Extracellular vesicles from retinal pigment epithelial cells expressing R345W-Fibulin-3 induce epithelial-mesenchymal transition in recipient cells.

We have shown previously that expression of R345W-Fibulin-3 induces epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. The purpose of the current study was to determine if extracellular vesicles (EVs) derived from RPE cells expressing R345W-Fibulin-3 mutation are sufficient to induce EMT in recipient cells. ARPE-19 cells were infected with luciferase-tagged wild-type (WT)- Fibulin-3 or luciferase-tagged R345W-Fibulin-3 (R345W) using lentiviruses. EVs were isolated from the media by ultracentrifugation or density gradient ultracentrifugation. Transmission electron microscopy and cryogenic electron microscopy were performed to study the morphology of the EVs. The size distribution of EVs were determined by nanoparticle tracking analysis (NTA). EV cargo was analysed using LC-MS/MS based proteomics. EV-associated transforming growth factor beta 1 (TGFß1) protein was measured by enzyme-linked immunosorbent assay. The capacity of EVs to stimulate RPE migration was evaluated by treating recipient cells with WT- or R345W-EVs. The role of EV-bound TGFß was determined by pre-incubation of EVs with a pan-TGFß blocking antibody or IgG control. EM imaging revealed spherical vesicles with two subpopulations of EVs: a group with diameters around 30 nm and a group with diameters over 100 nm, confirmed by NTA analysis. Pathway analysis revealed that members of the sonic hedgehog pathway were less abundant in R345W- EVs, while EMT drivers were enriched. Additionally, R345W-EVs had higher concentrations of TGFß1 compared to control. Critically, treatment with R345W-EVs was sufficient to increase EMT marker expression, as well as cell migration in recipient cells. This EV-increased cell migration was significantly inhibited by pre-incubation of EVs with pan-TGFß-neutralising antibody. In conclusion, the expression of R345W-Fibulin-3 alters the size and cargo of EVs, which are sufficient to enhance the rate of cell migration in a TGFß dependent manner. These results suggest that EV-bound TGFß plays a critical role in the induction of EMT in RPE cells.

Neurodegeneration, Neuroprotection and Regeneration in the Zebrafish Retina.

Neurodegenerative retinal diseases, such as glaucoma and diabetic retinopathy, involve a gradual loss of neurons in the retina as the disease progresses. Central nervous system neurons are not able to regenerate in mammals, therefore, an often sought after course of treatment for neuronal loss follows a neuroprotective or regenerative strategy. Neuroprotection is the process of preserving the structure and function of the neurons that have survived a harmful insult; while regenerative approaches aim to replace or rewire the neurons and synaptic connections that were lost, or induce regrowth of damaged axons or dendrites. In order to test the neuroprotective effectiveness or the regenerative capacity of a particular agent, a robust experimental model of retinal neuronal damage is essential. Zebrafish are being used more often in this type of study because their eye structure and development is well-conserved between zebrafish and mammals. Zebrafish are robust genetic tools and are relatively inexpensive to maintain. The large array of functional and behavioral tests available in zebrafish makes them an attractive model for neuroprotection studies. Some common insults used to model retinal disease and study neuroprotection in zebrafish include intense light, chemical toxicity and mechanical damage. This review covers the existing retinal neuroprotection and regeneration literature in the zebrafish and highlights their potential for future studies.

Descriptive analysis of Fibulin-3 and the extracellular vesicle marker, Alix, in drusen from a small cohort of postmortem human eyes.

Fibulin-3 (Fib3) is a secreted glycoprotein that is expressed in the retina and has been associated with drusen formation in age-related macular degeneration (AMD). The purpose of this study was to assess whether Fib3 is associated with extracellular vesicles (EVs) in drusen from non-diseased and AMD human donors. De-identified sections of human eyes were received from the National Disease Research Institute (NDRI, Philadelphia). Donor eyes were either non-diseased (no known ocular pathology) or had been diagnosed with AMD. Retinal cryostat sections were labeled with primary antibodies targeted to Fib3, Apolipoprotein E (ApoE; a drusen marker), and ALG-2 interacting protein X (Alix, an EV marker) for confocal imaging (Leica TCS SP8). Fib3-positive (Fib3+) puncta were detected on the apical region of the RPE layer and within large AMD drusen. Alix-positive (Alix+) puncta were also detected in a single AMD druse, where a number were Fib3+ and the remaining were Fib3-negative. Similarly, there were Fib3+ puncta that were Alix-negative. Fib3 and Alix also showed a degree of colocalization in the photoreceptor outer segments of the neural retina. Our data suggest that the Alix+ puncta are EV-rich populations that accumulate, together with Fib3, within the drusen matrix during AMD. The EV population is likely heterogeneous, such that there are sub-populations with different cargo content.

