Beltran, William

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  • Publication
    Age-Dependent Disease Expression Determines Remodeling of the Retinal Mosaic in Carriers of RPGR Exon ORFn15 Mutations
    (2009-08-01) Beltran, William; Aguirre, Gustavo D; Acland, Gregory M
    PURPOSE. To characterize the retinal histopathology in carriers of X-linked progressive retinal atrophy (XLPRA1 and XLPRA2), two canine models of X-linked retinitis pigmentosa caused, respectively, by a stop and a frameshift mutation in RPGRORF15. METHODS. Retinas of XLPRA2 and XLPRA1 carriers of different ages were processed for morphologic evaluation, TUNEL assay, and immunohistochemistry. Cell-specific markers were used to examine retinal remodeling events. RESULTS. A mosaic pattern composed of patches of diseased and normal retina was first detected in XLPRA2 carriers at 4.9 weeks of age. A peak of photoreceptor cell death led to focal rod loss; however, in these patches an increased density of cones was found to persist over time. Patches of disease gradually disappeared so that by 39 weeks of age the overall retinal morphology, albeit thinner, had improved lamination. In older XLPRA2 carriers (≥8.8 years), extended regions of severe degeneration occurred in the peripheral/mid-peripheral retina. In XLPRA1 carriers, opsin mislocalization and rare events of rod death were detected by TUNEL assay at 20 weeks of age; however, only patchy degeneration was seen by 1.4 years and was still apparent at 7.8 years. CONCLUSIONS. The time of onset and the progression of the disease differed between the two models. In the early-onset form (XLPRA2) the morphologic appearance of the retinal mosaic changed as a function of age, suggesting that structural plasticity persists in the early postnatal canine retina as mutant photoreceptors die. In the late-onset form (XLPRA1), patches of disease persisted until later ages.
  • Publication
    Acute and Protracted Cell Death in Light-Induced Retinal Degeneration in the Canine Model of Rhodopsin Autosomal Dominant Retinitis Pigmentosa
    (2017-01-01) Sudharsan, Raghavi; Aguirre, Gustavo D; Simone, Kristina M; Beltran, William A; Anderson, Nathan P
    Purpose To characterize a light damage paradigm and establish structural and immunocytochemical measures of acute and protracted light-induced retinal degeneration in the rhodopsin (RHO) T4R dog model of RHO–autosomal dominant retinitis pigmentosa (ADRP). Methods Retinal light damage was induced in mutant dogs with a 1-minute exposure to various light intensities (0.1–1.0 mW/cm2) delivered with a Ganzfeld stimulator, or by fundus photography. Photoreceptor cell death was assessed by TUNEL assay, and alterations in retinal layers were examined by histology and immunohistochemistry 24 hours and 2 weeks after light exposure. Detailed topographic maps were made to document changes in the outer retinal layers of all four retinal quadrants 2 weeks post exposure. Results Twenty-four hours post light exposure, the severity of photoreceptor cell death was dose dependent. Immunohistochemical analysis revealed disruption of rod outer segments, focal loss of the RPE integrity, and an increase in expression of endothelin receptor B in Müller cells with the two highest doses of light and fundus photography. Two weeks after light exposure, persistence of photoreceptor death, thinning of the outer nuclear layer, and induction of Müller cell gliosis occurred with the highest doses of light. Conclusions We have characterized outcome measures of acute and continuing retinal degeneration in the RHO T4R dog following light exposure. These will be used to assess the molecular mechanisms of light-induced damage and rescue strategies in this large animal model of RHO-ADRP.
