Autophagy in glaucoma: Crosstalk with apoptosis and its implications

Accepted Manuscript Title: Autophagy in glaucoma: Crosstalk with apoptosis and its implications Author: Yao Wang Changquan Huang Hongbing Zhang Renyi Wu PII: DOI: Reference:

S0361-9230(15)30002-2 http://dx.doi.org/doi:10.1016/j.brainresbull.2015.06.001 BRB 8861

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Please cite this article as: Yao Wang, Changquan Huang, Hongbing Zhang, Renyi Wu, Autophagy in glaucoma: Crosstalk with apoptosis and its implications, Brain Research Bulletin http://dx.doi.org/10.1016/j.brainresbull.2015.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Review Article Autophagy in glaucoma: Crosstalk with apoptosis and its implications

Yao Wang a,b,1, Changquan Huanga,1, Hongbing Zhang b, Renyi Wua,*

a

Eye Institute and Affiliated Xiamen Eye Center, Xiamen University, Fujian Provincial

Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian361102, China; b

Department of Ophthalmology, First Hospital of Xi’an, Shaanxi Institute of

Ophthalmology, Shaanxi Provincial Key Lab of Ophthalmology, Xi’an, Shaanxi710002, China.

*Corresponding author. Address: Xiamen University Affiliated Xiamen Eye Center, Xiahe Road 336, Xiamen, Fujian 361001, China; Tel: +86 592 2109085 Fax: +86 592 2024325. E-mail address: [email protected](R.Y. Wu);

1

These authors contributed equally to this paper.

Running title: Autophagy in glaucoma

1

Abstract Glaucoma is characterized by elevated intraocular pressure that causes progressive loss of retinal ganglion cells (RGCs). Autophagy is a lysosomal degradative process that updates the cellular components and plays an important role in cellular homeostasis. Recent studies have shown that autophagy is involved in the pathophysiological process of glaucoma. The role played by autophagy in glaucoma is complex, and conflicting evidence shows that autophagy promotes both RGC survival and death. The understanding of the major pattern of RGC loss and the crosstalk between autophagy and apoptosis remains limited in glaucoma. This review focuses on the relationship between autophagy and glaucoma, particularly on the influence of autophagy on apoptosis in glaucoma. Further research on autophagy in glaucoma may provide a novel understanding of the glaucoma pathology and novel treatment targets for glaucoma in the future.

Key words: Autophagy; Glaucoma; Retinal ganglion cell; Apoptosis.

2

Abbreviations: AP, acid phosphatase; Atg, autophagy-related genes; BAG3, Bcl-2-related anti-apoptotic gene 3; CTS, cathepsin; DR, death receptor; Iba1, a specific marker for activated microglia; IHC, immunofluorescence histochemistry; IOP, intraocular pressure; IPL, inner plexiform layer; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; 3-MA, 3-methyladenine; mTOR, mammalian target of rapamycin; MYOC, myocilin; OPTN, optineurin; PCD, programmed cell death; POAG, primary open angle glaucoma; Refs, references; RGCs, retinal ganglion cells; RGCL, retinal ganglion cell layer; ROS, reactive oxygen species; SQSTM1/p62, sequestosome; TBK1, TANK-binding kinase-1; TEM, transmission electron microscopy; TMCs, trabecular meshwork cells; TUNEL, terminal-deoxynucleotidyl transferase-mediated nick end labeling; WB, western blot; WDR36, WD-repeat domain 36.

Highlights ► Evidence shows that autophagy is involved in the pathological process of glaucoma. ►The crosstalk between autophagy and apoptosis in glaucoma is not fully understood. ►Evidence shows that autophagy can inhibit or enhance apoptosis in glaucomatous

RGCs. ►The modulation of autophagy may be a therapeutic option for glaucoma in the future.

