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Age-related macular degeneration (AMD) is a leading cause of vision loss with recent evidence indicating an important role for macroautophagy/autophagy in disease progression. In this study we investigate the efficacy of targeting autophagy for slowing dysfunction in a mouse model with features of early AMD. Mice lacking APOE (apolipoprotein E; B6.129P2-Apoesupitm1Unc/i/supJ/Arc) and C57BL/6 J- (wild-type, WT) mice were treated with metformin or trehalose in the drinking water from 5 months of age and the ocular phenotype investigated at 13 months. Control mice received normal drinking water. APOE-control mice had reduced retinal function and thickening of Bruch's membrane consistent with an early AMD phenotype. Immunohistochemical labeling showed reductions in MAP1LC3B/LC3 (microtubule-associated protein 1 light chain 3 beta) and LAMP1 (lysosomal-associated membrane protein 1) labeling in the photoreceptors and retinal pigment epithelium (RPE). This correlated with increased LC3-II:LC3-I ratio and alterations in protein expression in multiple autophagy pathways measured by reverse phase protein array, suggesting autophagy was slowed. Treatment of APOE-mice with metformin or trehalose ameliorated the loss of retinal function and reduced Bruch's membrane thickening, enhancing LC3 and LAMP1 labeling in the ocular tissues and restoring LC3-II:LC3-I ratio to WT levels. Protein analysis indicated that both treatments boost ATM-AMPK driven autophagy. Additionally, trehalose increased p-MAPK14/p38 to enhance autophagy. Our study shows that treatments targeting pathways to enhance autophagy have the potential for treating early AMD and provide support for the use of metformin, which has been found to reduce the risk of AMD development in human patients.bAbbreviations:/bAMD: age-related macular degeneration; AMPK: 5' adenosine monophosphate-activated protein kinase APOE: apolipoprotein E; ATM: ataxia telangiectasia mutated; BCL2L1/Bcl-xL: BCL2-like 1; DAPI: 4'-6-diamidino-2-phenylindole; ERG: electroretinogram; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; IS/OS: inner and outer photoreceptor segments; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; OCT: optical coherence tomography; ONL: outer nuclear layer; OPs: oscillatory potentials; p-EIF4EBP1: phosphorylated eukaryotic translation initiation factor 4E binding protein 1; p-MAPK14/p38: phosphorylated mitogen-activated protein kinase 14; RPE: retinal pigment epithelium; RPS6KB/p70 S6 kinase: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; TP53/TRP53/p53: tumor related protein 53; TSC2: TSC complex subunit 2; WT: wild type.

Diabetic retinopathy is the most feared complication for those with diabetes. Although visible vascular pathology traditionally defines the management of this condition, it is now recognised that a range of cellular changes occur in the retina from an early stage of diabetes. One of the most significant functional changes that occurs in those with diabetes is a loss of vasoregulation in response to changes in neural activity. There are several retinal cell types that are critical for mediating so-called neurovascular coupling, including Müller cells, microglia and pericytes. Although there is a great deal of evidence that suggests that Müller cells are integral to regulating the vasculature, they only modulate part of the vascular tree, highlighting the complexity of vasoregulation within the retina. Recent studies suggest that retinal immune cells, microglia, play an important role in mediating vasoconstriction. Importantly, retinal microglia contact both the vasculature and neural synapses and induce vasoconstriction in response to neurally expressed chemokines such as fractalkine. This microglial-dependent regulation occurs via the vasomediator angiotensinogen. Diabetes alters the way microglia regulate the retinal vasculature, by increasing angiotensinogen expression, causing capillary vasoconstriction and contributing to a loss of vascular reactivity to physiological signals. This article summarises recent studies showing changes in vascular regulation during diabetes, the potential mechanisms by which this occurs and the significance of these early changes to the progression of diabetic retinopathy.

