progenitor cells was initially favored. However, differentiation of ganglion cells as well as Muller Glia was significantly low even at later times. While it is possible that the differentiation of these cell types is not supported by NAC, we hypothesize that degeneration is beginning to occur in these retinas because at 11d PR fewer ganglion and ganglionic axons are present in the regenerated retina induced by FGF2 as well. The delivery method of NAC and FGF2 could account for the degeneration at 11d PR. For this study, NAC and FGF2 were both administered directly into the eyecup as opposed to delivery via heparin beads, which have been used in the past to deliver FGF2 (Spence et al., 2004). The heparin beads allow a slow release of FGF2, which sustains the treatment longer, resulting in the maintenance of the differentiated cells at 11 d PR (Spence et al., 2004). Since NAC is absorbed by heparin beads, this delivery system was not applicable to this study.

The results presented here not only show that NAC has the ability to induce retina regeneration and can therefore be a potential therapeutic treatment for retinal degenerative diseases but also increases our overall understanding about the role antioxidants play in the regulation of stem cells. Antioxidants work through diverse mechanisms, and this study shows that antioxidants can have different effects in different contexts as other antioxidants were unable induce retina regeneration. The fact that some antioxidants can specifically regulate the redox status through activation of signaling pathways is of great importance, not only because manipulation of signaling pathways can lead to induction of stem cells and a potential repair mechanism, but also to avoid off target effects from signaling molecules during future treatments involving antioxidants. In addition, the increase

48 in proliferation and volume in the optic tectum (OT) during our treatments was an interesting observation that needs to be further revisited, as it could be promising to evaluate if NAC can be used as a therapeutic approach in neural degenerative conditions.

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6. Figure and Legends Figure 1

Redox status in the ciliary margin in the chick eye post- retinectomy (PR). (A) Immunohistochemistry using the immunospin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) antibody on sections 6 hours (h) and 24 h post-retinectomy (PR) only as well as with the following treatments: FGF2, NAC, Hydrogen peroxide (H2O2), H2O2 + NAC. Scale bar is 125µm and applies to all. (B) Graphical representation of the ratio (intensity/area) of the signals detected with DMPO. Statistical analysis was performed using Dunnett multiple comparisons (n=3 to all). After 6h and 24h PR, the treatment with NAC significantly reduced the signal intensity compared to eyes receiving retinectomy only (Dunnett 6h **P=0.0031 and at 24h **P=0.0025), Dev by 24hPR (Dunnett **P=0.0070) or H2O2 (Dunnett at 6h *P=0.0310 and at 24h ***P˂0.0005). The intensity of the signal was also significantly lower in eyes treated with H2O2 +NAC compared to eyes treated with H2O2 alone at 24h PR (Dunnett **P=0.004). No significance was found between NAC and the eyes treated with FGF2 at 6 hr or 24 hr PR (Dunnett P=0.1, P=0.55 respectively), Dev by 6hPR (Dunnett P=0.25) nor with eyes comparing H2O2+NAC with H2O2 at 6h PR (Dunnett P= 0.33) or NAC compared with H2O2 +NAC at 6hPR (Dunnett P=0.24). Standard deviation (Stdev) and averages for each treatment are provided in table 1.

Figure 2

Presence of reactive oxygen species in the ciliary margin in the chick eye post- retinectomy (PR). (A) Reactive oxygen species quantification using CM-H2DCFDA. Confocal images of the ciliary margin with the different treatments. 6h PR (n=4), 6h PR+NAC (n=4) treated eyes, Negative control (n=2). Scale bar is 125µm and applies to all. (B) Reactive oxygen species quantification using CM-H2DCFDA. Confocal images of the ciliary margin with the different treatments 6h PR + hydrogen peroxide (H2O2) (n=8), 6h PR+ H2O2 +NAC (n=3). (C) Graphical representation of the ratio (intensity/area) of the signal after the oxidation of the CM-H2DCFDA. Statistical analysis was performed using Dunnett multiple comparisons, treatments with ret (P=1.00) or development (P=1.00) were not significant when compared with NAC (D) Graphical representation of the ratio (intensity/area) of the signal after the oxidation of the CM-H2DCFDA using Dunnett multiple comparisons showed treatments with H2O2 were not significant when compared with H2O2+NAC (P=0.9998). The use of CM-H2DCFDA has been shown to have high variability (Dikalov et al., 2014) and therefore is not a preferred method to measure ROS. However, we were able to observe a trend that supports the results with DMPO (Figure 1). Standard deviation (Stdev) and averages for each treatment are provided in table 2.