Contents 1 Introduction 2 Overview of autophagy 3

2.1 Formation of autophagy 2.2 Major functions of autophagy 2.3 Regulatory mechanisms of autophagy 3 Autophagy and glaucoma pathologies 3.1 Autophagy in the trabecular meshwork 3.2 Autophagy and mitophagy in glaucomatous RGCs 3.3 Autophagy in the optic nerve and the dendrites of RGCs 3.4 Autophagy and glial cell activation 3.5 Interactions between autophagy-related genes and the glaucoma gene OPTN 4 Crosstalk between autophagy and apoptosis in glaucoma 4.1 Autophagy versus apoptosis 4.2 Autophagy and apoptosis can coexist in the same damaged retinal neurons 4.3 Activation of autophagy may precede apoptosis in glaucoma 4.4 Connections between autophagic and apoptotic proteins 4.5 Mitochondria may be a connection point in the crosstalk between autophagy and apoptosis in glaucoma 4.6 Autophagy inhibits or enhances apoptosis in glaucomatous RGCs 5 Modulating autophagy as a potential alternative therapeutic strategy for neuroprotection in glaucoma 5.1 Possible mechanisms underlying the targeting of autophagy for glaucoma treatment 5.2 Pharmacological manipulation of autophagy 5.3 Genetic regulation of autophagy 4

6 Perspectives and conclusions Conflicts of interest Acknowledgments References

1 Introduction Glaucoma is a major cause of permanent blindness that afflicts more than 60 million people worldwide[70]. Glaucoma is a group of diseases characterized by the 5

progressive loss of retinal ganglion cells (RGCs) and their axons and results in subsequent visual field defects and vision loss. Abnormally elevated intraocular pressure (IOP) is one of the major risk factors for the occurrence and development of glaucoma. The current glaucoma therapies that seek to reduce IOP, including surgery, laser intervention, and medications, are unable to rescue RGCs from dying and/or dysfunction [2, 27, 68, 69]. The exploration and better understanding of the mechanisms underlying RGC death are essential and required for the prevention and treatment of glaucoma. Autophagy (auto-self, phagy-eating) is a vital phenomenon of life. Autophagy occurs widely in eukaryotic cells and is a highly evolutionarily conserved self-protection process that acts as an adaptive cellular response under conditions of nutritional

deficiency

[42,72].

Furthermore,

autophagy

is

a

cellular

degradation/recycling mechanism that plays roles in normal cell growth and development and some physiological and pathological processes. Autophagy is a lysosomal degradative process that eliminates damaged cellular constituents, including organelles and long-life proteins [50, 75]. Autophagy provides raw materials for the construction of intracellular components by recycling damaged or aging organelles, proteins and pathogens. Autophagy also regulates other important cellular activities, including apoptosis, inflammation, and adaptive immunity. Autophagy flux refers to the dynamic process of autophagy. It is the progress of the cargo through the autophagy system leading to its degradation in the lysosomes. Autophagy was recently reported to be associated with the development of some neurodegenerative diseases, particularly Alzheimer’s disease (AD). The pathology of 6

AD is characterized by the detrimental amyloid plaques (amyloid-β ptoteins) and neurofibrillary tangles (tau proteins), and autophagy is regarded as an intracellular hub for the removal of amyloid-β peptides and Tau aggregates. The induction of autophagy can be observed in the brain at early stages of AD, as well as in an AD transgenic mouse model [17, 43, 60, 76]. There are a few documents[11, 61, 63,74] discussing the relationship between autophagy and glaucoma, which is regarded as a neurodegenerative disorder[20]. However, the experimental results [61, 74] regarding whether autophagy exerts an activating or inhibitory effect on RGC death are inconsistent. This review summarizes the findings which focus on the relationship between autophagy and glaucoma and proposes that autophagy may play a role in the pathogenesis of glaucoma and that the modulation of autophagy may provide a novel treatment target for glaucoma.

