Vera L Bonilha

Cleveland Clinic · Department of Opthalmic Research

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated cont...

Somatic mitochondrial DNA (mtDNA) mutation accumulation has been observed in individuals with retinal degenerative disorders. To study the effects of aging and mtDNA mutation accumulation in the retina, a Polymerase gamma (POLG) deficiency model, the POLGD257A mutator mice (PolgD257A), was used. POLG is an enzyme responsible for regulating mtDNA re...

The goal of this study is to identify key transcriptomic markers within layers of the retina by individually profiling layers using cellular and subcellular spatial transcriptomics, additionally, comparing the results between each level. Both human and mouse retina samples, prepared fresh frozen and fixed frozen, are analyzed using the GeoMx® Digit...

Ophthalmic imaging instruments, including the confocal scanning laser ophthalmoscope and spectral-domain optical coherence tomography system, originally intended for revealing ocular microstructures in the human eye, have been deployed by vision researchers to evaluate the eyes of numerous small and large animal species for more than two decades. I...

Heparan-α-glucosaminide N-acetyltransferase (HGSNAT) participates in lysosomal degradation of heparan sulfate. Mutations in the gene encoding this enzyme cause mucopolysaccharidosis IIIC (MPS IIIC) or Sanfilippo syndrome type C. MPS IIIC patients exhibit progressive neurodegeneration, leading to dementia and death in early adulthood. Currently ther...

The retina and RPE cells are regularly exposed to chronic oxidative stress as a tissue with high metabolic demand and ROS generation. DJ-1 is a multifunctional protein in the retina and RPE that has been shown to protect cells from oxidative stress in several cell types robustly. Oxidation of DJ-1 cysteine (C) residues is important for its function...

Cub domain-containing protein 1 (CDCP1) is a surface protein highly expressed on the surface of many cancer cells, however, the distribution of CDCP1 in normal tissues and its potential roles in non-tumor cells are poorly understood. We previously reported that CDCP1 interacts with CD6, a surface marker of T cells, suggesting that it is a novel imm...

Purpose The purpose of this study is to assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection. Methods Eyes from seven COVID-19-positive and six similar age-matched control donors with a negative test for SARS-CoV-2 were assessed. Globes were evaluated ex vivo with m...

Rod-cone dystrophy (RCD), also known as retinitis pigmentosa, is a group of inherited disorders characterized by rod degeneration followed by degeneration of peripheral cones, leaving patients with tunnel vision in mid stages and blindness in the latest stages. Previous studies expressed microbial chloride pumps, in degenerating cone photoreceptors...

Long interspersed nuclear element-1 (L1)-mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA- induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched...

Purpose To assess for histopathological changes within the retina and the choroid and determine the long-term sequelae of the SARS-CoV-2 infection. Design Comparative analysis of human eyes. Subjects Eleven donor eyes from COVID-19 positive donors and similar age-matched donor eyes from patients with a negative test for SARS-CoV-2 were assessed....

Mutations in CLN3 lead to photoreceptor cell loss in CLN3 disease, a lysosomal storage disorder characterized by childhood-onset vision loss, neurological impairment, and premature death. However, how CLN3 mutations cause photoreceptor cell death is not known. Here, we show that CLN3 is required for phagocytosis of photoreceptor outer segment (POS)...

Stargardt macular degeneration (STGD) is a central blinding disease caused by loss of or dysfunctional ABCA4 transporter in both photoreceptors and retinal pigment epithelial (RPE) cells. Toxic bisretinoid-lipofuscin buildup in the RPE cells is a pathological hallmark of STGD patients and its mouse model, the Abca4−/−. These vitamin A-derived fluor...

Best disease (BD), also known as vitelliform macular dystrophy, is an inherited disease of the central retina caused by more than 300 pathogenic variants in the BEST1 gene. The phenotype of BD is variable, and there are just a few reports on the histopathology of eyes from donors with BD. Here, we describe the histopathological comparison of donor’...

Sorsby Fundus Dystrophy (SFD) is a rare inherited autosomal dominant macular degeneration caused by specific mutations in TIMP3. Patients with SFD present with pathophysiology similar to the more common Age-related Macular Degeneration (AMD) and loss of vision due to both choroidal neovascularization and geographic atrophy. Previously, it has been...