Human pluripotent stem cell (hPSC)-derived progenies are immature versions of cells, presenting a potential limitation to the accurate modelling of disease associated with maturity or age. Hence, it is important to characterise how closely cells used in culture resemble their native counterparts. In order to select appropriate points in time for RPE cultures to reflect native counterparts, we characterised the transcriptomic profiles of hPSC-derived retinal pigment epithelium (RPE) cells from 1- and 12-month cultures. We differentiated the human embryonic stem cell line H9 into RPE cells, performed single cell RNA-sequencing of a total of 16,576 cells, and analysed the resulting data to assess the molecular changes of RPE cells across these two culture time points. Our results indicate the stability of the RPE transcriptomic signature, with no evidence of an epithelial – mesenchymal transition, and with maturing populations of RPE observed with time in culture. Assessment of gene ontology pathways revealed that as cultures age, RPE cells upregulate expression of genes involved in metal binding and antioxidant functions. This might reflect an increased ability to handle oxidative stress as cells mature. Comparison with native human RPE data confirmed a maturing transcriptional profile of RPE cells in culture. These results suggest that in vitro long-term culture of RPE cells allow the modelling of specific phenotypes observed in native mature tissue. Our work highlights the transcriptional landscape of hPSC-derived RPE as they age in culture, which provides a reference for native and patient-samples to be benchmarked against.

There is increasing evidence for the vulnerability of specific retinal ganglion cell (RGC) types in those with glaucoma and in animal models. In addition, the P2X7-receptor (P2X7-R) has been suggested to contribute to RGC death following stimulation and elevated IOP, though its role in RGC dysfunction prior to death has not been examined. Therefore, we examined the effect of an acute, non-ischemic intraocular pressure (IOP) insult (50 mmHg for 30 min) on RGC function in wildtype mice and P2X7-R knockout (P2X7-KO) mice. We examined retinal function using electroretinogram recordings and individual RGC responses using multielectrode arrays, 3 days following acute IOP elevation. Immunohistochemistry was used to examine RGC cell death and P2X7-R expression in several RGC types. Acute intraocular pressure elevation produced pronounced dysfunction in RGCs; whilst other retinal neuronal responses showed lesser changes. Dysfunction at 3 days post-injury was not associated with RGC loss or changes in receptive field size. However, in wildtype animals, OFF-RGCs showed reduced spontaneous and light-elicited activity. In the P2X7-KO, both ON- and OFF-RGC light-elicited responses were reduced. Expression of P2X7-R in wildtype ON-RGC dendrites was higher than in other RGC types. In conclusion, OFF-RGCs were vulnerable to acute IOP elevation and their dysfunction was not rescued by genetic ablation of P2X7-R. Indeed, knockout of P2X7-R also caused ON-RGC dysfunction. These findings aid our understanding of how pressure affects RGC function and suggest treatments targeting the P2X7-R need to be carefully considered.

Photoreceptor death is an important contributor to irreversible vision loss worldwide. In this review, I outline our work examining the role that purines, such as adenosine triphosphate (ATP), have in normal retinal function and in retinal disease. Our work shows that the actions of ATP, mediated by P2X receptors, are expressed in various retinal layers including photoreceptor terminals, and when stimulated by excessive levels of ATP is associated with rapid death of neurons. Treatment with a compound that blocks the action of P2X and some P2Y receptors reduces photoreceptor death in a mouse model of retinal degeneration. Our observations not only provide a means for developing a potential treatment for reducing photoreceptor death, but also provides a novel way of studying the neural plasticity effects that develop in the inner retina following photoreceptor death. There are a range of inner retinal changes that could influence the effectiveness of retinal prostheses. Indeed, using an ATP-induced degeneration model, we established that the amount of electrical stimulation required to elicit a response in the visual cortex was affected by the level of glial scarring. However, changes in P2X7 receptor expression by OFF ganglion cells during retinal degeneration can also be exploited by photoswitches to restore light sensitivity to degenerated retinae. Finally, our work has also considered how P2X7 expression by innate immune cells, and its role as a scavenger receptor, contributes to age-related macular degeneration (AMD). Our results show that loss of P2X7 function is associated with thickening of Bruch's membrane as well as increased risk of advanced disease in people with AMD. Overall, our work over the last 20 years highlights the importance of purinergic signalling in normal retinal function and retinal disease and suggest that developing therapies that target P2X7 function could be of benefit for these diseases.

Glaucoma is a neurodegenerative disease characterised by progressive damage to the retinal ganglion cells (RGCs), the output neurons of the retina. RGCs are a heterogenous class of retinal neurons which can be classified into multiple types based on morphological, functional and genetic characteristics. This review examines the body of evidence supporting type-specific vulnerability of RGCs in glaucoma and explores potential mechanisms by which this might come about. Studies of donor tissue from glaucoma patients have generally noted greater vulnerability of larger RGC types. Models of glaucoma induced in primates, cats and mice also show selective effects on RGC types - particularly OFF RGCs. Several mechanisms may contribute to type-specific vulnerability, including differences in the expression of calcium-permeable receptors (for example pannexin-1, P2X7, AMPA and transient receptor potential vanilloid receptors), the relative proximity of RGCs and their dendrites to blood supply in the inner plexiform layer, as well as differing metabolic requirements of RGC types. Such differences may make certain RGCs more sensitive to intraocular pressure elevation and its associated biomechanical and vascular stress. A greater understanding of selective RGC vulnerability and its underlying causes will likely reveal a rich area of investigation for potential treatment targets.

