Ying Dun

Expression of the cystine-glutamate exchanger (xc-) in retinal ganglion cells and regulation by nitric oxide and oxidative stress.

The cystine-glutamate exchanger, system xc−, mediates the Na+-independent exchange of cystine into cells, coupled to the efflux of intracellular glutamate. System xc− plays a critical role in glutathione homeostasis. Early studies of brain suggested that system xc− was present primarily in astrocytes but not neurons. More recent work indicates that certain brain neurons have an active system xc−. In the retina, system xc− has been demonstrated in Müller and retinal pigment epithelial cells. We have recently suggested that two protein components of system xc−, xCT and 4F2hc, are present in ganglion cells of the intact retina. Here, we have used (1) molecular and immunohistochemical assays to determine whether system xc− is present in primary ganglion cells isolated from neonatal mouse retinas and (2) functional assays to determine whether its activity is regulated by oxidative stress in a retinal ganglion cell line (RGC–5). Primary mouse ganglion cells and RGC–5 cells express xCT and 4F2hc. RGC–5 cells take up [3H]glutamate in the absence of Na+, and this uptake is blocked by known substrates of system xc− (glutamate, cysteine, cystine, quisqualic acid). Treatment of RGC–5 cells with NO and reactive oxygen species donors leads to increased activity of system xc− associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. This is the first report of system xc− in primary retinal ganglion cells and RGC–5 cells. Oxidative stress upregulates this transport system in RGC–5 cells, and the process is associated with an increase in xCT mRNA and protein but no change in 4F2hc mRNA or protein.

In vivo protection against retinal neurodegeneration by sigma receptor 1 ligand (+)-pentazocine.

PURPOSE To evaluate the neuroprotective properties of the sigma receptor 1 (sigmaR1) ligand, (+)-pentazocine in an in vivo model of retinal neurodegeneration. METHODS Spontaneously diabetic Ins2(Akita/+) and wild-type mice received intraperitoneal injections of (+)-pentazocine for 22 weeks beginning at diabetes onset. Retinal mRNA and protein were analyzed by RT-PCR and Western blot analysis. Retinal histologic sections were measured to determine total retinal thickness, thicknesses of inner-outer nuclear and plexiform layers (INL, ONL, IPL, INL), and the number of cell bodies in the ganglion cell layer (GCL). Immunolabeling experiments were performed using antibodies specific for 4-hydroxynonenal and nitrotyrosine, markers of lipid peroxidation, and reactive nitrogen species, respectively, and an antibody specific for vimentin to view radial Müller fibers. RESULTS sigmaR1 mRNA and protein levels in the Ins2(Akita/+) retina were comparable to those in the wild-type, indicating that sigmaR1 is an available target during the disease process. Histologic evaluation of eyes of Ins2(Akita/+) mice showed disruption of retinal architecture. By 17 to 25 weeks after birth, Ins2(Akita/+) mice demonstrated approximately 30% and 25% decreases in IPL and INL thicknesses, respectively, and a 30% reduction in ganglion cells. In the (+)-pentazocine-treated group, retinas of Ins2(Akita/+) mice showed remarkable preservation of retinal architecture; IPL and INL thicknesses of (+)-pentazocine-treated Ins2(Akita/+) mouse retinas were within normal limits. The number of ganglion cells was 15.6 +/- 1.5 versus 10.4 +/- 1.2 cells/100 mum retinal length in (+)-pentazocine-treated versus nontreated mutant mice. Levels of nitrotyrosine and 4-hydroxynonenal increased in Ins2(Akita/+) retinas, but were reduced in (+)-pentazocine-treated mice. Retinas of Ins2(Akita/+) mice showed loss of the uniform organization of radial Müller fibers. Retinas of (+)-pentazocine-treated mice maintained the radial organization of glial processes. CONCLUSION Sustained (+)-pentazocine treatment in an in vivo model of retinal degeneration conferred significant neuroprotection, reduced evidence of oxidative stress, and preserved retinal architecture, suggesting that sigmaR1 ligands are promising therapeutic agents for intervention in neurodegenerative diseases of the retina.

