Nano-scale resolution of native retinal rod disk membranes reveals differences in lipid composition

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein–lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.

Protective Effect of Mitochondrially Targeted Peptide Against Oxidant Injury of Cone Photoreceptors Through Preventing Necroptosis Pathway

Retinopathy is an eye disease caused by the death of retinal cells in the macular area and the surrounding choroid. As the retinal rod cell dysfunction and death lead to the loss of night vision, the disease will lead to visual dysfunction and blindness as the disease progresses. Because of the irreversible nature of cell death, gene therapy has become a research hotspot in the field of retinopathy. But the technology is still in animal studies or clinical trials, and more research is needed to prove its feasibility. In this study, oxidative damage cell model was established and divided into a control group, H2O2 group, SS31 +NEC1 group, SS31 +H2O2 group, and SS31 +NEC1 +H2O2 group, for different interventions. The cell survival rate of the H2O2 group was significantly increased compared with those of the SS31 + H2O2 group, SS31 +NEC1 +H2O2 group, and NEC1 +H2O2 group. Nec1 combined treatment significantly reduced reactive oxygen species (ROS) production compared with that in the H2O2 group. The level of MDA in the SS31 group, Nec-1 group and combined treatment of SS31 +NEC1 group decreased significantly compared with the H2O2 group. The proportion of cells with decreased mitochondrial membrane potential in the H2O2 group significantly increased, and the rate of positivity for propidium iodide (PI) of 661W cells in the H2O2 group and the control group significantly increased. Nine hours after H2O2 treatment of 661W cells, the RIP3 expression level began to increase, and peaked at 24 h. The level of RIP3 in the H2O2 group was significantly increased, while this level was downregulated in the SS31 and NEC1 treatment groups. Therefore, this study suggests that SS31 has a partial protective effect on 661W cells by inhibiting necrosis, which has certain guiding significance for the treatment of retinal diseases.

Dissecting lipid contents in the distinct regions of native retinal rod disk membranes

ABSTRACTPhotoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to co-immunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample’s copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which were enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition provides a model for future studies of other complex cellular structures, and gives new insights into how intricate membrane structure and protein activity are balanced within the ROS.SUMMARYSander et al. have parsed the lipid composition of native-source photoreceptor disks and find large differences in fatty acid unsaturation and chain length between the center and rim regions. They selectively copurify membrane proteins and lipids from each region in SMALPs using nanobodies and antibodies.

Identification of PKCα-dependent phosphoproteins in mouse retina

Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKCα), the targets of PKCα phosphorylation in the retina have not been identified. PKCα activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKCα antibody and antibodies to phosphorylated PKC motifs. PKCα activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKCα-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKCα knockout (PKCα-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKCα-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKCα-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906.SignificanceRetinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKCα, though the specific mechanism by which PKCα modulates RBC physiology is unknown. This study examined PKCα phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKCα-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKCα-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKCα-dependent modulation of RBC physiology.HighlightsPKCα is a major source of phosphorylation in retinal RBC dendrites and its activity in RBCs is light dependent.Proteins showing differential phosphorylation between phorbol ester-treated wild type and PKCα-KO retinas belong to the following major functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein).The PKCα-dependent phosphoproteins, BORG4 and TPBG, are present in the synaptic layers of the retina and may be involved in PKCα-dependent modulation of RBC physiology.

Elementary response triggered by transducin in retinal rods

G protein-coupled receptor (GPCR) signaling is crucial for many physiological processes. A signature of such pathways is high amplification, a concept originating from retinal rod phototransduction, whereby one photoactivated rhodopsin molecule (Rho*) was long reported to activate several hundred transducins (GT*s), each then activating a cGMP-phosphodiesterase catalytic subunit (GT*·PDE*). This high gain at the Rho*-to-GT* step has been challenged more recently, but estimates remain dispersed and rely on some nonintact rod measurements. With two independent approaches, one with an extremely inefficient mutant rhodopsin and the other with WT bleached rhodopsin, which has exceedingly weak constitutive activity in darkness, we obtained an estimate for the electrical effect from a single GT*·PDE* molecular complex in intact mouse rods. Comparing the single-GT*·PDE* effect to the WT single-photon response, both in Gcaps−/− background, gives an effective gain of only ∼12–14 GT*·PDE*s produced per Rho*. Our findings have finally dispelled the entrenched concept of very high gain at the receptor-to-G protein/effector step in GPCR systems.