Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

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Many, if not most, optic neuropathies are untreatable. In some diseases affecting RGC axons, including chronic compressive optic neuropathy, acute optic neuritis, or papilledema, visual loss can be reversed when axonal damage is relieved, presumably because RGC death has not yet occurred. However, in glaucoma, the visual loss is almost always permanent, reflecting the fact that RGC loss is irreversible. Most research on optic neuropathies has focused on the pathophysiology specific to a particular disorder, e.g., elevated intraocular pressure for glaucoma, inflammation for optic neuritis, and ischemia for anterior ischemic optic neuropathy. However, optic neuropathies all share in common RGC axonal injury, except for the few disorders where the locus of injury is unknown (such as Leber’s hereditary optic neuropathy). Therefore, therapeutic inhibition of the molecular response of RGCs to axonal injury (i.e., neuroprotection) would be applicable to a wide variety of diseases of the optic nerve, independent of the mechanism by which the nerve is damaged.

THERE ARE MULTIPLE MECHANISMS BY WHICH AXONAL DAMAGE CAN TRANSDUCE AN INJURY SIGNAL

Several mechanisms have been hypothesized to mediate RGC death after optic nerve injury, including deprivation of neurotrophic factors from the target or other tissues, excitotoxicity from physiological or pathological levels of glutamate, free radical formation, increases in intra-axonal Ca++, accumulation of excess retrogradely transported macromolecules, and induction of p38 MAP kinase and other signaling molecules (12–22). Much research has focused on blocked retrograde transport of neurotrophic factors (23) or decreased levels of endogenous ocular neurotrophins (24,25). The normal developmental loss of RGCs is partly due to the competition for target-derived molecules, particularly brain-derived neurotrophic factor (BDNF) and other neurotrophins. It is thought that adult RGCs, like other neurons (26), are also dependent upon neurotrophic agents for their survival. Support for the role of neurotrophin dependence comes from experiments using identified neurotrophic factors to rescue axotomized neurons. Purified neonatal RGCs, which are axotomized during dissociation, can be kept alive for significant periods with a cocktail of factors, including BDNF, CNTF, forskolin, and insulin (27). Intraocular administration of certain neurotrophins (e.g., BDNF) delays RGC death after axotomy in adult rats (17,28) and cats (29), and in an experimental model of glaucoma (30). Gene delivery of BDNF to the retina or to the RGC itself also increases survival in experimental glaucoma (31,32), as does inhibition of apoptosis (33).

However, it is likely that RGC death after axotomy is mediated by additional mechanisms besides deprivation of neurotrophic factors. RGCs maintain viability for long periods of time during temporary neurotrophin deprivation, as when there is decreased axonal transport from a compressive optic neuropathy or papilledema, or even after axonal transection, suggesting that there are other mechanisms for signaling cell death, and possibly compensatory mechanisms for sustaining survival. Furthermore, retrograde axonal transport is rapid, and the subacute time-course by which RGCs die after axonal injury does not reflect the time-course of interrupted retrograde axonal transport. RGC axotomy induces changes in responsiveness to neurotrophins independent of neurotrophin deprivation (34). Most importantly, removal of the RGC axonal target,

and therefore, target-derived factors, causes very slow RGC death (35,36). Together, these findings suggest that axotomy can signal changes at the cell body independent of neurotrophin deprivation.

SUPEROXIDE IS AN INTRACELLULAR SIGNALING MOLECULE FOR RGC DEATH

Reactive oxygen species (ROS) serve as intracellular signaling molecules in a variety of cell types (37–40). For example, H2O2 mediates inhibition of dopamine release in the striatum (41) and inhibits bcl-xL expression (42), whereas O2− (superoxide anion) can signal cell proliferation (43) and hippocampal long-term potentiation, mediated by oxidation of protein kinase C (44). Particularly relevant to this chapter is work supporting superoxide generation in nerve growth factor (NGF) deprivation (45,46).