Purpose Age related macular degeneration (AMD) is the leading cause of irreversible vision loss in industrialised nations. Based on genetics, as well as proteome analysis of drusen, the role the innate immune system in the development and/or progression of the disease is well established. Mononuclear phagocytes, such as microglia and monocytes, play critical roles in innate immunity. Here, the role of retinal microglia in mediating normal retinal function, and how these cells change with age is discussed, so as to understand their role in the development and progression of AMD. Recent findings It is now known that microglia dynamically survey the neural environment, responding rapidly to even the most subtle neural injury. The dynamic and phagocytic roles of microglia can change with age contributing to alteration in the response of these cells to damage with age. Accumulation of innate immune cells in the subretinal space is a hallmark feature of the development of AMD, reflecting either an increase in migration of monocytes into the retina, or a failure of immune cell elimination from the retina. Furthermore, changes in phagocytic ability of immune cells could contribute to the accumulation of drusen deposits in the posterior eye. Summary An overview of how retinal microglia maintain retinal homeostasis under normal conditions is provided, and then how they contribute to each stage of AMD. In addition, circulating monocytes are altered in those with AMD, contributing to the overall inflammatory state. Understanding the role of cells of the innate immune system in AMD may uncover novel therapeutic targets with which to reduce either the development or progression of disease.

Significance This work identifies a role for microglia, the innate immune cells of the CNS, in the local control of the retinal vasculature and identifies deficits early in diabetes. Microglia contact neurons and vasculature and express several vasoactive agents. Activation of microglial fractalkine-Cx3cr1 signaling leads to capillary constriction and blocking the renin-angiotensin system (RAS) with candesartan abolishes microglial-mediated vasoconstriction in the retina. In early diabetes, reduced retinal blood flow is coincident with capillary constriction, increased microglial–vessel association, loss of microglial–capillary regulation, and altered microglial expression of the RAS pathway. While candesartan restores retinal capillary diameter early in diabetes, targeting of microglial–vascular regulation is required to prevent coincident dilation of large retinal vessels and reduced retinal blood flow., Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial–capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial–vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.

Reticular pseudodrusen (RPD) are subretinal deposits that, when observed with age-related macular degeneration (AMD), form a distinct phenotype, often associated with late-stage disease. To date, RPD genetic risk associations overlap six well-established AMD-risk regions. Determining RPD-specific underlying genetic causes by using adequate imaging methods should improve our understanding of the pathophysiology of RPD.

Purpose There is an urgent need for a more effective intervention to slow or prevent progression of age-related macular degeneration (AMD) from its early stages to vision-threatening late complications. Subthreshold nanosecond laser (SNL) treatment has shown promise in preclinical studies and a pilot study in intermediate AMD (iAMD) as a potential treatment. We aimed to evaluate the safety of SNL treatment in iAMD and its efficacy for slowing progression to late AMD. Design The Laser Intervention in Early Stages of Age-Related Macular Degeneration (LEAD) study is a 36-month, multicenter, randomized, sham-controlled trial. Participants Two hundred ninety-two participants with bilateral large drusen and without OCT signs of atrophy. Methods Participants were assigned randomly to receive Retinal Rejuvenation Therapy (2RT®; Ellex Pty Ltd, Adelaide, Australia) SNL or sham treatment to the study eye at 6-monthly intervals. Main Outcome Measures The primary efficacy outcome was the time to development of late AMD defined by multimodal imaging (MMI). Safety was assessed by adverse events. Results Overall, progression to late AMD was not slowed significantly with SNL treatment compared with sham treatment (adjusted hazard ratio [HR], 0.61; 95% confidence interval [CI], 0.33–1.14; P = 0.122). However, a post hoc analysis showed evidence of effect modification based on the coexistence of reticular pseudodrusen (RPD; adjusted interaction P = 0.002), where progression was slowed for the 222 participants (76.0%) without coexistent RPD at baseline (adjusted HR, 0.23; 95% CI, 0.09–0.59; P = 0.002), whereas an increased progression rate (adjusted HR, 2.56; 95% CI, 0.80–8.18; P = 0.112) was observed for the 70 participants (24.0%) with RPD with SNL treatment. Differences between the groups in serious adverse events were not significant. Conclusions In participants with iAMD without MMI-detected signs of late AMD, no significant difference in the overall progression rate to late AMD between those receiving SNL and sham treatment were observed. However, SNL treatment may have a role in slowing progression for those without coexistent RPD and may be inappropriate in those with RPD, warranting caution when considering treatment in clinical phenotypes with RPD. Our findings provide compelling evidence for further trials of the 2RT® laser, but they should not be extrapolated to other short-pulse lasers.