  • Publication
    Steroids Do Not Prevent Photoreceptor Degeneration in the Light-Exposed T4R Rhodopsin Mutant Dog Retina Irrespective of AP-1 Inhibition
    (2009-07-01) Beltran, William; Aguirre, Gustavo D; Gu, Danian; Pearce-Kelling, Sue; Li, Zexiao; Acland, Gregory M
    PURPOSE. AP-1 has been proposed as a key intermediate linking exposure to light and photoreceptor cell death in rodent light-damage models. Inhibition of AP-1 associated with steroid administration also prevents light damage. In this study the role of steroids in inhibiting AP-1 activation and/or in preventing photoreceptor degeneration was examined in the rhodopsin mutant dog model. METHODS. The dogs were dark adapted overnight, eyes dilated with mydriatics; the right eye was light occluded and the fundus of the left eye photographed (∼15–17 overlapping frames) with a fundus camera. For biochemical studies, the dogs remained in the dark for 1 to 3 hours after exposure. Twenty-four hours before exposure to light, some dogs were treated with systemic dexamethasone or intravitreal/subconjunctival triamcinolone. AP-1 DNA-binding activity was determined by electrophoresis mobility shift assay (EMSA) and phosphorylation of c-Fos and activation of ERK1/2 were determined by immunoblot analyses. The eyes were collected 1 hour and 2 weeks after exposure to light, for histopathology and immunocytochemistry. RESULTS. Inhibition of AP-1 activation, and phosphorylation of ERK1/2 and c-Fos were found after dexamethasone treatment in light-exposed T4R RHO mutant dog retinas. In contrast, increased AP-1 activity and phosphorylation of c-Fos and ERK1/2 were found in triamcinolone-treated mutant retinas. Similar extensive rod degeneration was found after exposure to light with or without treatment, and areas with surviving photoreceptor nuclei consisted primarily of cones. Only with systemic dexamethasone did the RPE cell layer remain. CONCLUSIONS. Intraocular or systemic steroids fail to prevent light-induced photoreceptor degeneration in the T4R RHO dog retina. Finding that systemic dexamethasone prevents AP-1 activation, yet does not prevent retinal light damage, further supports the hypothesis that AP-1 is not the critical player in the cell-death signal that occurs in rods.
  • Publication
    Intravitreal Injection of Ciliary Neurotrophic Factor (CNTF) Causes Peripheral Remodeling and Does Not Prevent Photoreceptor Loss in Canine RPGR Mutant Retina
    (2007-04-01) Beltran, William; Aguirre, Gustavo D; Wen, Rong; Acland, Gregory M
    Ciliary neurotrophic factor (CNTF) rescues photoreceptors in several animal models of retinal degeneration and is currently being evaluated as a potential treatment for retinitis pigmentosa in humans. This study was conducted to test whether CNTF prevents photoreceptor cell loss in XLPRA2, an early onset canine model of X-linked retinitis pigmentosa caused by a frameshift mutation in RPGR exon ORF15. Four different treatment regimens of CNTF were tested in XLPRA2 dogs. Under anesthesia, the animals received at different ages an intravitreal injection of 12 μg of CNTF in the left eye. The right eye served as a control and was injected with a similar volume of phosphate buffered saline (PBS). Ocular examinations were performed regularly during the treatment periods. At termination, the dogs were euthanatized, eyes collected and the retinas were processed for embedding in optimal cutting temperature (OCT) medium. The outer nuclear layer (ONL) thickness was evaluated on H&E sections and values in both CNTF- and PBS-treated eyes were compared. Morphologic alterations in the peripheral retina were characterized by immunohistochemistry using cell-specific markers. Cell proliferation in the retinas was examined on semi-thin plastic sections, and by BrdU pulse-labeling and Ki67 immunohistochemistry on cryosections. All CNTF-treated eyes showed early clinical signs of corneal epitheliopathy, subcapsular cataracts and uveitis. No statistically significant difference in ONL thickness was seen between the CNTF- and PBS-injected eyes. Prominent retinal remodeling that consisted in an abnormal increase in the number of rods, and in misplacement of some rods, cones, bipolar and Müller cells, was observed in the peripheral retina of CNTF-treated eyes. This was only seen when CNTF was in injected before the age at which the canine retina reaches full maturation. In XLPRA2 dogs, intravitreal injections of CNTF failed to prevent photoreceptors from undergoing cell death in the central and mid-peripheral retina. CNTF also caused ocular side-effects and morphologic alterations in the periphery that were consistent with cell dedifferentiation and proliferation. Our findings suggest that some inherited forms of retinal degeneration may not respond to CNTF's neuroprotective effects.
