Recharacterization of the RGC-5 Retinal Ganglion Cell Line

Bergen, Nicole J Van; Wood, J. K.; Chidlow, Glyn; Trounce, Ian A.; Casson, Robert J.; Ju, Won‐Kyu; Weinreb, Robert N.; Crowston, Jonathan G

doi:10.1167/iovs.09-3484

Cited by 160 publications

(157 citation statements)

References 20 publications

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“…Other antibodies include those labeling GFAP (Sigma-Aldrich), CD45 (Abcam), Mac-2 (ATCC), pNF (SMI31; Covance), NFH (Chemicon), and Iba1 (Wako). RGC-5 cells were obtained from N. Agarwal (University of North Texas Health Science Center, Fort Worth, TX), and sequencing of the Thy1 gene confirmed that they were of mouse origin, as reported (37). Flatmounted retinas were processed for in situ hybridization using digoxigeninor fluorescein-labeled riboprobes for γ-synuclein and Nfl, transcribed from cDNAs (IMAGE clones 1448798 and 4506903; OpenBiosystems), hydrolyzed, and detected using Cy3 tyramides (Perkin-Elmer) for single in situ hybridizations, and both Alexa 488 (Invitrogen) and Cy3 tyramides for double in situ hybridizations, as previously described (8).…”

Section: Mtz Astrocytes As Possible Mediators Of Axonal Injury In Glamentioning

confidence: 66%

Myelination transition zone astrocytes are constitutively phagocytic and have synuclein dependent reactivity in glaucoma

Nguyen

Soto

Kim

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

189

179

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Optic nerve head (ONH) astrocytes have been proposed to play both protective and deleterious roles in glaucoma. We now show that, within the postlaminar ONH myelination transition zone (MTZ), there are astrocytes that normally express Mac-2 (also known as Lgals3 or galectin-3), a gene typically expressed only in phagocytic cells. Surprisingly, even in healthy mice, MTZ and other ONH astrocytes constitutive internalize large axonal evulsions that contain whole organelles. In mouse glaucoma models, MTZ astrocytes further upregulate Mac-2 expression. During glaucomatous degeneration, there are dystrophic processes in the retina and optic nerve, including the MTZ, which contain protease resistant γ-synuclein. The increased Mac-2 expression by MTZ astrocytes during glaucoma likely depends on this γ-synuclein, as mice lacking γ-synuclein fail to up-regulate Mac-2 at the MTZ after elevation of intraocular pressure. These results suggest the possibility that a newly discovered normal degradative pathway for axons might contribute to glaucomatous neurodegeneration.DBA/2J mice | retinal ganglion cell | Sncg G laucoma, a neurodegenerative disorder that kills retinal ganglion cells (RGCs), affects more than 60 million people and is the second leading cause of blindness worldwide (1). In glaucomatous retinas, both astrocytes and Müller glia increase their reactivity (2, 3). Within the orbital portion of the optic nerve, astrocytes increase in number and reactivity (4). Within the optic nerve head (ONH), astrocytes also increase reactivity in glaucoma animal models and in the human disease (5). These ONH astrocytes are of particular interest as they enwrap axons at the location where damage would account for the arcuate vision field loss characteristic of glaucoma.Here, we identify a spatially discrete population of astrocytes within the ONH, those at the myelination transition zone (MTZ), which express the phagocytosis-related gene Mac-2. Surprisingly, astrocytes throughout the ONH including the MTZ phagocytose large axonal evulsions even in unaffected mice. Mac-2 expression is increased in optic nerve astrocytes upon injury and at the MTZ in two mouse models of glaucoma. In glaucomatous mice, there are protease resistant forms of γ-synuclein, including at the MTZ. Further, mice lacking γ-synuclein fail to up-regulate Mac-2 at the MTZ in response to increased intraocular pressure (IOP). These results suggest the possibility that failure to properly clear axon-derived material at the MTZ, including γ-synuclein, may contribute to axon loss in glaucoma.

