A Novel Therapeutic Strategy Targeting Photoreceptor Oxidative Damage in ABCR-related Retinal Degenerations

Research Plan Background Stargardt disease (STGD/FFM) is the most common hereditary recessive macular dystrophy (Blacharski, 1988) characterized by juvenile to young adult onset, central visual impairment, progressive bilateral atrophy of the macula and retinal pigment epithelium (RPE), with a frequent appearance of orange/yellow flecks distributed around the macula and/or the mid retinal periphery (Noble and Carr, 1971). A clinically similar retinal disorder, fundus flavimaculatus (FFM), often displays later ages of onset and slower progression. It has been suggested and demonstrated (Allikmets et al., 1997) that STGD and FFM represent allelic disorders. Mutations in the gene encoding an ATP-binding cassette (ABC) transporter (ABCR), mapping to chromosome 1p13-p21, have been found to be responsible of STGD (Allikmets et al., 1997). The ABCR gene is expressed exclusively and at high levels in the retina, in both rod and cone photoreceptors (Molday et al., 2000). A recent study by Weng et al. (1999) , investigating the molecular mechanisms underlying photoreceptor degeneration in ABCR knock-out mice, proposed that photoreceptors die as a consequence of 'poisoning' of the RPE by lipofuscin accumulation and loss of the RPE support role. Accumulation within the RPE cells of a compound, A2E, forming from condensation of phosphatydilethanlolamine and the all-trans-retinal released from photoactivated rhodopsin (Sparrow et al., 2000), probably leads in vivo to an increased absorption of blue lights and to phototoxic RPE cell damage. The mutation-induced disease may affect both rod and cone photoreceptors, at relatively early stages. In vitro studies (Sun and Nathans, 2001) also demonstrated that the ABCR itself is an efficient target of all-trans-retinal-mediated photooxidative damage.

Clinically, many reports have documented an abnormal functioning of both macular and peripheral cones, as well as rods, in STGD/FFM (Moloney et al., 1983; Lachapelle et al., 1990). There is also evidence (Lois et al., 2001) that STGD/FFM may be associated with different patterns of retinal dysfunction, with a selective involvement of macular function, or more widespread dysfunction involving cone and/or rod function, showing intrafamilial consistency. Characteristic abnormalities of dark adaptation (Aleman et al., 1999), involving a delayed post-bleach recovery, to the baseline sensitivity, of the last branch of the adaptation curve have been also described. Similar abnormalities in rod dark adaptation have been recently found in a mice heterozygous for a null mutation in the ABCR gene (Mata et al., 2001). Clinical evidence (Parisi et al., 2002) indicates that also the recovery of cone sensitivity after bleaching is severely impaired in STG/FF, suggesting that profoundly altered retinoid recycling, leading to photoxidative damage, specifically occurs in cone photoreceptors.

Recent experimental findings (Maccarone et al., 2008) indicate that Saffron, derived from the pistils of Crocus Sativus, may have a role as a retinal neuro-protectant against oxidative damage. Indeed, Saffron has been shown to be protective, for both morphology and function, in a rat model of light-induced photoreceptor degeneration. In this model, cell death is thought to result from oxidative stress induced by prolonged increase in oxygen tension and photooxidation. Saffron is an attractive candidate to be tested because the stigmata of Crocus sativus contain biologically high concentrations of interesting chemical compounds including crocin, crocetin (Giaccio, 2004), whose multiple C=C bonds give the antioxidant potential. Not to mention that its centuries-long use as spice, with no known ill effects increases the confidence in secure applicability. In addition it has been recently reported (Ochiai et al., 2007) that crocins are able to activate metabolic pathways to protect cells from apoptosis and to reduce light induced death in isolated photoreceptors (Laabich et al., 2006) , while crocetin (Giaccio, 2004) increases oxygen diffusivity through liquids, such as plasma. Considering the high metabolic rate of photoreceptors, the availability of oxygen may be a critical factor in protecting them from death. In addition, Kanakis et al. (Kanakis et al, 2007) showed that metabolites of antioxidant flavonoids bind directly to DNA and induce its partial conformation to beta-DNA, thereby protecting the cell from damage. Based on these observation it comes clear that Saffron extract does not act as a simple antioxidant. The peculiar characteristics of Saffron components support the hypothesis of an involvement of very different ways of action going from antioxidant activity to direct control of gene expression. These components may act in humans as protective agents against oxidative damage for the ageing retina, and may repair early photoreceptor damage associated with STG/FF, whose disease patho-physiology has been linked by experimental studies (Mata et al, 2001) to light-induced oxidative damage to the outer retina. In STG/FF eyes, at early disease stages, the normal number of cone photoreceptors is partially retained, although the cells might be dysfunctional. As a result of the rescuing effects of Saffron, the pool of damaged but viable photoreceptors could increase its response, resulting in improved retinal sensitivity.

The objective of the present project is to evaluate whether Saffron supplementation has a beneficial neuro-protective effect for the damaged retinas as consequence of ABCR mutation-STD/FF.

Clinical Protocol Patients A group of 30 STG/FF patients (14 males, 16 females, age range: 15-68 years) will be included in this study. Patients will meet the following inclusion criteria: 1. Macular and peripheral retinal degeneration with typical funduscopic lesions (retinal flecks) and a cone-rod pattern of retinal dysfunction, as determined by standard Ganzfeld electroretinography and dark-adapted fundus perimetry, and classic fundus appearance, 2. Relatively preserved central retinal function (visual field by Goldmann V/4e > 30°, corrected EDTRS visual acuity > 20/80) and stable central fixation as determined by a Visuskope, 3. Known genotype or genotype under study, 4. At least four follow-up clinical examination over the past three years, 5. No or minimal ocular media opacities, 6. No concomitant ocular (e.g. glaucoma, amblyopia) or systemic diseases. Informed consent for all patients and controls will be obtained after the aims and procedures of the study will be explained in detail.