Understanding of retinal degeneration through the lens of high throughput gene expression

The retina is comprised of an intricate network of neurons, including the light-sensing photoreceptors, and supporting glia. Retinal glia maintain tissue homeostasis, while the underlying retinal-pigmented epithelium (RPE) and vascular choroid synergise to ensures a constant nutrient supply and waste removal from the highly metabolically active photoreceptors. Photoreceptor degeneration is central to almost all retinal degenerative diseases, including the increasingly prevalent and currently untreatable Age-Related Macular Degeneration (AMD). Oxidative stress and retinal inflammation are established as drivers of degeneration and are instigated by retinal glia and immune cells from circulation and choroid. Although reductionist approaches and histological observations have unravelled some of the molecular players driving photoreceptor degeneration, the application of high-throughput methods is required to penetrate the bewildering molecular complexity of retinal degeneration. To this end, the work presented in this thesis leverages powerful RNA profiling technologies to uncover novel gene expression patterns in mRNA and regulatory microRNA (miRNA) underpinning retinal degeneration. This thesis integrates data from 7 bulk miRNA/mRNA datasets and 3 single-cell RNA sequencing datasets (scRNAseq) to probe the transcriptomes of (1) the whole degenerating retina, as well as (2) effector cells driving degeneration including Muller glia and choroidal melanocytes and (3) extracellular vesicles (EV) as follow: Published results [1] presented in Chapter 3 describe changes in the mRNA and miRNA in the mouse retina following retinal degeneration induced by photo-oxidative damage. miRNA are short, non-coding RNAs working within a protein complex (RNA-induced silencing complex - RISC) to repress the translation of their mRNA targets. This chapter demonstrates that retinal degeneration is underpinned by a shift in miRNA and mRNA expression towards a proinflammatory state. Simultaneously, retinal degeneration alters miRNA binding sites within pro-inflammatory glial mRNAs allowing targeting by RISC-bound, neuronal miRNA. Published results [2] in Chapter 4 explore the role of EVs in retinal degeneration. EVs are membrane-bound vesicles secreted by nearly all cells as a form of intercellular communication between anatomically separated cells. This chapter developed the first published method for EV isolation from mouse retinas. Using this method, retinal EVs were found to be enriched with neuronal miRNA, and retinal degeneration results in a depletion of EV bioavailability. Lastly, this chapter demonstrates that inhibition of EV biogenesis accelerates retinal degeneration and results in impaired miRNA trafficking. Chapter 5 the explores miRNA-mRNA interactions in Muller glia using data integration from miRNA expression and scRNAseq. These results demonstrate that Muller glia activate a transient gene expression programme driving proliferation and pluripotency early in degeneration, then a shift towards a sustained pro-inflammatory programme follows. Published work [3] in Chapter 6 focuses on the response of choroidal melanocytes to inflammation. The choroid is a reservoir of and entry portal for immune cells in retinal degeneration, yet the relevance of the melanocytes (non-immune, highly abundant choroidal cells) in retinal degeneration is unknown. This chapter demonstrates that choroidal melanocytes have extensive immunomodulatory properties that are activated by both inflammatory challenge and retinal degeneration. In summary, this thesis uncovers novel tissue-level and cell-level gene expression patterns and regulatory networks altered in retinal degeneration, potentially providing the impetus for future therapeutics tacking complex retinal degenerations such as AMD

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

IntroductionAge-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.MethodsC57BL/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.ResultsShort 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.ConclusionThese 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

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.This work would not have been possible without the support of the National Health and Medical Research Council of Australia (NHMRC: 1127705), Retina Australia, The Gordon and Gretel Bootes Foundation, and The ANU Translational Fellowshi

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.This work would not have been possible without the support of the National Health and Medical Research Council of Australia (NHMRC: 1127705), Retina Australia, The Gordon and Gretel Bootes Foundation and The ANU Translational Fellowshi

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

Decrease in plasma miR-27a and miR-221 after concussion in Australian football players

Introduction: Sports-related concussion (SRC) is a common form of brain injury that lacks reliable methods to guide clinical decisions. MicroRNAs (miRNAs) can influence biological processes involved in SRC, and measurement of miRNAs in biological fluids may provide objective diagnostic and return to play/recovery biomarkers. Therefore, this prospective study investigated the temporal profile of circulating miRNA levels in concussed male and female athletes. Methods: Pre-season baseline blood samples were collected from amateur Australian rules football players (82 males, 45 females). Of these, 20 males and 8 females sustained an SRC during the subsequent season and underwent blood sampling at 2-, 6- and 13-days post-injury. A miRNA discovery Open Array was conducted on plasma to assess the expression of 754 known/validated miRNAs. miRNA target identified were further investigated with quantitative real-time PCR (qRT-PCR) in a validation study. Data pertaining to SRC symptoms, demographics, sporting history, education history and concussion history were also collected. Results: Discovery analysis identified 18 candidate miRNA. The consequent validation study found that plasma miR-221-3p levels were decreased at 6d and 13d, and that miR-27a-3p levels were decreased at 6d, when compared to baseline. Moreover, miR-27a and miR-221-3p levels were inversely correlated with SRC symptom severity. Conclusion: Circulating levels of miR-27a-3p and miR-221-3p were decreased in the sub-acute stages after SRC, and were inversely correlated with SRC symptom severity. Although further studies are required, these analyses have identified miRNA biomarker candidates of SRC severity and recovery that may one day assist in its clinical management