Reticular pseudodrusen (RPD), or subretinal drusenoid deposits (SDD), refer to distinct lesions that occur in the subretinal space. Over the past three decades, their presence in association with age-related macular degeneration (AMD) has become increasingly recognized, especially as RPD have become more easily distinguished with newer clinical imaging modalities. There is also an increasing appreciation that RPD appear to be a critical AMD phenotype, where understanding their pathogenesis will provide further insights into the processes driving vision loss in AMD. However, key barriers to understanding the current evidence related to the independent impact of RPD include the heterogeneity in defining their presence, and failure to account for the confounding impact of the concurrent presence and severity of AMD pathology. This review thus critically discusses the current evidence on the prevalence and clinical significance of RPD and proposes a clinical imaging definition of RPD that will help move the field forward in gathering further key knowledge about this critical phenotype. It also proposes a putative mechanism for RPD formation and how they may drive progression to vision loss in AMD, through examining current evidence and presenting novel findings from preclinical and clinical studies.

Microglia, the resident immune cells of the central nervous system (CNS), were once considered quiescent cells that sat in readiness for reacting to disease and injury. Over the last decade, however, it has become clear that microglia play essential roles in maintaining the normal nervous system. The retina is an easily accessible part of the central nervous system and therefore much has been learned about the function of microglia from studies in the retina and visual system. Anatomically, microglia have processes that contact all synapses within the retina, as well as blood vessels in the major vascular plexuses. Microglia contribute to development of the visual system by contributing to neurogenesis, maturation of cone photoreceptors, as well as refining synaptic contacts. They can respond to neural signals and in turn release a range of cytokines and neurotrophic factors that have downstream consequences on neural function. Moreover, in light of their extensive contact with blood vessels, they are also essential for regulation of vascular development and integrity. This review article summarizes what we have learned about the role of microglia in maintaining the normal visual system and how this has helped in understanding their role in the central nervous system more broadly.

Retinitis pigmentosa is a family of inherited retinal degenerations associated with gradual loss of photoreceptors, that ultimately leads to irreversible vision loss. The Royal College of Surgeon's (RCS) rat carries a recessive mutation affecting mer proto-oncogene tyrosine kinase (merTK), that models autosomal recessive disease. The aim of this study was to understand the glial, microglial, and photoreceptor changes that occur in different retinal locations with advancing disease. Pigmented RCS rats (RCS-p+/LAV) and age-matched isogenic control rdy (RCS-rdy +p+/LAV) rats aged postnatal day 18 to 6 months were evaluated for in vivo retinal structure and function using optical coherence tomography and electroretinography. Retinal tissues were assessed using high resolution immunohistochemistry to evaluate changes in photoreceptors, glia and microglia in the dorsal, and ventral retina. Photoreceptor dysfunction and death occurred from 1 month of age. There was a striking difference in loss of photoreceptors between the dorsal and ventral retina, with a greater number of photoreceptors surviving in the dorsal retina, despite being adjacent a layer of photoreceptor debris within the subretinal space. Loss of photoreceptors in the ventral retina was associated with fragmentation of the outer limiting membrane, extension of glial processes into the subretinal space that was accompanied by possible adhesion and migration of mononuclear phagocytes in the subretinal space. Overall, these findings highlight that breakdown of the outer limiting membrane could play an important role in exacerbating photoreceptor loss in the ventral retina. Our results also highlight the value of using the RCS rat to model sectorial retinitis pigmentosa, a disease known to predominantly effect the inferior retina.

