Fusion of secretory vesicles isolated from rat liver

Secretory vesicles isolated from rat liver were found to fuse after exposure to Ca2+. Vescle fusion is characterized by the occurrence of twinned vesicles with a continuous cleavage plane between two vesicles in freeze-fracture electron microscopy. The number of fused vesicles increases with increasing Ca2+-concentrations and is half maximal around 10–6 m. Other divalent cations (Ba2+, Sr2+, and Mg2+) were ineffective. Mg2+ inhibits Ca2+-induced fusion. Therefore, the fusion of secretory vesiclesin vitro is Ca2+ specific and exhibits properties similar to the exocytotic process of various secretory cells. Various substances affecting secretionin vivo (microtubular inhibitors, local anethetics, ionophores) were tested for their effect on membrane fusion in our system. The fusion of isolated secretory vesicles from liver was found to differ from that of pure phospholipid membranes in its temperature dependence, in its much lower requirement for Ca2+, and in its Ca2+-specificity. Chemical and enzymatic modifications of the vesicle membrane indicate that glycoproteins may account for these differences

αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells

αB Crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether αB crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular αB crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that αB crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of αB crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced αB crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, αB crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that αB crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of αB crystallin to the basolateral side. In normal mouse retinal sections, αB crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous αB crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. αB Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for αB crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of αB crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for αB crystallin in ocular diseases

Histological Evaluation of Diabetic Neurodegeneration in the Retina of Zucker Diabetic Fatty (ZDF) Rats

In diabetes, retinal dysfunctions exist prior to clinically detectable vasculopathy, however the pathology behind these functional deficits is still not fully established. Previously, our group published a detailed study on the retinal histopathology of type 1 diabetic (T1D) rat model, where specific alterations were detected. Although the majority of human diabetic patients have type 2 diabetes (T2D), similar studies on T2D models are practically absent. To fill this gap, we examined Zucker Diabetic Fatty (ZDF) rats - a model for T2D - by immunohistochemistry at the age of 32 weeks. Glial reactivity was observed in all diabetic specimens, accompanied by an increase in the number of microglia cells. Prominent outer segment degeneration was detectable with changes in cone opsin expression pattern, without a decrease in the number of labelled elements. The immunoreactivity of AII amacrine cells was markedly decreased and changes were detectable in the number and staining of some other amacrine cell subtypes, while most other cells examined did not show any major alterations. Overall, the retinal histology of ZDF rats shows a surprising similarity to T1D rats indicating that despite the different evolution of the disease, the neuroretinal cells affected are the same in both subtypes of diabetes

Understanding the retinal basis of vision across species

The vertebrate retina first evolved some 500 million years ago in ancestral marine chordates. Since then, the eyes of different species have been tuned to best support their unique visuoecological lifestyles. Visual specializations in eye designs, large-scale inhomogeneities across the retinal surface and local circuit motifs mean that all species' retinas are unique. Computational theories, such as the efficient coding hypothesis, have come a long way towards an explanation of the basic features of retinal organization and function; however, they cannot explain the full extent of retinal diversity within and across species. To build a truly general understanding of vertebrate vision and the retina's computational purpose, it is therefore important to more quantitatively relate different species' retinal functions to their specific natural environments and behavioural requirements. Ultimately, the goal of such efforts should be to build up to a more general theory of vision

Pan-retinal characterisation of Light Responses from Ganglion Cells in the Developing Mouse Retina

International audienceWe have investigated the ontogeny of light-driven responses in mouse retinal ganglion cells (RGCs). Using a large-scale, high-density multielectrode array, we recorded from hundreds to thousands of RGCs simultaneously at pan-retinal level, including dorsal and ventral locations. Responses to di erent contrasts not only revealed a complex developmental pro le for ON, OFF and ON-OFF responses, but also unveiled di erences between dorsal and ventral RGC responses. At eye-opening, dorsal RGCs of all types were more responsive to light, perhaps indicating an environmental priority to nest viewing for pre-weaning pups. The developmental pro le of ON and OFF responses exhibited antagonistic behaviour, with the strongest ON responses shortly after eye-opening, followed by an increase in the strength of OFF responses later on. Further, we found that with maturation receptive eld (RF) center sizes decrease, spike-triggered averaged responses to white noise become stronger, and centers become more circular while maintaining di erences between RGC types. We conclude that the maturation of retinal functionality is not spatially homogeneous, likely re ecting ecological requirements that favour earlier maturation of the dorsal retina

Photopigment coexpression in mammals: comparative and developmental aspects

In mammals, each cone had been thought to contain only one single type of photopigment. It was not until the early 1990s that photopigment coexpression was reported. In the house mouse, the distribution of color cones shows a characteristic division. Whereas in the upper retinal field the ratio of short wave to middleto- long wave cones falls in the usual range (1:10), in the ventral retinal field M/L-pigment expression is completely missing. In the transitional zone, numerous dual cones are detectable (spatial coexpression). In other species without retinal division, dual cones appear during development, suggesting that M/L-cones develop from S-cones. Dual elements represent a transitory stage in M/L-cone differentiation that disappear with maturation (transitory coexpression). These two phenomena seem to be mutually exclusive in the species studied so far. In the comparative part of this report the retinal cone distribution of eight rodent species is reported. In two species dual cones appear in adult specimens without retinal division, and dual elements either occupy the dorsal peripheral retina, or make up the entire cone population. This is the first observation proving that all cones of a retina are of dual nature. These species are good models for the study of molecular control of opsin expression and renders them suitable sources of dual cones for investigations on the role and neural connections of this peculiar cone type. In the developmental part, the retinal maturation of other species is examined to test the hypothesis of transitory coexpression. In these species S-pigment expression precedes that of the M/L-pigment, but dual cones are either identified in a small number or they are completely missing from the developing retina. These results exclude a common mechanism for M/L-cone maturation: they either transdifferentiate from S-cones or develop independently