Loss of the ER membrane protein complex subunit Emc3 leads to retinal bipolar cell degeneration in aged mice.

Xiong Zhu,Xin Qi,Wenjing Liu,Wanli Tian,Zhilin Jiang,Xianjun Zhu,Shuzhen Li,Yeming Yang,Anand Swaroop

doi:10.1371/journal.pone.0238435

Abstract

The endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved protein complex involved in inserting the transmembrane domain of membrane proteins into membranes in the ER. EMC3 is an essential component of EMC and is important for rhodopsin synthesis in photoreceptor cells. However, the in vivo function of Emc3 in bipolar cells (BCs) has not been determined. To explore the role of Emc3 in BCs, we generated a BC-specific Emc3 knockout mouse model (named Emc3 cKO) using the Purkinje cell protein 2 (Pcp2) Cre line. Although normal electroretinography (ERG) b-waves were observed in Emc3 cKO mice at 6 months of age, Emc3 cKO mice exhibited reduced b-wave amplitudes at 12 months of age, as determined by scotopic and photopic ERG, and progressive death of BCs, whereas the ERG a-wave amplitudes were preserved. PKCa staining of retinal cryosections from Emc3 cKO mice revealed death of rod BCs. Loss of Emc3 led to the presence of the synaptic protein mGLuR6 in the outer nuclear layer (ONL). Immunostaining analysis of presynaptic protein postsynaptic density protein 95 (PSD95) revealed rod terminals retracted to the ONL in Emc3 cKO mice at 12 months of age. In addition, deletion of Emc3 resulted in elevated glial fibrillary acidic protein, indicating reactive gliosis in the retina. Our data demonstrate that loss of Emc3 in BCs leads to decreased ERG response, increased astrogliosis and disruption of the retinal inner nuclear layer in mice of 12 months of age. Taken together, our studies indicate that Emc3 is not required for the development of BCs but is important for long-term survival of BCs.

Highlights

The majority of the tdTomato-expressing cells were rod bipolar cells positive for the rod bipolar cell marker PKCα. This result is similar to the expression pattern (75% of the tdTomato-expressing bipolar cells were positive for PKCα) reported by Lu et al [24] (Fig 1A)
Since Purkinje cell protein 2 (Pcp2)-Cre is expressed in cone bipolar cells [24], as revealed by red tomato-positive cells not stained by a PKCα antibody (Fig 1A), we examined the ERG response under photopic conditions
The EMC is critical for the stable expression of multipass transmembrane proteins such as rhodopsin, and its loss causes retinal degeneration in Drosophila and mice [18, 19]

Summary

Introduction

To evaluate the physiological functions of Emc3 in retinas, ERG analysis was performed on control and Emc3 cKO mice at 6 and 12 months of age. At 6 months of age, under scotopic conditions, no visible changes were observed for the a-wave and b-wave amplitudes in Emc3 cKO mice (Fig 2A, left panels; Fig 3A and 3B).

Results

Conclusion