Multiomic integration reveals neuronal-extracellular vesicle coordination of gliotic responses in degeneration.

In the central nervous system (CNS), including in the retina, neuronal-to-glial communication is critical for maintaining tissue homeostasis including signal transmission, transfer of trophic factors, and in the modulation of inflammation. Extracellular vesicle (EV)-mediated transport of molecular messages to regulate these processes has been suggested as a mechanism by which bidirectional communication between neuronal and glial cells can occur. In this work we employed multiomics integration to investigate the role of EV communication pathways from neurons to glial cells within the CNS, using the mouse retina as a readily accessible representative CNS tissue. Further, using a well-established model of degeneration, we aimed to uncover how dysregulation of homeostatic messaging between neurons and glia via EV can result in retinal and neurodegenerative diseases. EV proteomics, glia microRNA (miRNA) Open Array and small RNA sequencing, and retinal single cell sequencing were performed, with datasets integrated and analysed computationally. Results demonstrated that exogenous transfer of neuronal miRNA to glial cells was mediated by EV and occurred as a targeted response during degeneration to modulate gliotic inflammation. Taken together, our results support a model of neuronal-to-glial communication via EV, which could be harnessed for therapeutic targeting to slow the progression of retinal-, and neuro-degenerations of the CNS.

Impairing Gasdermin D-mediated pyroptosis is protective against retinal degeneration.

BACKGROUND: Inflammasome activation and the subsequent release of pro-inflammatory cytokines including Interleukin 1ß (IL-1ß) have been widely reported to contribute to the progression of retinal degenerations, including age-related macular degeneration (AMD), the leading cause of blindness in the Western World. The role of Gasdermin D (GSDMD), a key executioner of pyroptosis following inflammasome activation, however, is less well-established. In this study we aimed to characterise the role of GSDMD in the healthy and degenerating retina, and uncover its role as a conduit for IL-1ß release, including via extracellular vesicle (EV)-mediated release. METHODS: GSDMD mutant and knockout mice, in vitro models of inflammation and a well-established in vivo model of retinal degeneration (photo-oxidative damage; PD) were utilised to explore the role and pathological contribution of GSDMD in regulating IL-1ß release and propagating retinal inflammation. RNA sequencing of whole retinas was used to investigate GSDMD-mediated inflammation during degeneration. The role of EVs in GSDMD-mediated IL-1ß release was investigated using nanoparticle tracking analysis, ELISA and EV inhibition paradigms. Finally, the therapeutic efficacy of targeting GSDMD was examined using GSDMD-specific siRNA. RESULTS: We identified in this work that mice deficient in GSDMD had better-preserved retinal function, increased photoreceptor survivability and reduced inflammation. RNA-Seq analysis revealed that GSDMD may propagate inflammation in the retina via NF-κB signalling cascades and release of pro-inflammatory cytokines. We also showed that IL-1ß was packaged and released via EV in a GSDMD-dependent manner. Finally, we demonstrated that impairing GSDMD function using RNAi or blocking EV release was able to reduce IL-1ß content in cell-free supernatant and EV. CONCLUSIONS: Taken together, these results suggest that pyroptotic pore-forming protein GSDMD plays a key role in the propagation of retinal inflammation, in particular via the release of EV-encapsulated IL-1ß. Targeting GSDMD using genetic or pharmacological inhibitors may pose a therapeutic opportunity to dampen inflammatory cascades and delay the progression of retinal degeneration.

Proteomics of Retinal Extracellular Vesicles: A Review into an Unexplored Mechanism in Retinal Health and AMD Pathogenesis.

Extracellular vesicles (EV) are nanosized delivery vehicles that participate in cell-to-cell communication through the selective transfer of molecular materials including RNA, DNA, lipids, and proteins. In the retina, the role of EV proteins is largely unclear, in part due to the lack of studies and the depth of proteomic analyses of EV cargo. This review summarizes the existing knowledge on retinal EV proteins and provides a comparative reanalysis of existing retinal EV proteomic datasets. Collective findings highlight that in homeostasis, the protein components of neural retinal and RPE-derived EV largely reflect the function of the host cells, while in disease RPE-EV protein composition becomes altered, favoring inflammatory modulation and potentially contributing to drusen formation. While these studies shed light on the potential roles of EV proteins in the neural retina and RPE, it is clear that comprehensive proteomic and molecular studies are required, in particular using in vivo models of retinal degenerations.