  • Publication
    Assessment of Visual Function and Retinal Structure Following Acute Light Exposure in the Light Sensitive T4R Rhodopsin Mutant Dog
    (2016-05-01) Iwabe, Simone; Aguirre, Gustavo D; Beltran, William A; Ying, Gui-shuang
    The effect of acute exposure to various intensities of white light on visual behavior and retinal structure was evaluated in the T4R RHO dog, a naturally-occurring model of autosomal dominant retinitis pigmentosa due to a mutation in the Rhodopsin gene. A total of 14 dogs (ages: 4–5.5 months) were used in this study: 3 homozygous mutant RHOT4R/T4R, 8 heterozygous mutant RHOT4R/+, and 3 normal wild-type (WT) dogs. Following overnight dark adaptation, the left eyes were acutely exposed to bright white light with a monocular Ganzfeld dome, while the contralateral right eye was shielded. Each of the 3 homozygous (RHOT4R/T4R) mutant dogs had a single unilateral light exposure (LE) to a different (low, moderate, and high) dose of white light (corneal irradiance/illuminance: 0.1 mW/cm2 , 170 lux; 0.5 mW/cm2 , 820 lux; or 1 mW/cm2 , 1590 lux) for 1min. All 8 heterozygous (RHOT4R/+) mutant dogs were exposed once to the same moderate dose of light. The 3 WT dogs had their left eyes exposed 1, 2, or 3 times to the same highest dose of light. Visual function prior to LE and at 2 weeks and 33 weeks after exposure was objectively assessed in the RHOT4R/T4R and WT dogs by using an obstacle-avoidance course. Transit time through the obstacle course was measured under different scotopic to photopic ambient illuminations. Morphological retinal changes were evaluated by non-invasive in vivo cSLO/sdOCT imaging and histology before and at several time-points (2–36 weeks) after light exposure. The analysis of the transit time through the obstacle course showed that no differences were observed in any of mutant or WT dogs at 2 weeks and 33 weeks post LE. The RHOT4R/T4R retina exposed to the lowest dose of white light showed no obvious changes in ONL thickness at 2 weeks, but mild decrease was noted 36 weeks after LE. The RHOT4R/T4R retina that received a moderate dose (showed an obvious decrease in ONL thickness along the superior and temporal meridians at 2 weeks post LE with more severe damage at 36 weeks post LE in all four meridians. The RHOT4R/T4R retina exposed to the high dose showed at 2 weeks after LE extensive ONL damage in all four meridians. This light intensity did not cause any retinal damage in WT dogs even after repeated (up to 3) LE. Analysis of ONL thickness in heterozygous mutant dogs exposed to the moderate dose of light confirmed the increased sensitivity to light damage of the superior/ tapetal retina, and the occurrence of an ongoing cell death process several weeks after the acute LE. In conclusion, a short single exposure to a dose of white light that is not retinotoxic in WT dogs causes in the T4R RHO retina an acute loss of ONL in the central to mid peripheral region that keeps progressing over the course of several weeks. However, this severe retinal damage does not affect visual behavior presumably because of islands of surviving photoreceptors found in the area centralis including the newly discovered canine fovea-like area, and the lack of damage to peripheral photoreceptors.
