Elevated retina-specific expression of the small heat shock protein, alphaA-crystallin, is associated with photoreceptor protection in experimental uveitis.
OBJECTIVE
During the early phase of experimental autoimmune uveitis (EAU), before macrophages infiltrate the retina and uvea, photoreceptor mitochondrial oxidative stress, nitration of photoreceptor mitochondrial proteins, and release of cytochrome c have been observed. However, no apoptosis has been detected during this phase. In this study, alphaA-crystallin upregulation in the retina and its antiapoptotic protective role were evaluated in early EAU.


METHODS
Gene microarrays were first used to identify upregulated genes in retinas with early EAU. Among highly upregulated crystallins, alphaA was confirmed by real-time polymerase chain reaction and Western blot, and the site of upregulation was localized by immunohistochemistry. The association of alphaA-crystallin to nitrated cytochrome c and interaction with a procaspase-3 subunit was assayed. Photoreceptor apoptosis in alphaA knockout mice was compared with that in wild-type animals with EAU, by using the terminal transferase dUTP nick-end labeling assay and polymerase chain reaction.


RESULTS
In early EAU, alphaA-crystallin was increased 33-fold, and the site of increase was localized to the photoreceptor inner segments. This crystallin suppressed apoptosis by associating with the nitrated cytochrome c and p24. The association with nitrated cytochrome c, in particular, appeared to be restricted to nitrated cytochrome c, and thus, no association of non-nitrated cytochrome c was detected. The knockout mice showed signs of EAU development early and showed apoptosis in the retina; no such changes were seen in the wild-type control animals.


CONCLUSIONS
alphaA-Crystallin is highly upregulated in the retina during early EAU. This upregulation is localized primarily in the photoreceptor inner segments, the site of mitochondrial oxidative stress. Further, in early EAU, the photoreceptors preferentially use alphaA-crystallin to suppress mitochondrial oxidative stress-mediated apoptosis.
Rao
Narsing A
NA
Doheny Eye Institute, Keck School of Mewdicine, University of Southern California, Los Angeles, CA 90033, USA. nrao@usc.edu
Saraswathy
Sindhu
S
Wu
Guey Shuang
GS
Katselis
George S
GS
Wawrousek
Eric F
EF
Bhat
Suraj
S
D000818:Animals; D017209:Apoptosis; D001327:Autoimmune Diseases / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004195:Disease Models, Animal; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D051379:Mice; D018345:Mice, Knockout; D020411:Oligonucleotide Array Sequence Analysis; D018384:Oxidative Stress; D020419:Photoreceptor Cells, Vertebrate / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D014605:Uveitis / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D038202:alpha-Crystallin A Chain / Q000235:genetics* / Q000378:metabolism
D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D038202:alpha-Crystallin A Chain; D045304:Cytochromes c; D053148:Caspase 3