Role of Epithelial-Mesenchymal Transition in Retinal Pigment Epithelium Dysfunction.

Retinal pigment epithelial (RPE) cells maintain the health and functional integrity of both photoreceptors and the choroidal vasculature. Loss of RPE differentiation has long been known to play a critical role in numerous retinal diseases, including inherited rod-cone degenerations, inherited macular degeneration, age-related macular degeneration, and proliferative vitreoretinopathy. Recent studies in post-mortem eyes have found upregulation of critical epithelial-mesenchymal transition (EMT) drivers such as TGF-ß, Wnt, and Hippo. As RPE cells become less differentiated, they begin to exhibit the defining characteristics of mesenchymal cells, namely, the capacity to migrate and proliferate. A number of preclinical studies, including animal and cell culture experiments, also have shown that RPE cells undergo EMT. Taken together, these data suggest that RPE cells retain the reprogramming capacity to move along a continuum between polarized epithelial cells and mesenchymal cells. We propose that movement along this continuum toward a mesenchymal phenotype be defined as RPE Dysfunction. Potential mechanisms include impaired tight junctions, accumulation of misfolded proteins and dysregulation of several key pathways and molecules, such as TGF-ß pathway, Wnt pathway, nicotinamide, microRNA 204/211 and extracellular vesicles. This review synthesizes the evidence implicating EMT of RPE cells in post-mortem eyes, animal studies, primary RPE, iPSC-RPE and ARPE-19 cell lines.

Expression of R345W-Fibulin-3 Induces Epithelial-Mesenchymal Transition in Retinal Pigment Epithelial Cells.

PURPOSE: To investigate the role of protein misfolding in retinal pigment epithelial (RPE) cell dysfunction, the effects of R345W-Fibulin-3 expression on RPE cell phenotype were studied. METHODS: Primary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of vascular endothelial growth factor (VEGF), was measured by Enzyme-linked immunosorbent assay (ELISA). Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells, and conversion to an epithelial-mesenchymal transition (EMT) phenotype was assessed by a migration assay. RESULTS: Compared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345W-Fibulin-3. In cells expressing mutant Fibulin-3, VEGF secretion was attenuated basally but not in the apical direction, whereas Fibulin-3 secretion was reduced in both the apical and basal directions. Retinal pigment epithelial signature genes were downregulated and multiple genes associated with EMT were upregulated in the mutant group. Migration assays revealed a faster recovery rate in ARPE-19 cells overexpressing R345W-Fibulin-3 compared to WT. CONCLUSIONS: The results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells.

A short period of dark-adaptation is sufficient to generate light-induced photoreceptor degeneration in pigmented zebrafish.

Light-induced retinal degeneration (LIRD) models are used to recapitulate the pathologies of retinal diseases that affect photoreceptors. Current LIRD models use a dark-adaptation period of 7-14 days followed by high-intensity light exposure. The purpose of this study was to determine whether photoreceptor damage and death would occur in pigmented zebrafish using a short period of dark-adaptation. Zebrafish were dark-adapted for 24 h and then exposed to constant high-intensity light for 48 h. Immunohistochemical analysis was performed on vertical retinal sections to assess damage and apoptosis. Photoreceptors exhibited structural damage, apoptosis, and cell loss after 24 and 48 h of light exposure as previously reported in studies using 7-14 day dark-adaption. Also, photoreceptors lost following light damage were regenerated after 28 days. These results suggest that a short period of dark-adaptation is sufficient for a LIRD model in pigmented zebrafish.

Adenosine receptor expression in the adult zebrafish retina.

