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Alecia K Gross, Evan R. Boitet, Guoxin Ying, Nicholas J Reish, Wolfgang Baehr; Developmental protein NUDC is crucial for rod maintenance and function. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4777.
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© ARVO (1962-2015); The Authors (2016-present)
Nuclear distribution protein C (NUDC) plays an essential role in mitosis and cytokinesis. Prior studies from our lab have shown NUDC interacts with rhodopsin and rab11a in rods. To determine if NUDC is critical for rod function, we have created transgenic X. laevis expressing either NUDC L280P or an shRNA directed against NUDC. Further, we generated a retina-specific Nudc knock-out mouse to identify its role in mouse retinal development.
Transgenic X. laevis tadpoles were prepared expressing mCherry-NUDC, NUDC L280P, or a bicistronic construct expressing EGFP and NUDC shRNA under the rod opsin promoter. Retina-specific Nudc knock-out mice were generated using Nudctm1a(KOMP)Mbp embryonic stem cells obtained from the UCDavis KOMP repository and bred with Six3-Cre mice. For tadpole immunohistochemistry (IHC), eyes were fixed, cryosectioned and labeled with antibodies against rhodopsin, transducin, and arrestin. Cells were stained with wheat germ agglutinin, DAPI and TUNEL. Scotopic electroretinography (ERG) was performed on 7 week-old Nudc-/- mice. IHC was performed on retinal cryosections from these mice and labeled with DAPI and antibodies against rhodopsin, PDE6, cone arrestin, and PKCα.
Expression of NUDC L280P or knock-down of Nudc causes slight mislocalization of rhodopsin and a strong staining pattern of rhodopsin at the base of the outer segments in X. laevis. Thionin staining of retina-specific Nudc knockout mouse whole retina sections revealed severe retinal degeneration involving all retina layers from photoreceptors to ganglion cells. Confocal IHC with DAPI, anti-rhodopsin and anti-PDE6 revealed reduced thickness of the mutant ONL, absence of OS, and presence of rhodopsin and PDE6 in the inner segments. However, rhodopsin and PDE did not mislocalize in the ONL. Labeling with anti-cone arrestin revealed the presence of cones lacking OS. The INL was reduced to 1-2 nuclear layers, and labeling with PKCα showed severe degeneration of rod bipolar cells. Ganglion cells are mostly absent. Scotopic ERG at 7 weeks was negative consistent with non-functional rods.
Here, we show NUDC has a critical role in rod cell maintenance, protein trafficking and homeostasis. It is essential for development and maintenance of all cells in the retina. The mouse knockout retina is severely degenerated, but rod, cone and bipolar cell remnants are present. The cause of degeneration is unknown.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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