Olfactomedin Domain-containing Proteins, Olfm1 and Olfm2, Interact with AMPA Receptors in the Retina

Abstract

Purpose: Olfactomedin 1 (Olfm1) and Olfactomedin 2 (Olfm2) are highly conserved secretory glycoproteins that are preferentially expressed in neuronal tissues of vertebrates during development and in adults. The Arg144Gln mutation in OLFM2 was reported as a possible disease-causing mutation in Japanese patients with open-angle glaucoma (Funayama et al., 2006). The main goal of this study was to elucidate the role of Olfm1 and Olfm2 proteins in the mouse retina and optic nerve.

Methods: Olfm1 knockout (KO) mice were described previously (Cheng et al., 2007). Olfm2 KO mice were produced by deleting exons 2-5 and replacing them with the beta-galactosidase gene. Optic nerves were assessed by axon quantification of semi-thin cross-sections and by electron microscopy. Retinal ganglion cell (RGC) death was evaluated by whole mount staining with NeuN and Brn3 antibodies. Conductivity of the optic nerve was evaluated by visual evoked potential test. Proteins interacting with Olfm1 were identified using shotgun proteomic analysis.

Results: Olfm1 and Olfm2 proteins are 60% identical. Both Olfm1 and Olfm2 are preferentially expressed in the RGCs. The level of Olfm1 expression is significantly higher than the level of Olfm2 expression in the developing and adult mouse retina. Olfm1 KO demonstrated a significant decrease in the amount of RGCs and a reduction in the amount of axons in the optic nerve with progression of age. Twelve month old Olfm1 KO mice showed a 25% reduction in a total cross-sectional area of the optic nerve. Olfm2 KO mice didn’t show a significant decrease in the number of RGCs and axons. Visual evoked potential tests revealed a significantly delayed latency and reduced amplitude in Olfm1 and Olfm2 KO mice, respectively. Proteins interacting with Olfm1 were precipitated from postnatal day one mouse brain using Olfm1-specific polyclonal antibodies. Shotgun sequencing of immunoprecipitates led to the identification of several synaptic proteins including GluR2, an AMPA receptor subunit. GluR2 is expressed and co-localized with Olfm1/2 in RGCs.

Conclusions: Our data suggest that Olfm1 and Olfm2 proteins play an important role in the development and function of RGCs and optic nerve. Interaction of Olfm1/2 with AMPA receptors may lead to the modulation of the receptor activity in the retina.

Keywords: 629 optic nerve • 531 ganglion cells