Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

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If we were able to extend the homeostatic capacity of retinal cells then it would be possible to substantially reduce cell death and lessen the risk of blindness in many retinal degenerative diseases. Two ways of doing so are to identify and potentiate the inherent neuroprotective mechanisms in the eye and/or to use factors that will allow the cells to withstand larger extremes of transient stress.

In this chapter, we will consider several classes of neuroprotective molecules that are strong candidates to increase the capacity of the retina to tolerate stress. These include neurotransmitters, steroids, fatty acids and their derivatives, and polypeptide neurotrophic (NT) factors, which can exert neuroprotective actions on a range of different target molecules. For example, some neurotransmitters and polypeptides activate membrane receptors, which can in turn trigger intracellular neuroprotective signals, whereas fatty acids and Co-enzyme Q can activate mitochondrial uncoupling proteins to alter abnormal amounts of reactive oxygen produced within a cell.

USING NEUROTRANSMITTERS AND THEIR RECEPTORS TO PROTECT RETINAL GANGLION CELLS

FROM GLAUCOMA-ASSOCIATED DEGENERATION

Neurotransmitters are molecules whose primary function is to facilitate the transmission of signals between neurons or from neurons to a target tissue. In some cases, however, excessive activity in neuronal tissues can lead to dangerously toxic concentrations of a specific neurotransmitter. This phenomenon was first reported in 1957 by Lucas and Newhouse, who showed that increased amounts of glutamate, above physiological levels, are toxic to the mammalian eye (1). The term “excitotoxicity” was coined from the observation that systemic injection of glutamate into neonatal mice led to destruction of the inner retinal layers, most notably the retinal ganglion cell (RGC) layer, which was evident by ultrastructural examination (2). Later, it was determined that glutamate caused depolarization of RGCs and that glutamate toxicity was associated with prolonged cation influx (3–6). Cation influxes are known to activate many intracellular pathways including excess transport of calcium into the mitochondria, with subsequent induction of permeability pores, release of cytochrome c, and initiation of an apoptotic cascade (7), as well as the production of nitric oxide and peroxide, both of which are highly toxic to cells (8,9). In addition, many intracellular signal transduction cascades are under the control of glutamate-induced cation influxes. Some of these, for example activation of p38 and ERK (extracellular-signal-regulated kinase) MAPKs, have been directly linked to the initiation of cell death signals (10–12).

Attempts to protect retinal ganglion cells from excitotoxicity by blocking glutamate receptors with specific antagonists has generated much interest. The problem with this approach, however, is that although general blocking of glutamate receptors may prevent cell death, it also prevents normal cell function including the transmission of visual signals through the ganglion cells. As an alternative to using strong glutamate receptor antagonists, several groups have explored the use of the weaker compound memantine, an N-methyl-D-aspartate (NMDA) receptor noncompetitive antagonist (13,14). Memantine blocks excessive activation of the NMDA receptor but does not block normal signaling. It is sufficiently antagonistic that it reduces calcium influx through the NMDA receptor to levels that are not toxic. The success of this approach in a variety

of model systems has led to clinical trials of memantine as a therapeutic agent for glaucoma, and its widespread use is awaiting Food and Drug Administration approval (15).

There is, however, clear evidence that some neurotransmitters can function as neuroprotective agents in the retina and other parts of the CNS. This is especially true for acetylcholine (ACh) where activation of the α-7 nicotinic ACh receptor (α-7 nAChR) by ACh has been linked to protection against glutamate-induced excitotoxicity in the brain (16–19). A similar effect has been found in the retina where ACh protects ganglion cells from high concentrations of glutamate (20). One possible mechanism by which it protects these cells is through activation of the α-7 nAChR followed by a calciumdependent activation of its intracellular signaling partners that are associated with the PI3-kinase and MAPK pathways (21,22).