Purpose: The present study investigated retinal glia and choroidal vessels in flatmounts and sections from individuals with clinically diagnosed Stargardt disease (STGD). Methods: Eyes from three donors clinically diagnosed with STGD were obtained through the Foundation Fighting Blindness (FFB). Genetic testing was performed to determine the dis...

Purpose: To describe the pathology of AMD in eyes with geographic atrophy (GA) using confocal scanning laser ophthalmoscopy (SLO) blue light autofluorescence (BAF), and near-infrared (IR) AF and to correlate it with the histology and immunohistochemistry analysis at the margins of the GA lesion. Methods: Enucleated, fixed eyes from seventeen don...

High levels of oxidative radicals generated by daily light exposure and high metabolic rate suggest that the antioxidant machinery of the retina and retinal pigment epithelium (RPE) is crucial for their survival. DJ-1 is a redox-sensitive protein that has been shown to have neuroprotective function in the brain in Parkinson’s disease and other neur...

Choroidal neovascularization (CNV) is the abnormal growth of blood vessels that sprout from the choroid vasculature and grow beneath and into the retina. The newly formed blood vessels in CNV often leak blood and fluid which deteriorates vision over time, eventually leading to blindness. In the present study, we examined the efficacy of intravenous...

Inherited retinal degenerations (IRDs) are characterized by the progressive loss of photoreceptors and represent one of the most prevalent causes of blindness among working-age populations. Cyclic nucleotide dysregulation is a common pathological feature linked to numerous forms of IRD, yet the precise mechanisms through which this contributes to p...

Sorsby’s fundus dystrophy (SFD) is an inherited blinding disorder caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP3) gene. The SFD pathology of macular degeneration with subretinal deposits and choroidal neovascularization (CNV) closely resembles that of the more common age-related macular degeneration (AMD). The objective o...

Retinal lesions in the posterior pole of laboratory mice occur due to native, developmental abnormalities or as a consequence of environmental or experimental conditions. In this study, we investigated the rate and extent of retinal lesions as a result of prolonged ocular exposure following general anesthesia. Following experimental preparation ind...

The observation of retinal lesions in the posterior pole of laboratory mice has been found to occur for many reasons, some of which are due to native, developmental abnormalities and those that are influenced by environmental or experimental conditions. Herein, we investigated the rate and extent of retinal lesions as a result of prolonged ocular e...

In the eye, the retina and RPE (retinal pigmented epithelium) are exposed to high levels of oxidative radicals generated by light exposure and high metabolic rate. Degeneration of these cells due to cumulative oxidative damage plays an important role in diseases like AMD (Age-related Macular Degeneration) and subsequent blindness. The underlying me...

Hypothesis: Hyperglycemia decreases angiotensin-(1-7), the endogenous counter-regulator of angiotensin II in the retina. Materials and methods: The distribution and levels of retinal angiotensin II (Ang II) and angiotensin-(1-7) (Ang-(1-7)) were evaluated by confocal imaging and quantitative immunohistochemistry during the development of strepto...

In the retina, oxidative stress can initiate a cascade of events that ultimately leads to a focal loss of RPE cells and photoreceptors, a major contributing factor in geographic atrophy. Despite these implications, the molecular regulation of RPE oxidative metabolism under physiological and pathological conditions remains largely unknown. DJ-1 func...

Noninvasive ocular imaging platforms are undeniably useful in identifying retinal abnormalities. The purpose of this study was to investigate a novel method for integrating information acquired from two independent imaging platforms, AF-SLO and SDOCT, in order to demonstrate retinal perturbations as a result of genetic or pharmacological manipulati...

[This corrects the article DOI: 10.1371/journal.pone.0067983.].

Uncropped blots for Figs 2 and 4. (ZIP)

Circadian clocks regulate various aspects of photoreceptor physiology, but their contribution to photoreceptor development and function is unclear. Cone photoreceptors are critical for color vision. Here, we define the molecular function of circadian activity within cone photoreceptors and reveal a role for the clock genes Bmal1 and Per2 in regulat...

Deimination refers to the conversion of protein-bound arginines into citrulline. It has been established as a posttranslational modification due to the lack of any known tRNA carrier for citrulline, as well as the presence of deiminases that are capable of catalyzing this modification in vitro. There is no known enzyme that can revert protein-bound...