Sigma Receptor 1 Modulates Endoplasmic Reticulum Stress in Retinal Neurons

PURPOSE To investigate the mechanism of σ receptor 1 (σR1) neuroprotection in retinal neurons. METHODS Oxidative stress, which is implicated in diabetic retinopathy, was induced in mouse primary ganglion cells (GCs) and RGC-5 cells, and the effect of the σR1 ligand (+)-pentazocine on pro- and anti-apoptotic and endoplasmic reticulum (ER) stress gene expression was examined. Binding of σR1 to BiP, an ER chaperone protein, and σR1 phosphorylation status were examined by immunoprecipitation. Retinas were harvested from Ins2Akita/+ diabetic mice treated with (+)-pentazocine, and the expression of ER stress genes and of the retinal transcriptome was evaluated. RESULTS Oxidative stress induced the death of primary GCs and RGC-5 cells. The effect was decreased by the application of (+)-pentazocine. Stress increased σR1 binding to BiP and enhanced σR1 phosphorylation in RGC-5 cells. BiP binding was prevented, and σR1 phosphorylation decreased in the presence of (+)-pentazocine. The ER stress proteins PERK, ATF4, ATF6, IRE1α, and CHOP were upregulated in RGC-5 cells during oxidative stress, but decreased in the presence of (+)-pentazocine. A similar phenomenon was observed in retinas of Ins2Akita/+ diabetic mice. Retinal transcriptome analysis of Ins2Akita/+ mice compared with wild-type revealed differential expression of the genes critically involved in oxidative stress, differentiation, and cell death. The expression profile of those genes was reversed when the Ins2Akita/+ mice were treated with (+)-pentazocine. CONCLUSIONS In retinal neurons, the molecular chaperone σR1 binds BiP under stressful conditions; (+)-pentazocine may exert its effects by dissociating σR1 from BiP. As stress in retinal cells increases, phosphorylation of σR1 is increased, which is attenuated when agonists bind to the receptor.

Endogenous Elevation of Homocysteine Induces Retinal Neuron Death in the Cystathionine-β-Synthase Mutant Mouse

PURPOSE To determine the effects of endogenous elevation of homocysteine on the retina using the cystathionine beta-synthase (cbs) mutant mouse. METHODS Retinal homocysteine in cbs mutant mice was measured by high-performance liquid chromatography (HPLC). Retinal cryosections from cbs(-/-) mice and cbs(+/-) mice were examined for histologic changes by light and electron microscopy. Morphometric analysis was performed on retinas of cbs(+/-) mice maintained on a high-methionine diet (cbs(+/-) HM). Changes in retinal gene expression were screened by microarray. RESULTS HPLC analysis revealed an approximate twofold elevation in retinal homocysteine in cbs(+/-) mice and an approximate sevenfold elevation in cbs(-/-) mice. Distinct alterations in the ganglion, inner plexiform, inner nuclear, and epithelial layers were observed in retinas of cbs(-/-) and 1-year-old cbs(+/-) mice. Retinas of cbs(+/-) HM mice demonstrated an approximate 20% decrease in cells of the ganglion cell layer (GCL), which occurred as early as 5-weeks after onset of the HM diet. Microarray analysis revealed alterations in expression of several genes, including increased expression of Aven, Egr1, and Bat3 in retinas of cbs(+/-) HM mice. CONCLUSIONS This study provides the first analysis of morphologic and molecular effects of endogenous elevations of retinal homocysteine in an in vivo model. Increased retinal homocysteine alters inner and outer retinal layers in cbs homozygous mice and older cbs heterozygous mice, and it primarily affects the cells of the GCL in younger heterozygous mice. Elevated retinal homocysteine alters expression of genes involved in endoplasmic reticular stress, N-methyl-d-aspartate (NMDA) receptor activation, cell cycle, and apoptosis.

Serine racemase expression and d-serine content are developmentally regulated in neuronal ganglion cells of the retina

d‐Serine, the endogenous ligand for the glycine modulatory binding site of the NMDA receptor, and serine racemase, the enzyme that converts l‐serine to d‐serine, have been reported in vertebrate retina; initial reports suggested that localization was restricted to Müller glial cells. Recent reports, in which d‐serine and serine racemase were detected in neurons of the brain, prompted the present investigation of neuronal expression of d‐serine and serine racemase in retina and whether expression patterns were developmentally regulated. RT‐PCR, in situ hybridization, western blotting, immunohistochemistry, and immunocytochemical methods were used to localize d‐serine and serine racemase in intact retina obtained from 1 to 3 day, 3 week, and 18 week mouse retinas and in primary ganglion cells harvested by immunopanning from neonatal mouse retina. Results of these analyses revealed robust expression of d‐serine and serine racemase in ganglion cells, both in intact retina and in cultured cells. The levels appear to be developmentally regulated with d‐serine levels being quite high in ganglion cells of neonatal retinas and decreasing rapidly postnatally. Serine racemase levels are also developmentally regulated, with high levels detected during the early postnatal period, but diminishing considerably in the mature retina. This represents the first report of neuronal expression of d‐serine and serine racemase in the vertebrate retina and suggests an important contribution of neuronal d‐serine during retinal development.