2 Overview of autophagy 2.1 Formation of autophagy Based on the pathways through which substrates are delivered to lysosomes and then degraded there, autophagy is categorized as macroautophagy, microautophagy, or chaperone-mediated autophagy. Macroautophagy is the main pathway and is generally referred to as autophagy. The formation process of macroautophagy consists of five sequential steps: (i) the cytoplasmic components form an independent membranous structure that turns into a phagophore during a process called membrane isolation; (ii) the phagophore then undergoes an elongation process; (iii) 7

degradable materials are swallowed and assimilated by the phagophore to form a mature autophagosome; (iv) the autophagosome is transported and fused with lysosomes to form an autophagolysosome; and (v) degradation occurs in the autophagolysosome due to the actions of lysosomal or hydrolytic enzymes[14, 16, 42, 51, 72].

2.2 Major functions of autophagy Recent studies have demonstrated that autophagy plays essential roles in the maintenance of cell homeostasis, adaptations to stress (e.g., starvation, endoplasmic reticulum stress, oxidative stress, hypoxia, etc.) [36, 56, 77, 78] and other physiological/pathophysiological processes, such as development, differentiation[42], anti-aging, elimination of microorganisms, cell death, tumor suppression and antigen presentation in the body[14, 48]. In responses to stress, the primary function of autophagy is thought to aid cellular survival by recycling damaged organelles and misfolded proteins to provide energy and substrates for reconstruction[44]. Disturbances in autophagy are associated with various diseases, including neurodegenerative disorders, such as Alzheimer's, Parkinson’s and Huntington’s diseases [25, 51, 67].

2.3 Regulatory mechanisms of autophagy The products of many autophagy-related genes (Atg) have been proven to play regulatory roles in certain steps of the initiation and/or progressionof autophagy [22, 96]. Starvation induces autophagy by inhibiting the expression of mammalian target of 8

rapamycin (mTOR) on the cell membrane. mTOR can forma multi-protein complex termed mTORC1 [28] that negatively regulates macromolecule substrate compounds, such as ULK1(named Atg1 in yeast and ULK1 in mammals) and Atg13, to inhibit autophagy [24, 49]. Energy loss activates ULK1 via the inhibition of mTORC1 and the activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK). Beclin 1 (Atg6 in yeast) is a specific autophagy gene that upregulates autophagy in response to various types of stimulation. Proteins of the anti-apoptotic Bcl-2 family (Bcl-2 and Bcl-XL) can combine with the functional domain of Beclin 1 to inhibit the combination of Beclin 1 with the type III PI3K complex[23, 62], which leads to the inhibition of Beclin 1-dependent autophagy. The HB3-only protein (a member of the Bcl-2 family) induces autophagic cell death by interfering with the interaction between Bcl-2/Bcl-XL and Beclin 1[46]. After the formation of a phagophore, the activation of two ubiquitin-like systems, namely the Atg5-Atg12 and microtubule-associated protein-1 light chain 3 (LC3, a homolog of the yeast protein Atg8) binding systems, is involved in the elongation of the phagophore membrane. The core protein of autophagy is the microtubule-associated protein light chain 3 (LC3). LC3-I is conjugated to phosphatidyl ethanolamine (PE) to form LC3-II (LC3-PE) via a ubiquitination-like enzymatic reaction[53]. LC3-II is specifically associated with autophagosome formation [29] and used as a marker of accumulation of autophagosomes. In contrast, the level of the sequestosome 1(SQSTM1)/p62 protein 7inversely correlates with autophagic activity[52].

