Retinal pigment epithelium defects in humans and mice with mutations in MYO7A: imaging melanosome-specific autofluorescence.

Vision loss in human hereditary retinal degenerations (RDs) is caused mainly by mutations in genes expressed in the photoreceptors, the retinal pigment epithelium (RPE), or both. In the Usher syndromes (USH), mutant genes also lead to inner ear dysfunction and cause debilitating bisensory disease. The success of human gene therapy in retinas of patients with LCA1–4 has paved the way for the application of this technique to other blinding disorders, such as the most common form of USH type 1, USH1B, which is caused by mutations in the myosin VIIa (MYO7A) gene.5,6 MYO7A is an unconventional myosin motor protein expressed both in retinal photoreceptors and the RPE.7,8 In photoreceptors, MYO7A appears to participate in opsin transport through the cilium, whereas in the RPE, one of its functions is in the movement of melanosomes. 9,10 Whether gene therapy of USH1B11,12 targets photoreceptors or RPE, or both, there is a need to monitor RPE-melanosome disease independent of photoreceptor disease and vision. RPE forms a thin monolayer between the photoreceptors and their blood supply, the choroid.13 Noninvasive evaluation of RPE health have depended on the lipofuscin granules found within these cells.14,15 Lipofuscin is a lipid and protein aggregate containing several distinct fluorophores that produce yellow-orange emission spectra when excited by blue light.14–16 Noninvasive imaging methods that take advantage of the autofluorescence (AF) of lipofuscin have enabled mapping and quantification of RPE disease and its progression in living human eyes with retinal degenerative diseases.15,17–20 However, conventional lipofuscin imaging involves bright short-wavelength illumination that may accelerate the natural history of RDs21 and possibly complicate outcomes of a clinical trial. Reduced short-wavelength illuminance methods are being developed. 20 RPE cells also contain abundant intracellular pigment granules, which correspond to the last stage of melanosome maturation—a process that starts early during gestation and is practically complete before 2 years of age in humans.22,23 Melanosomes in the RPE are thought to play a protective role by absorbing radiation, scavenging free radicals and quenching excited molecular states.22,24 Alternatively or additionally, melanosomes could mediate light-induced toxicity in the RPE.25 They may also contribute to protein degradation after the phagocytosis of outer segment disc membranes.22,26 Until recently, only indirect methods were available for imaging the abnormalities of melanin, based on absorption and scattering properties.27–30 Recently, a novel AF signal has been observed by noninvasive imaging of living human eyes with near-infrared (NIR) excitation light.20,31–38 It has been speculated that melanosomes contribute to the NIR-AF signal, but the validity of this hypothesis or the extent of the contribution from different fluorophores has not been defined. In the present study, AF imaging was used in USH1B patients and in Myo7a-null shaker1 mice. The results identify melanosomes of the RPE and choroid as the endogenous fluorophores that give rise to the NIR-AF signal and set the stage for further development of NIR-AF imaging to define and monitor subtle melanosome abnormalities in the RPE before and after therapeutic interventions.