To demonstrate changes observed in the photoreceptor layer of rodent animal models by spectral-domain optical coherence tomography (SD-OCT). Reflectivity changes observed by SD-OCT within the proximal outer segments (OS) are real, quantifiable entities that can provide insight into RPE-Photoreceptor status. These observations suggest that proximal...

Oxidative stress alters physiological function in most biological tissues and can lead to cell death. In the retina, oxidative stress initiates a cascade of events leading to focal loss of RPE and photoreceptors, which is thought to be a major contributing factor to geographic atrophy. Despite these implications, the molecular regulation of RPE oxi...

To evaluate the histopathology in donor eyes from patients with autosomal dominant retinitis pigmentosa (ADRP) caused by p.P23H, p.P347T and p.P347L rhodopsin ( RHO ) gene mutations. Eyes from a 72-year-old male (donor 1), an 83-year-old female (donor 2), an 80-year-old female (donor 3), and three age-similar normal eyes were examined macroscopical...

Purpose: To define the retinal pathology in a 3 year-old eye donor who died from complications of an undiagnosed genetic syndrome. Methods: Eyes were fixed and analyzed using macroscopic fundus photography (MF), confocal scanning laser ophthalmoscopy (cSLO) and spectral-domain optical coherence tomography (SD-OCT). Small areas from the perifovea...

To evaluate the retinal histopathology in donor eyes from patients with autosomal recessive retinitis pigmentosa (arRP) caused by EYS mutations. Eyes from a 72-year-old female (donor 1, family 1), a 91-year-old female (donor 2, family 2), and her 97-year-old sister (donor 3, family 2) were evaluated with macroscopic, scanning laser ophthalmoscopy (...

Background: The goal of this study was to define the histopathology of the retina in donor eyes from a patient with Stargardt disease (STGD1) due to compound mutations in the ABCA4 gene. Materials and methods: Eyes were obtained from a 66-year-old female and fixed within 18 hours postmortem. The fundi of the posterior globes were evaluated with...

Deimination refers to the posttranslational conversion of protein-bound arginines into protein-bound citrullines, catalyzed by peptidyl arginine deiminase (PAD) enzymes (Vossenaar et al. 2003). Lack of a carrier tRNA for citrulline has established deimination as a posttranslational modification. Deimination and citrullination are interchangeably us...

DJ-1 is a protein expressed in many tissues including the brain where it has been extensively studied due to its association with Parkinson's Disease (PD). DJ-1 was reported to function as an antioxidant, redox-sensitive molecular chaperone, and transcription regulator, which protected cells from oxidative stress by modifying signaling pathways tha...

DJ-1 is found in many tissues, including the brain, where it has been extensively studied due to its association with Parkinson's disease. DJ-1 functions as a redox-sensitive molecular chaperone and transcription regulator that robustly protects cells from oxidative stress. Retinal pigment epithelial (RPE) cultures were treated with H2O2 for variou...

Toxoplasma gondii infects both hematopoietic and nonhematopoietic cells and can cause cerebral and ocular toxoplasmosis, as a result of either congenital or postnatally acquired infections. Host protection likely acts at both cellular levels to control the parasite. CD40 is a key factor for protection against cerebral and ocular toxoplasmosis. We d...

Deimination is a form of protein posttranslational modification carried out by the peptidyl arginine deiminases (PADs) enzymes. PAD2 is the principal deiminase expressed in the retina. Elevated levels of PAD2 and protein deimination are present in a number of human neurological diseases, with or without ocular manifestation. To define the associati...

Age-related changes in the retina are often accompanied by visual impairment but their mechanistic details remain poorly understood. Proteomic studies were pursued toward a better molecular understanding of retinal pigment epithelium (RPE) aging mechanisms. RPE cells were isolated from young adults (3-4 month-old) and old (24-25 month-old) F344BN r...

List of proteins identified on old MV fraction of RPE cells. (XLS)

Networks identified on MV fractions in both young and old F344BN rat RPE. (DOC)

Lectin labeling of eyecups from young and old F344BN RPE. Both young (A, C) and old (B, D) eyecups with the exposed RPE were fixed and processed as wholemounts (A, B) or for cryosectioning (C, D). Whole mounts were labeled with WGA-FITC mounted on slides and observed in epifluorescence. 10 mm sections were labeled with WGA-FITC and nuclei were labe...

