Visualizing sphingomyelin in the retina

Abstract

Sphingomyelin (SM) is a major component of cellular membranes that is altered by retinal and optic nerve degenerations. However, our understanding of the influence of SM during degenerations is hampered by methodological limitations. Prior investigations have demonstrated the accumulation of SM to plasma membranes of cultured cells, using an enhanced green fluorescent non-toxic truncated form of lysenin (EGFP-NT-Lys), which is a protein that specifically binds to SM. Here, we used EGFP-NT-Lys and a permeabilization-free method for immunohistochemistry, which preserves membrane integrity, to demonstrate the accumulation of SM to the plasma membranes of retinal ganglion cells (RGCs) and retinal endothelial cells (RECs) of the intact mouse retina. To determine the sensitivity and selectivity of EGFP-NT-Lys for SM and SM species, we performed lipid dot blot assays. We found EGFP-NT-Lys is highly selective for SM and preferentially binds to longer-chain SMs. We confirmed that EGFP-NT-Lys labeling of SM is modifiable by treatment with the catabolic enzyme, sphingomyelinase. In addition, we verified EGFP-NT-Lys binding to SM by competition assays and EGFP. Confocal image analysis of immunofluorescence of RGC and REC markers and EGFP-NT-Lys labeling in flat mount mouse retinas revealed SM heavily accumulates within the retinal vasculature and around the perimeter of RGCs. Our data demonstrates that EGFP-NT-Lys combined with a permeabilization-free method for immunohistochemistry can be used to detect and quantify plasma membrane associated SM in defined cells of the intact retina.

Keywords: Lysenin; Retinal endothelial cells; Retinal ganglion cells; Sphingomyelin.