Sensitivity of staurosporine-induced differentiated RGC-5 cells to homocysteine

Purpose: Homocysteine is implicated in ganglion cell death associated with glaucoma. To understand mechanisms of homocysteine-induced cell death, we analyzed the sensitivity of the RGC-5 cell line, differentiated using staurosporine, to physiologically-relevant levels of the excitotoxic amino acid homocysteine. Methods: RGC-5 cells were differentiated 24 hr using 316 nM staurosporine and tested for expression of Thy 1.2 via immunodetection, RT-PCR, and immunoblotting. The sensitivity of staurosporine-differentiated RGC-5 cells to physiological levels of homocysteine (50, 100, 250 μM) and to high levels of homocysteine (1 mM), glutamate (1 mM), and oxidative stress (25 μM:10 mU/ml xanthine:xanthine oxidase) was assessed by TUNEL assay and by immunodetection of cleaved caspase-3. The sensitivity of undifferentiated RGC-5 cells to high (1, 5, and 10 mM) homocysteine was also examined. Results: Undifferentiated RGC-5 cells express Thy 1.2 mRNA and protein. Staurosporine-differentiated RGC-5 cells extend neurite processes and express Thy 1.2 after 24 hr differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine -differentiated RGC-5 cells with 50, 100, or 250 µM homocysteine did not alter neurite processes nor induce cell death (detected by TUNEL and active caspase-3) to a level greater than that observed in the control (non-homocysteine-treated, staurosporine-differentiated) cells. The 1 mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18 hr treatment of non-differentiated RGC-5 cells with 5 mM homocysteine decreased survival by 50%. Conclusions: RGC-5 cells differentiated for 24 hr with 316 nM staurosporine project robust neurite processes and are positive for ganglion cell markers consistent with a more neuronal phenotype than non-staurosporine-differentiated RGC-5 cells. However, concentrations of homocysteine known to induce ganglion cell death in vivo and in primary ganglion cells are not sufficient to induce death of RGC-5 cells, even when they are differentiated with staurosporine.

Expression and Function of System N Glutamine Transporters (SN1/SN2 or SNAT3/SNAT5) in Retinal Ganglion Cells

PURPOSE Glutamine transport is essential for the glutamate-glutamine cycle, which occurs between neurons and glia. System N, consisting of SN1 (SNAT3) and SN2 (SNAT5), is the principal mediator of glutamine transport in retinal Müller cells. Mediators of glutamine transport in retinal ganglion cells were investigated. METHODS The relative contributions of various transport systems for glutamine uptake (systems N, A, L, y+L, ASCT, and ATB(0,+)) were examined in RGC-5 cells based on differential features of the individual transport systems. mRNA for the genes encoding members of these transport systems were analyzed by RT-PCR. Based on these data, SN1 and SN2 were analyzed in mouse retina, RGC-5 cells, and primary mouse ganglion cells (GCs) by in situ hybridization (ISH), immunofluorescence (IF), and Western blotting. RESULTS Three transport systems--N, A, and L--participated in glutamine uptake in RGC-5 cells. System N was the principal contributor; systems A and L contributed considerably less. ISH and IF revealed SN1 and SN2 expression in the ganglion, inner nuclear, and photoreceptor cell layers. SN1 and SN2 colocalized with the ganglion cell marker Thy 1.2 and with the Müller cell marker vimentin, confirming their presence in both retinal cell types. SN1 and SN2 proteins were detected in primary mouse GCs. CONCLUSIONS These findings suggest that in addition to its role in glutamine uptake in retinal glial cells, system N contributes significantly to glutamine uptake in ganglion cells and, hence, contributes to the retinal glutamate-glutamine cycle.

Immunofluorescence analysis of the expression of Norpeg (Rai14) in retinal Müller and ganglion cells

Novel retinal pigment epithelial cell gene (Norpeg, Rai14), a developmentally regulated mouse gene, encodes a protein containing six ankyrin repeats and a coiled-coil domain. The objective of the present study was to characterize the Norpeg protein and to analyze its expression in mouse retina using an antibody preparation that we developed. The approximately 110kDa Norpeg was immunoprecipitated and characterized by mass spectrometry. Primary cultures of Müller and ganglion cells isolated from the mouse retina were found to express Norpeg when analyzed by immunoblotting and immunofluorescence microscopy. Immunofluorescence analysis of normal mouse retina sections demonstrated that Norpeg is expressed in cells of the ganglion cell layer, inner nuclear layer as well as in the retinal pigment epithelium. Immunoreactivity was also evident in the radial glial (Müller) cell fibers.