The presence of drusen is an important hallmark of age-related macular degeneration (AMD). Laser-induced regression of drusen, first observed over four decades ago, has led to much interest in the potential role of lasers in slowing the progression of the disease. In this article, we summarise the key insights from pre-clinical studies into the possible mechanisms of action of various laser interventions that result in beneficial changes in the retinal pigment epithelium/Bruch’s membrane/choriocapillaris interface. Key learnings from clinical trials of laser treatment in AMD are also summarised, concentrating on the evolution of laser technology towards short pulse, non-thermal delivery such as the nanosecond laser. The evolution in our understanding of AMD, through advances in multimodal imaging and functional testing, as well as ongoing investigation of key pathological mechanisms, have all helped to set the scene for further well-conducted randomised trials to further explore potential utility of the nanosecond and other subthreshold short pulse lasers in AMD.

Purpose Although rod function is known to be severely impaired in eyes with reticular pseudodrusen (RPD), it remains unknown whether this impairment is associated with a total loss of rod function or merely a delay in rod recovery. The purpose of the study was to determine rod functional recovery profiles after prolonged dark adaptation (DA) in eyes with age-related macular degeneration (AMD) and RPD. Design A cross-sectional, case-series study. Participants Subjects with AMD and RPD. Methods Retinal sensitivity was assessed simultaneously at 14 retinal locations within the central 12° in the study eye of each subject after the eye received approximately 20% bleach. Recovery of retinal sensitivity was monitored at regular intervals up to 30 minutes after bleach. If retinal sensitivity of all test points had not recovered to the rod criterion level (−3.0 log units of stimulus intensity) after 30 minutes of DA, monitoring recovery of retinal sensitivity was extended up to 24 hours of DA. Main Outcome Measures Rod functional recovery profile at each test point. Results Six AMD cases with RPD were included, aged 69 to 79 years, and visual acuity ranged from 20/20 to 20/25. All cases had a delay in rod functional recovery at many retinal locations, with test points within the central 6° most affected. The recovery rate was variable between retinal loci and between subjects, although RPD were present at all test locations. In 5 cases with stage 3 RPD, rod function recovered at all tested locations, but many locations took hours to do so. The case with stage 4 RPD had locations that failed to recover even after 24 hours of DA. Conclusions Eyes with AMD and RPD are associated with severe rod dysfunction throughout the macula; however, rod function does recover in most cases after an extended DA time. These findings suggest that the delay in rod recovery in eyes with RPD is, in most cases, associated with the impairment rather than the total loss of rod photoreceptor function. Stage 4 RPD may represent a point at which some rod photoreceptors are nonfunctional.

Because of complications and side effects, conventional laser therapy has taken a back seat to drugs in the treatment of macular diseases. Despite this, research on new laser modalities remains active. In particular, various approaches are being pursued to preserve and improve retinal structure and function. These include micropulsing, various exposure titration algorithms, and real-time temperature feedback control of short-pulse continuous wave lasers, and ultra-short-pulse nanosecond lasers. Some of these approaches are at the preclinical stage of development, whereas others are available for clinical use. Cell biology is providing important insights into the mechanisms of action of retinal laser treatment. We outline the technological bases of current laser platforms, their basic science, therapeutic concepts, clinical experience, and future directions for retinal laser treatment.

The presence of drusen in the posterior eye is a hallmark feature of the early stages of age-related macular degeneration and their size is an indicator of risk of progression to vision-threatening forms of the disease. Since the initial observations that laser treatment can resolve drusen, there has been great interest in whether laser treatment can be used to reduce the progression of age-related macular degeneration. In this article, we review the development of lasers for the treatment of those with age-related macular degeneration. We provide an overview of the clinical trial results that demonstrated drusen resolution but that had mixed effects on progression of disease. In addition, we provide a summary of the recent developments in pulsed lasers that are designed to reduce the energy applied to the posterior eye to provide the therapeutic effects of conventional continuous wave lasers while reducing the secondary tissue effects.

