**2. Apoptosis and necrosis in glaucoma**

Apoptosis and necrosis constitute the two major pathways to cell death. [12] In 1972, Kerr, Wyllie and Currie used the Greek term 'apoptosis' (from the Greek: dropping off of petals from plants) to describe a specific morphological aspect of cell death. [13] Apoptosis is ac‐ companied by rounding-up of the cell, reduction of cellular volume, chromatin condensa‐

tion, and engulfment by resident phagocytes. Apoptosis is the best-characterized type of programmed cell death, and these morphological changes are largely mediated by the acti‐ vation of the caspase family of cysteine proteases. [14] In contrast, 'necrosis' (from the Greek: death*)* is associated with a gain in cell volume, swelling of organelles, plasma mem‐ brane rupture and subsequent release of intracellular contents with ensuing inflammation. Until recently necrosis had been considered a passive, unregulated form of cell death. New evidence indicates that some forms of necrosis can be induced by regulated signal transduc‐ tion pathways such as those mediated by receptor interacting protein kinases (RIP Kinases). RIP kinases cross talk with caspases and lie downstream of cell death signals such as the Fas Ligand or the Tumor Necrosis Factor-α (TNF-α). [15] This programmed form of necrosis is termed programmed necrosis or necroptosis. [12, 16, 17]

Cysteine aspartate-specific proteases or caspases are central to the execution of apoptosis. Their activation occurs mainly through two distinct pathways: extrinsic and intrinsic (Fig. 1). [18] The extrinsic pathway is initiated by binding of extracellular death ligands such as TNF-α and Fas ligand to their cell-surface death receptors, TNF receptor and Fas. [19] The death domains of these receptors recruit adaptor molecules like Fas-associated death do‐ main (FADD) and caspase-8, forming the death inducing signaling complex (DISC). [20] The formation of DISC leads to activation of caspase-8, which in turn mediates cleavage of effec‐ tor caspases. The extrinsic pathway can cross-talk with the intrinsic pathway through cas‐ pase-8-mediated cleavage of Bid, aBH3-only member of the Bcl-2 family of proteins. [21, 22] Bid cleavage releases a truncated fragment that triggers the release of mitochondrial pro‐

Neuroprotection in Glaucoma http://dx.doi.org/10.5772/54294 181

The intrinsic pathway is mediated by mitochondria. [23] In response to intracellular and environmental stress, mitochondria release inter-membrane proteins such as cytochrome c and second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosisbinding protein with low pI (Diablo) into the cytosol. Released cytochrome c triggers the formation of an apoptosome along with apoptotic protease activating factor-1 (Apaf-1) and caspase-9 in the presence of ATP, which leads to caspase-9 activation. [24] Smac/Diablo en‐ hances caspase activation through the neutralization of inhibitor of apoptosis (IAP) (Fig. 1).

Necrosis is mainly regulated by a set of protein Kinases called RIP Kinases. RIP1 switches its function to a regulator of cell death when it is deubiquitinated by A20 or cylindromatosis (CYLD). [27, 28] Deubiquitination of RIP1 abolishes its ability to activate NF-κB after TNF-α stimulation, and leads to the formation of cytosolic DISC with FADD and caspase-8, the socalled complex II. [29] As described above in caspase signaling, DISC formation leads to cas‐ pase-8 activation and subsequent apoptosis. In contrast to TNF signaling, Fas directly recruits RIP1, FADD and caspase-8 to the plasma membrane and forms DISC (Fig. 2). [30] During apoptosis, RIP1 is cleaved and inactivated by caspases. [31] Although many cell lines are protected against death receptor-induced apoptosis by use of pan-caspase inhibitors, Vercammen and others found that, in mouse L929 fibrosarcoma cells, caspase inhibition does not prevent TNF- or Fas-induced cell death and the cells acquire a necrotic morpholo‐ gy. [32, 33] In 2000, Holler and others discovered that RIP1 kinase is a key molecule that in‐