Short exposure to photo-oxidative damage triggers molecular signals indicative of early retinal degeneration.

Introduction: Age-related macular degeneration (AMD) is the leading cause of blindness in the developed world, currently affecting over 350 billion people globally. For the most prevalent late-stage form of this disease, atrophic AMD, there are no available prevention strategies or treatments, in part due to inherent difficulties in early-stage diagnosis. Photo-oxidative damage is a well-established model for studying inflammatory and cell death features that occur in late-stage atrophic AMD, however to date has not been investigated as a potential model for studying early features of disease onset. Therefore, in this study we aimed to determine if short exposure to photo-oxidative damage could be used to induce early retinal molecular changes and advance this as a potential model for studying early-stage AMD. Methods: C57BL/6J mice were exposed to 1, 3, 6, 12, or 24h photo-oxidative damage (PD) using 100k lux bright white light. Mice were compared to dim-reared (DR) healthy controls as well as mice which had undergone long periods of photo-oxidative damage (3d and 5d-PD) as known timepoints for inducing late-stage retinal degeneration pathologies. Cell death and retinal inflammation were measured using immunohistochemistry and qRT-PCR. To identify retinal molecular changes, retinal lysates were sent for RNA sequencing, following which bioinformatics analyses including differential expression and pathway analyses were performed. Finally, to investigate modulations in gene regulation as a consequence of degeneration, microRNA (miRNA) expression patterns were quantified using qRT-PCR and visualized using in situ hybridization. Results: Short exposure to photo-oxidative damage (1-24h-PD) induced early molecular changes in the retina, with progressive downregulation of homeostatic pathways including metabolism, transport and phototransduction observed across this time-course. Inflammatory pathway upregulation was observed from 3h-PD, preceding observable levels of microglia/macrophage activation which was noted from 6h-PD, as well as significant photoreceptor row loss from 24h-PD. Further rapid and dynamic movement of inflammatory regulator miRNA, miR-124-3p and miR-155-5p, was visualized in the retina in response to degeneration. Conclusion: These results support the use of short exposure to photo-oxidative damage as a model of early AMD and suggest that early inflammatory changes in the retina may contribute to pathological features of AMD progression including immune cell activation and photoreceptor cell death. We suggest that early intervention of these inflammatory pathways by targeting miRNA such as miR-124-3p and miR-155-5p or their target genes may prevent progression into late-stage pathology.

Voluntary exercise modulates pathways associated with amelioration of retinal degenerative diseases.

Background: Exercise has been shown to promote a healthier and longer life and linked to a reduced risk of developing neurodegenerative diseases including retinal degenerations. However, the molecular pathways underpinning exercise-induced cellular protection are not well understood. In this work we aim to profile the molecular changes underlying exercise-induced retinal protection and investigate how exercise-induced inflammatory pathway modulation may slow the progression of retinal degenerations. Methods: Female C57Bl/6J mice at 6 weeks old were given free access to open voluntary running wheels for a period of 28 days and then subjected to 5 days of photo-oxidative damage (PD)-induced retinal degeneration. Following, retinal function (electroretinography; ERG), morphology (optical coherence tomography; OCT) and measures of cell death (TUNEL) and inflammation (IBA1) were analysed and compared to sedentary controls. To decipher global gene expression changes as a result of voluntary exercise, RNA sequencing and pathway and modular gene co-expression analyses were performed on retinal lysates of exercised and sedentary mice that were subjected to PD, as well as healthy dim-reared controls. Results: Following 5 days of PD, exercised mice had significantly preserved retinal function, integrity and reduced levels of retinal cell death and inflammation, compared to sedentary controls. In response to voluntary exercise, inflammatory and extracellular matrix integrity pathways were significantly modulated, with the gene expression profile of exercised mice more closely trending towards that of a healthy dim-reared retina. Conclusion: We suggest that voluntary exercise may mediate retinal protection by influencing key pathways involved in regulating retinal health and shifting the transcriptomic profile to a healthy phenotype.

Functional microRNA targetome undergoes degeneration-induced shift in the retina.