Figure 3

The antioxidant NAC induces retina regeneration in chick embryos. (A-C) Histological analysis of chick eyes collected 7dPR treated with (A) NAC (n=10), (B) FGF2 (n=10), or (C) retinectomy+ vector (PBS) (n=10). (D and E) Histological analysis of chick eyes collected 3dPR treated with (D) H2O2 + NAC or (E) H2O2. Scale bar in E is 250 µm and applies to all. (F) Quantitative analysis of the mean level of regeneration for each treatment: NAC (n=10), FGF2 (n=10). Statistical analysis was performed using nonparametric Mann-Whitney-Wilcoxon tests for both ciliary regeneration (CR) and retinal pigmented epithelium (RPE) transdifferentiation (TD). There was no significant difference in the regeneration induced from the CM by both treatments (Wilcoxon S=115.0, P=0.4813), however, FGF2 treated eyes showed a significant increase in RPE TD when compared to NAC (Wilcoxon S=138.0, *P=0.0107). (G) Quantitative analysis of the mean level of regeneration for each treatment: H2O2 + NAC (n=8), H2O2 (n=8). Area of regeneration was significantly larger in H2O2 + NAC (S=100.0, **P=0.002) when compared to H2O2. Standard deviation (Stdev) and averages for each treatment are provided in table 3.

Figure 4

NAC-treatment enhances cell proliferation after retina removal. (A) immunofluorescence using 5-ethynyl-2’-deoxyuridine click reaction (EdU) (green) and anti- PH3 (red) and from eyes collected 6h, 24h, or 3d PR with retinectomy only, or treated with NAC (n=3) or FGF2 (n=3). Scale bar is 125µm and applies to all. (B) Graphical representation of the mean level of Edu+ or (B) PH3+ cells after treatment with NAC for the indicated times. Statistical analysis was performed using Dunnett multiple comparisons. Treatment with NAC resulted in a significant number of EdU+ cells compared to eyes receiving retinectomy only at 6h, 24 h, and 3d PR (Dunnett for EdU + cells: *P6hPR=0.0170, **P24hPR =0.0061 and **P3dPR =0.0065; and for PH3 it was only M significant at 3dPR, Dunnett for PH3+ cells: P6hPR=0.3694, P24hPR =0.0587 and **P3dPR =0.0024). M=marginal significance. Standard deviation (Stdev) and averages for each treatment are provided in table 4.

Figure 5

Average number of cells that entered S phase labeled with (EdU) and apoptosis terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) (A) TUNEL (red) and click reaction EdU (green) from eyes collected 6h, 24h, or 3d PR with retinectomy only, or treated with NAC or FGF2. Scale bar in M is 125µm and applies to all. Arrows in yellow show some apoptotic cells. (B) Positive control for TUNEL (n=3 applies to all).

Figure 6

Cell differentiation during retinal regeneration. (A-X) Immunofluorescence for cell makers for the different cell types of the retina at 7dPR and 11dPR: (A-D) Visinin labeled photoreceptors, (E-H) Vimentin labeled Müller glia, (I-L) Brn3a labeled ganglion cells and NAPA 73 labeled ganglion axons, (M-P) Ap2 labeled amacrine cells, (Q-T) Lim 1/2 labeled horizontal cells, (U-X) Pax6 labeled amacrine and ganglion cells and Vsx2 labeled bipolar cells. Scale bar in X is 125µm and it applies to all panels. (Y) Graphical representation of the number of positive cells for each marker. After 7d PR the number of immunofluorescent+ cells present in NAC treated eyes was significantly lower compared to eyes treated with FGF2 for Visinin (*P=0.0118), Vimentin (***P=0.0006), Brn3a