3 Autophagy and glaucoma pathologies 9

3.1 Autophagy in the trabecular meshwork The trabecular meshwork (TM) is the reticulate structure of the anterior chamber angle in the eye and plays a vital role in the control of aqueous humor outflow. Aqueous humor is the fluid that produced in the eye and fills mainly the anterior chamber, the part of the anterior cavity of the eye. The anterior chamber angle is the angle formed by the inner cornea and the root of the iris, allows aqueous humor to drain out of the anterior chamber. Dysfunction of the TM can result in the obstruction of aqueous humor outflow and elevation of the IOP[87]. Using a model of mild chronic oxidative stress under high atmospheric O2 (40% O2), Porter et al. observed the decrease of autophagic activity in porcine TM cells. These researchers concluded that the decreased autophagic flux (indicator of autophagic activity) caused by oxidative stress may be one of the factors that leads to the progressive failure of cellular TM function with age and may be partially responsible for the pathogenesis of primary open angle glaucoma (POAG) [64]. In a recent study, Porter et al. [65] found that the biaxial static stretching (20% elongation) of cultured human TM cells increases the autophagic flux, as proven through electron microscopy and determination of the LC3II/I ratio. The perfusion of porcine eyes for 1 h under high pressure (30 mmHg) results in similar changes in the autophagic flux [65]. This study suggests that the activation of autophagy by stretching the TM cells is a physiological response that TM cells use to cope with mechanical forces [65]. Pulliero et al.[66] lately used fresh TM specimens from 28 healthy human donor eyes and demonstrated that the SQSTM 1/p62 protein levels of subjects over 60 years of age were lower than those of the younger subjects, which suggests that autophagy in human TM is upregulated with 10

age. The discrepancy between Porter’s [64] and Pullieroet’s [66] studies might reflect the difference in the autophagy activity either in different animal (including human) species, or under different stimulative circumstance (oxidative stimulation vs. normal physiological condition). The alteration in the autophagy activity in response to different stimuli needs further investigation, especially in the glaucoma patients.

3.2 Autophagy and mitophagy in glaucomatous RGCs Although it has been suggested that apoptosis may be the final pathway underlying RGC death in glaucoma [19, 31], increasing evidence indicates that non-apoptotic programmed cell death (PCD) is also responsible for RGC death [86, 91]. Based on a chronic hypertensive glaucoma model in rhesus monkeys, Deng et al.[11] reported that initially autophagic vacuoles and degraded autophagic vacuoles accumulate in the RGC layer and inner plexiform layer (IPL) and that the levels of lysosomal-associated membrane protein 1 (LAMP1), LC3B-II, LC3B-II/LC3B-I and Beclin 1 increase in the retina. Park et al.[61] found that the LC3-II/LC3-I ratio and the Beclin-1 levels are increased for eight weeks after IOP elevation in a Sprague Dawley rat model of chronic glaucoma and that these levels peak after four weeks. Additionally, autophagy is initially observed in the RGC dendrites and then in the cytoplasm. In a study conducted by Piras et al. [63], acid phosphatase (AP)-positive granules and LAMP1-positive vesicles were observed 12 and 24 h after retinal ischemia/reperfusion following high IOP in a rat model. In a mouse model of optic nerve axotomy, Rodriguez-Muela et al.[74] found that autophagy is rapidly activated, that autophagosomes are formed, and that the autophagy regulator Atg5 is upregulated 11

after five days. The term mitophagy is used to describe the selective removal of damaged and dysfunctional mitochondria through autophagy [93]. Under an electron microscope, mitochondria have been observed inside autophagosomes in the RGCs of retinas injured six days earlier through optic nerve axotomy in mouse[74] and one week after chronic IOP elevation in a rat model [61]. The factors that may account for RGC death induced by elevated IOP, such as ischemia, hypoxia, and excessive excitatory amino acids[32, 79], have been found to upregulate cell autophagy [4, 21, 39], which is indicative of the involvement of autophagy in the pathogenesis of glaucoma(Figure1).

3.3 Autophagy in the optic nerve and dendrites of RGCs Knöferle et al. [35] reported that autophagosomes are present in the axons of RGCs within 1 h of acute axonal injury in rats. The inhibition of autophagy interferes with injury-induced axonal degeneration. The autophagy levels are associated with the intra-axonal calcium levels. In another study, SQSTM1/p62 and LC3-II were found to be upregulated in the optic nerve, and it is revealed by observation with electron microscopy that autophagic vacuoles are present in the axons of the optic nerve of rats following IOP elevation[34]. Park et al.[61] confirmed that autophagosomes are deposited in the dendrites of RGCs in a chronic hypertensive glaucoma rat model and that autophagy is activated earlier in the dendrites of RGCs than in the cytoplasm of RGCs. Furthermore, in the same study, the researchers found that the autophagosomes in the dendrites of RGCs within the retinal inner-plexiform layer 12

contained organelles, which suggests that mitophagy may be active in the dendrites of RGCs.

