Zhiqian Dong

Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye

Two-photon excitation microscopy can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in subcellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all-trans-retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here, we report repetitive, dynamic imaging of these compounds in live mice through the pupil of the eye. By leveraging advanced adaptive optics, we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium by their characteristic localization, spectral properties and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light- and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions.Two-photon excitation microscopy (TPM) can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in sub-cellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all–trans–retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here we report repetitive, dynamic imaging of these compounds in live mice, through the pupil of the eye. Leveraging advanced adaptive optics we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium (RPE) by their characteristic localization, spectral properties, and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions.

Systems pharmacology identifies drug targets for Stargardt disease-Associated retinal degeneration

A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.

Two-photon microscopy reveals early rod photoreceptor cell damage in light-exposed mutant mice

Significance Identifying the sequence of events underlying light-induced pathology is important for understanding the mechanisms leading to retinal degeneration, and consequently for development of therapies against retinal diseases. In this study, we characterized the early phase of retinal degeneration using two-photon microscopy, mass spectroscopy, and genetically modified mice. We identified rod photoreceptors as the initial locus of degeneration. Primary changes included retinoid-dependent formation of fluorescent metabolic by-products within rod photoreceptor cells and a nearly three-fold expansion/swelling of rod outer segments. These changes were followed by secondary infiltration of microglia/macrophages to clear photoreceptor cell debris. Finally, we provide evidence that phagocytosis-mediated transfer of rod-derived toxic compounds to the retinal pigmented epithelium is required to elicit damage to that cell layer. Atrophic age-related and juvenile macular degeneration are especially devastating due to lack of an effective cure. Two retinal cell types, photoreceptor cells and the adjacent retinal pigmented epithelium (RPE), reportedly display the earliest pathological changes. Abca4−/−Rdh8−/− mice, which mimic many features of human retinal degeneration, allowed us to determine the sequence of light-induced events leading to retinal degeneration. Using two-photon microscopy with 3D reconstruction methodology, we observed an initial strong retinoid-derived fluorescence and expansion of Abca4−/−Rdh8−/− mouse rod cell outer segments accompanied by macrophage infiltration after brief exposure of the retina to bright light. Additionally, light-dependent fluorescent compounds produced in rod outer segments were not transferred to the RPE of mice genetically defective in RPE phagocytosis. Collectively, these findings suggest that for light-induced retinopathies in mice, rod photoreceptors are the primary site of toxic retinoid accumulation and degeneration, followed by secondary changes in the RPE.

QLT091001, a 9-cis-Retinal Analog, Is Well-Tolerated by Retinas of Mice with Impaired Visual Cycles

PURPOSE Investigate whether retinas of mice with impaired retinal cycles exposed to light or kept in the dark tolerate prolonged high-dose administration of QLT091001, which contains as an active ingredient, the 9-cis-retinal precursor, 9-cis-retinyl acetate. METHODS Four- to six-week-old Lrat(-/-) and Rpe65(-/-) mice (n = 126) as well as crossbred Gnat1(-/-) mice lacking rod phototransduction (n = 110) were gavaged weekly for 6 months with 50 mg/kg QLT091001, either after being kept in the dark or after light bleaching for 30 min/wk followed by maintenance in a 12-hour light ≤ 10 lux)/12-hour dark cycle. Retinal health was monitored by spectral-domain optical coherent tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) every other month and histological, biochemical, and visual functional analyses were performed at the end of the experiment. Two-photon microscopy (TPM) was used to observe retinoid-containing retinosome structures in the RPE. RESULTS Retinal thickness and morphology examined by SD-OCT were well maintained in all strains treated with QLT091001. No significant increases of fundus autofluorescence were detected by SLO imaging of any strain. Accumulation of all-trans-retinyl esters varied with genetic background, types of administered compounds and lighting conditions but retinal health was not compromised. TPM imaging clearly revealed maintenance of retinosomes in the RPE of all mouse strains tested. CONCLUSIONS Retinas of Lrat(-/-), Rpe65(-/-), and crossbred Gnat1(-/-) mice tolerated prolonged high-dose QLT091001 treatment well.

