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Maeda A.,Case Western Reserve University | Golczak M.,Case Western Reserve University | Chen Y.,Case Western Reserve University | Okano K.,Case Western Reserve University | And 7 more authors.
Nature Chemical Biology | Year: 2012

Vertebrate vision is initiated by photoisomerization of the visual pigment chromophore 11-cis-retinal and is maintained by continuous regeneration of this retinoid through a series of reactions termed the retinoid cycle. However, toxic side reaction products, especially those involving reactive aldehyde groups of the photoisomerized product, all-trans-retinal, can cause severe retinal pathology. Here we lowered peak concentrations of free all-trans-retinal with primary amine-containing Food and Drug Administration (FDA)-approved drugs that did not inhibit chromophore regeneration in mouse models of retinal degeneration. Schiff base adducts between all-trans-retinal and these amines were identified by MS. Adducts were observed in mouse eyes only when an experimental drug protected the retina from degeneration in both short-term and long-term treatment experiments. This study demonstrates a molecular basis of all-trans-retinal-induced retinal pathology and identifies an assemblage of FDA-approved compounds with protective effects against this pathology in a mouse model that shows features of Stargardt's disease and age-related retinal degeneration. © 2012 Nature America, Inc. All rights reserved.


Palczewska G.,Polgenix, Inc. | Maeda T.,Case Western Reserve University | Imanishi Y.,Case Western Reserve University | Sun W.,Polgenix, Inc. | And 6 more authors.
Nature Medicine | Year: 2010

Multiphoton excitation fluorescence microscopy (MPM) can image certain molecular processes in vivo. In the eye, fluorescent retinyl esters in subcellular structures called retinosomes mediate regeneration of the visual chromophore, 11-cis-retinal, by the visual cycle. But harmful fluorescent condensation products of retinoids also occur in the retina. We report that in wild-type mice, excitation with a wavelength of ∼730 nm identified retinosomes in the retinal pigment epithelium, and excitation with a wavelength of ∼910 nm revealed at least one additional retinal fluorophore. The latter fluorescence was absent in eyes of genetically modified mice lacking a functional visual cycle, but accentuated in eyes of older wild-type mice and mice with defective clearance of all-trans-retinal, an intermediate in the visual cycle. MPM, a noninvasive imaging modality that facilitates concurrent monitoring of retinosomes along with potentially harmful products in aging eyes, has the potential to detect early molecular changes due to age-related macular degeneration and other defects in retinoid metabolism. © 2010 Nature America, Inc. All rights reserved.


Palczewska G.,Polgenix, Inc. | Dong Z.,Polgenix, Inc. | Golczak M.,Case Western Reserve University | Hunter J.J.,Case Western Reserve University | And 5 more authors.
Nature Medicine | Year: 2014

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. © 2014 Nature America, Inc. All rights reserved.


Palczewska G.,Polgenix, Inc. | Golczak M.,Case Western Reserve University | Williams D.R.,University of Rochester | Hunter J.J.,University of Rochester | Palczewski K.,Case Western Reserve University
Investigative Ophthalmology and Visual Science | Year: 2014

Purpose. Noninvasive two-photon imaging of a living mammalian eye can reveal details of molecular processes in the retina and RPE. Retinyl esters and all-trans-retinal condensation products are two types of retinoid fluorophores present in these tissues. We measured the content of these two types of retinoids in monkey and human eyes to validate the potential of two-photon imaging for monitoring retinoid changes in human eyes. Methods. Two-photon microscopy (TPM) was used to visualize excised retina from monkey eyes. Retinoid composition and content in human and monkey eyes were quantified by HPLC and mass spectrometry (MS). Results. Clear images of inner and outer segments of rods and cones were obtained in primate eyes at different eccentricities. Fluorescence spectra from outer segments revealed a maximum emission at 480 nm indicative of retinols and their esters. In cynomolgus monkey and human retinal extracts, retinyl esters existed predominantly in the 11-cis configuration along with notable levels of 11-cis-retinol, a characteristic of cone-enriched retinas. Average amounts of di-retinoid-pyridinium-ethanolamine (A2E) in primate and human eyes were 160 and 225 pmol/eye, respectively. Conclusions. These data show that human retina contains sufficient amounts of retinoids for two-photon excitation imaging. Greater amounts of 11-cis-retinyl esters relative to rodent retinas contribute to the fluorescence signal from both monkey and human eyes. These observations indicate that TPM imaging found effective in mice could detect early age- and disease-related changes in human retina. © 2014 The Association for Research in Vision and Ophthalmology, Inc.


