Camasamudram Vijayasarathy

Inflammatory Cytokines Regulate MicroRNA-155 Expression in Human Retinal Pigment Epithelial Cells by Activating JAK/STAT Pathway

Inflammatory response of the retinal pigment epithelium plays a critical role in the pathogenesis of retinal degenerative diseases such as age-related macular degeneration. Our previous studies have shown that human retinal pigment epithelial (HRPE) cells, established from adult donor eyes, respond to inflammatory cytokines by enhancing the expression of a number of cytokines and chemokines. To investigate the role of microRNA (miRNA) in regulating this response, we performed microarray analysis of miRNA expression in HRPE cells exposed to inflammatory cytokine mix (IFN-γ+TNF-α+IL-1β). Microarray analysis revealed ∼11-fold increase in miR-155 expression, which was validated by real-time PCR analysis. The miR-155 expression was enhanced when the cells were treated individually with IFN-γ, TNF-α or IL-1β, but combinations of the cytokines exaggerated the effect. The increase in miR-155 expression by the inflammatory cytokines was associated with an increase in STAT1 activation as well as an increase in protein binding to putative STAT1 binding elements present in the MIR155 gene promoter region. All these activities were effectively blocked by JAK inhibitor 1. Our results show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT signaling pathway.

Synaptic pathology in retinoschisis knockout (Rs1-/y) mouse retina and modification by rAAV-Rs1 gene delivery.

PURPOSE At an early age, the retinoschisin knockout (Rs1-KO) mouse retina has progressive photoreceptor degeneration with severe disruption of the outer plexiform layer (OPL) that decreases at older ages. The electroretinogram (ERG) undergoes parallel changes. The b-wave amplitude from bipolar cells is reduced disproportionately to the photoreceptor a-wave at young but not at older ages. The protein expression and morphology of the OPL in Rs1-KO mice was investigated at different ages, to explore the role of the synaptic layer in these ERG changes. METHODS Retinas of wild-type (Wt) and Rs1-KO mice from postnatal day (P)7 to 12 months were evaluated by light and electron microscopy (EM) and biochemistry. PSD95 (postsynaptic density protein), mGluR6 (metabotropic glutamate receptor subtype 6), retinoschisin (Rs1), the Müller cell proteins glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), the bipolar cell marker protein kinase C alpha (PKCalpha), and the horizontal cell marker calbindin were localized by immunofluorescence and immuno-EM. Levels of PSD95 and mGluR6 were determined by quantitative Western blot. Rs1-KO mice treated by intravitreous injection of rAAV(2/2)-CMV-Rs1 in one eye at P14 were evaluated at 8 months by full-field scotopic ERG responses and retinal immunohistochemistry. RESULTS Rs1 was associated with the outer surface of synaptic membranes in wild-type (Wt) retinas. PSD95 and mGluR6 were juxtaposed in the OPL of the Rs1-KO retinas by P14, implying that synaptic structures are formed. Light microscopic retinal morphology was similar in Wt and Rs1-KO at P14, but by P21, the OPL was disrupted in Rs1-KO, and some PSD95 and mGluR6 was mislocalized in the outer nuclear layer (ONL). GFAP expression spanned all retinal layers. EM showed synaptic structures adjacent to photoreceptor nuclei. PSD95 and mGluR6 levels were normal at 1 month on Western blot but declined to 59% (P < 0.001) and 55% (P < 0.05) of Wt, respectively, by 4 months. Levels thereafter showed no further reduction out to 12 months. Eyes injected with AAV-Rs1 were studied at 8 months by immunohistochemistry and had higher expression of PSD95 and mGluR6 and less GFAP expression compared with fellow untreated eyes. CONCLUSIONS In the Rs1-KO mouse, retinal layer formation and synaptic protein expression in the OPL is normal up to P14, implying normal development of synaptic connections. Aberrant localization of synaptic proteins by P21 indicates that displacement of developing and/or mature synapses contributes to the b-wave reduction at young ages, when photoreceptor numbers and synaptic protein levels are normal. The subsequent decline in PSD95 and mGluR6 between 1 and 12 months in Rs1-KO retina mirrors the course of b-wave change and provides evidence of causal relationship between the ERG and OPL changes. These findings and the improved structural integrity of the OPL and b-wave amplitude after Rs1 gene transfer therapy provide a cellular and molecular basis for interpreting the changes in retinal signaling in this model.