(***P=0.0008), Ap2 (*P=0.0123), Lim1/2 (*P=0.0119), Pax6 (**P=0.0026) and Vsx2 (**P=0.0051) and marginal for Napa 73 (MP =0.0672). After 11 d PR the number of immunofluorescent+ cells present in NAC treated eyes was significantly lower compared to eyes treated with FGF2 for Vimentin (***P=0.0009), Ap2 (*P=0.0415) and Lim1/2 (*P=0.0115), marginal for Visinin (MP =0.0641), but was not significant for Brn3a (P=0.9721), Napa 73 (P=0.1300), Pax6 (P=0.7596) and Vsx2 (P=0.2537). The number of immunofluorescent+ cells was also compared between 7dPR with 11d PR in the NAC treated eyes, and there was significance difference for Vimentin (*P=0.0120), Pax6 (***P=0.0002) and Vsx2 (***P=0.0001), marginal for Ap2 (MP =0.0529), but not for Visinin (P=0.1141), Brn3a (P=0.1360), Napa 73 (P=0.3078), and Lim1/2 (P=1.00). The significance is not shown in the graph for the comparison of 7d PR and 11d PR NAC treated eyes. Note that the Pax6+ cells used for the comparisons were only the ones present in the INL excluding horizontal cells and ganglion cells, so it is representative of amacrine cells. Mixed model ANOVA was used for the statistical analysis (n=3 applies to all). Standard deviation (Stdev) and averages for each treatment are provided in table 5.

Figure 7

NAC induces the expansion of progenitor cell and induces progenitor cell markers during chick retina regeneration. Immunofluorescence for (A) the cell surface antigen SSEA1 indicates the presence of progenitor cells in the anterior and posterior retina at 7d PR. Eyes were treated with NAC (n=3) or FGF2 (n=3). (B) Double immunofluorescence for the transcription factors, Vsx2 (red) and Pax6 (green) indicates the presence of progenitor cells in the anterior and posterior retina at 7d PR in eyes treated with NAC or FGF2. Scale bar in A is 125µm and it applies to all panels in A and for B is 60µm and applies for all panels in B. (C and D) Quantitative analysis of the mean presence SSEA-1 positive cells and the co-expression of Vsx2/Pax6 in NAC and FGF2 treated eyes at 7d PR. There is a significant difference in the anterior region of the eye between eyes treated with NAC and FGF2 for both markers (SSEA1: (F(1,4)=212.64, ***P=0.0001); Vsx2/Pax6: (F(1,4)=14.77, *P=0.0184)). In the posterior region, there was a significant difference in eyes treated with NAC for Vsx2/Pax6 (F(1,4)=8.82, *P=0.0412) but not for SSEA1 (F(1,4)=4.62, P=0.0981). Mixed model ANOVA was used for the statistical analysis. Standard deviation (Stdev) and averages for each treatment are provided in table 6.

Figure 8

Lin28 and Sox2 progenitor markers profile during NAC and FGF2 induced retina regeneration. (A) Immunofluorescence staining for the ribonucleoprotein Lin28. 3dPR Lin 28 in green is present in the nucleus during the NAC-induced regeneration, however after 7dPR Lin28 is mainly present in the cytoplasm for both NAC and FGF2 induced regeneration (n=4); no differences were observed in the amount of expression. (B) Immunofluorescence staining for the transcription factor Sox2, after 7dPR. There was no significant difference between the FGF2-induced or NAC-induced regeneration. CR=Ciliary margin regeneration, L=lens; RPE= retinal pigmented epithelium; TD= Transdifferentiation; ANT: Anterior (n=3 applies to all). Scale bar in A is 120µm (applies to all in A), and in B is 60 µm (applies to all in B).

Figure 9

Increased levels of GSH are not required during NAC-induced retina regeneration. (A) Schematic representation of the GHS pathway indicating the inhibitory mechanism of L-Buthionine sulphoximine (BSO) on γ- glutamyl-cysteine synthase, and dietyl maleate (DEM). (B) Graphical representation of the GSH/GSSH ratio in the CM after 6h and 24h PR in eyes treated with DMSO (n=10), NAC (n=10), NAC + BSO (n=10), and NAC + DEM (n=10). The use of the inhibitors decreased the levels of GHS in presence of NAC. Values were: at 6h PR with NAC: GSH/GSSH= 29:1, after 24h PR: NAC: GSH/GSSH=9:1, compared to untreated controls after 6h PR GSH/GSSH=1:4, after 24h PR GSH/GSSH=0.2:8). The use of the inhibitors decreased the levels of GHS in presence of NAC. At 6hPR BSO+NAC: GSH/GSSH=1:2.33, DEM+NAC: GSH/GSSH=1:8, at 24hPR BSO+NAC: GSH/GSSH=2.8:2, DEM+NAC: GSH/GSSH=1:4. (C) Histological analysis of eyes collected 3dPR and treated with NAC and BSO, DEM, or DMSO. Magnification bar in E is 125µm and it applies to all panels. (D) Statistical analysis was performed using Dunnett multiple comparisons. Quantitative analysis of the regenerated area showing no statistical significance in regeneration between DMSO+NAC and BSO+NAC (P=0.949) nor with DMSO+NAC and DEM+NAC (P=0.4639). Standard deviation (Stdev) and averages for each treatment are provided in table 7.