Adenosine is an endogenous nucleoside in the central nervous system that acts on adenosine receptors. These are G protein-coupled receptors that have four known subtypes: A1, A2A, A2B, and A3 receptors. In the present study, we aimed to map the location of the adenosine receptor subtypes in adult wild-type zebrafish retina using in situ hybridization and immunohistochemistry. A1R, A2AR, and A2BR mRNA were detected in the ganglion cell layer (GCL), the inner nuclear layer (INL), the outer nuclear layer (ONL), and the outer segment (OS). A3R mRNA was detected in the GCL, ONL, and OS. A1R-immunoreactivity was expressed as puncta in the INL and in the outer plexiform layer (OPL). A1Rs were located within the cone pedicle and contiguous to horizontal cell tips in the OPL. A2AR-immunoreactivity was expressed as puncta in the GCL, inner plexiform layer (IPL), INL, and outer retina. A2AR puncta in the outer retina were situated around the ellipsoids and nuclei of cones, and weakly around the rod nuclei. A1Rs and A2ARs were clustered around ON cone bipolar cell terminals and present in the OFF lamina of the INL but were not expressed on mixed rod/cone response bipolar cell terminals. A2BR-immunoreactivity was mainly localized to the Müller cells, while A3Rs were found to be expressed in retinal ganglion cells of the GCL, INL, ONL, and OS. In summary, all four adenosine receptor subtypes were localized in the zebrafish retina and are in agreement with expression patterns shown in retinas from other species.

Control of Neuronal Ryanodine Receptor-Mediated Calcium Signaling by Calsenilin.

Calsenilin is a calcium ion (Ca2+)-binding protein involved in regulating the intracellular concentration of Ca2+, a second messenger that controls multiple cellular signaling pathways. The ryanodine receptor (RyR) amplifies Ca2+ signals entering the cytoplasm by releasing Ca2+ from endoplasmic reticulum (ER) stores, a process termed calcium-induced calcium release (CICR). Here, we describe a novel mechanism, in which calsenilin controls the activity of neuronal RyRs. We show calsenilin co-localized with RyR2 and 3 in the ER of mouse hippocampal and cortical neurons using immunocytochemistry. The underlying protein-protein interaction between calsenilin and the RyR was determined in mouse central nervous system (CNS) neurons using immunoprecipitation studies. The functional relevance of this interaction was assayed with single-channel electrophysiology. At low physiological Ca2+ concentrations, calsenilin binding to the cytoplasmic face of neuronal RyRs decreased the RyR's open probability, while calsenilin increased the open probability at high physiological Ca2+ concentrations. This novel molecular mechanism was studied further at the cellular level, where faster release kinetics of caffeine-induced Ca2+ release were measured in SH-SY5Y neuroblastoma cells overexpressing calsenilin. The interaction between calsenilin and neuronal RyRs reveals a new regulatory mechanism and possibly a novel pharmacological target for the control of Ca2+ release from intracellular stores.

Quantification of Changes in Visual Function During Disease Development in a Mouse Model of Pigmentary Glaucoma.

PURPOSE: We investigated the relationship between visual parameters that are commonly affected during glaucomatous disease progression with functional measures of retina physiology using electroretinography and behavioral measures of visual function in a mouse model of glaucoma. Electroretinogram components measuring retinal ganglion cell (RGC) responses were determined using the non-invasive Ganzfeld flash electroretinography (fERG) to assess RGC loss in a mouse model of glaucoma. METHODS: Intraocular pressure (IOP), behaviorally assessed measures of visual function, namely visual acuity and contrast sensitivity as well as fERG responses were recorded in 4- and 11-month-old male DBA/2 mice. Scotopic threshold response (STR) and photopic negative response components as well as oscillatory potentials (OPs) were isolated from fERG responses and correlated with IOP, optomotor reflex measurements, and RGC counts. RESULTS: The 11-month-old DBA/2 mice had significantly elevated IOP, reduced visual performance, as assessed behaviorally, significant RGC loss, deficits in standardized fERG responses, reduced STRs, and differences in OP amplitudes and latencies, when compared with 4-month-old mice of the same strain. STRs and OPs correlated with some visual and physiological parameters. In addition, elevated IOP and RGC loss correlated positively with measures of visual function, specifically with surrogate measures of RGC function derived from fERG. CONCLUSIONS: Our data suggest that RGC function as well as interactions of RGCs with other retinal cell types is impaired during glaucoma. In addition, a later OP wavelet denoted as OP4 in this study was identified as a very reproducible indicator of loss of visual function in the glaucoma mouse model.

