Photoreceptors in a mouse model of Leigh syndrome are capable of normal light-evoked signaling

Mitochondrial dysfunction is an important cause of heritable vision loss. Mutations affecting mitochondrial bioenergetics may lead to isolated vision loss or life-threatening systemic disease, depending on a mutations severity. Primary optic nerve atrophy resulting from death of retinal ganglion cells is the most prominent ocular manifestation of mitochondrial disease. However, dysfunction of other retinal cell types has also been described, sometimes leading to a loss of photoreceptors and retinal pigment epithelium that manifests clinically as pigmentary retinopathy. A popular mouse model of mitochondrial disease that lacks NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4), a subunit of mitochondrial complex I, phenocopies many traits of the human disease Leigh syndrome, including the development of optic atrophy. It has also been reported that ndufs4-/- mice display diminished light responses at the level of photoreceptors or bipolar cells. By conducting electroretinography (ERG) recordings in live ndufs4-/- mice, we now demonstrate that this defect occurs at the level of retinal photoreceptors. We found that this deficit does not arise from retinal developmental anomalies, photoreceptor degeneration, or impaired regeneration of visual pigment. Strikingly, the impairment of ndufs4-/- photoreceptor function was not observed in ex vivo ERG recordings from isolated retinas, indicating that photoreceptors with complex I deficiency are intrinsically capable of normal signaling. The difference in electrophysiological phenotypes in vivo and ex vivo suggests that the energy deprivation associated with severe mitochondrial impairment in the outer retina renders ndufs4-/- photoreceptors unable to maintain the homeostatic conditions required to operate at their normal capacity

Increased proteasomal activity supports photoreceptor survival in inherited retinal degeneration

Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed that one such condition is an insufficiency of the ubiquitin–proteasome system to process misfolded or mistargeted proteins in affected photoreceptor cells. We now report that retinal degeneration in mice can be significantly delayed by increasing photoreceptor proteasomal activity. The largest effect is observed upon overexpression of the 11S proteasome cap subunit, PA28α, which enhanced ubiquitin-independent protein degradation in photoreceptors. Applying this strategy to mice bearing one copy of the P23H rhodopsin mutant, a mutation frequently encountered in human patients, quadruples the number of surviving photoreceptors in the inferior retina of 6-month-old mice. This striking therapeutic effect demonstrates that proteasomes are an attractive target for fighting inherited blindness

Increased proteasomal activity supports photoreceptor survival in inherited retinal degeneration

Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed that one such condition is an insufficiency of the ubiquitin–proteasome system to process misfolded or mistargeted proteins in affected photoreceptor cells. We now report that retinal degeneration in mice can be significantly delayed by increasing photoreceptor proteasomal activity. The largest effect is observed upon overexpression of the 11S proteasome cap subunit, PA28α, which enhanced ubiquitin-independent protein degradation in photoreceptors. Applying this strategy to mice bearing one copy of the P23H rhodopsin mutant, a mutation frequently encountered in human patients, quadruples the number of surviving photoreceptors in the inferior retina of 6-month-old mice. This striking therapeutic effect demonstrates that proteasomes are an attractive target for fighting inherited blindness

Full genome comparison and characterization of avian H10 viruses with different pathogenicity in Mink (Mustela vison) reveals genetic and functional differences in the non-structural gene

<p>Abstract</p> <p>Background</p> <p>The unique property of some avian H10 viruses, particularly the ability to cause severe disease in mink without prior adaptation, enabled our study. Coupled with previous experimental data and genetic characterization here we tried to investigate the possible influence of different genes on the virulence of these H10 avian influenza viruses in mink.</p> <p>Results</p> <p>Phylogenetic analysis revealed a close relationship between the viruses studied. Our study also showed that there are no genetic differences in receptor specificity or the cleavability of the haemagglutinin proteins of these viruses regardless of whether they are of low or high pathogenicity in mink.</p> <p>In poly I:C stimulated mink lung cells the NS1 protein of influenza A virus showing high pathogenicity in mink down regulated the type I interferon promoter activity to a greater extent than the NS1 protein of the virus showing low pathogenicity in mink.</p> <p>Conclusions</p> <p>Differences in pathogenicity and virulence in mink between these strains could be related to clear amino acid differences in the non structural 1 (NS1) protein. The NS gene of mink/84 appears to have contributed to the virulence of the virus in mink by helping the virus evade the innate immune responses.</p

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

The prion protein in normal cells and disease

Transmissible spongiform encephalopathies (TSEs) are also known as prion diseases. The unusual infectious agent is composed largely, if not entirely, of a proteinase resistant aberrantly folded isoform of the prion protein (PrPSc). PrPSc, through an unknown mechanism, is believed to impose its aberrant conformation onto the host-encoded cellular prion protein, PrPC. The infectious particle is designated a "prion", an acronym for proteinaceous infectious particle, to distinguish it from conventional pathogens such as viruses and bacteria. The essential role of PrPC in the pathogenesis of prion diseases motivated the detailed study of the cellular processing, turnover and release of bovine PrPC (boPrPC) performed in this thesis. BoPrPC was found to be subjected to two distinct proteolytic cleavage events, generating an N-terminal and a C-terminal boPrPC fragment. Both PrP fragments were released from the cell. Moreover, in normal bovine brain a C-terminal fragment was found, suggesting that similar proteolytic processing events occur in vivo. The finding that boPrPC in addition to a protease-mediated release also was released in association with exosomes, provide important information in relation to functional aspects of PrPC and possible roles of exosome-associated PrP in pathogenesis of prion diseases. Taken together, these results indicate that release of boPrPC from cells represent an important step in the normal cellular processing of boPrPC. Nor98 is a recently recognised prion disease in sheep. The molecular characterization of the Nor98 prion showed that the unique PK-resistant PrP fragments present in Nor98-affected sheep display striking similarities to those reported from individuals affected by the human prion disorder Gerstmann-Sträussler-Scheinker syndrome (GSS). Interestingly, GSS is always associated with amino acid substitutions in the PrP. Differently, no disease-causing changes in the PrP of Nor98-affected sheep have been found in the affected sheep in Sweden. These findings together with observations of a distinct epidemiology, suggest that Nor98 could be the result of a spontaneous conversion of PrPC into PrPSc, similar to that proposed for sporadic Creutzfeldt-Jakob disease and sporadic fatal insomnia

