Loss of Fas Receptor Function Preserves Photoreceptor Structure and Function in Two Mouse Models of Inherited Retinal Degeneration.

Purpose: The genetic heterogeneity of inherited retinal degeneration (IRD) has limited the development of mutation-specific therapies, necessitating the development of therapeutic approaches targeting broadly shared pathophysiologic pathways. The Fas receptor has been reported as a contributor to retinal cell death and inflammation in a wide variety of ocular diseases. The purpose of this study was to assess targeting the Fas pathway as a novel mutation-independent approach to improve photoreceptor survival in IRD. Methods: We examined the effects of genetic inactivation of the Fas receptor on retinal degeneration in two distinct IRD mouse models, P23H and rd10. The Fas-lpr mouse, which contains a functionally inactive Fas receptor, was crossed with the P23H and rd10 mice to generate P23H/Fas-lpr and rd10/Fas-lpr mice. Fas activation, photoreceptor survival and retinal function were assessed. Results: We detected elevated levels of Fas receptor and microglial activation in the retinas of both P23H and rd10 mice. Inactivation of Fas in these two IRD models (P23H/Fas-lpr and rd10/Fas-lpr mice) resulted in reduced cell death, increased photoreceptor survival, improved retinal function, and reduced microglial activation and inflammatory cytokine production. Conclusions: The protective effect of a nonfunctional Fas receptor in two different mouse models of retinal degeneration suggests that whereas the individual IRD mutation may be specific, the retina's response to the different stressors appears to be shared and driven by Fas. Reducing Fas activity might represent a potential mutation-independent therapeutic approach to preserve retinal structure and function in patients with IRD.

Correlation of vascular change and cognitive impairment in age-related macular degeneration patients.

Age-related macular degeneration (AMD) is one of the leading causes of blindness among the elderly. However, the correlation between vascular change and cognitive impairment in AMD disease is still unknown. In our study, we investigate the blood flow change among different layers of the retina in the AMD eye and normal fellow eye of AMD patients and its influence with patients' cognition. Our study applies optical coherence tomography angiography (OCTA) to assess the blood flow of the retina in AMD patients and the healthy controls (HCs). Magnetic resonance imaging (MRI) and Montreal Cognitive Assessment (MoCA) were performed to evaluate the cognitive change of the individuals. The results showed that deep capillary plexus density, superficial capillary plexus density, retina thickness and retinal nerve fiber layer thickness deduction existed in both eyes of the AMD patient compared with the HCs. The reduced vessel density in the choroidal layer only existed in the AMD eye of the patients while the fellow eye of patients and HCs did not change much. Furthermore, the AMD patient got a lower MoCA score compared to the HCs. Our results illustrate that the fellow eye of the AMD patient underwent vessel density change, which may lead to the early stage of AMD. The lower score of the MoCA test in AMD patients refers to the cognitive impairment. These findings show the significance of taking actions to prevent the progress of AMD in the fellow eye, as well as paying more attention to the development of cognitive impairment of these patients.

Shifting the balance of autophagy and proteasome activation reduces proteotoxic cell death: a novel therapeutic approach for restoring photoreceptor homeostasis.

The P23H variant of rhodopsin results in misfolding of the protein, and is a common cause of the blinding disease autosomal dominant retinitis pigmentosa (adRP). We have recently demonstrated that degeneration of photoreceptor cells in retinas of P23H mice is due to the endoplasmic reticulum stress (ERS)-induced activation of autophagy that leads to a secondary proteasome insufficiency and activation of cell death pathways. We propose that this increased level of autophagy flux relative to proteasome activity, which we term the A:P ratio, represents a marker of altered photoreceptor cell homeostasis, and that therapies aimed at normalizing this ratio will result in increased photoreceptor cell survival. To test this postulate, we treated P23H mice with a chemical chaperone (4-phenylbutyric acid) to improve rhodopsin folding, or with a selective phosphodiesterase-4 inhibitor (rolipram) to increase proteasome activity. P23H mice treated with either of these agents exhibited reduced ERS, decreased autophagy flux, increased proteasome activity, and decreased activation of cell death pathways. In addition, rates of retinal degeneration were decreased, and photoreceptor morphology and visual function were preserved. These findings support the conclusion that normalizing the A:P ratio, either by reducing the ERS-induced activation of autophagy, or by increasing proteasome activity, improves photoreceptor survival, and suggest a potential new therapeutic strategy for the treatment of adRP caused by protein folding defects.