BACKGROUND: MicroRNA (miRNA) play a significant role in the pathogenesis of complex neurodegenerative diseases including age-related macular degeneration (AMD), acting as post-transcriptional gene suppressors through their association with argonaute 2 (AGO2) - a key member of the RNA Induced Silencing Complex (RISC). Identifying the retinal miRNA/mRNA interactions in health and disease will provide important insight into the key pathways miRNA regulate in disease pathogenesis and may lead to potential therapeutic targets to mediate retinal degeneration. METHODS: To identify the active miRnome targetome interactions in the healthy and degenerating retina, AGO2 HITS-CLIP was performed using a rodent model of photoreceptor degeneration. Analysis of publicly available single-cell RNA sequencing (scRNAseq) data was performed to identify the cellular location of AGO2 and key members of the microRNA targetome in the retina. AGO2 findings were verified by in situ hybridization (RNA) and immunohistochemistry (protein). RESULTS: Analysis revealed a similar miRnome between healthy and damaged retinas, however, a shift in the active targetome was observed with an enrichment of miRNA involvement in inflammatory pathways. This shift was further demonstrated by a change in the seed binding regions of miR-124-3p, the most abundant retinal AGO2-bound miRNA, and has known roles in regulating retinal inflammation. Additionally, photoreceptor cluster miR-183/96/182 were all among the most highly abundant miRNA bound to AGO2. Following damage, AGO2 expression was localized to the inner retinal layers and more in the OLM than in healthy retinas, indicating a locational miRNA response to retinal damage. CONCLUSIONS: This study provides important insight into the alteration of miRNA regulatory activity that occurs as a response to retinal degeneration and explores the miRNA-mRNA targetome as a consequence of retinal degenerations. Further characterisation of these miRNA/mRNA interactions in the context of the degenerating retina may provide an important insight into the active role these miRNA may play in diseases such as AMD.

Inhibition of microRNA-155 Protects Retinal Function Through Attenuation of Inflammation in Retinal Degeneration.

Although extensively investigated in inflammatory conditions, the role of pro-inflammatory microRNAs (miRNAs), miR-155 and miR-146a, has not been well-studied in retinal degenerative diseases. We therefore aimed to explore the role and regulation of these miRNA in the degenerating retina, with a focus on miR-155. C57BL/6J mice were subjected to photo-oxidative damage for up to 5 days to induce focal retinal degeneration. MiR-155 expression was quantified by qRT-PCR in whole retina, serum, and small-medium extracellular vesicles (s-mEVs), and a PrimeFlow™ assay was used to identify localisation of miR-155 in retinal cells. Constitutive miR-155 knockout (KO) mice and miR-155 and miR-146a inhibitors were utilised to determine the role of these miRNA in the degenerating retina. Electroretinography was employed as a measure of retinal function, while histological quantification of TUNEL+ and IBA1+ positive cells was used to quantify photoreceptor cell death and infiltrating immune cells, respectively. Upregulation of miR-155 was detected in retinal tissue, serum and s-mEVs in response to photo-oxidative damage, localising to the nucleus of a subset of retinal ganglion cells and glial cells and in the cytoplasm of photoreceptors. Inhibition of miR-155 showed increased function from negative controls and a less pathological pattern of IBA1+ cell localisation and morphology at 5 days photo-oxidative damage. While neither dim-reared nor damaged miR-155 KO animals showed retinal histological difference from controls, following photo-oxidative damage, miR-155 KO mice showed increased a-wave relative to controls. We therefore consider miR-155 to be associated with the inflammatory response of the retina in response to photoreceptor-specific degeneration.

MicroRNA-223 Regulates Retinal Function and Inflammation in the Healthy and Degenerating Retina.