List of proteins identified on young MV fraction of RPE cells. (XLS)

List of proteins identified on young CB fraction of RPE cells. (XLS)

Networks identified on CB fractions in both young and old F344BN rat RPE. (DOC)

Age-related changes in RPE fractions networks. Functional pathway and network analyses of RPE fractions were generated through the use of Ingenuity Pathway Analysis. Displayed are network analyses of protein interactions involved in lipid metabolism, molecular transport and small molecules biochemistry pathways in both young and old MV (A, B) and C...

List of proteins identified on old CB fraction of RPE cells. (XLS)

Confocal scanning laser ophthalmoscope (SLO) and spectral-domain optical coherence tomography (OCT) provide a comprehensive diagnostic assessment in the clinic. Here, we describe the use of SLO and OCT as the initial assessment to both screen "normal" postmortem donor eyes for retinal lesions and characterize retinal lesions in eyes with suspected...

Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51. Eyes were enucleated within 13.5 hours after death. Repre...

To define the retinal pathology in an 88-year-old male affected with Goldmann-Favre syndrome with a 2 bp 5' A>C splice site mutation in the NR2E3 gene. Retinal tissue from the macula and periphery was processed for immunohistochemistry. Perimacular retina was processed for transmission electron microscopy. Cryosections were studied by indirect immu...

To compare mechanical and ethanol epithelial removal with respect to myofibroblast development and haze formation after photorefractive keratectomy (PRK). Seventeen rabbits underwent mechanical or ethanol debridement, and the opposite eye of each rabbit served as an unwounded control. In both groups, the epithelium was removed with a spatula and di...

To define the retinal pathology in a 91 year-old affected matriarch of a three-generation choroideremia family with multiple manifesting carriers. Tissue from three different retinal areas was processed for immunohistochemistry. The macular area was processed for transmission electron microscopy. Cryosections were studied by indirect immunofluoresc...

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and i...

As the retinal pigment epithelium (RPE) ages, a number of structural changes occur, including loss of melanin granules, increase in the density of residual bodies, accumulation of lipofuscin, accumulation of basal deposits on or within Bruch's membrane, formation of drusen (between the basal lamina of the RPE and the inner collagenous layer of Bruc...

Increased deimination and peptidyl arginine deiminase type 2 (PAD2) expression has been observed in age-related neurodegenerative diseases without discrimination between their aging and disease component. Here, we describe reduced levels of deimination commensurate with reduced protein, mRNA and activity of peptidylarginine deiminase type 2 in the...

Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidati...

The two cellular targets of interest in age-related macular degeneration (AMD) are the photoreceptors and the RPE. However, the mechanisms involved in AMD pathology are not yet fully understood. In the present report, we extend our previous studies on semenogelin proteins (Sgs) in normal human retina and compare these with the distribution in retin...

Glucose utilization and glycogen metabolism by human retinal pigment epithelium (RPE) cultures with high transepithelial resistance maintained on porous Millicell polycarbonate filters, were quantified by fluorophore-assisted carbohydrate electrophoresis (FACE). Glucose uptake was more efficient at the apical surface of the RPE. The utilization of...

Semenogelin I and II are the major proteins present in semen coagulum. In the present study, semenogelin I and II were detected in human RPE lysates by proteomic analysis. We further analyzed the expression of these proteins in the retinal cells in vivo and in vitro. Western blots detected semenogelin I and II in both RPE and neural retina while th...

Proteomic analyses of normal and glaucomatous human optic nerve were pursued for insights into the molecular pathology of primary open-angle glaucoma (POAG). Peptidyl arginine deiminase 2 (PAD2), an enzyme that converts protein arginine to citrulline, was found only in POAG optic nerve and was probed further for a mechanistic role in glaucoma. Prot...

Ezrin, a member of the ezrin/moesin/radixin (ERM) family, localizes to microvilli of epithelia in vivo, where it functions as a bridge between actin filaments and plasma membrane proteins. In the eye, ezrin has been localized to both apical microvilli of Müller cells and retinal pigment epithelium (RPE) apical microvilli and basal infoldings. In th...