ROS Signal Apoptosis

Although ROS can directly injure cell proteins and cause necrotic cell death (47), consistent with a downstream role (48), more recently, ROS have been shown to signal apoptosis, which supports an upstream role. Examples include measurement of an early burst of superoxide before cytochrome c release in hippocampal neurons treated with glutamate (49) and inhibition of cytochrome c release in NGF-deprived sympathetic neurons treated with N-acetyl-l-cysteine (50). Another control point is S-nitrosylation of mitochondrial caspases (51). Although ROS generation can result from activation of the caspase cascade, there is evidence that ROS can activate caspase activation in models of cerebral ischemia (52).

ROS Signal RGC Death

We and others have shown that ROS, and particularly, superoxide, are factors that signal RGC death after axonal injury (53–56). Specific ROS scavengers and hypoxia reduce the death of cultured neonatal RGCs after axotomy (57), and RGC survival is dependent on their redox state, with greatest survival observed under mildly reducing conditions (55,58,59).

ROS Signal Cell Death by Thiol Oxidation

ROS transduce intracellular signals through several mechanisms. Protein targets for ROS transduction most commonly have a redox-sensitive moiety, often a cysteine thiol, at the active site. Proteins can also be covalently modified by ROS, e.g., S-nitrosylation with NO+, S-nitration with peroxynitrite, or glutathiolation with glutathione. Redox modulation of vicinal cysteine thiols is an efficient means of modulating protein function, as the oxidative cross-linking results in a disulfide bond that can dramatically change the conformation of the active site (60,61). Some targets for ROSmediated cysteine oxidation are probably involved in the induction of apoptosis, e.g., creatine kinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (62). Other examples of specific targets of ROS include tyrosine phosphatases [by H2O2 (63)], aconitases [by superoxide (64,65)], the NMDA receptor (66), signal transducers Akt and ERK1/2 (67), transcription factors [e.g., OxyR in Escherichia coli (68) and Nrf2Keap1 in mammals (69)], and even ROS scavengers, e.g., members of the periredoxin

family (70). Finally, our own work, described in the following section, demonstrates that inhibiting thiol oxidation blocks RGC death after axotomy.

REDUCTION OF OXIDIZED THIOLS INHIBITS RGC DEATH AFTER AXOTOMY IN VITRO AND IN VIVO

If thiol oxidation is important for RGC death after axonal injury, then drugs that reduce thiols would be expected to inhibit RGC death, as we have shown (55). Dithiothreitol (DTT) is an example of the most commonly used class of thiol-reducing agents in biochemical procedures. However, DTT, like -mercaptoethanol and similar agents, itself contains oxidizable thiols, chelates, metal ions (e.g. Cu++), and is not highly stable at physiological temperatures and pH (71). Alkylated phosphines represent a class of reducing agents, first described more than 60 years ago, which efficiently reduce oxidized thiols but do not themselves undergo thiol oxidation. Initial trialkylphosphines (72) were poorly soluble in water and thus not relevant to biological systems. The discovery that Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) is water soluble and a potent thiol-reducing agent (73) led to its widespread use in biochemistry. It is highly stable at even elevated temperatures (74) and at neutral pH (75,76), and unlike DTT, is not a strong metal-chelating agent (77). For these reasons, it has characteristics suitable for use as a pharmacological agent. However, it is polar and therefore does not cross membranes well, and the phosphine is highly reactive with oxygen. For these reasons, a modified TCEP molecule might be even more biologically active.

We have previously shown that some (but not all) drugs that scavenge or otherwise interfere with ROS increase RGC survival. For example, there is significantly greater RGC survival in the presence of catalase compared with that of control medium, whereas neither MnTMPyP nor Ebselen enhanced RGC survival. Culture in 95% N2/5% CO2 resulted in significantly slowed death than in standard (95% air/5% CO2) conditions (55), suggesting a requirement for O2 in signaling or executing apoptosis. The effects of ROS appeared to be due to shifts in the redox potential, as RGC survival was dependent on redox state, with greatest survival under mildly reducing conditions (55).