  • Publication
    Exclusion of the Unfolded Protein Response in Light-Induced Retinal Degeneration in the Canine T4R RHO Model of Autosomal Dominant Retinitis Pigmentosa
    (2015-02-19) Genini, Sem; Aguirre, Gustavo D; Marsilli, Stefania; Beltran, William; Sudharsan, Raghavi; Gingrich, Jeremy
    Purpose To examine the occurrence of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) following acute light damage in the naturally-occurring canine model of RHO-adRP (T4R RHOdog). Methods The left eyes of T4R RHOdogs were briefly light-exposed and retinas collected 3, 6 and 24 hours later. The contra-lateral eyes were shielded and used as controls. To evaluate the time course of cell death, histology and TUNEL assays were performed. Electron microscopy was used to examine ultrastructural alterations in photoreceptors at 15 min, 1 hour, and 6 hours after light exposure. Gene expression of markers of ER stress and UPR were assessed by RT-PCR, qRT-PCR and western blot at the 6 hour time-point. Calpain and caspase-3 activation were assessed at 1, 3 and 6 hours after exposure. Results A brief exposure to clinically-relevant levels of white light causes within minutes acute disruption of the rod outer segment disc membranes, followed by prominent ultrastructural alterations in the inner segments and the initiation of cell death by 6 hours. Activation of the PERK and IRE1 pathways, and downstream targets (BIP, CHOP) of the UPR was not observed. However increased transcription of caspase-12 and hsp70 occurred, as well as calpain activation, but not that of caspase-3. Conclusion The UPR is not activated in the early phase of light-induced photoreceptor cell death in the T4R RHO model. Instead, disruption in rods of disc and plasma membranes within minutes after light exposure followed by increase in calpain activity and caspase-12 expression suggests a different mechanism of degeneration.
  • Publication
    rAAV2/5 Gene-Targeting to Rods: Dose-Dependent Efficiency and Complications Associated With Different Promoters
    (2010-09-01) Beltran, William; Aguirre, Gustavo D; Boye, Sanford L; Boye, Shannon E; Chiodo, Vince A; Lewin, Alfred S; Hauswirth, William W
    A prerequisite for using corrective gene therapy to treat humans with inherited retinal degenerative diseases that primarily affect rods is to develop viral vectors that target specifically this population of photoreceptors. The delivery of a viral vector with photoreceptor tropism coupled with a rod-specific promoter is likely to be the safest and most efficient approach to target expression of the therapeutic gene to rods. Three promoters that included a fragment of the proximal mouse opsin promoter (mOP), the human G-protein-coupled receptor protein kinase 1 promoter (hGRK1), or the cytomegalovirus immediate early enhancer combined with the chicken β actin proximal promoter CBA were evaluated for their specificity and robustness in driving GFP reporter gene expression in rods, when packaged in a recombinant adeno-associated viral vector of serotype 2/5 (AAV2/5), and delivered via subretinal injection to the normal canine retina. Photoreceptor-specific promoters (mOP, hGRK1) targeted robust GFP expression to rods, whereas the ubiquitously expressed CBA promoter led to transgene expression in the retinal pigment epithelium, rods, cones and rare Müller, horizontal and ganglion cells. Late onset inflammation was frequently observed both clinically and histologically with all three constructs when the highest viral titers were injected. Cone loss in the injected regions of the retinas that received the highest titers occurred with both the hGRK1 and CBA promoters. Efficient and specific rod transduction, together with preservation of retinal structure was achieved with both mOP and hGRK1 promoters when viral titers in the order of 1011 vg ml–1 were used.
  • Publication
    Development and Validation of a Canine-Specific Profiling Array to Examine Expression of Pro-Apoptotic and Pro-Survival Genes in Retinal Degenerative Diseases
    (2012-01-01) Genini, Sem; Beltran, William
    We developed an expression profiling array to examine pro-apoptotic and pro-survival genes in dog retinal degeneration models. Gene-specific canine TaqMan assays were developed and included in a custom real-time quantitative reverse transcription-PCR (qRT-PCR) array. Of the 96 selected genes, 93 belonged to known relevant pro-apoptotic and pro-survival pathways, and/or were positive controls expressed in retina, while three were housekeeping genes. Ingenuity Pathway Analysis (IPA) showed that the selected genes belonged to expected biological functions (cell death, cell-mediated immune response, cellular development, function, and maintenance) and pathways (death receptor signaling, apoptosis, TNFR1 signaling, and induction of apoptosis by HIV1). Validation of the profiling array was performed with RNA extracted from cultured MDCK cells in the presence or absence of treatment with 10 μM staurosporin for 5 or 10 h. The vast majority of the genes showed positive amplifications, and a number of them also had fold change (FC) differences > ±3 between control and staurosporin-treated cells. To conclude, we established a profiling array that will be used to identify differentially expressed genes associated with photoreceptor death or survival in canine models of retinal degenerative diseases with mutations in genes that cause human inherited blindness with comparable phenotypes.