Figure 10

Erk1/2 are activated during the regeneration induced with NAC. (A) Immunofluorescence for the presence of pErk (red) at 6 and 24h PR, 3d and 7d PR. (B) Quantitative analysis of the pErk+ cells at 6h, 24h, 3d and 7d PR (intensity of the signal/area). Statistical analysis was done using Dunnett multiple comparisons. After 6hPR, 24h PR, 3d PR and 7d PR no significance in the intensity of pErk signal was observed when compared with FGF2 (Dunnett P6h=0.40, P24h=0.81, P3d=0.38, P7d=0.25). (n=3 applies to all). Scale bar is 125 µm and applies to all panels in A. Standard deviation (Stdev) and averages for each treatment are provided in table 8.

Figure 11

(A) Histological analysis 3dPR. Regeneration was inhibited in presence of the MAPK- inhibitor but not in presence of the FGFR inhibitor. Scale bar in A is 250 µm and applies to all panels (B) Quantitative analysis of embryos that regenerated in presence of the different inhibitors at 3d PR. The Statistical analysis was done using Dunnett multiple comparisons. There was a high significance in the DMSO+NAC (n=9) and PD98+NAC (n=9) (***P=0.0008), there was no significance with DMSO+NAC (n=9) and PD17+NAC (n=15) (P=0.4973). There was a high significant difference in the DMSO+FGF2 (n=9) among PD17+FGF (n=11) (***P˂0.0001) and PD98+FGF (n=8) (**P=0.0013). Standard deviation (Stdev) and averages for each treatment are provided in table 9.

Figure 12

Use of N-acetyl amino acids similar to NAC without the thiol group. (A) Histological analysis after 3d PR of the different N-acetyl amino acids that mimic NAC structure without thiol group (NAS) and some functions (NAG). The structures are represented below (n=10 applies to all). Scale bar in C and E is 180 µm and it applies to A- B, and to D respectively.

Figure 13

A model for the induction of chick retina regeneration by NAC. Arrows in yellow represent the different roles of the amino thiol antioxidant NAC. Arrows and line in red represent the different molecules that feed into the specific roles of NAC. When the GSH synthesis role of NAC was tested through the use of specific inhibitors, regeneration still occurred. The use of another N-acetyl amino acid, N-acetylglycine (NAG), which can feed into the GSH synthesis pathway, did not result in regeneration. When different molecules that can scavenge ROS were used, no regeneration occurred. In addition, N-acetylserine (NAS), which is similar in structure to NAC but lacks the thiol group in carbon 3, and instead has a hydroxyl group, was unable to induce regeneration. Our data suggests that the thiol group from NAC is essential for the activation of pErk and subsequent induction of retina regeneration.

Figure 14

Effects of NAC in the optic tectum. (A) For this analysis chick embryos were used at 7dPR +/- NAC or 11d Dev. Below the histological image of the optic tectum (OT). Scale bar represents 250µm and applies to all histology panels.

Figure 15

Increased presence of different optic tectum markers. Immunohistochemistry of the OT in 7dPR+/- NAC or 11d chick embryos. Pax7 (green) represents optic tectum cells that are proliferating, Meis2 (red) labels cells from the OT. Scale bar represents 125µm and applies to all panels.

7. Tables

Table 1

Analysis Variable: Redox status

TIME Treatment N AVERAGE Std dev OBS

RET 3 0.0026315 0.0017059

FGF2 3 0.000760132 0.000362493

6hPR NAC 3 0.000655126 0.000375836

H2O2 3 0.0021426 0.0011127

H2O2+NAC 3 0.0016462 0.000814778

RET 3 0.0018287 0.0010334

FGF2 3 0.000963721 0.000363413

24hPR NAC 3 0.00052964 0.000630145

H2O2 3 0.0027564 0.000721233

H2O2+NAC 3 0.0014007 0.000475977

Table 2

Quantification of the levels of ROS with CM-H2DCFDA

Analysis variable: ROS CM-H2DCFDA

TREATMENT N Obs Average Std dev

DEV 4 0.000316609 0.00279614 NAC 4 0.000147232 0.00279614 RET 4 0.000477965 0.00279614

H202+NAC 3 0.000671651 0.00322870 H2OH 8 0.003839900 0.00197717

Table 3

Regeneration analysis

Analysis Variable: Area of regeneration TREATMENT N Obs Variable Mean Std Dev N FGF 10 CM 4849.87 2260.13 10 TD 3235.43 2000.25 10 NAC 10 CM 3775.49 1532.00 10 TD 847.49 949.13 10