Melanopsin retinal ganglion cells are not labeled in Thy-1YFP-16 transgenic mice.

Retinal ganglion cells (RGCs) that express the photopigment melanopsin (mRGCs) are photosensitive and initiate the non-image-forming pathway, where the majority of their axons terminate in the suprachiasmatic nucleus (SCN). RGCs only make up approximately half of the cells in the ganglion cell layer of the retina; therefore, it is important to be able to distinguish them from other cell types. The transgenic Thy-1 YFP mouse line 16 (Thy-1 YFP-16) expresses yellow-fluorescent protein (YFP) in projection neurons, including RGCs. Our objective was to determine whether mRGCs are labeled with YFP in Thy-1 YFP-16 transgenic mice. Paraformaldehyde-fixed retinal wholemounts and frozen vertical sections were prepared from Thy-1 YFP-16 mice and fluorescently labeled with rabbit anti-melanopsin and guinea-pig anti-RNA binding protein with multiple splicing to identify mRGCs and total RGCs, respectively. Thy-1 YFP-16 mouse brains were sectioned coronally and imaged to view RGC axonal projections to the SCN. Confocal images of retinal preparations show that the majority (∼89%) of mRGCs are not YFP-positive in Thy-1 YFP-16 mice, where ∼11% expressed a weak fluorescent signal. In addition, there are almost no YFP-positive axons present in the SCN of coronal brain sections. We conclude that the majority of mRGC somas and axons are not labeled with YFP in the transgenic Thy-1 YFP-16 mouse line; therefore, this mouse model may not suitable for research involving mRGC visual pathways.

Changes in Retinal N-Acylethanolamines and their Oxylipin Derivatives During the Development of Visual Impairment in a Mouse Model for Glaucoma.

Neurons are especially susceptible to oxidative damage, which is increasingly implicated in neurodegenerative disease. Certain N-acylethanolamines (NAEs) have been shown to protect neurons from oxidative stress. Since glaucoma may be considered a neurodegenerative disorder and the survival of retinal neurons could also be influenced by N-acylethanolamines, our goal was to quantify changes in certain N-acylethanolamine species and their oxylipin derivatives in the retina of a mouse model for glaucoma. We also sought to identify relationships between these and parameters of glaucoma disease development, specifically intraocular pressure, visual acuity, and contrast sensitivity. Five N-acylethanolamine species and three NAE oxylipin derivatives were quantified in retina from young and aged DBA/2Crl mice. N-Acylethanolamines and NAE-oxylipins in retinal extracts were quantified against deuterated standards by isotope dilution gas chromatography-mass spectrometry. Levels (nmol/g dry weight) of N-arachidonoylethanolamine (anandamide; NAE 20:4) were significantly (p = 0.008) decreased in aged (2.875 ± 0.6702) compared to young animals (5.175 ± 0.971). Conversely, the anandamide oxylipin, 15(S)-HETE ethanolamide (15(S)-HETE EA), was significantly (p = 0.042) increased in aged (0.063 ± 0.009) compared to young animals (0.039 ± 0.011). Enzymatic depletion of the anandamide pool by 15-lipoxygenase and consequent accumulation of 15(S)-HETE ethanolamine may contribute to decreased visual function in glaucomatous mice. Since N-acylethanolamines effectively attenuate glaucoma pathogenesis and associated visual impairment, our data provides additional rationale and novel targets for glaucoma therapies.

Psychophysical testing in rodent models of glaucomatous optic neuropathy.

