Increased expression of ceruloplasmin in the retina following photic injury.
OBJECTIVE
Oxidative stress plays a role in the photic injury model of retinal degeneration and in age-related macular degeneration. Our preliminary microarray analysis of retinal gene expression upon photic injury suggested increased expression of ceruloplasmin, a ferroxidase that could reduce retinal oxidative stress. Patients with acerul oplasminemia have retinal degeneration, indicating that ceruloplasmin is necessary for maintenance of retinal health. The purpose of this study was to determine whether retinal ceruloplasmin is upregulated following photo-oxidation, to localize ceruloplasmin protein, and to determine which ceruloplasmin isoform is present in the retina.


METHODS
Balb/c mice were exposed to bright white light for seven hours. TUNEL labeling was used to detect photoreceptor apoptosis. At several intervals after the light injury, retinal ceruloplasmin was studied by quantitative PCR, immunohistochemistry, and western analysis. Expression of the secreted and expression of the membrane-anchored glycosyl phosphatidyl inositol (GPI) linked forms of ceruloplasmin were assesed in rat retina using primers specific for each form. Vitreous ceruloplasmin was detected by immunohistochemistry in Balb/c mouse eyes and by western analysis of aspirated vitreous from post-mortem human eyes.


RESULTS
Retinal ceruloplasmin mRNA was upregulated eight-fold following photic injury. Ceruloplasmin protein was detected throughout normal retinas by immunohistochemistry, with a specific increase in Muller cell labeling following photic injury. Western analysis confirmed an increase in ceruloplasmin protein following photic injury and revealed eight-fold more ceruloplasmin protein in normal retina than in brain. The mRNAs for both the secreted and GPI linked forms of ceruloplasmin were detected by RT-PCR in the retina. Ceruloplasmin protein was detected by western analysis of normal human vitreous and was increased in mouse vitreous following photic injury.


CONCLUSIONS
Ceruloplasmin, a retinal ferroxidase, is upregulated at the mRNA and protein levels upon light damage. The increased protein is primarily in Muller cells. Ceruloplasmin is considerably more abundant in retina than in brain. The retina expresses both the GPI-linked and secreted forms of ceruloplasmin, and since vitreous ceruloplasmin increases following photic injury, some of the retinal ceruloplasmin may be secreted into the vitreous. Ceruloplasmin may protect the retina from oxidative stress by decreasing the amount of ferrous iron available to produce reactive oxygen species.
F. M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, PA, USA.
Lin
L
Chen
Tzvete
T
Dentchev
Robert
R
Wong
Paul
P
Hahn
Rong
R
Wen
Jean
J
Bennett
Joshua L
JL
Dunaief
D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000528:radiation effects; D015153:Blotting, Western; D002570:Ceruloplasmin / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D020287:In Situ Nick-End Labeling; D008027:Light; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000378:metabolism; D011833:Radiation Injuries, Experimental / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D012160:Retina / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects*; D012162:Retinal Degeneration / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D014822:Vitreous Body / Q000378:metabolism
D012333:RNA, Messenger; D002570:Ceruloplasmin