Analysis Variable: Area of regeneration at CM TREATMENT N Obs Mean Std Dev N

H2O2 8 127.4737375 53.5441258 8 H2O2+NAC 8 1740.53 841.6131142 8

Table 4

Proliferation analysis

Analysis Variable: Proliferation

Location Marker TIME Treatment N AVERAGE Std dev OBS

6hPR FGF2 3 55.2222222 19.7915058

NAC 3 34.7777778 3.532914

RET 3 15.7777778 17.3344017

CM EdU 24hPR FGF2 3 45.3333333 8.5114302

NAC 3 75.4444444 29.4285072

RET 3 9.3333333 4.3333333

3h PR FGF2 3 124.6666667 25.9272486

NAC 3 133.4444444 26.3509083

RET 3 16.8333333 2.5658007

6hPR FGF2 3 7.666667 1.20185

NAC 3 6.111111 5.315944

RET 3 6.777778 4.350394

24hPR FGF2 3 8.555556 6.500712

NAC 3 7.333333 2

CM PH3 RET 3 2.222222 1.835857

3h PR FGF2 3 23.33333 1.885618

NAC 3 23.66667 4.358899

RET 3 2.277778 1.058476

Table 5

Cell differentiation analysis

Analysis Variable: Cell differentiation

Time Marker Treatment N Obs Average Std dev

Visinin FGF2 3 118.39 20.63

NAC 3 48.78 15.12

Vimentin FGF2 3 104.39 16.82

NAC 3 38.61 9.15

Brn3 FGF2 3 68.22 17.11

NAC 3 22.06 5.85

Napa FGF2 3 56.89 20.04

7d PR NAC 3 12.89 2.79

Ap2 FGF2 3 97.86 34.19

NAC 3 44.33 5.2

Lim 1/2 FGF2 3 27.67 9.78

NAC 3 11.17 3.51

Pax6 FGF2 3 103.22 10.38

NAC 3 52.97 16.69

Vsx2 FGF2 3 67.92 26.35

NAC 3 28.67 5.51

Visinin FGF2 3 144.17 33.57

NAC 3 85.33 48.06

Vimentin FGF2 3 176.39 31.25

NAC 3 70.39 20.94

Brn3 FGF2 3 15.56 4.86

NAC 3 15.22 3.73

Napa FGF2 3 19.5 3.91

NAC 3 10.5 9.01

Ap2 FGF2 3 132.56 22.5

11d PR NAC 3 77.72 25.31

Lim 1/2 FGF2 3 27.94 10.54

NAC 3 11.17 3.51

Pax6 FGF2 3 141 13.93

NAC 3 149.72 27.26

Vsx2 FGF2 3 155 20.88

NAC 3 119.67 18.34

Table 6

Progenitor markers analysis.