INTRODUCTION: MicroRNAs (miRNAs) are small, non-coding RNA molecules that have powerful regulatory properties, with the ability to regulate multiple messenger RNAs (mRNAs) and biological pathways. MicroRNA-223-3p (miR-223) is known to be a critical regulator of the innate immune response, and its dysregulation is thought to play a role in inflammatory disease progression. Despite miR-223 upregulation in numerous neurodegenerative conditions, largely in cells of the myeloid lineage, the role of miR-223 in the retina is relatively unexplored. Here, we investigated miR-223 in the healthy retina and in response to retinal degeneration. METHODS: miR-223-null mice were investigated in control and photo-oxidative damage-induced degeneration conditions. Encapsulated miR-223 mimics were intravitreally and intravenously injected into C57BL/6J wild-type mice. Retinal functional responses were measured using electroretinography (ERG), while extracted retinas were investigated by retinal histology (TUNEL and immunohistochemistry) and molecular analysis (qPCR and FACS). RESULTS: Retinal function in miR-223-/- mice was adversely affected, indicating that miR-223 may be critical in regulating the retinal response. In degeneration, miR-223 was elevated in the retina, circulating serum, and retinal extracellular vesicles. Conversely, retinal microglia and macrophages displayed a downregulation of miR-223. Further, isolated CD11b+ inflammatory cells from the retinas and circulation of miR-223-null mice showed an upregulation of pro-inflammatory genes that are critically linked to retinal inflammation and progressive photoreceptor loss. Finally, both local and systemic delivery of miR-223 mimics improved retinal function in mice undergoing retinal degeneration. CONCLUSION: miR-223 is required for maintaining normal retinal function, as well as regulating inflammation in microglia and macrophages. Further investigations are required to determine the targets of miR-223 and their key biological pathways and interactions that are relevant to retinal diseases. Future studies should investigate whether sustained delivery of miR-223 into the retina is sufficient to target these pathways and protect the retina from progressive degeneration.

Small-Medium Extracellular Vesicles and Their miRNA Cargo in Retinal Health and Degeneration: Mediators of Homeostasis, and Vehicles for Targeted Gene Therapy.

Photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (AMD). However, the molecular mechanisms underlying these biological processes are largely unknown. Extracellular vesicles (EV) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. EVs, including exosomes, encapsulate and transfer microRNA (miRNA) to recipient cells and in this way can modulate the environment of recipient cells. Dysregulation of EVs however is correlated to a loss of cellular homeostasis and increased inflammation. In this work we investigated the role of isolated retinal small-medium sized EV (s-mEV) which includes exosomes in both the healthy and degenerating retina. Isolated s-mEV from normal retinas were characterized using dynamic light scattering, transmission electron microscopy and western blotting, and quantified across 5 days of photo-oxidative damage-induced degeneration using nanotracking analysis. Small RNAseq was used to characterize the miRNA cargo of retinal s-mEV isolated from healthy and damaged retinas. Finally, the effect of exosome inhibition on cell-to-cell miRNA transfer and immune modulation was conducted using systemic daily administration of exosome inhibitor GW4869 and in situ hybridization of s-mEV-abundant miRNA, miR-124-3p. Electroretinography and immunohistochemistry was performed to assess functional and morphological changes to the retina as a result of GW4869-induced exosome depletion. Results demonstrated an inverse correlation between s-mEV concentration and photoreceptor survivability, with a decrease in s-mEV numbers following degeneration. Small RNAseq revealed that s-mEVs contained uniquely enriched miRNAs in comparison to in whole retinal tissue, however, there was no differential change in the s-mEV miRNAnome following photo-oxidative damage. Exosome inhibition via the use of GW4869 was also found to exacerbate retinal degeneration, with reduced retinal function and increased levels of inflammation and cell death demonstrated following photo-oxidative damage in exosome-inhibited mice. Further, GW4869-treated mice displayed impaired translocation of photoreceptor-derived miR-124-3p to the inner retina during damage. Taken together, we propose that retinal s-mEV and their miRNA cargo play an essential role in maintaining retinal homeostasis through immune-modulation, and have the potential to be used in targeted gene therapy for retinal degenerative diseases.

Upregulation of pannexin-1 hemichannels explains the apparent death of the syncytiotrophoblast during human placental explant culture.

