Pathogenesis of ganglion ‘‘cell death’’ in glaucoma and neuroprotection: focus on ganglion cell axonal mitochondria

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Introduction

Glaucoma, or glaucomatous optic neuropathy, is a chronic neurodegenerative disease characterized by a progressive loss of retinal ganglion cells (Quigley, 1999; Whitmore et al., 2005). The disease is associated with a specific remodeling of the optic nerve head. Primary open-angle glaucoma (POAG) constitutes the majority of all forms of glaucoma where the iris position is not affected. Traditionally, glaucoma has been viewed as a disease of elevated intraocular pressure (IOP). Excessive elevation of IOP can cause compression of retinal ganglion cell axons at the optic nerve head to affect axonal transport and alter the appropriate nutritional requirements for ganglion cell survival. Blood flow in the optic nerve head is also reduced because of compression of blood vessels and/or altered perfusion occurring when IOP is moderately elevated. Compelling evidence therefore exists to show that raised IOP can be the cause of visual loss in glaucoma. This is supported by the finding that the lowering IOP is often linked with the prevention of visual loss (Weinreb and Khaw, 2004).

A substantial number of glaucoma patients do not have raised IOP, however, and often lowering of elevated IOP does not result in the prevention of visual loss (Flammer and Orgu¨l, 1998; Weinreb and Khaw, 2004). Moreover, not all ocular

hypertensive patients have glaucoma. Clearly, raised IOP is not synonymous with loss of vision in all glaucoma patients. Factors other than raised IOP therefore contribute to loss of vision in glaucoma. However, unequivocal proof of risk factors other than raised IOP causing loss of vision in glaucoma remains a problem. A number of potential risk factors (Fig. 1) have been identified that include fluctuation of IOP, aging, family history, severe myopia, central cornea thickness, hypertension, hypotension, vasospasm, hemorheology, immune system, diabetes mellitus, sleep disturbances, family history, and light (Flammer et al., 2002; Pache and Flammer, 2006, Osborne et al., 2001, 2006). In addition, a number of candidate genes have also been associated with the risk of developing glaucoma (Abu-Amero et al., 2006).

Retinal ganglion cells and mitochondria

Retinal ganglion cell axons are unmyelinated within the ocular globe and have now been shown to have varicosities that are rich in mitochondria. These axons make desmosomeand hemidesmo- some-like junctions with other axons and glial cells (Wang et al., 2003; Carelli et al., 2004). The mitochondria-enriched axon varicosities have been interpreted as functional sites with local

high-energy demand, possibly relevant for signal transmission. Significantly, immunocytochemical labeling of cytochrome c oxidase, a marker related to the intensity of oxidative phosphorylation, is greatest in the nerve fiber layer and prelaminar– laminar ganglion cell axons within the globe. It also decreases drastically in the myelin posterior to the lamina. Thus, there is an asymmetrical distribution of mitochondria along the ganglion cell axons, with the unmyelinated regions within the globe containing an enriched population of mitochondria (Bristow et al., 2002; Carelli et al., 2004). Clearly, an alteration in the functional status of these ganglion cell axon mitochondria will influence ganglion cell survival, and the question arises as to whether this phenomenon plays a part in the pathogenesis of optic neuropathies such as glaucoma.

Mitochondria are constantly produced in neuronal soma and transported to their terminals where they participate in synaptic transmission and undergo fusion and fission (Chan, 2006). One explanation for the different number of mitochondria in the prelaminar and postlaminar regions of a ganglion cell axon is that mitochondria are transported more slowly in the prelaminar region as they are required to provide a lot of energy to propagate an action potential along an unmyelinated axon. If this is the case, then prelaminar axonal transport would vary between different ganglion cells depending on the length of unmyelinated axon within the globe. A reduction in nutrient/oxygen supply to the optic nerve head, for example, caused by raised IOP, will ultimately affect the efficiency of energy production by mitochondria in the prelaminar region, and as a consequence axonal transport is affected. There is good evidence to suggest that ganglion cell axonal transport is attenuated in glaucoma (Levy, 1976). Moreover, reduced nutrient/oxygen supply to the optic nerve head region might not affect all ganglion cells in the same way, with some being energetically compromised to a greater extent than others. This might be partially dependent on the length and number of mitochondria in a ganglion cell axon within the globe.