Norepinephrine is another neurotransmitter linked to neuroprotection throughout the CNS (23,24). Many of the protective effects of this transmitter appear to be mediated by the α-2-adrenergic receptor (25,26). Activation of α-2-adrenergic receptors, which are expressed on RGCs and other cells in the inner nuclear layer, has been associated with neuroprotective outcomes in the retina (27). When the norepinephrine agonist, brimonidine, engages with the α-2-adrenergic receptors, it induces strong protection of RGCs in many models of glaucoma and in ischemia-reperfusion models of general retinal damage (28,29). Brimonidine is effective at preserving retinal ganglion cells after optic nerve crush when given either at the time of or 14 h before injury. Pharmacologically active concentrations of brimonidine can be achieved in the retina (>2 nM) when this compound is applied to the ocular surface. One response of the retina to brimonidine is increased expression of the brain-derived NT factor (BDNF) by RGCs (30). Because brimonidine also reduces intraocular pressure, it is being studied as a potentially useful agent to treat glaucoma.

The approach of modulating the actions of specific neurotransmitters or the actions of their cognate receptors for neuroprotective ends is less appropriate for macular degeneration and other photoreceptor-associated retinal degenerations because these cells are primary sensory neurons that do not receive synaptic input. There are other classes of factors, however, that can promote survival of photoreceptors, which are more relevant for these conditions.

STEROIDS AND LIPIDS AS NEUROPROTECTIVE FACTORS FOR RETINAL DEGENERATIONS

Two steroid hormones most typically associated with reproductive function are estrogen and progesterone. Estrogen affects differentiation and neurite outgrowth of neurons and is protective against several toxic insults including oxidative stress, glutamate excitotoxicity, hypoglycemia, and ischemia (31–37). In addition, an observed increase in risk for degenerative eye diseases in postmenopausal women is reduced with estrogen treatment (38). The classical method by which estrogen works upon entry into a cell is that it binds to its cognate receptor, this receptor–ligand complex then dimerizes, translocates to the nucleus, and interacts with an estrogen receptor binding motif on the promoter regions of many genes to activate gene expression. One mechanism by which estrogen may exert neuroprotective effects in the retina is by promoting expression of antiapoptotic and neuroprotective molecules, such as BCL-2 and thioredoxin (39,40).

Estrogen may also act upstream at the level of the plasma membrane to induce activation of cell survival signals associated with the ERK/MAPK and PI3 kinase/Akt cascades (41,42).

Although progesterone can also reduce the death of retinal neurons resulting from global ischemia and glutamate excitotoxicity, there is less extensive evidence for the molecular mechanisms of the neuroprotective actions of this hormone (43). Larger scale studies to elucidate the effects of these hormones on eye diseases have been limited by the use of synthetic steroids. For example, hormone replacement therapies using synthetic progestins have complicated the studies because these molecules are not neuroprotective and may even be toxic (44).

There is now mounting evidence that fatty acids may also contribute to the survival of neurons in conditions of stress. This is not entirely surprising because omega-3 fatty acids are natural body constituents and found in high concentrations in the retina, particularly in photoreceptors. Two of the most common polyunsaturated fatty acids in the retina are arachidonic acid (AA) and docosahexanoic acid (DHA) (45). These molecules serve as building blocks for the synthesis of eicosanoids and docosanoids respectively, when released from phospholipids by the action of phospholipases (46). AA protects RGCs from cell death induced by high levels of glutamate but can be toxic when used above concentrations of 10 M (47). Whether AA itself is the active neuroprotective compound or whether it needs to be converted into prostaglandins or leukotrienes for activity is still being determined.

This neuroprotective compound NPD1 (10,17S-docosatriene) is formed from DHA by the action of a 15-lipoxygenase-like enzyme (46,48). NPD1 is neuroprotective in a wide range of in vivo and in vitro systems including ischemia-reperfusion or chemically induced oxidative stress. In patients suffering from Alzheimer’s disease, the concentration of NPD1 is significantly reduced in brain regions undergoing degeneration suggesting that it may play a role in augmenting neurodegenerative processes (49). NPD1 triggers increased expression of a number of key anti-apoptotic molecules including Bcl-2, Bcl-xl, and Bfl- 1(A1). It also reduces the expression of pro-inflammatory molecules suggesting that its neuroprotective role may be mediated by these actions (50). Therefore, strategies that can modulate in vivo levels of NPD1 has the potential to be effective in reducing neuronal degeneration although, so far, it has been difficult to regulate ocular levels of its precursor, DHA, by dietary supplementation.

