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Ushananthini Shanmugalingam, Abigail E. Morris, Patrice D. Smith; Age-dependent post-translational modification of neuronal activity-regulated pentraxin (Narp) in the mouse retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):119.
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© ARVO (1962-2015); The Authors (2016-present)
Neuronal activity-regulated pentraxin (Narp) expression varies in the mouse eye; however, very little is known about the developmental regulation of Narp in the visual system. The goal of this study was to determine the expression pattern and post-translational modification of Narp protein across retinal development.
Immunofluorescence was used to determine the time course of Narp protein expression in the retina. The localization of Narp in glia versus retinal ganglion cells (RGCs) was determined by double immunofluorescence with specific markers of RGCs or glial cell markers. Briefly, fresh enucleated embryonic 18 (E18), postnatal 7, postnatal 14 and adult mouse eyes (n=4) were perfused and then fixed in 4% paraformaldehyde, and 14μm tissue sections were collected using a cryostat. Sections were immunostained with β-III-tubulin (βIIIT), RNA binding protein with multiple splicing (RBPMS), glial fibrillary acidic protein (GFAP) and Narp. The number of Narp-positive cells was quantified in the retina and co-labeled cells were analyzed. To examine post translational modification state of Narp, protein samples from the different developmental stages (n=4) were denatured and treated with N-glycosidase F (PNGase F), an enzyme which specifically deglycosylates N-glycoproteins. Previous work has shown that Narp can undergo N-glycosylation, however the developmental regulation of this modification is not known. SDS-PAGE was used to determine the expression levels of glycosylated Narp in protein samples isolated from the retina.
Narp is predominantly co-localized with β-III- tubulin in the ganglion cell layer, suggesting an RGC-specific expression pattern of Narp in the retina. Western blot analysis of E18 retina samples resulted in a unique band that was not present in adult retina samples. PNGase F treatment of protein samples resulted in a lower band that likely represents the deglycosylated Narp form. Interestingly, the prominent low molecular weight band was predominantly expressed in the adult samples.
Narp is predominantly expressed in RGCs and is developmentally regulated. Narp glycosylation (post-translational modification) is differentially regulated across development of the retina.
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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