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Section: Mtz Astrocytes As Possible Mediators Of Axonal Injury In Glamentioning

confidence: 66%

Myelination transition zone astrocytes are constitutively phagocytic and have synuclein dependent reactivity in glaucoma

Nguyen

Soto

Kim

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

189

179

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“…RGC-5 is a mouse ganglion cell line (20,21). 661W and RGC-5 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin (Meiji Seika Kaisha, Ltd., Tokyo), and 100 μg/mL streptomycin (Meiji Seika Kaisha) under a humidified atmosphere of 95% air and 5% CO 2 at 37°C.…”

Section: W Photoreceptor Cells and Rgc-5 Culturesmentioning

confidence: 99%

Role of Oxidative Stress in Retinal Photoreceptor Cell Death in N-Methyl-N-nitrosourea–Treated Mice

et al. 2012

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Abstract. This study aimed to investigate whether oxidative stress contributes to retinal cell death in a mouse model of photoreceptor degeneration induced by N-methyl-N-nitrosourea (MNU). We measured in vitro MNU-induced radical production in retinal cell cultures of murine 661W photoreceptor-derived cells; RGC-5, a mouse ganglion cell line; and primary retinal cells. The addition of MNU induced oxidative radical generation in 661W and primary retinal cells, but not in RGC-5 cells. Edaravone, a free radical scavenger, at 1 μM reduced MNU-induced radical production in 661W and primary retinal cells. To induce in vivo retinal photoreceptor degeneration in mice, we administered 60 mg/kg MNU by intraperitoneal injection. We intravenously administered 1 mg/kg edaravone immediately and at 6 h after the MNU injection. Retinal photoreceptor degeneration was evaluated by measuring the thickness of the outer nuclear layer (ONL) by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and by oxidative stress markers. MNU caused photoreceptor cell loss at 7 days after administration. Edaravone inhibited ONL thinning and reduced TUNEL-positive cells and the oxidative stress markers. These findings indicate that MNU leads to selective photoreceptor degradation via oxidative stress in vitro and in vivo and may help to understand the pathogenic mechanism of retinitis pigmentosa.

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“…In this study, we determined the involvement of UPP and autophagy in processing of the endogenous optineurin in RGC5 cells, a neuronal cell type recently shown to be of mouse origin (31) and an established model for RGCs (31,32), as well as neuronal rat adrenal pheochromocytoma PC12 cells (33). The processing of overexpressed wild type optineurin and E50K mutant protein was also studied to test the hypothesis that, similar to other neurodegenerative diseases, UPP function is compromised and autophagy is induced with elevated level or mutation of aggregate-prone optineurin.…”

mentioning

confidence: 99%

Processing of Optineurin in Neuronal Cells

Shen

Ying

Qiu

et al. 2011

Journal of Biological Chemistry

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Optineurin is a gene linked to amyotrophic lateral sclerosis, Paget disease of bone, and glaucoma, a major blinding disease. Mutations such as E50K were identified in glaucoma patients. We investigated herein the involvement of ubiquitin-proteasome pathway (UPP) and autophagy, two major routes for protein clearance, in processing of optineurin in a retinal ganglion cell model line RGC5 and neuronal PC12 cells. It was found that the endogenous optineurin level in neuronal cells was increased by treatment of proteasomal inhibitor but not by autophagic and lysosomal inhibitors. Multiple bands immunoreactive to anti-ubiquitin were seen in the optineurin pulldown, indicating that optineurin was ubiquitinated. In cells overexpressing wild type and E50K optineurin, the level of the proteasome regulatory ␤5 subunit (PSMB5, indicative of proteasome activity) was reduced, whereas that for autophagy marker microtubule-associated protein 1 light chain 3 was enhanced compared with controls. Autophagosome formation was detected by electron microscopy. The foci formed after optineurin transfection were increased upon treatment of an autophagic inhibitor but were decreased by treatment of an inducer, rapamycin. Moreover, the level of optineurin-triggered apoptosis was reduced by rapamycin. This study thus provides compelling evidence that in a normal homeostatic situation, the turnover of endogenous optineurin involves mainly UPP. When optineurin is up-regulated or mutated, the UPP function is compromised, and autophagy comes into play. A decreased PSMB5 level and an induced autophagy were also demonstrated in vivo in retinal ganglion cells of E50K transgenic mice, validating and making relevant the in vitro findings.

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Recharacterization of the RGC-5 Retinal Ganglion Cell Line

Abstract: Investigators using cells designated as RGC-5 should confirm the species to be of rat origin and retinal-specific marker expression before considering their use as retinal ganglion-like cells.

Cited by 160 publications

References 20 publications

Myelination transition zone astrocytes are constitutively phagocytic and have synuclein dependent reactivity in glaucoma

Myelination transition zone astrocytes are constitutively phagocytic and have synuclein dependent reactivity in glaucoma

Role of Oxidative Stress in Retinal Photoreceptor Cell Death in N-Methyl-N-nitrosourea–Treated Mice

Processing of Optineurin in Neuronal Cells

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