3.4 Autophagy and glial cell activation The retina contains three types of glial cells: astrocytes, microglia, and Müller cells. Microglial activation and microglia-derived neurotoxic mediators have been shown to play detrimental roles in glaucoma[5, 6, 38, 57], and the inhibition of microglial activation has been proven to protect RGCs from death in animal models of glaucoma[58, 59, 98]. Su et al.[88] demonstrated that the autophagy activator rapamycin significantly inhibits the expression of Iba1 (a specific marker of activated microglia) and activates NF-κB expression in a rat model of chronic hypertension. Additionally, Tezel et al.[92] reported a marked upregulation of mTOR, Atg3 and Atg7 in retinal astrocytes in an experimental rat model of glaucoma. The activation of autophagy in glaucoma is summarized in Table 1.

3.5 Interactions between autophagy-related genes and the glaucoma gene OPTN The OPTN gene encodes the optineurin protein and is expressed in the ocular tissues of the trabecular meshwork, the non-pigmented ciliary epithelium of the ciliary body and the retina [73]. Optineurin serves as one of the autophagy receptors by interacting with LC3 [95] and may play a neuroprotective role in glaucoma. A mutant OPTN gene has been identified as a causative gene in normal-tension glaucoma and adult-onset primary open-angle glaucoma [1,73]. Several glaucoma-associated 13

mutants of optineurin have been found. However, only the E50K and M98K variants of optineurin induce more retinal cell death than wild-type optineurin[9, 85]. Sirohi et al. [85] showed that the knockdown of Atg5 can prevent M98K-OPTN-induced cell death and that M98K mutations in OPTN are associated with autophagy in RGCs. Studies conducted by Chalasan et al.[8] have revealed that the overexpression of E50K-OPTN but not wild-type optineurin inhibits autophagy flux in retinal cells and that this inhibition is mediated by TBC1D17 (a GTPase-activating protein). Furthermore, E50K-OPTN-induced RGC death is reduced by the autophagy activator rapamycin, which further supports an association between E50K-OPTN and autophagy. The opposite effect of M98K-OPTN and E50L-OPTN on the autophagy activity in the retinal cells suggests that those two variants signal to cell death by engaging different effectors, reflecting the complexity and heterogenicity in the pathogenesis of glaucoma. The clinical significance of these findings needs to be further explored.

4 Crosstalk between autophagy and apoptosis in glaucoma 4.1 Autophagy versus apoptosis Autophagy and apoptosis are two distinct self-destructive processes that play essential roles in determining cell fate. Autophagy (self-eating) is the type II form of PCD. Apoptosis (self-killing) is a type I form of PCD and is characterized by morphological changes, including chromatin condensation, fragmentation, and the formation of apoptotic bodies. Apoptosis is executed by activated caspases, which are a family of cysteine proteases that participate in signaling cascades[12]. Apoptotic activation is categorized primarily into the extrinsic and intrinsic pathways. The former 14

is initiated by cell-surface receptors that are termed death receptors (DRs), and the latter is activated by various intracellular stimuli, such as hypoxia, growth-factor deprivation, oxidative stress and DNA damage[55].

4.2 Autophagy and apoptosis can coexist in the same damaged retinal neurons Increasing evidence indicates that apoptosis and autophagy may coexist in the same cell [13], that their pathways share common upstream signals, and that their functional relationship is complex[47]. Previous studies have shown that autophagy and apoptosis can coexist in the same damaged RGC in a rhesus monkey model of chronic hypertensive glaucoma[11] and in a rat model that mimics POAG [63]. Piras et al. [63] further showed that autophagy and apoptosis do not necessarily overlap and can occur independently in the POAG model.