Processing of visual information begins in the retina, with photoreceptors converting light stimuli into neural signals. Ultimately, signals are transmitted to the brain through signaling networks formed by interneurons, namely bipolar, horizontal and amacrine cells providing input to retinal ganglion cells (RGCs), which form the optic nerve with their axons. As part of the chronic nature of glaucomatous optic neuropathy, the increasing and irreversible damage and ultimately loss of neurons, RGCs in particular, occurs following progressive damage to the optic nerve head (ONH), eventually resulting in visual impairment and visual field loss. There are two behavioral assays that are typically used to assess visual deficits in glaucoma rodent models, the visual water task and the optokinetic drum. The visual water task can assess an animal's ability to distinguish grating patterns that are associated with an escape from water. The optokinetic drum relies on the optomotor response, a reflex turning of the head and neck in the direction of the visual stimuli, which usually consists of rotating black and white gratings. This reflex is a physiological response critical for keeping the image stable on the retina. Driven initially by the neuronal input from direction-selective RGCs, this reflex is comprised of a number of critical sensory and motor elements. In the presence of repeatable and defined stimuli, this reflex is extremely well suited to analyze subtle changes in the circuitry and performance of retinal neurons. Increasing the cycles of these alternating gratings per degree, or gradually reducing the contrast of the visual stimuli, threshold levels can be determined at which the animal is no longer tracking the stimuli, and thereby visual function of the animal can be determined non-invasively. Integrating these assays into an array of outcome measures that determine multiple aspects of visual function is a central goal in vision research and can be realized, for example, by the combination of measuring optomotor reflex function with electroretinograms (ERGs) and optical coherence tomography (OCT) of the retina. These structure-function correlations in vivo are urgently needed to identify disease mechanisms as potential new targets for drug development. Such a combination of the experimental assessment of the optokinetic reflex (OKR) or optomotor response (OMR) with other measures of retinal structure and function is especially valuable for research on GON. The chronic progression of the disease is characterized by a gradual decrease in function accompanied by a concomitant increase in structural damage to the retina, therefore the assessment of subtle changes is key to determining the success of novel intervention strategies.

Differential up-regulation of Vesl-1/Homer 1 protein isoforms associated with decline in visual performance in a preclinical glaucoma model.

Glaucoma is a multifactorial progressive ocular pathology, clinically presenting with damage to the retina and optic nerve, ultimately leading to blindness. Retinal ganglion cell loss in glaucoma ultimately results in vision loss. Vesl/Homer proteins are scaffolding proteins that are critical for maintaining synaptic integrity by clustering, organizing and functionally regulating synaptic proteins. Current anti-glaucoma therapies target IOP as the sole modifiable clinical parameters. Long-term pharmacotherapy and surgical treatment do not prevent gradual visual field loss as the disease progresses, highlighting the need for new complementary, alternative and comprehensive treatment approaches. Vesl/Homer expression was measured in the retinae of DBA/2J mice, a preclinical genetic glaucoma model with spontaneous mutations resulting in a phenotype reminiscent of chronic human pigmentary glaucoma. Vesl/Homer proteins were differentially expressed in the aged, glaucomatous DBA/2J retina, both at the transcriptional and translational level. Immunoreactivity for the long Vesl-1L/Homer 1c isoform, but not of the immediate early gene product Vesl-1S/Homer 1a was increased in the synaptic layers of the retina. This increased protein level of Vesl-1L/Homer 1c was correlated with phenotypes of increased disease severity and a decrease in visual performance. The increased expression of Vesl-1L/Homer 1c in the glaucomatous retina likely results in increased intracellular Ca(2+) release through enhancement of synaptic coupling. The ensuing Ca(2+) toxicity may thus activate neurodegenerative pathways and lead to the progressive loss of synaptic function in glaucoma. Our data suggest that higher levels of Vesl-1L/Homer 1c generate a more severe disease phenotype and may represent a viable target for therapy development.

N-Palmitoylethanolamine depot injection increased its tissue levels and those of other acylethanolamide lipids.

N-Palmitoylethanolamine (NAE 16:0) is an endogenous lipid signaling molecule that has limited water solubility, and its action is short-lived due to its rapid metabolism. This poses a problem for use in vivo as oral administration requires a high concentration for significant levels to reach target tissues, and injection of the compound in a dimethyl sulfoxide- or ethanol-based vehicle is usually not desirable during long-term treatment. A depot injection of NAE 16:0 was successfully emulsified in sterile corn oil (10 mg/kg) and administered in young DBA/2 mice in order to elevate baseline levels of NAE 16:0 in target tissues. NAE 16:0 levels were increased in various tissues, particularly in the retina, 24 and 48 hours following injections. Increases ranged between 22% and 215% (above basal levels) in blood serum, heart, brain, and retina and induced an entourage effect by increasing levels of other 18 carbon N-Acylethanolamines (NAEs), which ranged between 31% and 117% above baseline. These results indicate that NAE 16:0 can be used as a depot preparation, avoiding the use of inadequate vehicles, and can provide the basis for designing tissue-specific dosing regimens for therapies involving NAEs and related compounds.