Molecular pharmacodynamics of emixustat in protection against retinal degeneration

Emixustat is a visual cycle modulator that has entered clinical trials as a treatment for age-related macular degeneration (AMD). This molecule has been proposed to inhibit the visual cycle isomerase RPE65, thereby slowing regeneration of 11-cis-retinal and reducing production of retinaldehyde condensation byproducts that may be involved in AMD pathology. Previously, we reported that all-trans-retinal (atRAL) is directly cytotoxic and that certain primary amine compounds that transiently sequester atRAL via Schiff base formation ameliorate retinal degeneration. Here, we have shown that emixustat stereoselectively inhibits RPE65 by direct active site binding. However, we detected the presence of emixustat-atRAL Schiff base conjugates, indicating that emixustat also acts as a retinal scavenger, which may contribute to its therapeutic effects. Using agents that lack either RPE65 inhibitory activity or the capacity to sequester atRAL, we assessed the relative importance of these 2 modes of action in protection against retinal phototoxicity in mice. The atRAL sequestrant QEA-B-001-NH2 conferred protection against phototoxicity without inhibiting RPE65, whereas an emixustat derivative incapable of atRAL sequestration was minimally protective, despite direct inhibition of RPE65. These data indicate that atRAL sequestration is an essential mechanism underlying the protective effects of emixustat and related compounds against retinal phototoxicity. Moreover, atRAL sequestration should be considered in the design of next-generation visual cycle modulators.

Heterologous Expression and Purification of the Serotonin Type 4 Receptor from Transgenic Mouse Retina

Recent breakthroughs in the solution of X-ray structures for G protein-coupled receptors (GPCRs) with diffusible ligands have employed extensively mutated or recombined receptor fusion proteins heterologously expressed in conventional in vitro cell-based systems. While these advances now show that crystallization of non-rhodopsin members of this superfamily can be accomplished, the use of radically modified proteins may limit the relevance of the derived structures for precision-guided drug design. To better enable the study of native GPCR structures, we report here efforts to engineer an in vivo expression system that harnesses the photoreceptor system of the retina to express heterologous GPCRs with native human sequences in a biochemically homogeneous and pharmacologically functional conformation. As an example, we show that the human 5HT4 receptor, when placed under the influence of the mouse opsin promoter and an opsin rod outer segment (ROS) targeting sequence, localized to ROS of transgenic mouse retina. The resulting receptor protein was uniformly glycosylated and pharmacologically intact as demonstrated by immunoblotting and radioligand binding assays. Upon solubilization, the retinal 5HT4 receptor retained the binding properties of its initial state in retinal membranes. With the engineered T7 monoclonal epitope sequence, the solubilized receptor was easily purified by one-step immunoaffinity chromatography and the purified receptor in detergent solution preserved its ligand binding properties. This expression method may prove generally useful for generating functional, high-quality GPCR protein.

Expansion of First-in-Class Drug Candidates That Sequester Toxic All-Trans-Retinal and Prevent Light-Induced Retinal Degeneration

All-trans-retinal, a retinoid metabolite naturally produced upon photoreceptor light activation, is cytotoxic when present at elevated levels in the retina. To lower its toxicity, two experimentally validated methods have been developed involving inhibition of the retinoid cycle and sequestration of excess of all-trans-retinal by drugs containing a primary amine group. We identified the first-in-class drug candidates that transiently sequester this metabolite or slow down its production by inhibiting regeneration of the visual chromophore, 11-cis-retinal. Two enzymes are critical for retinoid recycling in the eye. Lecithin:retinol acyltransferase (LRAT) is the enzyme that traps vitamin A (all-trans-retinol) from the circulation and photoreceptor cells to produce the esterified substrate for retinoid isomerase (RPE65), which converts all-trans-retinyl ester into 11-cis-retinol. Here we investigated retinylamine and its derivatives to assess their inhibitor/substrate specificities for RPE65 and LRAT, mechanisms of action, potency, retention in the eye, and protection against acute light-induced retinal degeneration in mice. We correlated levels of visual cycle inhibition with retinal protective effects and outlined chemical boundaries for LRAT substrates and RPE65 inhibitors to obtain critical insights into therapeutic properties needed for retinal preservation.

Synergistically acting agonists and antagonists of G protein–coupled receptors prevent photoreceptor cell degeneration