BACKGROUND: It has been reported that during the culture of human placental explants, the syncytiotrophoblast dies between 3 and 24 h and is then replaced within 48 h by a new syncytiotrophoblast layer formed by the fusion of underlying cytotrophoblasts. Most frequently the death of the syncytiotrophoblast is indicated by the uptake of nuclear stains such as propidium iodide (PI). This process is reportedly similar in both early and late gestation placental explants. METHODS: We cultured first trimester placental explants for up to 48 h and tested membrane intactness by exposure to PI. Connexin and pannexin mRNAs were quantified by RT-PCR and protein levels determined by immunofluorescence. The syncytiotrophoblast membrane leak was determined by culturing explants in the presence of hemichannel blockers. Extrusion of extracellular vesicles from the syncytiotrophoblast was quantified. RESULTS: Nuclei of the syncytiotrophoblast were stained with PI following approximately 4 h of culture and this was prevented by culturing the explants with pannexin-1 blockers. Expression of pannexin-1 hemichannels increased during explant culture (p = 0.0027). Extracellular vesicles were most abundantly extruded from the explants during the first 3 h of culture and the temporal pattern of extrusion was unaltered by blocking hemichannels. DISCUSSION: We show the mechanism of uptake of nuclear non-viability stains into the syncytiotrophoblast during explant culture is via upregulation of pannexin 1 hemichannels. Contrary to suggestions by some, the production of extracellular vesicles from cultured placental explants is not an in vitro artefact resulting from the apparent death of the syncytiotrophoblast in explant cultures.

Caspase-1-dependent inflammasomes mediate photoreceptor cell death in photo-oxidative damage-induced retinal degeneration.

Activation of the inflammasome is involved in the progression of retinal degenerative diseases, in particular, in the pathogenesis of Age-Related Macular Degeneration (AMD), with NLRP3 activation the focus of many investigations. In this study, we used genetic and pharmacological approaches to explore the role of the inflammasome in a mouse model of retinal degeneration. We identify that Casp1/11-/- mice have better-preserved retinal function, reduced inflammation and increased photoreceptor survivability. While Nlrp3-/- mice display some level of preservation of retinal function compared to controls, pharmacological inhibition of NLRP3 did not protect against photoreceptor cell death. Further, Aim2-/-, Nlrc4-/-, Asc-/-, and Casp11-/- mice show no substantial retinal protection. We propose that CASP-1-associated photoreceptor cell death occurs largely independently of NLRP3 and other established inflammasome sensor proteins, or that inhibition of a single sensor is not sufficient to repress the inflammatory cascade. Therapeutic targeting of CASP-1 may offer a more promising avenue to delay the progression of retinal degenerations.

IL-1 Family Members Mediate Cell Death, Inflammation and Angiogenesis in Retinal Degenerative Diseases.

Inflammation underpins and contributes to the pathogenesis of many retinal degenerative diseases. The recruitment and activation of both resident microglia and recruited macrophages, as well as the production of cytokines, are key contributing factors for progressive cell death in these diseases. In particular, the interleukin 1 (IL-1) family consisting of both pro- and anti-inflammatory cytokines has been shown to be pivotal in the mediation of innate immunity and contribute directly to a number of retinal degenerations, including Age-Related Macular Degeneration (AMD), diabetic retinopathy, retinitis pigmentosa, glaucoma, and retinopathy of prematurity (ROP). In this review, we will discuss the role of IL-1 family members and inflammasome signaling in retinal degenerative diseases, piecing together their contribution to retinal disease pathology, and identifying areas of research expansion required to further elucidate their function in the retina.

Photoreceptor Survival Is Regulated by GSTO1-1 in the Degenerating Retina.

Purpose: Glutathione-S-transferase omega 1-1 (GSTO1-1) is a cytosolic glutathione transferase enzyme, involved in glutathionylation, toll-like receptor signaling, and calcium channel regulation. GSTO1-1 dysregulation has been implicated in oxidative stress and inflammation, and contributes to the pathogenesis of several diseases and neurological disorders; however, its role in retinal degenerations is unknown. The aim of this study was to investigate the role of GSTO1-1 in modulating oxidative stress and consequent inflammation in the normal and degenerating retina. Methods: The role of GSTO1-1 in retinal degenerations was explored by using Gsto1-/- mice in a model of retinal degeneration. The expression and localization of GSTO1-1 were investigated with immunohistochemistry and Western blot. Changes in the expression of inflammatory (Ccl2, Il-1ß, and C3) and oxidative stress (Nox1, Sod2, Gpx3, Hmox1, Nrf2, and Nqo1) genes were investigated via quantitative real-time polymerase chain reaction. Retinal function in Gsto1-/- mice was investigated by using electroretinography. Results: GSTO1-1 was localized to the inner segment of cone photoreceptors in the retina. Gsto1-/- photo-oxidative damage (PD) mice had decreased photoreceptor cell death as well as decreased expression of inflammatory (Ccl2, Il-1ß, and C3) markers and oxidative stress marker Nqo1. Further, retinal function in the Gsto1-/- PD mice was increased as compared to wild-type PD mice. Conclusions: These results indicate that GSTO1-1 is required for inflammatory-mediated photoreceptor death in retinal degenerations. Targeting GSTO1-1 may be a useful strategy to reduce oxidative stress and inflammation and ameliorate photoreceptor loss, slowing the progression of retinal degenerations.

