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N. P. Turunen, K. Tuppurainen, V. Karjalainen, U. Näpänkangas; BH3-Only Proteins Noxa and Puma Are Expressed in Normal Rat Retina and Upregulated After Optic Nerve Transection. Invest. Ophthalmol. Vis. Sci. 2007;48(13):561.
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Puma and Noxa are members of the BH3-only-family of proteins that regulate apoptosis under a variety of physiological conditions and mediate neural precursor cell death. We studied whether they are expressed in adult rat retina and if their expression is increased after optic nerve injury.
The left optic nerve of adult Sprague-Dawley rats (N = 48) was transected to induce loss of retinal ganglion cells. The animals were sacrificed 6 h, 12 h, 24 h, 3 d, 7 d or 14 d and their retinas were collected for analysis. Total RNA was extracted with Trizol, analyzed for purity, and cDNA was produced. The mRNA levels of Puma and Noxa, as well as of their regulator p53, were determined using real time PCR with SYBR Green assay. Untreated animals were used as controls and the expression levels were normalized against beta-actin. Protein expression was studied by immunofluorescence in Fluoro-Gold labeled retinas.
Puma and Noxa mRNA and small amounts of protein can be detected in normal adult rat retina. The relative level of Puma mRNA increases slightly already 6 h after injury, peaks at 24 hrs returns to normal levels by day 14. Noxa mRNA is increased 2-fold 24 hrs after injury, but remains at normal levels in other time points.
Small amounts of Puma and Noxa mRNA can be detected in normal adult rat retina. Optic nerve transection induces an upregulation of Puma and Noxa, both proapoptotic members of BH3-only proteins, in rat retina suggesting that they play a role in the regulation of apoptosis shortly after axonal injury.
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