Mechanism of Fibronectin Binding to Human Trabecular Meshwork Exosomes and Its Modulation by Dexamethasone.

Exosomes are emerging as important mediators of cell-matrix interactions by means of specific adhesion proteins. Changes in the tissue-specific exosomal protein expression may underlie pathological conditions whereby extracellular matrix turnover and homeostasis is disrupted. Ocular hypertension due to extracellular matrix accumulation in the trabecular meshwork is a hallmark of glucocorticoid-induced glaucoma. In the trabecular meshwork, exosomal fibronectin mediates cell matrix interactions at cellular structures called "invadosomes". Trabecular meshwork cells use invadosomes to turn over their surrounding matrix and maintain passageways for flow of aqueous humor. In this study, we observed that human trabecular meshwork explants treated with dexamethasone released exosomes with significantly reduced amounts of fibronectin bound per exosome. Further, we found that exosome-fibronectin binding is heparan sulfate-dependent, consistent with our observation that trabecular meshwork exosomes are enriched in the heparin/heparan sulfate binding annexins A2 and A6. In this way, dexamethasone-treated explants released exosomes with a significant reduction in annexin A2 and A6 per exosome. Interestingly, we did not detect exosomal matrix metalloproteinases, but we identified abundant dipeptidyl peptidase 4, a serine protease whose activity was reduced on exosomes isolated from dexamethasone-treated explants. Together, our findings demonstrate mechanistically how corticosteroid-induced alterations in exosomal adhesion cargo and properties can account for the pathological matrix accumulation seen in many glaucoma patients

Molecular interaction between fibronectin and heparan sulfate on trabecular meshwork (TM) exosome surfaces.

<p>(A) Serum-free media conditioned by human TM cells for 2.5 hours was split into 7 equal portions and purified heparan sulfate was added at increasing log₁₀ concentrations. Mixtures were incubated for 1 hour on a rocker at room temperature. Exosomes were then isolated from each sample and Fn content was assessed by dot blot. The intensity of Fn staining from 3 biological replicates (cell strains isolated from 3 different human donor eyes) were measured with ImageJ. The bar graph data is the mean±SEM of the 3 replicates. One representative dot blot is shown underneath. *p<0.05, [heparan sulfate] vs no heparan sulfate, Student’s t-test. (B) A dose-response curve was generated from the data shown in A (3 biological replicates). When no exogenous heparan sulfate was added, an endogenous concentration on the exosome of 1pM was used to generate the curve and estimate an EC₅₀ value. <b>C,</b> Conditioned media was split into 3 equal portions. Exosome-depleted FBS (10% final v:v) was added to two portions. Exosomes were prepared, resuspended in PBS with or without a bacterial heparanase II and incubated at 34°C for 1 hour with periodic shaking (approx. 10 minute intervals). Fn content of exosomes was assessed by western blot. Blot shown is representative of 3 biological replicates from cell strains isolated from different donor eyes.</p

Treatment of human trabecular meshwork (TM) explants with dexamethasone (Dex) reduces the amount of exosome bound fibronectin (Fn).

<p>TM explants were dissected from human donor eyes and split in half. Each half was cultured for 72 hours in control (Ctrl) media or media containing 100nM dexamethasone (Dex). Conditioned media from control and Dex treated TM explants was probed for fibronectin (Fn) and myocilin (Myoc) by western blot. Band intensity was normalized to dry TM tissue weight. Dex treatment increased the secretion of (A) Fn and (B) Myoc in the conditioned media (mean ± SD, n = 3). Exosomes were isolated, sized and counted by nanoparticle tracking analysis. (C) Dex treatment does not alter the mean or (D) mode size of exosomes released by human TM explants (control n = 6, Dex n = 6). (E) Dex treatment does not significantly alter the amount of exosomes in the conditioned media (p = 0.1654, Student’s t-test, n = 6). (F) Equal volumes of exosomes were analyzed for Fn content by western blot from six donors (6 control, 6 Dex treated). Fn band intensity was determined using ImageJ and the intensity was normalized to the number of exosomes loaded per lane. The representative bands shown are from a single donor, control = 3.39E+08 particles and Dex = 3.49E+08 particles. Bar graph is the mean±SEM of six control and Dex treated Fn band intensities normalized to the number of particles per lane. *p<0.05, control vs Dex, n = 6, Student’s t-test. Box-and-whisker plots in A, B and C are the max, 3^rd quartile, median, 1^st quartile and min.</p