Autophagosome immunoisolation from GFP-LC3B mouse tissue.

We describe a protocol for rapid and efficient enrichment of autophagosomes from various tissues of the GFP-LC3 mouse. In order to increase the number of autophagosomes, we block autophagy flux in the GFP-LC3 mouse tissue with a single intraperitoneal injection of leupeptin 4-5 h before tissue harvesting. We homogenize dissected tissue samples using a Dounce homogenizer followed by passing the slurry through needles of different sizes to dissociate the cells and disrupt their outer membranes. The post-nuclear supernatant fraction of the cell lysate is further centrifuged and the supernatant fraction is discarded to remove residual cytosolic GFP-LC3 that is not associated with autophagosomes. The pellet fraction is resuspended and incubated with magnetic microbeads coated with anti-GFP antibodies for 1 h on ice. The lysate-bead mixture is then applied to a column that is placed in a magnetic separator. After washes, the autophagosome fraction is eluted from the column for morphological and protein analysis. Abbreviations: EDTA: ethylenediaminetetraacetic acid; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LC3: microtubule-associated protein 1 light chain 3 beta; MES: 4-morpholineethanesulfonic acid (MES); SQSTM1: sequestosome 1; TEM: transmission electron microscopy.

Inhibiting autophagy reduces retinal degeneration caused by protein misfolding.

Mutations in the genes necessary for the structure and function of vertebrate photoreceptor cells are associated with multiple forms of inherited retinal degeneration. Mutations in the gene encoding RHO (rhodopsin) are a common cause of autosomal dominant retinitis pigmentosa (adRP), with the Pro23His variant of RHO resulting in a misfolded protein that activates endoplasmic reticulum stress and the unfolded protein response. Stimulating macroautophagy/autophagy has been proposed as a strategy for clearing misfolded RHO and reducing photoreceptor death. We found that retinas from mice heterozygous for the gene encoding the RHOP23H variant (hereafter called P23H) exhibited elevated levels of autophagy flux, and that pharmacological stimulation of autophagy accelerated retinal degeneration. In contrast, reducing autophagy flux pharmacologically or by rod-specific deletion of the autophagy-activating gene Atg5, improved photoreceptor structure and function. Furthermore, proteasome levels and activity were reduced in the P23H retina, and increased when Atg5 was deleted. Our findings suggest that autophagy contributes to photoreceptor cell death in P23H mice, and that decreasing autophagy shifts the degradation of misfolded RHO protein to the proteasome and is protective. These observations suggest that modulating the flux of misfolded proteins from autophagy to the proteasome may represent an important therapeutic strategy for reducing proteotoxicity in adRP and other diseases caused by protein folding defects.

Problem/case-based learning with competition introduced in severe infection education: an exploratory study.

BACKGROUND: Problem/case-based learning (PCBL) is one of the most commonly used educational methods in medical schools. AIM: To further improve PCBL in clinical course of severe infection by introducing competition mode. METHODS: Two classes of medical students were divided into two groups by class-based simple randomization and were taught the course of severe infection by PCBL. A team-based competition was introduced in the study group (n = 35) while not in the control group (n = 36). After the course, four closely associated references were recommended. All the students were notified about a group consultation on a similar case. In the final examination, a case with severe infection complicated with infectious shock was presented for the students to analyze and resolve listed questions. Their performances were qualitatively evaluated to justify the effectiveness of the competition-based PCBL. RESULTS: The students in the study group were more active and initiative in case discussion and interaction, in referring to case-related articles and attending clinical group-consultation. They had better performance in the case analysis in the final examination. The typical case analysis test easily figured out more excellent students in the study group. CONCLUSIONS: The PCBL with competition mode introduced in is an effective approach to guide students to fully understand the clinical diagnoses and treatment of severe infection. It also prompts medical students' initiative in referring to case-related articles and attending group-consultation, both of which are essential to equip medical students with sufficient competency for clinical practice.