Spatial and Temporal Expression of AIPL1 and NUB1 Protein in Human Retina

Abstract

Abstract: : Purpose: Mutations in the AIPL1 gene lead to Leber congenital amaurosis (LCA), the most severe form of retinal dystrophy with the earliest age of onset. AIPL1 is expressed exclusively in the rod photoreceptors of the adult human retina extending from the photoreceptor inner segments to the pedicles in the outer plexiform layer. Recently, yeast two hybrid analysis has identified the NUB1 protein as an AIPL1 interacting protein. We have investigated the relative spatial and temporal expression of AIPL1 and NUB1 in the developing human retina to gain a better insight into the role that these proteins play in the development of vision. Methods: Peptide directed antisera were generated against human NUB1. The antisera were tested in Western blotting using a panel of human tissue samples and cell lines. Immunohistochemical localization of AIPL1 and NUB1 in human fetal and adult paraffin-embedded sections were performed with the previously characterized AIPL1 antibody and the NUB1 antibody respectively. Results: The NUB1 antibody labelled a single band of 60 kDa apparent molecular weight on western blots. Western analysis of a human tissue panel and cell line panel indicated that this 60 kDa species was expressed ubiquitously in all tissues and cell lines investigated, but was enriched in the human retina and Y79 retinoblastoma cell line. Both AIPL1 and NUB1 were detected in the human retina by immunohistochemistry. In the adult and fetal human retina, NUB1 was detected in the nuclei of all the retinal neural layers from central to peripheral retina. In addition, NUB1 was detected in the nuclei of other cells in adult and fetal eyes. In contrast, AIPL1 was detected only in the presumptive outer nuclear layer of the early developing human retina with a spatial gradient of expression from central to peripheral retina. With increasing age, AIPL1 was also detected in the developing rod inner segments and synaptic pedicles. In addition, the spatial gradient of AIPL1 detection gradually increased towards the peripheral retina from the central retina until AIPL1 was detected with equal intensity in rods throughout the adult human retina. Conclusions: The expression of AIPL1 closely follows the spatial and temporal pattern of photoreceptor development, whereas NUB1 is expressed in all cell types. The early detection of AIPL1 and NUB1 in the developing human retina suggests a potential role for these proteins in retinal development.

Keywords: retinal degenerations: cell biology • chaperones • retinal development