Ernesto Barron

The unfolded protein response regulator GRP78/BiP is required for endoplasmic reticulum integrity and stress-induced autophagy in mammalian cells

In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3′-kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3–Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.

Retinal Nerve Fiber Layer Thickness in Normal Human Eyes

PURPOSE To measure the histologic thickness of the retinal nerve fiber layer (RNFL) in normal human eyes. METHODS Human eyes were obtained at autopsy within 6 hours postmortem. The retina was dissected into four quadrants and serially sectioned in historesin. The RNFL thickness was measured histologically in all four quadrants at the disc margin and at regular intervals from the disc margin. Measurements of the RNFL thickness also were obtained at the fovea and in the retinal periphery. RESULTS Ten eyes of ten white individuals were studied. Age (mean +/- standard deviation) was 53.1 +/- 19.6 years (range, 18-76 years). For the eyes studied, the disc area (mean +/- standard deviation) and cup:disc ratio (mean +/- standard deviation) were 1.92 +/- 0.1 mm2 and 0.3 +/- 0.08, respectively. Mean superior, inferior, nasal, and temporal RNFL thickness at the disc margin was 405, 376, 372, and 316 microns, respectively. In all four quadrants, the RNFL thickness decreased with increasing distance from the disc margin. The average superior and inferior RNFL thickness was inversely related to age (P = 0.033, P = 0.097, respectively). The average RNFL thickness was not related to disc area. The average RNFL thickness just superior, inferior, nasal, and temporal to the foveola was 27, 34, 26, and 12 microns, respectively. The average RNFL thickness just posterior to the ora serrata in the superior, inferior, nasal, and temporal retinal periphery was 8 to 11 microns. CONCLUSION The peripapillary RNFL thickness in humans is thicker than that seen in nonhuman primates. The thinnest peripapillary RNFL is in the region of the papillomacular bundle. These data can be used to determine the accuracy of NFL analyzers in obtaining in vivo RNFL thickness measurements.

A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.

We provide our detailed, standardized in vitro protocol for the culture and differentiation of human retinal pigment epithelial (RPE) cells into a highly polarized and functional monolayer. Disruption of the polarized RPE function plays an important role in the pathogenesis of common blinding disorders of the retina. The availability of this polarized RPE monolayer allows for reproducible evaluation of RPE function, modeling of RPE dysfunction in retinal disease and in vitro evaluation of new therapies. The protocol, which takes approximately 6 weeks to complete, describes the culture of RPE from human fetal donor eyes, the differentiation of these cells into a polarized monolayer with high transepithelial resistance and morphologic characteristics that mimic the RPE monolayer in vivo. By modifying the procedure for initial isolation of pure RPE cells and the culture conditions used in existing protocols, we have established a standardized protocol that provides highly reproducible RPE monolayers from the same donor eye.

Novel growth factors involved in the pathogenesis of proliferative vitreoretinopathy.

Aims To determine whether hepatocyte growth factor (HGF) and connective tissue growth factor (CTGF) are expressed in human specimens of proliferative vitreoretinopathy (PVR) and to propose a model of PVR pathogenesis based upon the known activities of these growth factors.Methods Immunohistochemical methods (ABC Elite) were used to demonstrate the presence of HGF and CTGF in cryostat sections of five human PVR membranes.Results In each of the five PVR membranes, stromal cells were immunohistochemically positive for both HGF and CTGF. Based upon this information and the known actions of these growth factors, a model of PVR pathogenesis was developed. In this model, injury of the retina induces an inflammatory response that upregulates HGF expression inducing the formation of multilayered groups of migratory retinal pigment epithelial cells (RPE). These RPE, present in a provisional extracellular matrix, come in contact with vitreous containing TGF-β. The TGF-β is activated, upregulating expression of CTGF. Under the influence of TGF-β and CTGF, RPE become myofibroblastic and fibrosis ensues. Retinal traction induces further detachment continuing the cycle of retinal injury.Conclusions HGF and CTGF are expressed in PVR membranes and may play important roles in the pathogenesis of PVR. The expression and function of these growth factors should be critically examined in human PVR specimens, in in vitro cultures of RPE, and in animal models of PVR.

αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells

αB Crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether αB crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular αB crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that αB crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of αB crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced αB crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, αB crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that αB crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of αB crystallin to the basolateral side. In normal mouse retinal sections, αB crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous αB crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. αB Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for αB crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of αB crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for αB crystallin in ocular diseases.

Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis

Neurodegenerative diseases are often associated with dysfunction in protein quality control. The endoplasmic reticulum (ER), a key site for protein synthesis, senses stressful conditions by activating the unfolded protein response (UPR). In this study we report the creation of a novel mouse model in which GRP78/BiP, a major ER chaperone and master regulator of UPR, is specifically eliminated in Purkinje cells (PCs). GRP78-depleted PCs activate UPR including the induction of GRP94, PDI, CHOP and GADD34, feedback suppression of eIF2α phosphorylation and apoptotic cell death. In contrast to current models of protein misfolding in which an abnormal accumulation of ubiquitinated protein is prominent, cytosolic ubiquitin staining is dramatically reduced in GRP78-null PCs. Ultrastructural evaluation reveals that the ER shows prominent dilatation with focal accumulation of electron-dense material within the ER. The mice show retarded growth and severe motor coordination defect by week 5 and cerebellar atrophy by week 13. Our studies uncover a novel link between GRP78 depletion and reduction in cytosolic ubiquitination and establish a novel mouse model of accelerated cerebellar degeneration with basic and clinical applications.

Polarized Secretion of PEDF from Human Embryonic Stem Cell–Derived RPE Promotes Retinal Progenitor Cell Survival

PURPOSE Human embryonic stem cell-derived RPE (hES-RPE) transplantation is a promising therapy for atrophic age-related macular degeneration (AMD); however, future therapeutic approaches may consider co-transplantation of hES-RPE with retinal progenitor cells (RPCs) as a replacement source for lost photoreceptors. The purpose of this study was to determine the effect of polarization of hES-RPE monolayers on their ability to promote survival of RPCs. METHODS The hES-3 cell line was used for derivation of RPE. Polarization of hES-RPE was achieved by prolonged growth on permeable inserts. RPCs were isolated from 16- to 18-week-gestation human fetal eyes. ELISA was performed to measure pigment epithelium-derived factor (PEDF) levels from conditioned media. RESULTS Pigmented RPE-like cells appeared as early as 4 weeks in culture and were subcultured at 8 weeks. Differentiated hES-RPE had a normal chromosomal karyotype. Phenotypically polarized hES-RPE cells showed expression of RPE-specific genes. Polarized hES-RPE showed prominent expression of PEDF in apical cytoplasm and a marked increase in secretion of PEDF into the medium compared with nonpolarized culture. RPCs grown in the presence of supernatants from polarized hES-RPE showed enhanced survival, which was ablated by the presence of anti-PEDF antibody. CONCLUSIONS hES-3 cells can be differentiated into functionally polarized hES-RPE cells that exhibit characteristics similar to those of native RPE. On polarization, hES-RPE cells secrete high levels of PEDF that can support RPC survival. These experiments suggest that polarization of hES-RPE would be an important feature for promotion of RPC survival in future cell therapy for atrophic AMD.

