Protein 4.1G-mediated membrane trafficking is essential for correct rod synaptic location in the retina and for normal visual function

Abstract

Purpose: Retinal neural circuits form three distinct layers: the outer nuclear layer (ONL), the inner nuclear layer (INL), and the ganglion cell layer (GCL). In the ONL, rod photoreceptor cell bodies are broadly distributed throughout the ONL, while cone photoreceptor cell bodies are arrayed on the outer side of the retina. Photoreceptor axon terminals are located at relatively restricted positions in the OPL, indicating that photoreceptor axon terminal location is regulated in a genetically fixed manner, although the distance between each cell body and synaptic terminal varies among photoreceptor cells. Despite the molecular mechanisms for triad synapse formation having been extensively studied, the molecular basis and functional importance of OPL formation at a fixed laminar location throughout the retina during development remains unexplored.

Methods: Protein 4.1 family genes, 4.1R, 4.1N, 4.1G, and 4.1B in vertebrates, encode membrane-cytoskeletal proteins, which anchor a transmembrane protein to the actin-spectrin cytoskeleton. In the current study, we found that 4.1G is predominantly expressed in rod photoreceptor cells in the retina. We generated 4.1G-null mutant mice and analyzed their retinal phenotypes.

Results: We observed ectopic rod photoreceptor terminals in the ONL and aberrant neurite sprouting from bipolar cells and horizontal cells into the ONL of 4.1G^−/− mice. By mass spectrometric analysis, we identified AP3B2, a component of the adaptor protein complex 3 (AP3) in neuronal cells, as a 4.1G-binding protein. AP3B2 is involved in synaptic vesicle formation from endosomes in the membrane trafficking pathway. The Ap3b2^−/− retina exhibited ectopic synapse formation and VGLUT1 mislocalization phenotypes that were similar to those observed in the 4.1G^−/− retina. In the developing 4.1G^−/− retina, synaptic component proteins such as CtBP2 and Pikachurin were aberrantly localized and/or aggregated, suggesting that vesicle transport to axon terminals is impaired in rods during development.

Conclusions: The current study show that the membrane trafficking system mediated by the 4.1G-AP3 complex is required for the formation of correct synapse location, normal visual function, and neuronal survival, leading us to come up with a novel notion that synapse location in the retinal circuitry has physiological significance.