Caveolae and their associated structural proteins, the caveolins, are specialized plasmalemmal microdomains involved in endocytosis and compartmentalization of cell signaling. We examined the expression and distribution of caveolae and caveolins in retinal pigment epithelium (RPE), which plays key roles in retinal support, visual cycle, and acts as...

The visual cycle is the complex enzymatic retinoid-processing involved in regenerating bleached rod and cone visual pigments.1 Central to visual cycle physiology is the cellular retinaldehyde-binding protein (CRALBP), a 36kDa cytosolic protein with high affinity for 11-cis-retinal and 11-cis-retinol. CRALBP is expressed in retinal pigment epitheliu...

The RPE performs highly specialized, unique functions essential for homeostasis of the neural retina. These include phagocytosis of photoreceptors shed outer segments, directional transport of nutrients into and removal of waste products from photoreceptor cells and visual pigment transport and regeneration. All of these functions involve the RPE a...

To define the distribution of the red/green and blue opsins in cones from donor eyes from an affected member of a clinically well-characterized family with an autosomal dominant form of cone dystrophy. Tissue was fixed and processed for immunohistochemistry. Cryosections were studied by indirect immunofluorescence, using well-characterized antibodi...

Trypanosoma cruzi can modulate a large number of host intracellular responses during its invasion. GTPases such as RhoA, Rac1 and Cdc42 are examples of molecules that could be activated at this moment and trigger changes in the pattern of F-actin cytoskeleton leading to the formation of structures like stress fibers, lamellipodium and fillopodium,...

The etiology of primary open angle glaucoma, a leading cause of age-related blindness, remains poorly defined, although elevated intraocular pressure (IOP) contributes to the disease progression. To better understand the mechanisms causing elevated IOP from aqueous humor circulation, we pursued proteomic analyses of trabecular meshwork (TM) from gl...

Purpose: The progressive accumulation of lipofuscin in the retinal pigment epithelium (RPE), correlates with the pathogenesis of age–related macular degeneration (AMD). We seek a better molecular understanding of the sources and consequences of lipofuscin accumulation, including the protein content of lipofuscin. Methods: Human RPE lipofuscin was...

Polarized epithelial cells are characterized by displaying compartmentalized functions associated with differential distribution of transporters, structural proteins, and signaling molecules on their apical and basolateral surfaces. Their apical surfaces frequently elaborate microvilli, which vary in structure according to the specific type and fun...

The interaction of cellular retinaldehyde-binding protein (CRALBP) with ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50) in retinal pigment epithelium (RPE) microsomes has led to the hypothesis that a retinoid-processing protein complex exists in apical RPE. Mouse RPE apical processes were isolated on wheat germ agglutinin-coated agar...

One fundamental step of Leishmania-macrophage interaction is the phase of parasite internalization through an endocytic process, with the formation of the parasitophorous vacuole (PV). The present study analyzed this process using two approaches. First, to investigate the host cell proteins which take part in this compartment, the macrophage surfac...

Identification of binding partners for ezrin, an actin-binding protein crucial for morphogenesis of apical microvilli and basolateral infoldings in RPE cells. Rat eyes, rat primary RPE, the rat RPE-J cell line, and a clonal line of RPE-J cells transfected with human ezrin cDNA were analyzed by immunofluorescence microscopy and immunoblot. Immunoflu...

A large body of knowledge has been accumulated in the last two decades on the nature of the mechanisms responsible for the polarization of epithelial cells (Rodriguez- Boulan and Nelson, 1989; Rodriguez-Boulan and Powell, 1992). Three fundamental types of mechanisms have emerged that contribute to epithelial polarity (Figure 7.1): (1) intracellular...

Ezrin, a member of the ezrin/radixin/moesin (ERM) family, localizes to microvilli of epithelia in vivo, where it bridges actin filaments and plasma membrane proteins. Here, we demonstrate two specific morphogenetic roles of ezrin in the retinal pigment epithelium (RPE), i.e., the formation of very long apical microvilli and of elaborate basal infol...

We have characterized comparatively the subcellular distributions of caveolins-1 and -2, their interactions and their roles in caveolar formation in polarized epithelial cells. In Fischer rat thyroid (FRT) cells, which express low levels of caveolin-2 and no caveolin-1, caveolin-2 localizes exclusively to the Golgi complex but is partially redistri...