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Athanasios Vachtsevanos, Stuart Peirson, Aarti Jagannath, Steve Hughes, Georgia Perganta, Robert E. MacLaren; Application Of RNAi To Unravel The Melanopsin Phototransduction Pathway. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2564.
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© ARVO (1962-2015); The Authors (2016-present)
It has been demonstrated that retinal photoreception among mammals extends beyond rods and cones to include a small number of intrinsically photosensitive retinal ganglion cells (pRGCs), which are capable of responding to light due to expression of the melanopsin (OPN4) photopigment. OPN4 may have therapeutic potential if ectopically expressed in the degenerate retina in cases where photoreceptors are lost, but the molecules involved in this OPN4 light induced transduction cascade are less well characterized. We therefore sought to probe further the mechanism of OPN4 mediated light sensitivity by siRNA medicated knock down of specific molecules in the aged rd mouse, which is almost exclusively dependent on the OPN4 pathway for light sensitivity.
We generated siRNA probes against the long transcript variant of murine Opn4 mRNA (UCG CAC UGA UUG UCA UUC UUC UCU U) and tested these probes on the murine Neuro2A (N2a) cell line. Mice on the C3H/HeN background harboring the rd mutation were aged to 80 days. At this point virtually all surviving cone cells are lost - similar to end-stage retinitis pigmentosa in humans. siRNA was given by intravitreal injection into one eye and pupillometry was assessed combined with molecular analyses at 1, 3 and 5 days.
Reverse transcription polymerase chain reaction (RT-PCR) analysis in N2a cells confirmed Opn4 knockdown and immunolabelling techniques identified >85% silencing with siRNA OPN4. Pupillometry in the mouse was also affected by the siRNA injections in vivo and this was correlated to silencing of the murine Opn4 at all time points. We also used immunolabelling to identify the distribution of the cation channel TRPC3, which interacts with melanopsin in vivo.
We present a reproducible in vivo model in which siRNA induced silencing of the melanopsin pathway can be assessed by pupillometry and compared to levels of mRNA and protein at specific timepoints. Probes against other putative second messengers, such as TRPC3, may unravel the molecular interactions of this pathway. This may help in future to induce light sensitivity in other retinal neurons, which may benefit patients who are completely blind from photoreceptor loss.
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