In 2005, Degterev, Yuan, and others using chemical library screening, identified small com‐ pounds named necrostatins that specifically inhibit death receptor- mediated necrosis. [16] Necrostatins have been shown to specifically inhibit RIP1 kinase phosphorylation during necrosis without affecting death receptor-induced NF-κB activation. [35] RIP1 kinase activi‐ ty appears to be important for necrosome formation, as necrostatin-1 abolishes the forma‐ tion of the RIP1-RIP3 complex and RIP3 kinase phosphorylation during necrosis. [36, 37] Cho and others propose that another unknown kinase activated by RIP1 may mediate RIP3 phosphorylation, based on the findings that ectopically expressed RIP1 does not phosphory‐ late RIP3. [36] The activities of RIP1 and RIP3 may be mutually regulated in a necrosome signaling complex. RIPK activation leads likely to increased reactive oxygen species (ROS) production. Activated RIP3 interacts with metabolic enzymes such as glycogen phosphory‐

teins, thereby initiating the intrinsic caspase cascade as described below.

duces necrotic cell death mediated by death receptors. [34]

[25, 26]

**Figure 1.** The extrinsic pathway is initiated by binding of death ligands such as TNF-α and Fas ligand to their cell-sur‐ face death receptors such as TNF receptor and Fas. The death domains of these receptors recruit adaptor molecules like FADD and caspase-8, which leads to the activation of caspase-8. Activated caspase-8 cleaves the effector caspases such as caspase-3, thereby activating them and inducing apoptosis. The extrinsic pathway interacts with the intrinsic pathway via caspase-8-mediated cleavage of Bid. The intrinsic pathway is initiated by release of mitochondrial inter‐ membrane proteins such as cytochrome c and Smac/Diablo into the cytosol. Released cytochrome c forms an apopto‐ some with Apaf-1 and caspase-9, which leads to caspase-9 activation. Smac/Diablo enhances caspase activation through the neutralization of IAP proteins.

Cysteine aspartate-specific proteases or caspases are central to the execution of apoptosis. Their activation occurs mainly through two distinct pathways: extrinsic and intrinsic (Fig. 1). [18] The extrinsic pathway is initiated by binding of extracellular death ligands such as TNF-α and Fas ligand to their cell-surface death receptors, TNF receptor and Fas. [19] The death domains of these receptors recruit adaptor molecules like Fas-associated death do‐ main (FADD) and caspase-8, forming the death inducing signaling complex (DISC). [20] The formation of DISC leads to activation of caspase-8, which in turn mediates cleavage of effec‐ tor caspases. The extrinsic pathway can cross-talk with the intrinsic pathway through cas‐ pase-8-mediated cleavage of Bid, aBH3-only member of the Bcl-2 family of proteins. [21, 22] Bid cleavage releases a truncated fragment that triggers the release of mitochondrial pro‐ teins, thereby initiating the intrinsic caspase cascade as described below.

tion, and engulfment by resident phagocytes. Apoptosis is the best-characterized type of programmed cell death, and these morphological changes are largely mediated by the acti‐ vation of the caspase family of cysteine proteases. [14] In contrast, 'necrosis' (from the Greek: death*)* is associated with a gain in cell volume, swelling of organelles, plasma mem‐ brane rupture and subsequent release of intracellular contents with ensuing inflammation. Until recently necrosis had been considered a passive, unregulated form of cell death. New evidence indicates that some forms of necrosis can be induced by regulated signal transduc‐ tion pathways such as those mediated by receptor interacting protein kinases (RIP Kinases). RIP kinases cross talk with caspases and lie downstream of cell death signals such as the Fas Ligand or the Tumor Necrosis Factor-α (TNF-α). [15] This programmed form of necrosis is

**Figure 1.** The extrinsic pathway is initiated by binding of death ligands such as TNF-α and Fas ligand to their cell-sur‐ face death receptors such as TNF receptor and Fas. The death domains of these receptors recruit adaptor molecules like FADD and caspase-8, which leads to the activation of caspase-8. Activated caspase-8 cleaves the effector caspases such as caspase-3, thereby activating them and inducing apoptosis. The extrinsic pathway interacts with the intrinsic pathway via caspase-8-mediated cleavage of Bid. The intrinsic pathway is initiated by release of mitochondrial inter‐ membrane proteins such as cytochrome c and Smac/Diablo into the cytosol. Released cytochrome c forms an apopto‐ some with Apaf-1 and caspase-9, which leads to caspase-9 activation. Smac/Diablo enhances caspase activation

termed programmed necrosis or necroptosis. [12, 16, 17]

180 Glaucoma - Basic and Clinical Aspects

through the neutralization of IAP proteins.