4.3 Activation of autophagy may precede apoptosis in glaucoma In aluminum-insulted astrocytes, autophagy processes occur before neuronal apoptosis[99]. In a mouse optic nerve axotomy model, autophagy has been observed three days after injury, whereas RGC apoptosis peaks five days after optic nerve transection [74]. Similarly, in a chronic glaucoma model, autophagy activation occurs early after IOP elevation and is followed by RGC loss[61]. Taken together, these findings demonstrate that autophagy may be initially activated by stress to exert cytoprotective effects until the stress reaches a threshold at which apoptosis is activated.

15

4.4 Connections between autophagic and apoptotic proteins Autophagy shares several common regulatory elements with apoptosis [47, 94]. SQSTM 1/p62 and Beclin1 are thought to be two crucial autophagy proteins that are common to autophagy and apoptosis[18]. The SQSTM 1/p62 protein can be incorporated into the completed autophagosome [52], and Beclin 1 participates in autophagosome formation[30]. SQSTM1/p62 interacts directly with several apoptotic proteins, including caspase-8 [54], and binding with caspase-8 may activate and enhance apoptosis[26]. Beclin1 can interact and combine with the anti-apoptotic Bcl-2 protein[18]. Calpain and caspase-3 may inhibit autophagy by destroying the autophagy-specific factors Atg4D, Beclin 1, and Atg5 [36]. Additionally, activated autophagic Beclin1 can co-localize with apoptotic caspase-3 [71]. In a chronic hypertensive glaucoma model in the rhesus monkey, increased Beclin1 expression has been observed to occur simultaneously with elevations in the levels of apoptotic caspase-3 and cleaved caspase-3, as demonstrated by western blot analyses[88].

4.5 Mitochondria may be a connection point in the crosstalk between autophagy and apoptosis in glaucoma Mitochondria are primarily energy-generating organelles and play a critical role in the maintenance of cellular homeostasis[10]. Elevated ROS (produced by mitochondria) not only participates in mitochondria-mediated apoptosis[10, 37] but also modulates the autophagy process [45, 81, 82] by regulating Atg4 activity[83]. Mitochondrial dysfunction leads to the induction of autophagy, which exerts its cytoprotective mechanism to clear the damaged mitochondria[33]. In contrast, a 16

common manifestation of mitochondrial dysfunction is oxidative stress (including the production of ROS), and ROS production occurs in the pathogenesis of glaucoma[90]; thus, mitochondrial dysfunction may play a primary role in the initial susceptibility to the development of glaucoma [41]. Taken together, these findings suggest that mitochondria may be a nexus at which autophagy interacts with apoptosis in the pathological process of glaucoma.

4.6 Autophagy inhibits or enhances apoptosis in glaucomatous RGCs Autophagy may play a dual role in cell survival and death [7, 89, 97]. Conflicting evidence indicates that autophagy may inhibit apoptosis and hence promote cell survival, but other lines of evidence indicate that autophagy may act as a stimulator of apoptosis [61, 74]. Rodriguez-Muela et al.[74] demonstrated that autophagy can promote the survival of RGCs following optic nerve transection in mice. In contrast, in a chronic glaucoma model, Park et al. [61] reported that the activation of autophagy results in the apoptosis of RGCs, as determined through double-labeled LC3B and TUNEL(terminal-deoxynucleotidyl transferase-mediated nick end labeling) staining.

4.7 Functional relationship between autophagy and apoptosis in glaucoma The proposed model regarding the relationship between autophagy and apoptosis is illustrated in Figure 2. Several possible pathways may be included in this process: (i) under normal physiological conditions, the base line level of autophagy allows the turnover of damaged organelles and long-lived proteins; (ii) in the early stage of glaucoma (mild stress), the autophagic pathway functions as an adaptive response to 17

stress; (iii)under conditions of severe or protracted stress, the stress increases to a threshold at which apoptosis is activated, the inhibition of autophagy may also lead to the exacerbation of cell death; (iv)and the activation of autophagy may promote cell survival via the inhibition of apoptosis; and the overactivation of autophagy can either (v) promote apoptosis or(vi) commit the cells to undergo autophagic cell death.