Extracellular matrix and oxidative stress regulate human retinal pigment epithelium growth

Age-related macular degeneration (AMD), the most common cause of vision loss with ageing, is characterised by degeneration of the photoreceptors and retinal pigment epithelium (RPE) and changes in the extracellular matrix (ECM) underlying the RPE. The pathogenesis of AMD is still not fully understood. In this study we investigated the in vitro growth and function of primary human RPE cells in response to different ECM substrates, including nitrite-modified ECM. We initially confirmed the presence of disorganised retinal glial and photoreceptor cells, marked retinal cytoplasmic and Bruch's membrane expression of nitro-tyrosine (an oxidative stress marker) and increased numbers of Iba1+ macrophages/microglia in human donor eye sections (aged and AMD) using multimarker immunohistochemistry (n = 3). Concurrently, we utilised two-photon microscopy to reveal topographical changes in flatmounts of RPE-associated ECM and in the underlying choroid of aged and AMD donor eyes (n = 3). To recapitulate these observations in vitro, we then used primary human RPE cells to investigate how different ECM proteins, including nitrite cross-linked RPE-secreted ECM, modified RPE cell growth and function. Collagen I or IV increased RPE attachment and spreading two-to three-fold, associated with significantly increased cell migration and proliferation, consistent with a preferential interaction with these matrix substrates. Primary human RPE cells grown on collagen I and IV also showed increased secretion of pro-inflammatory cytokines, MCP-1 and IL-8. Nitrite-modification of RPE-secreted ECM (simulating ageing of Bruch's membrane) significantly reduced in vitro RPE attachment to the ECM and this was mitigated with collagen IV coating of the modified ECM. Taken together, our observations confirm the importance of RPE-ECM interactions for normal RPE growth and function, and for inducing RPE secretion of pro-inflammatory cytokines. Furthermore, the findings are consistent with ageing and/or oxidative stress-induced disruption of RPE-ECM interactions contributing to the pathogenesis of AMD

The Effects of Honey Sulfonamides on Immunological and Hematological Parameters in Wistar Rats

Sulfonamides are among the most used drugs in beekeeping due to their effectiveness, despite their long-term persistence in tissues. Bee honey containing such residues poses numerous risks to human health. The aim of the study was to evaluate the effects on immunological and hematological parameters of Wistar rats produced by sulfonamide residues in bee honey, through the evaluation of various blood parameters such as triiodothyronine and thyroxine levels, hematocrit, hemoglobin, red blood cell count and mean corpuscular hemoglobin concentration in a given volume of erythrocytes following administration of sulfonamide-containing honey. The hematological and immunological parameters showed significant variations in the group of rats that had been fed with honey spiked with sulfonamides compared to the control group. Changes in hematological indices were demonstrated in terms of a significant reduction in the number of erythrocytes, the amount of hemoglobin, and the value of hematocrit, thus confirming the induction of anemia in the tested group. Investigation of thyroid function through the analysis of triiodothyronine (T3) and thyroxine (T4) and their ratio showed a very significant decrease in plasma thyroxine levels in laboratory rats that were fed sulfonamide-spiked honey compared to the control group. The mean T3 concentration decreased from 0.70 ± 0.14 ng/dL to 0.34 ± 0.03 ng/dL, while the mean T4 concentration was reduced from 4.50 ± 0.30 μg/dL to 3.32 ± 0.21 μg/dL, thus demonstrating toxic effects on thyroid function. In sum, the presence of sulfonamides induced significant changes in the evaluated parameters indicating that the consumption of contaminated honey samples represents a high risk factor for thyroid dysfunction with potentially serious health impacts

In Vitro Antioxidant, Antitumor and Photocatalytic Activities of Silver Nanoparticles Synthesized Using <i>Equisetum</i> Species: A Green Approach

The ethanolic extracts of three Equisetum species (E. pratense Ehrh., E. sylvaticum L. and E. telmateia Ehrh.) were used to reduce silver ions to silver nanoparticles (AgNPs). The synthesized AgNPs were characterized using UV-Vis spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), Energy Dispersive X-ray (EDX), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS) measurements. FTIR data revealed the functional groups of biomolecules involved in AgNPs synthesis, such as O-H, C-H, C=O, C-O, and C-C. EDX spectroscopy was used to highlight the presence of silver, while DLS spectroscopy provided information on the mean diameter of AgNPs, that ranged from 74.4 to 314 nm. The negative Zeta potential values (−23.76 for Ep–AgNPs, −29.54 for Es–AgNPs and −20.72 for Et–AgNPs) indicate the stability of the obtained colloidal solution. The study also focused on establishing the photocatalytic activity of AgNPs, which is an important aspect in terms of removing organic dyes from the environment. The best photocatalytic activity was observed for AgNPs obtained from E. telmateia, which degraded malachite green in a proportion of 97.9%. The antioxidant action of the three AgNPs samples was highlighted comparatively through four tests, with the best overall antioxidant capacity being observed for AgNPs obtained using E. sylvaticum. Moreover, the biosynthesized AgNPs showed promising cytotoxic efficacy against cancerous cell line MG63, the AgNPs obtained from E. sylvaticum L. providing the best result, with a LD50 value around 1.5 mg/mL