MicroRNA-124 Dysregulation is Associated With Retinal Inflammation and Photoreceptor Death in the Degenerating Retina.

Purpose: We sought to determine the role and retinal cellular location of microRNA-124 (miR-124) in a neuroinflammatory model of retinal degeneration. Further, we explored the anti-inflammatory relationship of miR-124 with a predicted messenger RNA (mRNA) binding partner, chemokine (C-C motif) ligand 2 (Ccl2), which is crucially involved in inflammatory cell recruitment in the damaged retina. Methods: Human AMD donor eyes and photo-oxidative damaged (PD) mice were labeled for miR-124 expression using in situ hybridization. PDGFRa-cre RFP mice were used for Müller cell isolation from whole retinas. MIO-M1 immortalized cells and rat primary Müller cells were used for in vitro analysis of miR-124 expression and its relationship with Ccl2. Therapeutic efficacy was tested with intravitreal administration of miR-124 mimic in mice, with electroretinography used to determine retinal function. IBA1 immunohistochemistry and photoreceptor row counts were used for assessment of inflammation and cell death. Results: MiR-124 expression was correlated with progressive retinal damage, inflammation, and cell death in human AMD and PD mice. In addition, miR-124 expression was inversely correlated to Ccl2 expression in mice following PD. MiR-124 was localized to both neuronal-like photoreceptors and glial (Müller) cells in the retina, with a redistribution from neurons to glia occurring as a consequence of PD. Finally, intravitreal administration of miR-124 mimics decreased retinal inflammation and photoreceptor cell death, and improved retinal function. Conclusions: This study has provided an understanding of the mechanism behind miR-124 in the degenerating retina and demonstrates the usefulness of miR-124 mimics for the modulation of retinal degenerations.

The use of the vaccinia virus complement control protein (VCP) in the rat retina.

The complement system is highly implicated in both the prevalence and progression of Age-Related Macular Degeneration (AMD). Complement system inhibitors therefore have potential therapeutic value in managing excessive activation of the complement pathways in retinal degenerations. The vaccinia virus complement control protein (VCP) has been shown to be effective as a complement inhibitor in neuroinflammatory models including traumatic brain injury and spinal cord injury. We aimed to investigate the potential of VCP as a therapeutic molecule for retinal degenerations. In this study, we investigated the effect, localisation and delivery of VCP to the rodent retina. Complement inhibition activity of VCP was tested using a hemolytic assay. Photoreceptor cell death, inflammation and retinal stress were assayed to determine if any retinal toxicity was induced by an intravitreal injection of VCP. The effect of VCP was investigated in a model of photo-oxidative retinal degeneration. Localisation of VCP after injection was determined using a fluorescein-tagged form of VCP, as well as immunohistochemistry. Finally, a copolymer resin (Elvax) was trialled for the slow-release delivery of VCP to the retina. We found that a dose equivalent to 20µg VCP when intravitreally injected into the rat eye did not cause any photoreceptor cell death or immune cell recruitment, but led to an increase in GFAP. In photo-oxidative damaged retinas, there were no differences in photoreceptor loss, retinal stress (Gfap) and inflammation (Ccl2 and C3) between VCP and saline-injected groups; however, Jun expression was reduced in VCP-treated retinas. After VCP was injected into the eye, it was taken up in all layers of the retina but was cleared within 1-3 hours of delivery. This study indicates that a method to sustain the delivery of VCP to the retina is necessary to further investigate the effect of VCP as a complement inhibitor for retinal degenerations.