The intrinsic pathway is mediated by mitochondria. [23] In response to intracellular and environmental stress, mitochondria release inter-membrane proteins such as cytochrome c and second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosisbinding protein with low pI (Diablo) into the cytosol. Released cytochrome c triggers the formation of an apoptosome along with apoptotic protease activating factor-1 (Apaf-1) and caspase-9 in the presence of ATP, which leads to caspase-9 activation. [24] Smac/Diablo en‐ hances caspase activation through the neutralization of inhibitor of apoptosis (IAP) (Fig. 1). [25, 26]

Necrosis is mainly regulated by a set of protein Kinases called RIP Kinases. RIP1 switches its function to a regulator of cell death when it is deubiquitinated by A20 or cylindromatosis (CYLD). [27, 28] Deubiquitination of RIP1 abolishes its ability to activate NF-κB after TNF-α stimulation, and leads to the formation of cytosolic DISC with FADD and caspase-8, the socalled complex II. [29] As described above in caspase signaling, DISC formation leads to cas‐ pase-8 activation and subsequent apoptosis. In contrast to TNF signaling, Fas directly recruits RIP1, FADD and caspase-8 to the plasma membrane and forms DISC (Fig. 2). [30] During apoptosis, RIP1 is cleaved and inactivated by caspases. [31] Although many cell lines are protected against death receptor-induced apoptosis by use of pan-caspase inhibitors, Vercammen and others found that, in mouse L929 fibrosarcoma cells, caspase inhibition does not prevent TNF- or Fas-induced cell death and the cells acquire a necrotic morpholo‐ gy. [32, 33] In 2000, Holler and others discovered that RIP1 kinase is a key molecule that in‐ duces necrotic cell death mediated by death receptors. [34]

In 2005, Degterev, Yuan, and others using chemical library screening, identified small com‐ pounds named necrostatins that specifically inhibit death receptor- mediated necrosis. [16] Necrostatins have been shown to specifically inhibit RIP1 kinase phosphorylation during necrosis without affecting death receptor-induced NF-κB activation. [35] RIP1 kinase activi‐ ty appears to be important for necrosome formation, as necrostatin-1 abolishes the forma‐ tion of the RIP1-RIP3 complex and RIP3 kinase phosphorylation during necrosis. [36, 37] Cho and others propose that another unknown kinase activated by RIP1 may mediate RIP3 phosphorylation, based on the findings that ectopically expressed RIP1 does not phosphory‐ late RIP3. [36] The activities of RIP1 and RIP3 may be mutually regulated in a necrosome signaling complex. RIPK activation leads likely to increased reactive oxygen species (ROS) production. Activated RIP3 interacts with metabolic enzymes such as glycogen phosphory‐ lase (PYGL), glutamate-ammonia ligase (GLUL) and glutamate dehydrogenase 1 (GLUD1). [38] Activation of these enzymes eventually stimulates the Krebs cycle and oxidative phos‐ phorylation, thereby increasing mitochondrial ROS production. Secondly, after TNF-α stim‐ ulation, RIP1 forms a complex with TNFR, Riboflavin kinase, and NADPH oxidase 1. [39, 40] NADPH oxidase is the best characterized non-mitochondrial source of ROS and forms a membrane bound enzyme complex with p22phox and Rac. [41] Thirdly, RIP1 kinase acti‐ vates autophagic degradation of catalase, which converts hydrogen peroxide to water and oxygen, thereby increasing ROS accumulation. [42] More recently, activation of the ne‐ crosome has shown to interact with the mixed lineage kinase domain-like (MLKL) and phosphoglycerate mutase 5 (PGAM5) resulting in the fusion of mitochondria and necrotic cell death. [43, 44]

**Figure 3.** Schematic of changes in animal models of high IOP mediated optic nerve damage. A, In normal IOP micro‐ glia are quiescent and cells are in normal state. B, Elevated IOP leads to increased numbers of activated microglia with amoeboid morphology around the optic nerve head. These microglia appear to secrete TNF-α leading to RGC death. Other molecules, including FasL on microglia, nitric oxide (NO), and reactive oxygen species (ROS) may also play a role

Neuroprotection in Glaucoma http://dx.doi.org/10.5772/54294 183

In chronic glaucoma, apoptosis of retinal ganglion cells has been shown as the main path‐ way to cell death. [2, 45, 46] The exact mechanism though is not clear. Since a significant proportion of patients who suffer from glaucoma have high IOP, it has been hypothesized that high IOP induces stress to retinal ganglion cells either directly [47, 48] or indirectly to their axons at the lamina cribrosa [49] thus leading to apoptosis. However, although high IOP has been thought to be the main causative factor, the fact that glaucoma can occur in the presence of IOP within the normal range, while can be absent in a subset of subjects with high IOP indicates that the underlying etiology of this disease remains unknown and in es‐