Padayatti P.,Polgenix, Inc. | Palczewska G.,Polgenix, Inc. | Sun W.,Polgenix, Inc. | Palczewski K.,Case Western Reserve University | Salom D.,Polgenix, Inc.
Biochemistry | Year: 2012

Second-order nonlinear optical imaging of chiral crystals (SONICC), which portrays second-harmonic generation (SHG) by noncentrosymmetric crystals, is emerging as a powerful imaging technique for protein crystals in media opaque to visible light because of its high signal-to-noise ratio. Here we report the incorporation of both SONICC and two-photon excited fluorescence (TPEF) into one imaging system that allows visualization of crystals as small as ∼10 μm in their longest dimension. Using this system, we then documented an inverse correlation between the level of symmetry in examined crystals and the intensity of their SHG. Moreover, because of blue-green TPEF exhibited by most tested protein crystals, we also could identify and image SHG-silent protein crystals. Our experimental data suggest that the TPEF in protein crystals is mainly caused by the oxidation of tryptophan residues. Additionally, we found that unspecific fluorescent dyes are able to bind to lysozyme crystals and enhance their detection by TPEF. We finally confirmed that the observed fluorescence was generated by a two-photon rather than a three-photon process. The capability for imaging small protein crystals in turbid or opaque media with nondamaging infrared light in a single system makes the combination of SHG and intrinsic visible TPEF a powerful tool for nondestructive protein crystal identification and characterization during crystallization trials. © 2012 American Chemical Society.


Orban T.,Case Western Reserve University | Palczewska G.,Polgenix, Inc. | Palczewski K.,Case Western Reserve University
Journal of Biological Chemistry | Year: 2011

Levels of many hydrophobic cellular substances are tightly regulated because of their potential cytotoxicity. These compounds tend to self-aggregate in cytoplasmic storage depots termed lipid droplets/bodies that have well defined structures that contain additional components, including cholesterol and various proteins. Hydrophobic substances in these structures become mobilized in a specific and regulated manner as dictated by cellular requirements. Retinal pigmented epithelial cells in the eye produce retinyl ester-containing lipid droplets named retinosomes. These esters are mobilized to replenish the visual chromophore, 11-cis-retinal, and their storage ensures proper visual function despite fluctuations in dietary vitamin A intake. But it remains unclear whether retinosomes are structures specific to the eye or similar to lipid droplets in other organs/tissues that contain substances other than retinyl esters. Thus, we initially investigated the production of these lipid droplets in experimental cell lines expressing lecithin:retinol acyltransferase, a key enzyme involved in formation of retinyl ester-containing retinosomes from all-trans-retinol. We found that retinosomes and oleate-derived lipid droplets form and co-localize concomitantly, indicating their intrinsic structural similarities. Next, we isolated native retinosomes from bovine retinal pigmented epithelium and found that their protein and hydrophobic small molecular constituents were similar to those of lipid droplets reported for other experimental cell lines and tissues. These unexpected findings suggest a common mechanism for lipid droplet formation that exhibits broad chemical specificity for the hydrophobic substances being stored. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.


Salom D.,Polgenix, Inc. | Padayatti P.S.,Polgenix, Inc. | Palczewski K.,Polgenix, Inc. | Palczewski K.,Case Western Reserve University
Methods in Cell Biology | Year: 2013

Oligomerization is one of several mechanisms that can regulate the activity of G protein-coupled receptors (GPCRs), but little is known about the structure of GPCR oligomers. Crystallography and NMR are the only methods able to reveal the details of receptor-receptor interactions at an atomic level, and several GPCR homodimers already have been described from crystal structures. Two clusters of symmetric interfaces have been identified from these structures that concur with biochemical data, one involving helices I, II, and VIII and the other formed mainly by helices V and VI. In this chapter, we describe the protocols used in our laboratory for the crystallization of rhodopsin and the β2-adrenergic receptor (β2-AR). For bovine rhodopsin, we developed a new purification strategy including a (NH4)2SO4-induced phase separation that proved essential to obtain crystals of photoactivated rhodopsin containing parallel dimers. Crystallization of native bovine rhodopsin was achieved by the classic vapor-diffusion technique. For β2-AR, we developed a purification strategy based on previously published protocols employing a lipidic cubic phase to obtain diffracting crystals of a β2-AR/T4-lysozyme chimera bound to the antagonist carazolol. © 2013 Elsevier Inc.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.87M | Year: 2012