Systems pharmacology reveals a combination of GPCR-targeted drugs that prevent retinal degeneration. Preventing blindness Loss of photoreceptors in the retina results in visual impairment and eventually blindness. Light can damage the retina through processes that involve G protein–coupled receptors (GPCRs). Chen et al. took a systems pharmacology approach to identify combinations of drugs that activate or inhibit specific GPCRs to prevent light-induced retinal damage in a mouse model of progressive retinal degeneration. This approach identified a photoreceptor-protecting combination of FDA-approved drugs that activated the Gi/o-coupled dopamine receptors D2R and D4R, inhibited the Gs-coupled dopamine receptor D1R, and inhibited the Gq-coupled α1A-adrenergic receptor. This study not only provides a potential therapeutic strategy for preventing blindness due to retinal degeneration but also suggests that systems pharmacology approaches could lead to the discovery of new combinations of available drugs to promote therapeutic changes in signaling networks. Photoreceptor cell degeneration leads to visual impairment and blindness in several types of retinal disease. However, the discovery of safe and effective therapeutic strategies conferring photoreceptor cell protection remains challenging. Targeting distinct cellular pathways with low doses of different drugs that produce a functionally synergistic effect could provide a strategy for preventing or treating retinal dystrophies. We took a systems pharmacology approach to identify potential combination therapies using a mouse model of light-induced retinal degeneration. We showed that a combination of U.S. Food and Drug Administration–approved drugs that act on different G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) exhibited synergistic activity that protected retinas from light-induced degeneration even when each drug was administered at a low dose. In functional assays, the combined effects of these drugs were stimulation of Gi/o signaling by activating the dopamine receptors D2R and D4R, as well as inhibition of Gs and Gq signaling by antagonizing D1R and the α1A-adrenergic receptor ADRA1A, respectively. Moreover, transcriptome analyses demonstrated that such combined GPCR-targeted treatments preserved patterns of retinal gene expression that were more similar to those of the normal retina than did higher-dose monotherapy. Our study thus supports a systems pharmacology approach to identify treatments for retinopathies, an approach that could extend to other complex disorders.

Receptor MER Tyrosine Kinase Proto-oncogene (MERTK) Is Not Required for Transfer of Bis-retinoids to the Retinal Pigmented Epithelium

Accumulation of bis-retinoids in the retinal pigmented epithelium (RPE) is a hallmark of aging and retinal disorders such as Stargardt disease and age-related macular degeneration. These aberrant fluorescent condensation products, including di-retinoid-pyridinium-ethanolamine (A2E), are thought to be transferred to RPE cells primarily through phagocytosis of the photoreceptor outer segments. However, we observed by two-photon microscopy that mouse retinas incapable of phagocytosis due to a deficiency of the c-Mer proto-oncogene tyrosine kinase (Mertk) nonetheless contained fluorescent retinoid condensation material in their RPE. Primary RPE cells from Mertk−/− mice also accumulated fluorescent products in vitro. Finally, quantification of A2E demonstrated the acquisition of retinal condensation products in Mertk−/− mouse RPE prior to retinal degeneration. In these mice, we identified activated microglial cells that likely were recruited to transport A2E-like condensation products to the RPE and dispose of the dying photoreceptor cells. These observations demonstrate a novel transport mechanism between photoreceptor cells and RPE that does not involve canonical Mertk-dependent phagocytosis.

Serum levels of lipid metabolites in age-related macular degeneration

Age‐related macular degeneration (AMD) is a neurodegenerative disease that causes adult‐onset blindness. There are 2 forms of this progressive disease: wet and dry. Currently there is no cure for AMD, but several treatment options have started to emerge making early detection critical for therapeutic success. Analysis of the eyes of Abca4‐/‐Rdh8‐/‐ mice that display light‐induced retinal degeneration indicates that 11‐cis‐retinal and docosahexaenoic acid (DHA) levels were significantly decreased as compared with the eyes of control dark‐adapted C57BL/6J mice. In addition, exposure to intense light correlated with higher levels of prostaglandin G2 in the eyes of Abca4‐/‐Rdh8‐/‐ mice. Intense light exposure also lowered DHA levels in the eyes of wild‐type C57BL/6J mice without discernible retinal degeneration. Analysis of human serum from patients with AMD recapitulated these dysregulated DHA levels and revealed dysregulation of arachidonic acid (AA) levels as well (∼32% increase in patients with AMD compared with average levels in healthy individuals). From these observations, we then built a statistical model that included levels of DHA and AA from human serum. This model had a 74% probability of correctly identifying patients with AMD from controls. Addition of a genetic analysis for one of the most prevalent amino acid substitutions in the age‐related maculopathy susceptibility 2 gene linked to AMD, Ala69→Ser, did not improve the statistical model. Thus, we have characterized a reliable method with the potential to detect AMD without a genetic component, paving the way for a larger‐scale clinical evaluation. Our studies on mouse models along with the analysis of human serum suggest that our small molecule‐based model may serve as an effective tool to estimate the risk of developing AMD.—Orban, T., Johnson, W. M., Dong, Z., Maeda, T., Maeda, A, Sakai, T., Tsuneoka, H., Mieyal, J. J., Palczewski, K. Serum levels of lipid metabolites in age‐related macular degeneration. FASEB J. 29, 4579‐4588 (2015). www.fasebj.org