5 Modulating autophagy as a potential alternative therapeutic strategy for neuroprotection in glaucoma 5.1 Possible mechanisms underlying the targeting of autophagy for glaucoma treatment The targeting of autophagy for glaucoma treatment may involve multiple mechanisms, including the following: (i) enhancing the recycling of damaged organelles or misfolded proteins, which is one of the primary functions of autophagy, and further processing to maintain bioenergetic homeostasis, including sustained ATP production[76]; (ii) clearing damaged and dysfunctional mitochondria to alleviate the oxidative effects induced by ROS production[74, 76] rather than directly preventing the production of ROS or eliminating the excess ROS; (iii) modulating cell apoptosis to protect retinal cells, particularly RGCs[61, 74]; and (iv) inhibiting the activation of retinal microglial cells to reduce the release of neurotoxic mediators, such as TNF-α, NO, and glutamate[88, 92].

5.2 Pharmacological manipulation of autophagy A variety of potential therapeutic strategies for the rescue of RGCs have been 18

tested in different animal models. These include manipulating cellular signal pathways, genetic modifications, the prevention of RGC death, and RGC replacement via transplantation[3]. Nevertheless, a novel approach for the delay or prevention of retinal injury would be to adjust the mammalian target of rapamycin (mTOR, a key downstream molecule of the PI3K/Akt pathway) signaling pathway[3]. Rapamycin is a macrolide antibiotic that was first developed as an antifungal agent and is a well-recognized autophagy inducer via the inhibition of mTOR [84]. A recent study indicated

that

rapamycin

markedly

promotes

RGC

survival

and

exhibits

neuroprotective effects in a rat chronic glaucoma model [88]. However, it should be careful when consider rapamycin as a specific activator of autophagy, since rapamycin is also a well-known immune suppressor and the inhibition of mTOR with rapamycin may has much broader effects than induction of autophagy level. Lithium also induces autophagy through an mTOR-independent pathway and has been proposed to be beneficial in the treatment of neurodegenerative diseases [80]. The autophagic inhibitor 3-methyladenine (3-MA) delays neuronal cell loss by inhibiting the induction of autophagosomes in the early stages of neurodegenerative disease [40]. In addition, novel potential methods for autophagy regulation, including small-molecule screening [15, 100] and the synthesis of an alogues of Atg proteins, are being explored [67].

5.3 Genetic regulation of autophagy The genetic downregulation of autophagy to ameliorate RGC death has been tested in animal models. Rodriguez-Muela et al. [74] found that RGC death is upregulated in Atg4B--/-- and Atg5-knockout mice compared with wild-type mice. These 19

results suggest that the genetic modification of autophagic activity may represent a potential intervention for the neuroprotection of RGCs in the future.

6 Perspectives and conclusions Many unknown puzzles regarding the relationship between autophagy and glaucoma remain and need to be further explored. How autophagy is initiated, and how does it progress? What are the regulatory pathways of autophagy? What is the relationship between autophagic activity and functional recovery of the optic nerve? What are the complex molecular mechanisms that underlie the interaction between apoptosis and autophagy in glaucoma? Better understanding of autophagy in RGCs will surely benefit the prevention and treatment of glaucoma. We propose that the dual role of autophagy during glaucoma progression and treatment may depend on how autophagy is induced, the animal model used, the cell type, the glaucoma stage, the stage of autophagy, the time points used for the detection of autophagy, and even genomic alterations. Notably, when targeting autophagy to protect glaucomatous RGCs, the type of inducer and the progression of glaucoma should be considered because autophagy may be a “double-edged sword” that can act as a cyto-protector or a cyto-killer. The targeting of autophagy may provide novel therapeutic strategies for the treatment of glaucoma, which may include the following mechanisms: protecting retinal cells from death by inhibiting apoptosis, recycling damaged proteins and organelles for reconstruction and energy production, clearing damaged mitochondria to alleviate the oxidative damage caused by ROS production, and inhibiting the 20

activation of retinal microglial cells.