Analysis Variable: Progenitor markers

Marker Treatment N Obs Variable Average Std dev N

FGF2 3 ANT 13.444 5.966 3

SSEA1 POST 21.000 10.408 3

NAC 3 ANT 66.444 2.009 3

POST 53.556 24.089 3

FGF2 3 ANT 51.778 8.514 3

Vsx2/Pax6 POST 9.222 4.07 3

NAC 3 ANT 112.000 21 3

POST 45.333 20.033 3

Table 7

GSH inhibitor analysis

Analysis variable: Inhibitors GSH

TREATMENT N Obs Average Std dev

BSO+NAC 10 1757.57 1431.39

DEM +NAC 9 2056.80 957.29

NAC+DMSO 9 1711.61 977.04

Table 8 pErk analysis

Analysis Variable: Cell signaling, pErk

Time Treatment N Obs Average Std dev

6hPR FGF2 6 0.011089 0.002038

NAC 6 0.019846 0.024485

24hPR FGF2 8 0.034895 0.018601

NAC 6 0.030957 0.006291

3d PR FGF2 8 0.050305 0.056038

NAC 6 0.023901 0.021824

7d PR FGF2 8 0.022876 0.016742

NAC 6 0.050626 0.056053

Table 9 pErk1/2 and FGFR inhibitor analysis

Analysis variable: pErk inhibitors

TREATMENT N Obs Average Std dev

PD173074+NAC 15 1391.24 900.26

PD98059+NAC 9 415.25 600.08

DMSO+NAC 9 1711.61 977.05

PD173074+FGF2 11 475.31 241.61

PD98059+FGF2 8 678 554.82

DMSO+FGF2 9 1683.12 9500.04

Table 10

Definition of asterisks according P values

P Value Number of asterisks

˃0.07 NS=No significant

≤0.06 M=Marginal

≤0.05 *

≤0.001 **

≤0.0001 ***

CHAPTER 4

CONCLUSIONS

1. Final remarks

The chicken embryo is a feasible model to study retina regeneration because it is accessible for surgical manipulations and it is amendable to follow up this biological process in vivo step by step. The developmental stages are very well documented (Hamburger and Hamilton, 1951). Also, the ability to use many different technologies makes the chick one of the most versatile experimental systems available (Stern, 2005). Additionally, the chicken genome has been sequenced (Wallis et al., 2004). The chick model has several advantages over mammalian systems for in-vivo studies. These include cost-effectiveness, ease of manipulation and the availability of mosaic and transgenic procedures. Furthermore, the chick can be manipulated with both viral vectors and electroporations during many developmental stages (Coleman, 2008). Importantly the chick has the ability to regenerate its retina when completely removed during a small window of development by activating stem/progenitor cells or by the reprogramming of the RPE. All major retina cell types regenerate within 7 days post-retinenctomy as long as a source of growth factors is present. Different studies on retina regeneration have been performed in this model, exploring different signaling pathways including FGF (Spence et al., 2007), BMP (Haynes., et al 2007), and Wnt (Zhu et al., 2013) signaling. In this dissertation, I describe two more molecules/signaling pathways capable of inducing retina regeneration via the CM. These are C3a/C3aR signaling (Haynes et al., 2013) and NAC/MAPK signaling (paper in under review, Echeverri et al., 2016).

Understanding the different cellular programs and pathways involved in the activation of the stem cells and cell reprograming is important knowledge in order to induce regeneration in organisms that have very limited capability to regenerate like humans. Chapter 2 describes the role of the complement component C3a in the process of retina regeneration. We described that C3a, which is part of the innate immune system, was able to activate stem/progenitor cells housed in the CM of the embryonic chick eye. The innate immune system has different roles: it is the first line of defense after insults (i.e. pathogens) and it is important for homeostasis during wound repair. Recently, it has been described that in different organisms, the immune system contributes to regeneration of damaged tissues

83 including limbs, skeletal muscle, heart and nervous system (Aurora and Olson, 2014).

After damage, mammals respond in different ways: compensatory growth of the remaining tissue, activation of residual precursor cells or through the formation of a scar. Regeneration requires correct coordination of multiple processes, including: scavenging of cellular debris (Paolicelli et al., 2011), immune modulation (Arnold et al., 2007), angiogenesis (Lucas et al., 2010) and innervation of the newly formed tissue (Aurora and Olson, 2014). The complement component C3 has been reported to be expressed during newt limb and lens regeneration (Del Rio-Tsonis et al., 1998; Kimura et al., 2003), and to induce proliferation in mouse liver following injury (Strey et al., 2003). Our results show the relevance of C3a in the induction of regeneration in the chick embryo (Haynes et al., 2013). We were able to demonstrate that C3a binds to its own receptor, activates MAPK followed by STAT3 and in turn some target genes such as TNFα and IL-6 were up-regulated or and Wnt2b down-regulated. The activity of C3a in the induction of the chick retina regeneration was independent of FGF2 (Haynes et al., 2013).

In Chapter 3 we describe for the first time that the antioxidant NAC is able to induce chick retina regeneration in an FGFR independent manner. NAC is versatile antioxidant, which plays diverse roles: it is a free radical scavenger, a GSH precursor, and cysteine precursor with special chemical properties conferred by its cysteinyl thiol.