Vascular Basement Membrane Pathology and Alzheimer's Disease

ABSTRACT: We have previously demonstrated that the capillary vascular basement membrane (VBM) is pathologically altered in Alzheimers disease (AD). This microangiopathy is highlighted by the immunocytochemical localization of the three principal intrinsic VBM components: heparan sulfate proteoglycan, collagen type IV, and laminin. These three VBM components also immunolabel amyloid deposits and senile plaque‐associated glial processes. The present study examines the ultrastructure of the VBM in one brain region severely affected (temporal gyrus) and one relatively spared (cerebellum) from the lesions of AD in both AD and neurological control cases. The cross‐sectional area as well as the width of the VBM were found to be greater in AD cortical capillaries. In addition, we found ultrastructural evidence for the activation of microglial‐related perivascular cells, and their apparent extravasation through the VBM, findings consistent with the hypothesis that these cells are being recruited as part of a disease‐related immune response. The recruitment of these “resting” microglial‐like cells from their intra‐VBM location to plaques and tangles in AD may explain (1) the thickening and vacuolization of the VBM; (2) the specificity of this VBM alteration to brain regions where there are plaques and tangles; and (3) the source of some of the large number of activated microglia in these affected areas. Thus, while VBM alterations may not be specific to AD, these changes appear to be specifically related to the disease process.

Connective Tissue Growth Factor as a Mediator of Intraocular Fibrosis

PURPOSE To investigate the role of connective tissue growth factor (CTGF) in the pathogenesis of proliferative vitreoretinopathy (PVR). METHODS Expression of CTGF was evaluated immunohistochemically in human PVR membranes, and the accumulation of CTGF in the vitreous was evaluated by ELISA. The effects of CTGF on type I collagen mRNA and protein expression in RPE were assayed by real-time PCR and ELISA, and migration was assayed with a Boyden chamber assay. Experimental PVR was induced in rabbits with vitreous injection of RPE cells plus rhCTGF; injection of RPE cells plus platelet derived-growth factor, with or without rhCTGF, or by injection of RPE cells infected with an adenoviral vector that expressed CTGF. RESULTS CTGF was highly expressed in human PVR membranes and partially colocalized with cytokeratin-positive RPE cells. Treatment of RPE with rhCTGF stimulated migration with a peak response at 50 ng/mL (P < 0.05) and increased expression of type I collagen (P < 0.05). There was a prominent accumulation of the N-terminal half of CTGF in the vitreous of patients with PVR. Intravitreous injection of rhCTGF alone did not produce PVR, whereas such injections into rabbits with mild, nonfibrotic PVR promoted the development of dense, fibrotic epiretinal membranes. Similarly, intravitreous injection of RPE cells infected with adenoviral vectors that overexpress CTGF induced fibrotic PVR. Experimental PVR was associated with increased CTGF mRNA in PVR membranes and accumulation of CTGF half fragments in the vitreous. CONCLUSIONS The results identify CTGF as a major mediator of retinal fibrosis and potentially an effective therapeutic target for PVR.

Isolation and culture of rat retinal microvessel endothelial cells using magnetic beads coated with antibodies to PECAM-1.

PURPOSE To isolate retinal endothelial cells (RECs) from Lewis rats using magnetic beads coated with antibodies to rat platelet-endothelial cell adhesion molecule-1 (PECAM-1) and to characterize the cultured RECs. METHODS Magnetic beads were coated with anti-rat PECAM-1 antibodies. Retinas were obtained from Lewis rat eyes. After the retinas were digested with collagenase, they were incubated with the antibody-coated beads with agitation. RECs that stuck to the beads were collected with a magnetic particle concentrator and cultured in fibronectin coated wells. The characteristics of the RECs were examined by immunohistochemical study utilizing von Willebrands Factor, acetylated low-density lipoprotein uptake and transmission electron microscopy. RESULTS The cells isolated using the PECAM-1-coated magnetic-bead technique formed a contact-inhibited cobblestone monolayer that stained positive for von Willebrands Factor. These cells revealed low-density lipoprotein uptake. Ultrastructurally, the isolated cells exhibited pinocytic vesicles and a high density of intercellular junctions without fenestrations. CONCLUSION These results indicate that the isolated cells were vascular endothelial cells showing both morphologic and functional characteristics of retinal vascular endothelium. The magnetic-bead technique was useful for isolating high purity RECs that can be cultured to study the physiological, immunological and biochemical role of the endothelium in various ocular diseases.