To investigate this, we studied the effects of the sulfhydryl reducing agents DTT or TCEP (55). RGCs were retrogradely labeled by stereotactic injection of the fluorescent tracer DiI into the superior colliculi of postnatal day 2–4 Long-Evans rats. At postnatal day 7–9, the animals were killed by decapitation and the retinas dissociated with papain and plated on poly-l-lysine-coated 96-well flat-bottom tissue culture plates in medium containing various additives. RGCs were identified by the presence of retrogradely transported cytoplasmic DiI and cell viability assessed with calcein-AM.

Both DTT and TCEP significantly increased RGC survival. Surprisingly, optimal levels of TCEP resulted in survival at 72 h equivalent to the neurotrophins BDNF and ciliary neurotrophic factor (CNTF) combined, and survival at 6 and 14 days was greater than with BDNF and CNTF (55).

Based on these in vitro results, we tested the hypothesis that TCEP would protect RGCs following optic nerve crush in vivo (78). RGCs of postnatal day 2–4 Long-Evans rats were retrogradely labeled with DiI, as before. At approximately 8 weeks of age, the left optic nerve of each rat was crushed with fine forceps approximately 2 mm behind

Fig. 1. PB1, a borane-protected phenyl-di(carboxymethylester)phosphine.

the globe. At the same time, 4 μl of 850 mM TCEP dissolved in balanced salt solution (BSS), or BSS alone, was injected into the vitreous at the pars plana. The right eye served as an unoperated, uninjected control. Eight days after crush, animals were killed and retinal whole mounts prepared and stained with the lectin Bandeiraea simplifolia (BSL-1) to distinguish contaminating microglia (34). An observer masked to treatment or presence of crush then counted DiI-labeled ganglion cells at fixed retinal locations.

The mean number of surviving RGCs in the TCEP group was 1250 ± 156 cells/mm2 (see Fig. 1A). This was significantly greater than the mean number of surviving RGCs in the BSS group, which was 669 ± 109 cells/mm2 (p = 0.00819). In the unoperated control eyes, the mean number of surviving RGCs was 1686 ± 93.7 cells/mm2, which is comparable to the average number of RGCs (1710 ± 73 cells/mm2) reported by Klöcker et al. (79) in their study using DiI to label RGCs. The number of RGCs in the BSS group was significantly lower than the control group (p < 0.001). In eyes with intravitreal injections without optic nerve crush, the mean number of RGCs in the TCEP group was 1781 ± 81.7 cells/mm2 and in the BSS group was 1689 ± 176 cells/mm2 (see Fig. 1B). There was no significant difference in the survival of RGCs in eyes receiving BSS or TCEP injections without the optic nerve crush injury (p = 0.70). Moreover, neither of the groups receiving injections differed significantly from the control eyes, which had a mean RGC count of 1764 ± 278 cells/mm2.

NOVEL AGENTS THAT REDUCE OXIDIZED THIOLS PROTECT RGCS IN VITRO AT LOW CONCENTRATIONS (80)

The two previous studies demonstrated that (i) a thiol-reducing agent TCEP potently prevented RGC death in vitro to a degree equivalent to the combined effect of BDNF and CNTF, and (ii) TCEP injected intravitreally into adult rats inhibited RGC death after optic nerve crush. However, the concentrations required for RGC neuroprotection with this molecule were on the order of 100 μM, which is unlikely to be pharmacologically available through typical (e.g., topical or transscleral) routes. The TCEP molecule, being highly polar, does not cross cell membranes well, and the extracellular stability of the compound is low.

In response to these problems, we formed a collaboration with Ronald T. Raines, PhD, of the Department of Biochemistry at the University of Wisconsin, with the goal of synthesizing modified versions of the TCEP molecule that could be effective at lower concentrations. Goals in modifications were protecting it from reactivity in the extracellular compartment, supporting transmembrane diffusion, and providing for a side-group that could be cleaved once intracellular, and thus maintaining a suitable intracellular concentration. Molecules were characterized by NMR spectroscopy and mass spectrometry.

