Involvement of ER stress in retinal cell death.
OBJECTIVE
To clarify whether endoplasmic reticulum (ER) stress is involved in retinal cell death, using cultured retinal ganglion cells (RGC-5, a rat ganglion cell line transformed with E1A virus), and transgenic mice ER stress-activated indicator (ERAI) mice carrying a human XBP1 and venus a variant of green fluorescent protein (GFP) fusion gene.


METHODS
RGC-5 damage was induced by tunicamycin, and cell viability was measured by double nuclear staining (Hoechst 33342 and either YO-PRO-1 or propidium iodide). The expressions of glucose-regulated protein 78(GRP78)/BiP, the phosphorylated form of eukaryotic initiation factor 2alpha (p-eIF2alpha), and C/EBP-homologous (CHOP) protein after tunicamycin (in vitro or in vivo) or N-methyl-D-aspartate (NMDA; in vivo) treatment were measured using immunoblot or immunostaining. ERAI mice carrying the F-XBP1-DBD-venus expression gene were used to monitor ER-stress in vivo. Twenty-four hours after intravitreal injection of tunicamycin or NMDA, or after raising intraocular pressure (IOP), the retinal fluorescence intensity was visualized in anesthetized animals using an ophthalmoscope and in retinal flatmount or cross-section specimens using laser confocal microscopy.


RESULTS
Treatment with tunicamycin induced apoptotic cell death in RGC-5 and also induced production of ER stress-related proteins (BiP, the phosphorylated form of eIF2alpha, and CHOP protein). In vivo, tunicamycin induced retinal ganglion cell (RGC) loss and thinning of the inner plexiform layer, 7 days after intravitreal injection. In flatmounted retinas of ERAI mice, the fluorescence intensity arising from the XBP-1-venus fusion protein, indicating ER-stress activation, was increased at 24 h after tunicamycin, NMDA, or IOP elevation. In transverse cross-sections from ERAI mice, the fluorescence intensity was first increased in cells of the ganglion cell and inner plexiform layers at 12 and 24 h, respectively, after NMDA injection, and it was localized to ganglion and amacrine cells at 12 and 24 h, respectively, and to microglial cells at 72 h. BiP and CHOP were increased at 12 h after NMDA injection, and the increases persisted for the remainder of the 72 h observation period.


CONCLUSIONS
These data indicate that ER-stress may play a pivotal role in RGC death, whether induced by NMDA or IOP elevation.
Shimazawa
Masamitsu
M
Department of Biofunctional Molecules, Gifu Pharmaceutical University, Gifu, Japan. shimazawa@gifu-pu.ac.jp <shimazawa@gifu-pu.ac.jp>
Inokuchi
Yuta
Y
Ito
Yasushi
Y
Murata
Hiroshi
H
Aihara
Makoto
M
Miura
Masayuki
M
Araie
Makoto
M
Hara
Hideaki
H
D025042:Amacrine Cells / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins / Q000235:genetics; D004721:Endoplasmic Reticulum; D000091342:Endoplasmic Reticulum Chaperone BiP; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D014644:Genetic Variation; D049452:Green Fluorescent Proteins / Q000235:genetics; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007267:Injections; D007429:Intraocular Pressure; D051379:Mice; D008822:Mice, Transgenic; D017628:Microglia / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D016202:N-Methylaspartate / Q000008:administration & dosage / Q000494:pharmacology; D009687:Nuclear Proteins / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D012160:Retina / Q000473:pathology / Q000503:physiopathology*; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000473:pathology; D013312:Stress, Physiological / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D051742:Transcription Factor CHOP / Q000378:metabolism; D014157:Transcription Factors; D014415:Tunicamycin / Q000008:administration & dosage / Q000494:pharmacology; D014822:Vitreous Body; D000071717:X-Box Binding Protein 1
D004268:DNA-Binding Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D018690:Excitatory Amino Acid Agonists; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; C000717248:Hspa5 protein, mouse; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C519634:XBP-1 venus fusion protein; C000605543:XBP1 protein, human; C000605544:Xbp1 protein, mouse; C000605545:Xbp1 protein, rat; D014415:Tunicamycin; D051742:Transcription Factor CHOP; D049452:Green Fluorescent Proteins; D016202:N-Methylaspartate