Depleting Rac1 in mouse rod photoreceptors protects them from photo-oxidative stress without affecting their structure or function

In nonphagocytic cells, Rac1 is a component of NADPH oxidase that produces reactive oxygen species [Ushio-Fukai M (2006) Sci STKE 2006:re8]. Rac1 is expressed abundantly in mammalian retinal photoreceptors, where it is activated in response to light stimuli [Balasubramanian N, Slepak VZ (2003) Curr Biol 13:1306–1310]. We used Cre-LoxP conditional gene targeting to knock down Rac1 expression in mouse rod photoreceptors and found protection against light-induced photoreceptor death compared with WT litter-mates. We also found a similar protective effect on rods using apocynin, which inhibits NADPH oxidase activity. These results implicate both neuronal Rac1 and NADPH oxidase in cell death in this model of CNS degeneration. Studies in which dominant-mutants of Rac1 were expressed in transgenic Drosophila species demonstrated that Rac1 is a key regulator of photoreceptor morphogenesis and polarity [Chang HY, Ready DF (2000) Science 290:1978–1980]. However, we found that diminished Rac1 expression in mouse rods had no effect on retinal structure or function examined by light microscopy, electron microscopy, rhodopsin measurement, electroretinogram activity, and visual acuity, indicating rod outer segment morphogenesis proceeded normally in Rac1 conditional knockout mice. The lack of structural or functional effect of Rac1 depletion on photoreceptors, but protection under conditions of stress, indicate that the Rac1 pathway warrants exploration as a target for therapy in retinal neurodegenerative diseases.

Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-RS1 transfer

Strategies aimed at invoking synaptic plasticity have therapeutic potential for several neurological conditions. The human retinal synaptic disease X-linked retinoschisis (XLRS) is characterized by impaired visual signal transmission through the retina and progressive visual acuity loss, and mice lacking retinoschisin (RS1) recapitulate human disease. Here, we demonstrate that restoration of RS1 via retina-specific delivery of adeno-associated virus type 8-RS1 (AAV8-RS1) vector rescues molecular pathology at the photoreceptor-depolarizing bipolar cell (photoreceptor-DBC) synapse and restores function in adult Rs1-KO animals. Initial development of the photoreceptor-DBC synapse was normal in the Rs1-KO retina; however, the metabotropic glutamate receptor 6/transient receptor potential melastatin subfamily M member 1-signaling (mGluR6/TRPM1-signaling) cascade was not properly maintained. Specifically, the TRPM1 channel and G proteins Gαo, Gβ5, and RGS11 were progressively lost from postsynaptic DBC dendritic tips, whereas the mGluR6 receptor and RGS7 maintained proper synaptic position. This postsynaptic disruption differed from other murine night-blindness models with an electronegative electroretinogram response, which is also characteristic of murine and human XLRS disease. Upon AAV8-RS1 gene transfer to the retina of adult XLRS mice, TRPM1 and the signaling molecules returned to their proper dendritic tip location, and the DBC resting membrane potential was restored. These findings provide insight into the molecular plasticity of a critical synapse in the visual system and demonstrate potential therapeutic avenues for some diseases involving synaptic pathology.

N-(4-hydroxyphenyl)retinamide induces apoptosis in human retinal pigment epithelial cells : Retinoic acid receptors regulate apoptosis, reactive oxygen species generation, and the expression of heme oxygenase-1 and Gadd153

N‐(4‐hydroxyphenyl)retinamide (4HPR, fenretinide), a retinoic acid (RA) derivative and a potential cancer preventive agent, is known to exert its chemotherapeutic effects in cancer cells through induction of apoptosis. Earlier work from our laboratory has shown that relatively low concentrations of 4HPR induce neuronal differentiation of cultured human retinal pigment epithelial (ARPE‐19) cells (Chen et al., 2003, J Neurochem 84:972–981). However, at higher concentrations of 4HPR, these cells showed morphological changes including cell shrinkage and cell death. Here we demonstrate that ARPE‐19 cells treated with 4HPR exhibit a dose‐ and time‐dependent induction of apoptosis as evidenced by morphological changes, mono‐ and oligonucleosome generation, and increased activity of caspases 2 and 3. The 4HPR‐induced apoptosis as well as the activation of caspases 2 and 3 were blocked by both retinoic acid receptors (RAR) pan‐antagonists, AGN193109 and AGN194310, and by an RARα‐specific antagonist AGN194301. 4HPR treatment also increased reactive oxygen species (ROS) generation in ARPE‐19 cells in a time‐dependent manner as determined from the oxidation of 2′,7′‐dichlorofluorescin. In addition, the increase in the expression of heme oxygenase‐1 (HO‐1), a stress response protein, and the growth arrest and DNA damage‐inducible transcription factor 153 (Gadd153) in response to the ROS generation were also blocked by these receptor antagonists. Pyrrolidine dithiocarbamate (PDTC), a free‐radical scavenger, inhibited 4HPR‐induced ROS generation, the expression of its downstream mediator, Gadd153, and apoptosis in the pretreated cells. Therefore, our results, clearly demonstrate that 4HPR induces apoptosis in ARPE‐19 cells and that RARs mediate this process by regulating ROS generation as well as the expression of Gadd153 and HO‐1. J. Cell. Physiol. 209: 854–865, 2006.