Recent studies have proven that mitochondrial respiratory chain enzymes such as favin and

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cytochrome oxidases are able to absorb light maximally around 440–450 nm, and as a consequence, contribute to the generation of reactive oxygen species (ROS) in cells (Chen et al., 1992, 2003; King et al., 2004; Godley et al., 2005; Osborne et al., 2006). The interaction of light with ganglion cell axon mitochondria in healthy cells probably results in a generation of ROS, which is removed by the appropriate scavenging mechanisms. However, when ganglion cells are in an energetic compromised state, normal scavenging mechanisms might not be adequate. The proposition has therefore been made that light becomes a risk factor to energetic compromised cells in glaucoma. Laboratory studies on cells in culture provide support for this notion (Osborne et al., 2006, 2008; Lascaratos et al., 2007).

Possible causes for ganglion cell death in glaucoma

It is now clear that the ganglion cell death in glaucoma is not solely caused by raised IOP. It is also evident that, in order to develop additional methods of treatment other than the lowering of IOP, it is necessary to understand the pathogenesis of the disease. From our present knowledge, it is tempting to conclude that impairment in the quality of blood flow in the microcirculation associated with the optic nerve head region is the factor initiating retinal changes that leads to loss of vision in glaucoma (Fig. 1), although it remains to be unequivocally demonstrated that optic nerve head blood flow is compromised in glaucoma. Present logic suggests that sustained or intermittent changes in the microcirculation in the optic nerve head region is likely to occur because of raised or fluctuation changes in IOP, aging, vasospasm, hemorheology, hypotension, or hypertension. This could result in variable ischemia/ reperfusion and/or hypoxic insults to ganglion cell axons and glial cells in the optic nerve head region. This idea is supported by the finding that HIF-1a, activated by a reduced oxygen supply, is upregulated in pathological retinas from glaucoma subjects (Tezel and Wax, 2004).

How might ischemic insults to retinal ganglion cell axons and glia in the optic nerve head region

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cause the death of ganglion cells at different times in the life of a glaucoma patient? We have hypothesized that in the initial stages of the disease, ganglion cells will be forced to survive at a lower energetic status because of the altered optic nerve head blood flow (Fig. 2) and that as the disease progresses, individual ganglion cells will be at risk of a combination of different types of additional secondary insults. As a result, ganglion cells will die at different times. We have also proposed that secondary insults originate from light entering the eye (Osborne et al., 2006) and from altered function to glial cells (astrocytes, Mu¨ller cells, and microglia), originating from abnormal blood flow in the optic nerve head region that is also the cause for ganglion cells being energetically compromised (Figs. 1 and 2). This is based partly on experimental studies that have shown that a variety of potentially toxic substances (glutamate, TNF-a, serine, nitric oxide, and potassium) become elevated in the extracellular retinal spaces when retinal glial cell function

is affected (see Tezel, 2006). Moreover, in pathological tissues, an upregulation of substances like nitric oxide synthase (involved in the formation of nitric oxide) (Neufeld et al., 1999) and TNF-a (see Tezel, 2006) has been reported. Also, an elevation of glutamate occurs in the vitreous humor of glaucoma patients (Dryer et al., 1996). Elevation of these substances will affect the survival of all retinal neurones by having membrane effects that may be receptor mediated. However, their effects on retinal ganglion cells will be particularly detrimental because these cells are in an already compromised energetic state. For example, elevated extracellular levels of glutamate will depolarize excessively all cells containing NMDA-type receptors, but the ganglion cells, being at a compromised energetic state, will not be able to compensate as efficiently as other neurones for this excessive depolarization, causing them to become dysfunctional and eventually die by apoptosis. This hypothesis therefore predicts that the trigger(s) for different ganglion cells dying by