DESCRIPTION (provided by applicant): Polgenix, Inc. is developing an imaging instrument, the 2-photon ophthalmoscope , or 2PO , with the capability for non-invasive, repetitive and high-resolution imaging of biochemical processes within human retina.This device will allow early detection of age- and disease- related changes in the eye, long before pathological manifestations of retinal disease become discernable by existing methods. Such real-time retinal imaging will also be critical for evaluationof various therapies for retinal pathology. Thus, patients susceptible to retinal disease could be diagnosed with 2PO and treated well before vision loss occurs. Once developed, the instrument will be used initially for ophthalmic drug screening and establishing objective biomarkers for retinal disease in animal models. Then it will be employed for clinical trials and ultimately for monitoring retinal health and the impact of therapy in patients. Early application of the device toward the development of effective ophthalmic therapies and clinical diagnosis will be leveraged through collaboration with experts in biochemical processes in the eye and high-resolution adaptive optics ophthalmic imaging from Case Western Reserve University and the University of Rochester, respectively. PUBLIC HEALTH RELEVANCE: We seek to develop a novel instrument for noninvasive imaging of the back of the eye with sub-cellular resolution. The instrument will visualize and quantify the age or disease related changes in the biochemical processes within human retina. Our goal is to further understanding of the biochemistry of vision and to enable rapid evaluation of the impact of therapeutic interventions aimed at retinal diseases at the earliest stages, before retina is damaged and vision is irreparably diminished.


Patent
Polgenix, Inc. | Date: 2015-02-05

A method of determining retinal degeneration of photoreceptors and/or the retinal pigment epithelium (RPE) of a subject includes measuring two-photon induced fluorescence inner and/or outer segments of the photoreceptor cells and/or retinal pigment epithelium to assess photoreceptor cell death and retinal pigment epithelium cell death or degeneration.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 188.13K | Year: 2010

DESCRIPTION (provided by applicant): Our long-term goal is to develop a clinical instrument, the 2-photon ophthalmoscope, for non-invasive, high-resolution and repetitive imaging of biochemical processes within the human retina. Such an instrument will have tremendous potential for early detection of age and disease related changes in the eye, long before pathological manifestations of retinal disease become obvious. This real-time retinal imaging instrument will also be critical for rapid evaluation of various pharmacological agents used to treat retinal pathologies. The method has the great advantage of imaging endogenous retinoid fluorophores in their native state without the need for additional staining. In Phase I, we seek to answer whether two-photon excitation based imaging can track age related changes in the retina and then whether a 2-photon ophthalmoscope, with the ultimate goal of clinical instrument can be made. We are proposing three specific aims: (1) Determine whether two-photon excitation imaging can be used to monitor age related changes in human retina; (2) Image the eye of a living monkey to determine the feasibility of two-photon adaptive optics ophthalmoscope system for non-invasive, in vivo imaging of human retinal pigment epithelium cells; and (3) Determine if the size and cost of the instrument could be reduced by replacing the Ti:Sapphire modelocked laser with a femtosecond fiber laser to validate commercialization plans. Once these aims are fulfilled, in Phase 2 we will use the derived data to adapt the two-photon adaptive optics ophthalmoscope for imaging human eyes in vivo and characterize two-photon fluorescence in eyes affected by retina diseases. In addition, the use of the fiber laser and micro-electro- mechanical systems deformable mirror technology, in adaptive optics design, promises to reduce the cost and allow the physical footprint of the instrument to be kept small, greatly aiding potential commercialization. PUBLIC HEALTH RELEVANCE: We seek to develop a novel instrument for noninvasive imaging of the back of the eye with sub-cellular resolution. The instrument will visualize the age or disease related changes in the biochemical processes within human retina, specifically retinoid cycle. Our goal is to further understanding of the biochemistry of vision to allow for rapid evaluation of various pharmacological interventions to prevent retinal degeneration and other pathologies at the early stages, before the retina degenerates and vision is irreparably damaged.

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