Fig. 2. Mechanism of ester cleavage to help maintain higher intracellular concentration.

A first-generation molecule had inadequate stability and will not be discussed further. The second-generation molecule, named PB1, is a borane-protected phenyldi(carboxymethylester)phosphine (see Fig. 1). It has several desirable qualities for a pharmacologically active thiol-reducing agent:

1)The borane protects the phosphine from oxidation and thus stabilizes the molecule. It also probably makes it less polar, and thus more likely to cross cell membranes or the corneal epithelium. This is because the molecule is a zwitterion, with both a negative and positive charge. In the unprotected state without the borane (i.e., unprotected), the molecule would have no charge and be less polar. Although the phosphine in the unprotected molecule could presumably be protonated (giving it a positive charge and making it more polar), this is unlikely to happen at physiological pH because the pKa of the phosphine is likely below pH 7, and thus would be without a charge at neutral pH.

2)The phenyl group has two likely effects. First, it is very non-polar and should increase the molecule’s ability to cross cell membranes. Second, it delocalizes the electrons on the phosphine by resonance, so that even after borane deprotection the phosphine is less reactive. Again, this lower reactivity is an advantage because phosphines normally react very rapidly with oxygen gas.

3)The methyl esters should be cleaved by cytosolic esterases, yielding a polar intracellular intermediate that would be less likely to cross cell membranes, and thus would maintain an intracellular concentration gradient (see Fig. 2).

We tested the effect of this molecule on RGC survival in vitro, using the same methodology as described above for studying TCEP. RGCs were retrogradely labeled with DiI, cultured in the presence of varying concentrations of PB1, and viability assessed at 72 and 144 h. There was a significant effect on inhibition of RGC death after axotomy by dissociation, with detectable increased survival at 1 nM at 72 h and 10 pM at 144 h (see Fig. 3). Concentrations of 10 μM or higher were toxic at 6 days.

We also tested non-borane-protected triphenylphosphines (see Fig. 4) and found RGC neuroprotection in concentrations of 10–100 μM. This higher concentration range is presumably due to their higher reactivity to oxygen, given the lack of borane protection, and the lack of the ester linkage, which means that they are not concentrated intracellularly.

SUMMARY AND OUTLOOK FOR THE FUTURE

TCEP, a phosphine that is a thiol reductant, is neuroprotective for RGCs in vitro and in vivo. Phosphines designed to be more cell permeable, protected from oxidation, and able to be retained within cells, can be synthesized and are neuroprotective of RGCs in vitro at nanomolar concentrations.

Fig. 3. PB1 protects RGCs from axotomy-induced death in vitro at picomolar to nanomolar concentrations.

Fig. 4. Phenyl-substituted phosphines, which are water soluble but less neuroprotective than borane-protected carboxylic acid alkyl ester phosphines.

The ultimate goal is to find novel neuroprotective therapies for glaucoma and other optic neuropathies, most of which are difficult to treat or treatable or with current treatments that are incompletely effective. The most optimistic case would be that one or more lead compounds had a good safety profile, were effective in a small-scale randomized clinical trial, and could be tested in a large-scale Phase III clinical trial, possibly in collaboration with a pharmaceutical industry partner.

The eventual goal of this research is to translate it to treatment of human disease. It is difficult to predict the efficacy of the redox modulatory compounds in animal models. If molecules that are highly protective of RGCs in rat models of optic nerve crush and ocular hypertension can be identified, then the next steps would be the design of toxicology and pharmacokinetics studies, followed by small-scale clinical trials.

ACKNOWLEDGMENTS

Canada Research Chair Program, National Institutes of Health, Research to Prevent Blindness, Retina Research Foundation. A patent for the use of the compounds described in this chapter has been assigned to the Wisconsin Alumni Research Foundation.