ANTIOXIDANTS OFFER PROTECTION TO PHOTORECEPTORS: MITOCHONDRIAL UNCOUPLING PROTEINS

Elevated levels of reactive oxygen species (ROS) are toxic and their accumulation in a tissue causes damage to membranes, proteins, and DNA eventually leading to death of the cell (51,52). The retina is particularly sensitive to the increased ROS generated through the high metabolic activity of photoreceptors and RGCs (53). In accord with this, there is evidence that antioxidants provide protection for photoreceptors by reducing intracellular levels of ROS generated by oxidative stress (54).

Intracellular generation of reactive oxygen species is a natural function of mitochondria because the proton gradient that drives production of adenosine triphosphate (ATP) produces reactive oxygen species as a byproduct. The greater the rate at which mitochondria

Fig. 1. Schematic illustration of ATP synthase functioning in the absence of UCP2 activity

(A), and in the presence of active UCP2 (B). The proton gradient drives ATP synthase and ATP production. If the proton gradient decreases as a result of the UCP2 activity, ATP production decreases, energy is dissipated in the form of heat (thermogenesis), superoxide (O2) production is decreased, and calcium efflux is increased. However, despite decreased ATP production by individual mitochondria, overall the neurons will have more ATP available, because uncoupling is accompanied by mitochondrial proliferation. (Adapted from ref. 66.)

produce ATP, the greater is the endogenous production of ROS. Increased intracellular levels of ROS can lessen the ability of a cell to tolerate additional ROS generated extracellularly in tissues exposed to oxidative stress.

The intracellular levels of ROS however, can be reduced by uncoupling proteins (UCPs), located in the inner membrane of the mitochondria, which serve as intracellular antioxidants (Fig. 1). The primary function of these proteins is to allow hydrogen ions to leak from the intermembrane space into the matrix of the mitochondria and in this way dissipate the energy in the form of heat (55–60). By decreasing the driving force of ATP synthase, the enzyme that catalyzes ATP synthesis, UCPs reduce the amount of ATP and ROS produced (61,62).

The most well-characterized UCP, is UCP1, which is expressed solely in brown adipose tissue and is mainly responsible for thermogenesis in small rodents (55,63). Brown adipose tissue is virtually insignificant for normal physiology in primates and, until recently, little attention was paid to the action of uncoupling proteins in other tissues of the body. In the last few years, however, several other members of the UCP family have been identified and found to promote partial uncoupling of oxidation from phosphorylation in vitro. The five putative UCPs differ greatly in tissue distribution and regulation and may have distinct physiological roles. UCP2, UCP4, and BMCP1 are predominantly expressed in the central nervous system, including the retina, but are also detected in muscle, spleen, and adipose tissue. UCP1 and UCP3 are expressed only in peripheral tissues (56–60).

The relevance of these uncoupling proteins to neurodegenerative processes has been shown in studies that linked increased activity of UCP2 with protection of cells from

seizure-induced excitotoxicity or injury induced by MPTP (1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine) in several models of neurodegenerative diseases (64,65). Further support for a role of UCPs in cell death is that UCP2 overexpressing mice show an increased number of RGCs owing to decreased programmed cell death in the early postnatal stages of development (66).

UCPs can be regulated by a number of factors, most importantly by their co-factors co-enzyme Q (CoQ) and fatty acids. Although studies have not yet separated the general antioxidant function of CoQ from its specific effects on mitochondrial UCPs there is a general view that most antioxidants including the CoQ offer some protection for photoreceptors.

POLYPEPTIDES THAT PROMOTE SURVIVAL

OF NEURONS IN THE CNS

The other major class of factors that promote survival of neurons in the CNS is a group of polypeptides that also have trophic influence and other functions during normal development. Four of these are currently considered to have the most impact on neurons in the eye. These are BDNF, ciliary NT factor (CNTF), glial-derived NT factor (GDNF), and pigment epithelium-derived factor (PEDF).

