SRT647 and SRT501 are neuroprotective for RGCs during acute optic neuritis, as shown by the fact that each SIRT1 activator attenuates RGC loss in a dose-dependent manner. SRT647 and SRT501 are chemically distinct from each other and represent direct and indirect activators of SIRT1. As such, the findings reported with both agents in this animal model help confirm the role of SIRT1 activation as important for neuroprotection in CNS demyelinating disease. SRT501 also provides significantly longer term neuroprotection for RGCs at day 30, suggesting that the increased RGC survival is not merely delayed at day 14 but can have long-lasting neuroprotective effects. This level of neuroprotection was seen with multiple- and single-dose drug administration, demonstrating that treatment at day 11, after optic nerve inflammation begins,15
provides effective neuroprotection. This is important because it suggests that treatment may be effective when patients are expected to present with clinical symptoms. No change in RGC number was found in control eyes treated with SRT647 or SRT501, demonstrating that these compounds are not toxic to RGCs. No systemic adverse effects were observed, further suggesting that these compounds are well tolerated.
Importantly, the fact that SRT501-treated eyes with optic neuritis also demonstrated no reduction in RGC numbers when retrograde labeling was performed after optic neuritis began (2 days before mice were killed) suggests that SRT501 neuroprotection supports RGC survival and maintains intact RGC axons with functional axonal transport. These results suggest that active optic nerve inflammation does not interfere with axonal transport in this model. Unfortunately, because of photoreceptor receptor degeneration in SJL/J mice, visual function could not be assessed in these mice, though we have previously shown that the RGCs are not affected in control eyes.15
However, treatment of C57BL/6 mice with SRT647 or SRT501 did not alter ERG responses, demonstrating that, at least in this strain, the compounds are not toxic to photoreceptors or overall retinal function.
The mechanism of neuroprotection by SRT647 and SRT501 likely involves SIRT1 deacetylase activity. This is suggested by the fact that two structurally and mechanistically distinct SIRT1 activators had significant neuroprotective effects and is confirmed by the finding that sirtinol, a known SIRT1 inhibitor, blocked the RGC protection. SIRT1 is expressed in virtually all cell types and localizes to cell nuclei,41
raising a concern that the use of a SIRT1 inhibitor may have significant toxicity. Fortunately, at the concentration used, sirtinol did not have any detectable retinal toxicity, similar to the SIRT1 activators.
Specific substrates of SIRT1 mediating RGC survival are not yet known. Future examination of specific substrates will be useful to identify focused downstream targets for potential therapeutic intervention. The sirtuin family of deacetylases, including SIRT1, is known to act on proteins involved in apoptosis, such as p5342,43
and on structural proteins, including α
alteration of which may lead to axonal degeneration. Apoptosis has been shown previously to mediate RGC loss in a chronic EAE optic neuritis model16
and in a spontaneous optic neuritis model,17
and we have found similar apoptotic cell death in relapsing EAE optic neuritis (unpublished observations, 2006), making apoptotic proteins likely candidates for the downstream targets of SIRT1-mediated RGC neuroprotection. Alternatively, SIRT1 regulation of cellular stress responses may mediate neuroprotection because mitochondrial oxidative stress is known to induce neuronal damage in experimental optic neuritis.46
Interestingly, SRT501 exerts significant neuroprotective effects at day 11, but not at day 9, despite the fact that some optic nerve inflammation begins by day 9.15
This suggests that the molecular pathways underlying RGC loss in this model are not yet activated or present for SIRT1 to act on until a couple of days after inflammation develops.
Optic neuritis and other MS lesions are characterized by inflammation and demyelination.1,2
Current therapies include immunosuppression with steroids and immunomodulation with interferon beta and glatiramer acetate.1
These treatments target the inflammatory component but have limited efficacy in preventing relapse or progression of disease. In addition, evidence is limited to suggest immunomodulatory therapies prevent neuronal damage and long-term disability in MS patients.10,11
Although recently described immunomodulatory therapy with natalizumab alone or in combination with interferon beta did reduce the progression of disability in MS patients,47,48
effects were not complete, and adverse effects might have limited the use of this combination therapy. In EAE rats, glatiramer acetate had neuroprotective effects for RGCs when given before optic neuritis developed, but no effect was found when treatment was started at the onset of disease.49
Neuroprotective therapies that work through nonimmunomodulatory mechanisms may have tremendous clinical benefits. The neuroprotective effects of SRT647 and SRT501 occurred without suppression of the inflammatory infiltration of the optic nerve because incidence of optic neuritis did not differ between placebo and treatment groups and was similar to the previously reported incidence.15
In addition, localized treatment with intravitreal injections did not reduce spinal cord inflammation, as evidenced by the lack of effect on clinical EAE score. The ability of SIRT1 to prevent RGC loss without reducing inflammation suggested that the therapeutic effects of SIRT1 activators had a strong potential to be additive or synergistic to disease-modifying effects of current immunomodulatory therapies. Future studies will evaluate the effects of immunomodulatory therapies in optic neuritis in combination with SIRT1 activators. Synergistic effects of two mechanistically distinct immunomodulatory therapies have been shown in EAE,50
demonstrating the usefulness of studying combination therapies in this MS model.
The potent neuroprotective effects of SIRT1 activators suggest an important potential therapy for optic neuritis and probably for other diseases leading to RGC death, such as ischemic, toxic, and traumatic optic neuropathies and glaucoma. SIRT1 activators did not reduce inflammation; hence, they may be neuroprotective in these noninflammatory-mediated optic nerve diseases. Neuroprotective effects of SIRT1 activation have been demonstrated in other noninflammatory neurologic disease models. SIRT1 activity reduces neuronal death induced by the Huntington gene mutation32
and prevents degeneration of axotomized dorsal root ganglion neurons.31
The current route of administration of SRT501 and SRT647, by intravitreal injection, may be useful for optic nerve insults that have a limited time course and treatment period, such as optic neuritis and ischemic optic neuropathy, though less invasive routes would be preferred. For chronic RGC loss in diseases such as glaucoma, topical or oral formulations with ocular penetration would be ideal; such formulations are being developed.
SIRT1 activation also has great potential for preventing neuronal loss throughout the central nervous system in patients with MS. Clinical trials that include patients with optic neuritis have helped shape the current clinical practice patterns for use of steroids and immunomodulators in MS.51–53
Development of SIRT1 activators with good systemic bioavailability will likely lead to new MS therapies that prevent long-term disability. Future studies will examine the intraocular penetration and potential neuroprotection of systemic SIRT1-activating drugs.
Overall, the current studies demonstrate that SIRT1 activation prevents RGC loss in optic neuritis through SIRT1 family enzymatic activity. SIRT1 activators are well tolerated without RGC toxicity and provide neuroprotection even in the presence of active inflammation. SRT501 and other SIRT1 activators have important therapeutic potential for preventing permanent neuronal loss and disability from optic neuritis and MS and may have a therapeutic role in preventing vision loss in other optic nerve diseases.