Age-related macular degeneration (AMD) is a leading cause of blindness, and dry AMD has no effective treatment. Retinal constructs comprising retinal pigment epithelium (RPE) cells supported by electrospun scaffolds have been investigated to treat dry AMD. However, electrospun scaffolds studied to-date do not mimic the structural microenvironment of human Bruch’s membrane (BM), essential for native-like RPE monolayers. The aim of this study was to develop a structurally biomimetic scaffold designed to support a functional RPE monolayer, comprising porous, electrospun nanofibrous membranes (ENMs), coated with laminin, mimicking the inner collagenous layer (ICL) and basal RPE lamina respectively, the cell supporting layers of the BM. In vitro evaluation showed 70 nm PLLA ENMs adsorbed high amounts of laminin and supported functional RPE monolayers, exhibiting 3D polygonal-cobblestone morphology, apical microvilli, basal infoldings, high transepithelial resistance (TER), phagocytic activity and expression of signature RPE markers. 70 nm PLLA ENMs were successfully implanted into the subretinal space of RCS-rdy+p+/LAV rats, also commonly know as rdy rats. At week 4, in the absence of immunosuppressants, implanted PLLA ENMs were surrounded by a significantly low number of activated microglial cells, compared to week 1, indicating no adverse long-term immune response. In conclusion, we successfully designed and tested ENMs emulating the RPE cell supporting layers of the BM, and found 70 nm PLLA ENMs to be best suited as scaffolds for fabricating retinal constructs. Statement of Significance Age related macular degeneration (AMD) is a leading cause of vision loss in the developed world, with an increasing number of people suffering from blindness or severe visual impairment. Transplantation of retinal pigment epithelium (RPE) cells supported on a synthetic, biomimetic-like Bruch’s membrane (BM) is considered a promising treatment. However, the synthetic scaffolds used do not mimic the microenvironment of the RPE cell supporting layers, required for the development of a functional RPE monolayer. This study indicated that porous, laminin coated, 70 nm PLLA ENMs supported functional RPE monolayers, exhibiting 3D polygonal-cobblestone morphology, apical microvilli, basal infoldings, high transepithelial resistance (TER), phagocytic activity and expression of signature RPE markers. These findings indicate the potential clinical use of porous, laminin coated, 70 nm PLLA ENMs in fabricating retinal constructs aimed at treating dry AMD.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in those older than 80 years. Understanding the mechanisms that cause this condition or its progression is critical for developing novel treatments. Here we summarize our studies evaluating the role of purine, adenosine triphosphate (ATP), in early AMD as well as photoreceptor loss and have also provided some insights to our investigations of a new laser treatment for those with early AMD. One of the receptors that are activated by ATP, P2X7, is expressed by neurons and immune cells and has a different function in each cell type. In neurons, P2X7 receptors form a ligand-gated ion channel, whereas on immune cells P2X7 receptors act as a scavenger receptor. These distinct functions have provided new insights to the mechanisms of AMD. On the one hand, high concentrations of ATP can cause photoreceptor death, most likely via stimulation of P2X7 receptors localized on photoreceptor terminals. On the other hand, P2X7 receptors mediate removal of dead and dying cells by monocytes. By understanding the fundamental cell biological changes that occur in patients and animal models of disease, we have uncovered mechanisms that may help us manage and treat patients in the future.

Age-related macular degeneration (AMD) is a leading cause of irreversible, severe vision loss in Western countries. Recently, we identified a novel pathway involving P2X7 receptor scavenger function expressed on ocular immune cells as a risk factor for advanced AMD. In this study, we investigate the effect of loss of P2X7 receptor function on retinal structure and function during aging. P2X7-null and wild-type C57bl6J mice were investigated at 4, 12, and 18 months of age for macrophage phagocytosis activity, ocular histological changes, and retinal function. Phagocytosis activity of blood-borne macrophages decreased with age at 18 months in the wild-type mouse. Lack of P2X7 receptor function reduced phagocytosis at all ages compared to wild-type mice. At 12 months of age, P2X7-null mice had thickening of Bruchs membrane and retinal pigment epithelium dysfunction. By 18 months of age, P2X7-null mice displayed phenotypic characteristics consistent with early AMD, including Bruchs membrane thickening, retinal pigment epithelium cell loss, retinal functional deficits, and signs of subretinal inflammation. Our present study shows that loss of function of the P2X7 receptor in mice induces retinal changes representing characteristics of early AMD, providing a valuable model for investigating the role of scavenger receptor function and the immune system in the development of this age-related disease.

Adenosine triphosphate (ATP) is actively transported into vesicles for purinergic neurotransmission by the vesicular nucleotide transporter, VNUT, encoded by the gene, solute carrier 17, member 9 (SLC17A9). In this chapter, methods are described for fluorescent labeling of VNUT positive cells and quantification of vesicular ATP release using live cell imaging. Directions for preparation of viable dissociated neurons and cellular labeling with an antibody against VNUT and for ATP containing synaptic vesicles with fluorescent ATP markers, quinacrine or MANT-ATP, are detailed. Using confocal microscope live cell imaging, cells positive for VNUT can be observed colocalized with fluorescent ATP vesicular markers, which occur as discrete puncta near the cell membrane. Vesicular release, stimulated with a depolarizing, high potassium physiological saline solution induces ATP marker fluorescence reduction at the cell membrane and this can be quantified over time to assess ATP release. Pretreatment with the voltage gated calcium channel blocker, cadmium, blocks depolarization-induced membrane fluorescence changes, suggesting that VNUT-positive neurons release ATP via calcium-dependent exocytosis. This technique may be applied for quantifying vesicular ATP release across the peripheral and central nervous system and is useful for unveiling the intricacies of purinergic neurotransmission.