We tested different antioxidants and similar molecules to NAC, and, in all the cases regeneration failed. These results elucidated the mechanism of action of NAC during regeneration. We found that NAC modified the levels of protein oxidation and increased the levels of redox status through the increase in the levels of GSH, whereas, the synthesis of GSH was not essential in the process of regeneration. This means that NAC induced regeneration in a GSH-independent manner. Previous reports have shown that through the changes in the redox status, NAC can modify the activity of certain proteins such as the MAPK enzymes (Kim et al., 2001). As a matter of fact, NAC induced the activation of the pErk1/2 (a MAPK member), this activation was necessary for the induction of retina regeneration. In support of our work, NAC has been found to increase the survival-promoting actions in PC12 cells (Ferrar et al., 1995) independently of its antioxidant/radical scavenger property (Yan et al., 1995), using its thiol-disulfide exchange activity as a reductant (Kim et al., 2001).

Another important fact is the enhancement in the expansion of the stem/progenitor cells located in the CM during NAC-induced retina regeneration. These results are very important to understand the relevance of the redox status in the process of self-renewal. Stem cells are the main source of cells that replenish damaged tissues after any insult, but the capacity to replenish those cells diminishes with age. A better understanding of the factors involved in regeneration will improve the quality of life of patients with the need of organs or tissues.

2. Questions of immediate interest

Regeneration is a fascinating field, which have caught the attention for centuries. The studies described here help to understand different pathways and mechanisms that can be used as potential treatment. However, several questions remain unanswered:

 Can NAC or C3a have deleterious effects in older individuals if used as a treatment, predisposing to developing cancer?

 Can NAC induce epigenetic modifications, if it is known that some DNA methy-transferases are redox sensitive?

 Which will be a good method to continuously deliver NAC/C3a into the eye to get a better understanding of cell differentiation in the retina?

 Is the redox status different in organisms that have the ability to regenerate Vs organisms with limited capabilities?

 Which are the specific characteristics that an ideal thiol molecule should have to induce regeneration?

Answering these questions will help provide better approaches in the field of regeneration through the use of iPSC cells, stem cells, biomaterials, etc. Such knowledge will be essential for future treatments in diseases such as Parkinson, Alzheimer, and heart failure, among others that require organ or tissue transplants.

3. Future directions

Chapter 2 and chapter 3 address two different ways to activate the stem/progenitor cells in the ciliary margin of the chick eye. Chapter 2 refers to the regeneration

85 induced with C3a, a peptide that has different roles in addition to the immune function. Chapter 3 refers to an antioxidant that is involved in modifying the redox status which in turn can be affected by cellular damage (ie. inflammation) or aging. C3a as well as NAC have diverse functions, but it is clear that in both cases the activation of the MAPK pathway was essential to induce regeneration. If it would be possible to specifically turn on/off this pathway we will be certain that this pathway is essential and sufficient to induce regeneration.

Another interesting question, which is the most common among those who study regeneration is: why mammals have lost the capability to regenerate? Is it evolution? From an evolutionary standpoint, what advantages do mammals gain to compensate for their inability to regenerate tissue, organs, and limbs? Proteomic analysis and genomic stability analysis would be a great start point to understand all these questions, and our knowledge and technology is advancing so fast that maybe in the next 10 years we can answer some of those questions in a wet lab.

The study of DNA repair mechanisms in organisms that have high (i.e. Planarian and Hydra) versus limited (i.e. Mammals) regeneration abilities would be another hotspot area of study. It is not clear if lower vertebrates have less error prone DNA repair mechanisms in comparison with higher vertebrates, these differences might help to understand why lower vertebrates rarely develop cancer.

Additionally, lower vertebrates have low cysteine content in proteins in comparison with higher vertebrates (Miseta and Csutora, 2000), which make us wonder if this (higher cysteine content) is an evolutionary trait that was gained as a response of the increased metabolic needs in higher vertebrates, since the amino acid cysteine is one of the main redox sensors in the cells (Klomsiri et al., 2011).

Finally, it was interesting that FGF2 was able to modify the redox status in the injured chick eye. Previous reports have showed that the redox state is modulated by different molecules that can alter the balance between self-renewal and differentiation, such is the case for growth factors such as FGF2 that promote a more reduced state to enhance self-renewal and other that promote a more oxidized state to enhance cell differentiation (Smith et al, 2000). Our results show that FGF2 as well as NAC were able to modify the redox status. FGF2 is a well- known growth factor that is capable of inducing regeneration, and therefore the redox status seems to be necessary for the process of regeneration.

4. References

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