Molecular Mechanisms Leading to Null-protein Product from Retinoschisin (RS1) Signal-sequence Mutants in X-linked Retinoschisis (XLRS) Disease

Retinoschisin (RS1) is a cell‐surface adhesion molecule expressed by photoreceptor and bipolar cells of the retina. The 24‐kDa protein encodes two conserved sequence motifs: the initial signal sequence targets the protein for secretion while the larger discoidin domain is implicated in cell adhesion. RS1 helps to maintain the structural organization of the retinal cell layers and promotes visual signal transduction. RS1 gene mutations cause X‐linked retinoschisis disease (XLRS) in males, characterized by early‐onset central vision loss. We analyzed the biochemical consequences of several RS1 signal‐sequence mutants (c.1A>T, c.35T>A, c.38T>C, and c.52G>A) found in our subjects. Expression analysis in COS‐7 cells demonstrates that these mutations affect RS1 biosynthesis and result in an RS1 null phenotype by several different mechanisms. By comparison, discoidin‐domain mutations generally lead to nonfunctional conformational variants that remain trapped inside the cell. XLRS disease has a broad heterogeneity in general, but subjects with the RS1 null‐protein signal‐sequence mutations are on the more severe end of the clinical phenotype. Results from the signal‐sequence mutants are discussed in the context of the discoidin‐domain mutations, clinical phenotypes, genotype–phenotype correlations, and implications for RS1 gene replacement therapy. Hum Mutat 31:1251–1260, 2010. Published 2010 Wiley‐Liss, Inc.

Probing potassium channel function in vivo by intracellular delivery of antibodies in a rat model of retinal neurodegeneration.

Inward rectifying potassium (Kir) channels participate in regulating potassium concentration (K+) in the central nervous system (CNS), including in the retina. We explored the contribution of Kir channels to retinal function by delivering Kir antibodies (Kir-Abs) into the rat eye in vivo to interrupt channel activity. Kir-Abs were coupled to a peptide carrier to reach intracellular epitopes. Functional effects were evaluated by recording the scotopic threshold response (STR) and photopic negative response (PhNR) of the electroretinogram (ERG) noninvasively with an electrode on the cornea to determine activity of the rod and cone pathways, respectively. Intravitreal delivery of Kir2.1-Ab coupled to the peptide carrier diminished these ERG responses equivalent to dimming the stimulus 10- to 100-fold. Immunohistochemistry (IHC) showed Kir2.1 immunostaining of retinal bipolar cells (BCs) matching the labeling pattern obtained with conventional IHC of applying Kir2.1-Ab to fixed retinal sections postmortem. Whole-cell voltage-clamp BC recordings in rat acute retinal slices showed suppression of barium-sensitive Kir2.1 currents upon inclusion of Kir2.1-Ab in the patch pipette. The in vivo functional and structural results implicate a contribution of Kir2.1 channel activity in these electronegative ERG potentials. Studies with Kir4.1-Ab administered in vivo also suppressed the ERG components and showed immunostaining of Müller cells. The strategy of administering Kir antibodies in vivo, coupled to a peptide carrier to facilitate intracellular delivery, identifies roles for Kir2.1 and Kir4.1 in ERG components arising in the proximal retina and suggests this approach could be of further value in research.

Long-term 12 year follow-up of X-linked congenital retinoschisis.

