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M.L. Woodruff, Z. Wang, G.L. Fain, J. Lem; Constitutive Activation and Photoreceptor Degeneration in RPE65 Knockout Mice . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3553.
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Purpose: RPE65 is localized to the RPE and is essential for the regeneration of 11-cis retinal. Rods in animals deficient in this protein have abnormally low levels of rhodopsin, are desensitized, and slowly degenerate. Since these animals have normal levels of opsin, which can by itself produce a low level of constitutive activation of visual transduction, we asked whether this activation might be responsible for the degeneration in RPE65-/- animals. Methods: RPE65-/- were mated with transducin alpha (Trα) -/- mice. Electrical recording and Ca measurement were made from rods of 6-12 wk animals (dark-adapted for at least 3 hours) in bicarbonate-buffered medium at 35o-37oC with methods as previously described (J. Physiol. 542:843-854, 2002). Results: Rods in RPE65-/- mice show some degeneration by 28 weeks and a considerable loss of photoreceptor nuclei by 40 weeks. In RPE65/Trα double knockout animals, degeneration is greatly reduced, indicating that transduction is in some way responsible for cell death in the RPE65-/- mice. Rod responses in RPE65-/- mice had smaller saturating light responses than for wild-type, indicating that an abnormally large fraction of the channels were closed in darkness. The channels could be reopened (and responses made larger) either with 100 µM IBMX or by regenerating with 11-cis retinal. Response sensitivity was also depressed in RPE65-/- rods (2.8 x 10-5 pAΦ-1µm2) in comparison to normal rods (1.2 pAΦ-1µm2), but this sensitivity loss could be largely reversed by treatment with chromophore (1.7 x 10-1 pAΦ-1µm2). The dark outer segment Ca2+i averaged 250 nM in wild-type rods (n=40) but only 140 nM in RPE65-/- animals (n=7), also consistent with partial channel closure in darkness. The Ca2+i in RPE65/Trα double knockout animals (n=12) was not significantly different from that in wild-type. Conclusions: These experiments show that photoreceptor degeneration in animals deficient in RPE65 is the result of constitutive activation of the visual cascade, probably produced by the apoprotein opsin. Consistent with this conclusion, the rods in these animals have smaller light responses and less dark free calcium, indicating that a fraction of the cyclic-nucleotide gated channels that would normally be open in darkness are closed in RPE65-/- photoreceptors.
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