Purpose To test the hypothesis that the capacity for retinal ganglion cells to functionally recover from chronic IOP elevation is dependent on the duration of IOP elevation. Methods IOP elevation was induced in one eye in anesthetized (isoflurane) adult C57BL6/J mice using a circumlimbal suture. Sutures were left in place for 8 and 16 weeks (n = 30 and 28). In two other groups the suture was cut after 8 and 12 weeks (n = 30 and 28), and ganglion cell function (electroretinography) and retinal structure (optical coherence tomography) were assessed 4 weeks later. Ganglion cell density was quantified by counting RBPMS (RNA-binding protein with multiple splicing)-stained cells. Results With IOP elevation (∼10 mm Hg above baseline), ganglion cell function declined to 75% ± 8% at 8 weeks and 59% ± 4% at 16 weeks relative to contralateral control eyes. The retinal nerve fiber layer was thinner at 8 (84% ± 4%) and 16 weeks (83% ± 3%), without a significant difference in total retinal thickness. Ganglion cell function recovered with IOP normalization (suture removal) at week 8 (97% ± 7%), but not at week 12 (73% ± 6%). Ganglion cell loss was found in all groups (-8% to -13%). Conclusions In the mouse circumlimbal suture model, 12 weeks of IOP elevation resulted in irreversible ganglion cell dysfunction, whereas retinal dysfunction was fully reversible after 8 weeks of IOP elevation.

The retina is known to have a local renin-angiotensin system (RAS) and dysfunction in the RAS is often associated with diseases of the retinal vasculature that cause irreversible vision loss. Regulation of the retinal vasculature to meet the metabolic needs of the tissues occurs through a mechanism called neurovascular coupling, which is critical for maintaining homeostatic function and support for neurons. Neurovascular coupling is the process by which support cells, including glia, regulate blood vessel calibre and blood flow in response to neural activity. In retinal vascular diseases, this coupling mechanism is often disrupted. However, the role that angiotensin II (Ang II), the main effector peptide of the RAS, has in regulating both the retinal vasculature and neurovascular coupling is not fully understood. As components of the RAS are located on the principal neurons, glia and blood vessels of the retina, it is possible that Ang II has a role in regulating communication and function between these three cell types, and therefore the capacity to regulate neurovascular coupling. This review focuses on components of the RAS located on the retinal neurovascular unit, and the potential of this system to contribute to blood flow modulation in the healthy and compromised retina.

Purines, when present in the extracellular space, can mediate fast neurotransmission in the retina and central nervous system. Over the last decade there has been emerging evidence for the expression of P2X and P2Y receptors in a range of retinal neuronal subtypes. These results have highlighted important roles for purines in modulating specific retinal circuits, including the rod pathway and amacrine cell circuits. Traditionally, synaptic release of adenosine triphosphate (ATP) involves the novel anion vesicular nucleotide transporter, VNUT, which has recently been identified in a single wide-field amacrine cell population. In addition, nontraditional, conductive mechanisms of release have also been described in the retina. In the synapse, the enzymes involved in rapid degradation of purines are present in both plexiform layers of the retina. A role for P2X receptors in retinal diseases has also emerged recently. High concentrations of ATP lead to photoreceptor loss, through mechanisms involving P2X7 receptors. In addition, activation of P2X7 receptors is associated with activation of the inflammasome, a protein complex important for the release of proinflammatory cytokines. P2X receptors, especially P2X7, are emerging as targets to combat retinal disease.