Purpose: To investigate the retinal structure and function during the progression of X-linked retinoschisis (XLRS) from childhood to adulthood. Methods: Ten patients clinically diagnosed with XLRS were investigated at 6–15 years of age (mean age 9 years) with a follow-up 8 to 14 years later (mean 12 years). The patients underwent regular ophthalmic examination as well as testing of best corrected visual acuity (BCVA), visual field (VF) and assessment of full-field electroretinography (ERG) during their first visit. During the follow-up, the same clinical protocols were repeated. In addition, macular structure and function was examined with multifocal electroretinography (mfERG) and optical coherence tomography (OCT). The patients were 18–25 years of age (mean age 21 years) at the follow-up examination. All exons and exon-intron boundaries of RS1-gene were sequenced for gene mutations in 9 out of the 10 patients. Results: Best corrected VA and VF were stable during this follow-up period. No significant progression in cone or rod function could be measured by full-field ERG. Multifocal electroretinography and OCT demonstrated a wide heterogeneity of macular changes in retinal structure and function at the time of follow-up visit. Three different mutations were detected in these nine patients, including a known nonsense mutation in exon 3, a novel insertion in exon 5 and an intronic mutation at 5′ splice site of intron 3. Conclusions: Clinical follow-up (mean 12 years) of ten young XLRS patients (mean age of 9 years) with a typical congenital retinoschisis phenotype revealed no significant decline in retinal function during this time period. MfERG and OCT demonstrated a wide variety of macular changes including structure and dysfunction. The XLRS disease was relatively stable during this period of observation and would afford opportunity for therapy studies to judge benefit against baseline and against the fellow eye.

Retinoschisin (RS1) interacts with negatively charged lipid bilayers in the presence of Ca2+: an atomic force microscopy study.

Retinoschisin (RS1) is a retina-specific secreted protein encoding a conserved discoidin domain sequence. As an adhesion molecule, RS1 preserves the retinal cell architecture and promotes visual signal transduction. In young males, loss-of-function mutations in the X-linked retinoschisis gene (RS1) cause X-linked retinoschisis, a form of progressive blindness. Neither the structure of RS1 nor the nature of its anchoring and organization on the plasma membranes is fully understood. The discoidin C2 domains of coagulation factors V and VIII are known to interact with extracellular phosphatidylserine (PS). In this study we have used atomic force microscopy (AFM) to study the interactions of murine retinoschisin (Rs1) with supported anionic lipid bilayers in the presence of Ca(2+). The bilayers consisting of a single lipid, PS, and mixtures of lipids with or without PS were used. Consistent with previous X-ray diffraction studies, AFM imaging showed two distinct domains in pure PS bilayers when Ca(2+) was present. Upon Rs1 adsorption, these PS and PS-containing mixed bilayers underwent fast and extensive reorganization. Protein localization was ascertained by immunolabeling. AFM imaging showed the Rs1 antibody bound exclusively to the calcium-rich ordered phase of the bilayers pointing to the sequestration of Rs1 within those domains. This was further supported by the increased mechanical strength of these domains after Rs1 binding. Besides, changes in bilayer thickness suggested that Rs1 was partially embedded into the bilayer. These findings support a model whereby the Rs1 protein binds to PS in the retinal cell plasma membranes in a calcium-dependent manner.

Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human

Papillorenal syndrome (PRS, also known as renal-coloboma syndrome) is an autosomal dominant disease characterized by potentially-blinding congenital optic nerve excavation and congenital kidney abnormalities. Many patients with PRS have mutations in the paired box transcription factor gene, PAX2. Although most mutations in PAX2 are predicted to result in complete loss of one alleles function, three missense mutations have been reported, raising the possibility that more subtle alterations in PAX2 function may be disease-causing. To date, the molecular behaviors of these mutations have not been explored. We describe a novel mouse model of PRS due to a missense mutation in a highly-conserved threonine residue in the paired domain of Pax2 (p.T74A) that recapitulates the ocular and kidney findings of patients. This mutation is in the Pax2 paired domain at the same location as two human missense mutations. We show that all three missense mutations disrupt potentially critical hydrogen bonds in atomic models and result in reduced Pax2 transactivation, but do not affect nuclear localization, steady state mRNA levels, or the ability of Pax2 to bind its DNA consensus sequence. Moreover, these mutations show reduced steady-state levels of Pax2 protein in vitro and (for p.T74A) in vivo, likely by reducing protein stability. These results suggest that hypomorphic alleles of PAX2/Pax2 can lead to significant disease in humans and mice.