  • Publication
    Immunolocalization of Ciliary Neurotrophic Factor Receptor α (CNTFRα) in Mammalian Photoreceptor Cells
    (2005-04-01) Beltran, William; Aguirre, Gustavo D; Rohrer, Hermann
    PURPOSE: To characterize the site of expression of the α subunit of the receptor for ciliary neurotrophic factor (CNTFRα) in the retina of a variety of mammalian species, and determine whether CNTFRα is localized to photoreceptor cells. METHODS: The cellular distribution of CNTFRα (protein) was examined by immunocytochemistry in the adult retinas of several mammalian species that included mouse, rat, dog, cat, sheep, pig, horse, monkey, and human. Developing retinas from 3-day-old and 6-day-old rats were also included in this study. The molecular weight of CNTFRα in rat, dog, cat, pig, and human retinas was determined by immunoblotting. RESULTS: CNTFRα immunolabeling was present in the retina of all species. A common pattern was observed in all species, and represented labeling of the nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL). CNTFRα did not immunolocalize to photoreceptor cells in both adult and developing rodent retinas, but was consistently observed in both rods and cones of non-rodent species. The molecular weight of CNTFRα in mammalian retinas was approximately 61-64 kDa. CONCLUSIONS: These findings highlight a significant difference in the expression of CNTFRα in the retina of rodent and non-rodent mammalian species. The expression of CNTFRα by rods and cones in non-rodent species may suggest a direct mechanism of action if CNTF administration results in photoreceptor rescue.
  • Publication
    Successful Arrest of Photoreceptor and Vision Loss Expands the Therapeutic Window of Retinal Gene Therapy to Later Stages of Disease
    (2015-10-27) Beltran, William A; Cideciyan, Artur V; Iwabe, Simone; Swider, Malgorzata; Kosyk, Mychajlo S; McDaid, Kendra S; Martynyuk, Inna; Ying, Gui-shuang; Shaffer, James; Deng, Wen-Tao; Jacobson, Samuel G; Boye, Sanford L; Lewin, Alfred S; Hauswirth, William W
    Inherited retinal degenerations cause progressive loss of photoreceptor neurons with eventual blindness. Corrective or neuroprotective gene therapies under development could be delivered at a predegeneration stage to prevent the onset of disease, as well as at intermediate-degeneration stages to slow the rate of progression. Most preclinical gene therapy successes to date have been as predegeneration interventions. In many animal models, as well as in human studies, to date, retinal gene therapy administered well after the onset of degeneration was not able to modify the rate of progression even when successfully reversing dysfunction. We evaluated consequences of gene therapy delivered at intermediate stages of disease in a canine model of X-linked retinitis pigmentosa (XLRP) caused by a mutation in the Retinitis Pigmentosa GTPase Regulator (RPGR) gene. Spatiotemporal natural history of disease was defined and therapeutic dose selected based on predegeneration results. Then interventions were timed at earlier and later phases of intermediate-stage disease, and photoreceptor degeneration monitored with noninvasive imaging, electrophysiological function, and visual behavior for more than 2 y. All parameters showed substantial and significant arrest of the progressive time course of disease with treatment, which resulted in long-term improved retinal function and visual behavior compared with control eyes. Histology confirmed that the human RPGR transgene was stably expressed in photoreceptors and associated with improved structural preservation of rods, cones, and ON bipolar cells together with correction of opsin mislocalization. These findings in a clinically relevant large animal model demonstrate the long-term efficacy of RPGR gene augmentation and substantially broaden the therapeutic window for intervention in patients with RPGR-XLRP.