Conflicts of interest The authors have no conflicts of interest to declare.

Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No. 81170841) and the Xiamen Science & Technology Project (Nos. 3502Z20116011 and 3502Z20134040), Xiamen, Fujian Province, China.

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Figure Legends Figure 1. A schematic demonstration of the relationship between the activation of autophagy or mitophagy (mitochondrial autophagy) and the RGC death pathway in glaucoma. Stimulating factors, such as ischemia, hypoxia and excessive excitatory amino acids, can induce RGCs to undergo autophagy, and these factors may also be involved in the mechanisms of pathological injury to the RGCs in glaucoma, which implies the existence of some connection between glaucoma and autophagy.

IOP, intraocular 25

pressure; RGCs, retinal ganglion cells.

Figure 2. Schematic illustration of the proposed effects of autophagy on RGCs and the functional relationship between autophagy and apoptosis in glaucoma. Several possible conditions may be included in this process: (i) the basal level of autophagy (under normal physiological conditions); (ii) mild stress (in the early stage of glaucoma); and (iii-vi) under conditions of severe or protracted stress (moderate or advanced glaucoma), autophagy is inhibited, activated, or overactivated (excessive-self-digestion). RGCs, 26

retinal ganglion cells.

27

Table 1 A summary of autophagy in glaucoma pathologies. Type of cell TM cells

RGCs

Retinal glial cells

Retinal astrocytes

Techniques

Refs.

-Porcine TM cells (Mild chronic oxidative stress under high atmospheric oxygen, oxygen concentration 40%)

Disease model

WB(LC3-II/I, SQSTM1/P62, LAMP1);

[64]

-Human TM cells (Biaxial static stretching, 20% elongation)

WB (LC3-I, LC3II);

[65]

-Porcine TM cells (Eyes were perfused for one hour in high pressure, 30mmHg)

Stretch-induced autophagy in the TMCs unrelated with mTOR and BAG3

[65]

-Human (TM specimens from 28 healthy corneal donors) -Rhesus monkeys (Chronic hypertensive glaucoma model)

WB(LC3-I,LC3II, SQSTM1/P62)

[66]

TEM(autophagic vacuoles); IHC; WB (LAMP1, Beclin-1, LC3B-II/LC3B-I)

[11]

-Sprague Dawley rat (Chronic hypertensive glaucoma model)

TEM(autophagosomes); IHC;WB(Beclin-1, LC3-II/LC3-I,)

[61]

-Wistar rat (Retinal ischemia/reperfusion following high intraocular pressure)

IHC; WB(LC3, LAMP1)

[63]

-GFP-LC3 transgenic mice, Atg4B -/- mice (Optic nerve axotomy)

TEM(autophagosomes); WB(LC3-II/LC3-I, QSTM1/P62) Autophagy inducer inhibits Iba1, NF-kB

[74]

-Sprague Dawley rat (Chronic ocular hypertension model)

Autophagy inducer inhibits Iba1, NF-kB

[88]

-Brown Norway rats (IOP elevation by hypertonic saline injections into episcleral veins)

WB(Atg 3, Atg 7, mTOR)

[92]

TEM (autophagosomes)

[61]

TEM (autophagosomes)

[35]

-Mouse microglia BV2 cells

Dendrites of RGCs-Sprague Dawley rat (Chronic hypertensive glaucoma model) Axons of RGCs

-Wistar rats (acute axonal degeneration)

[88]

28

Optic nerve

-Wistar rats (IOP elevation)

WB(LC3II, SQSTM1/P62); TEM(autophagic vacuoles)

[34]

Abbreviations: Atg, autophagy-related genes; GFP, green fluorescent protein; mTOR, mammalian target of rapamycin; IHC, immunofluorescence histochemistry; IOP, intraocular pressure; Refs, references; TEM, transmission electron microscopy; TMCs, trabecular meshwork cells; RGCs,etinal ganglion cells; WB, western blot.

29