Mechanisms believed to cause stress to retinal ganglion cells and to initiate the apoptotic cascade include: biomechanical stress [52, 53], excitotoxicity [54-57], tissue hypoxia [58, 59], altered nutritional blood supply [60, 61], mitochondrial dysfunction [62-65], Müller glial cell activation [66], protein misfolding [67-69], oxidative stress [70, 71], dysfunctional autoim‐

Animal experimental models in glaucoma research are produced by inducing either an ele‐ vation in intraocular pressure or damage to the axons of retinal ganglion cells. [77] Several

in RGC death. Changes in blood supply and ischemia also contribute to the death of RGCs.

munity [72], neurotrophin deprivation [73, 74], and inflammation. [75, 76]

sence fail to fully fulfill Koch's postulates [50, 51].

**3. Animal models**

#### **Figure 2. Schematic of the RIPK signaling pathway.**

A, In response to TNF-α stimulation, RIP1 is recruited to TNFR and forms a membrane associated complex I with TRADD, TRAF2/5 and cIAP1/2, which in turn leads to polyubiquitination of RIP1 and pro-survival NF-κB activation. B, RIP1 switches function to a regulator of cell death when RIP1 is unubiquitinated by A20 or CYLD. Deubiquitination of RIP1 leads to the formation of cytosolic DISC with FADD and caspase-8, the so-called complex II. In contrast to TNF signaling, Fas stimulation directly forms DISC. Activation of caspase-8 in DISC leads to apoptosis induction. During apoptosis, RIP1 is cleaved and inactivated by caspase-8. C, In conditions where caspases are blocked or cannot be acti‐ vated efficiently, RIP1 binds to RIP3, and both RIP1 and RIP3 kinases are phosphorylated at the RIP1-RIP3 complex. RIP1 kinase phosphorylation is critical for necrosis induction. In response to TNF-α, RIP1 binds to NADPH oxidase 1 and produces superoxide. Activated RIP3 binds to PYGL, GLUL and GLUD1 and increases the production of mitochondrial ROS. ROS overproduction leads to mitochondrial dysfunction, resulting in the release of mitochondrial pro-death pro‐ teins. Activation of the necrosome has been shown to interact with mixed lineage kinase domain-like (MLKL) and phosphoglycerate mutase 5 (PGAM5) resulting in the fusion of mitochondria and necrotic cell death

**Figure 3.** Schematic of changes in animal models of high IOP mediated optic nerve damage. A, In normal IOP micro‐ glia are quiescent and cells are in normal state. B, Elevated IOP leads to increased numbers of activated microglia with amoeboid morphology around the optic nerve head. These microglia appear to secrete TNF-α leading to RGC death. Other molecules, including FasL on microglia, nitric oxide (NO), and reactive oxygen species (ROS) may also play a role in RGC death. Changes in blood supply and ischemia also contribute to the death of RGCs.

In chronic glaucoma, apoptosis of retinal ganglion cells has been shown as the main path‐ way to cell death. [2, 45, 46] The exact mechanism though is not clear. Since a significant proportion of patients who suffer from glaucoma have high IOP, it has been hypothesized that high IOP induces stress to retinal ganglion cells either directly [47, 48] or indirectly to their axons at the lamina cribrosa [49] thus leading to apoptosis. However, although high IOP has been thought to be the main causative factor, the fact that glaucoma can occur in the presence of IOP within the normal range, while can be absent in a subset of subjects with high IOP indicates that the underlying etiology of this disease remains unknown and in es‐ sence fail to fully fulfill Koch's postulates [50, 51].

Mechanisms believed to cause stress to retinal ganglion cells and to initiate the apoptotic cascade include: biomechanical stress [52, 53], excitotoxicity [54-57], tissue hypoxia [58, 59], altered nutritional blood supply [60, 61], mitochondrial dysfunction [62-65], Müller glial cell activation [66], protein misfolding [67-69], oxidative stress [70, 71], dysfunctional autoim‐ munity [72], neurotrophin deprivation [73, 74], and inflammation. [75, 76]