All four polypeptides are produced at multiple sites in the brain and in other nonneural tissues. All four are also expressed in the normal retina, though by different cell types. Immunocytochemical studies show that BDNF is localized to ganglion cells and other cells of the inner retina (67). CNTF, on the other hand, is found primarily in Müller glia and astrocytes (68). Similarly, the major sites of synthesis of GDNF in the retina are glial cells (69,70). PEDF is synthesized in the retinal pigment epithelial (RPE), Müller glia, and ganglion cells of the retina, as well as cells in the ciliary body (71). These patterns of expression have been defined in the normal retina and may differ in conditions of retinal injury or disease where all four polypeptides are upregulated as part of a homeostatic response. As discussed later in this chapter, expression of these factors by various retinal cell types including microglia and vascular endothelial cells in injury may be an important component in limiting damage to the retina.

All four polypeptides have been tested extensively for their neuroprotective properties in a wide range of in vitro and in vivo models. Some of these, such as the axotomy model of neurodegeneration, have been studied extensively but are outside the scope of this chapter. Here we will only review the actions of these polypeptides on RGCs injured by the excitotoxin glutamate, to all retinal neurons damaged by oxidative stress, and to photoreceptors induced to degenerate by mutations or by excessive light exposure.

Neuroprotective Polypeptides Impede Glutamate Excitotoxicity

As discussed in a previous section, excessive amounts of glutamate in the nervous system can kill neurons. Cell death is proportional to the concentration of the excitotoxin present as is clearly shown in studies in which dissociated cultures of neurons are treated with micromolar concentrations of glutamate (72).

Both BDNF and GDNF counteract glutamate excitotoxicity by reducing NMDA- receptor-mediated Ca2+ influx through an ERK-dependent pathway, as shown in a number of different culture models (73,74). GDNF has additional autocrine actions in the retina

Fig. 2. Neuroprotective polypeptides protect RPE cells from H2O2-induced cell death. In each histogram, combinations of two neuroprotective factors were used for each point. The increased survival given by increasing doses of PEDF is show from left to right. The increased survival induced by BDNF (A) or CNTF (B) is shown from front to back. Even at saturating doses of one factor, additional protection can be given by the addition of a second factor.

where it increases expression of the GLAST glutamate transporter in glia and in so doing may help reduce glutamate levels and glutamate excitotoxity (75). A number of studies have shown that nanomolar concentrations of PEDF allow cells to withstand the toxic influence of glutamate, which would otherwise induce apoptotic cell death in many neurons. (reviewed in ref. 71.)

Neuroprotective Polypeptides Reduce Oxidative Stress-Related Damage

As we have already discussed, one of the most common causes of neuronal death and a possible contributing factor to many forms of retinal degeneration is oxidative stress. One way of experimentally inducing oxidative stress to test the neuroprotective efficacy of a compound is to treat cells with low concentrations of hydrogen peroxide, a naturally occurring toxic byproduct of visual transduction. Many of the NT polypeptides that reduce the death of neurons that are challenged with toxic levels of glutamate also shield against oxidative stress (Fig. 2). In one study, we observed that when retinal neurons were pretreated with PEDF, they develop resistance to moderate concentrations of hydrogen peroxide (76). It is important to note that protection only occurred when cells were pretreated for at least an hour with PEDF and even after pretreatment, high concentrations of hydrogen peroxide were still toxic.

Similarly, others have shown that BDNF offers protection to photoreceptors and RGCs from oxidative stress (77–79). It has been suggested that BDNF does so by reducing endogenous production of ROS, possibly by its actions at the level of the mitochondria (80,81). It is interesting to speculate that uncoupling proteins may be direct or indirect targets for BDNF actions in reducing oxidative damage. Both GDNF and CNTF can prevent neuronal injury caused by oxidative stress as shown in many models of CNS injury (76,82,83). Such findings support the idea that the damage caused by oxidative stress can be reduced by the endogenous neuroprotective mechanisms controlled by these four polypeptides.