Inherited retinal degeneration such as retinitis pigmentosa (RP) is associated with photoreceptor loss and concomitant morphological and functional changes in the inner retina. It is not known whether these changes are associated with changes in the density and distribution of synaptic inputs to retinal ganglion cells (RGCs). We quantified changes in ganglion cell density in rd1 and age-matched C57BL/6J-(wildtype, WT) mice using the immunocytochemical marker, RBPMS. Our data revealed that following complete loss of photoreceptors, (∼3months of age), there was a reduction in ganglion cell density in the peripheral retina. We next examined changes in synaptic inputs to A type ganglion cells by performing double labeling experiments in mice with the ganglion cell reporter lines, rd1-Thy1 and age-matched wildtype-Thy1. Ribbon synapses were identified by co-labelling with CtBP2 (RIBEYE) and conventional synapses with the clustering molecule, gephyrin. ON RGCs showed a significant reduction in RIBEYE-immunoreactive synapse density while OFF RGCs showed a significant reduction in the gephyrin-immmunoreactive synapse density. Distribution patterns of both synaptic markers across the dendritic trees of RGCs were unchanged. The change in synaptic inputs to RGCs was associated with a reduction in the number of immunolabeled rod bipolar and ON cone bipolar cells. These results suggest that functional changes reported in ganglion cells during retinal degeneration could be attributed to loss of synaptic inputs.

The biologically important metals such as zinc, copper and iron play key roles in retinal function, yet no study has mapped the spatio-temporal distribution of retinal biometals in healthy or diseased retina. We investigated a natural mouse model of retinal degeneration, the Cln6nclf mouse. As dysfunctional metabolism of biometals is observed in the brains of these animals and deregulated metal homeostasis has been linked to retinal degeneration, we focused on mapping the elemental distribution in the healthy and Cln6nclf mouse retina with age. Retinal and RPE elemental homeostasis was mapped in Cln6nclf and C57BL6/J mice from 1 to 8 months of age using X-ray Fluorescence Microscopy at the Australian Synchrotron. In the healthy retina, we detected a progressive loss of phosphorus in the outer nuclear layer and significant reduction in iron in the inner segments of the photoreceptors. Further investigation revealed a unique elemental signature for each retinal layer, with high areal concentrations of iron and sulfur in the photoreceptor segments and calcium, phosphorus, zinc and potassium enrichment predominantly in the nuclear layers. The analysis of retinae from Cln6nclf mice did not show significant temporal changes in elemental distributions compared to age matched controls, despite significant photoreceptor cell loss. Our data therefore demonstrates that retinal layers have unique elemental composition. Elemental distribution is, with few exceptions, stably maintained over time in healthy and Cln6nclf mouse retina, suggesting conservation of elemental distribution is critical for basic retinal function with age and is not modulated by processes underlying retinal degeneration.

Age-related macular degeneration and glaucoma are the commonest causes of irreversible vision loss in industrialized countries. The purine ATP is known to regulate a range of cellular functions in the retina via its action on P2 receptors, especially the P2X7 receptor. Although agents that attenuate P2X7 receptor function have been in development for many years, no compound is currently approved for the treatment of eye disease. However, newer compounds that cross the blood-brain barrier could have potential to reduce vision loss. This review will outline recent information relating to the role of P2X7 in age-related macular degeneration and glaucoma and, subsequently, we will discuss recent developments for attenuating P2X7 receptor function.

Purpose Vision loss caused by photoreceptor death represents one of the first symptoms in neuronal ceroid lipofuscinosis, a condition characterized by accumulation of intracellular waste. Cln6nclf mice have a naturally occurring mutation in ceroid-lipofuscinosis neuronal (CLN) protein 6 and are a model of this disorder. In order to identify the effect intracellular waste (lipofuscin) accumulation plays in driving retinal degeneration, the time course of degeneration was carefully characterized functionally using the electroretinogram and structurally using histology. Methods Cln6nclf and C57BL/6J, wild-type, mice were studied at postnatal day 18 (P18), P30, P60, P120, and P240, and retinal degeneration was correlated with changes in the retinal pigment epithelial (RPE) and neuronal autophagy-lysosomal pathways using super-resolution microscopy. Results In Cln6nclf mice there was significant loss of rod photoreceptor function at P18, prior to photoreceptor nuclei loss at P60. In contrast, cone pathway function was not affected until P240. The loss of rod photoreceptor function correlated with significant disruption of the autophagy-lysosomal degradation pathways within photoreceptors, but not in the RPE or other retinal neurons. Additionally, there was cytosolic accumulation of P62 and undigested mitochondrial-derived, ATP synthase subunit C in the photoreceptor layers of Cln6nclf mice at P30. Conclusions These results suggest that rod photoreceptors have an increased sensitivity to disturbances in the autophagy-lysosomal pathway and the subsequent failure of mitochondrial turnover, relative to other retinal cells. It is likely that primary failure of the rod photoreceptors rather than the RPE or other retinal neurons underlies the early visual dysfunction that occurs in the Cln6nclf mouse model.

Retinal dopamine is released by a specialized subset of amacrine cells in response to light and has a potent influence on how the retina responds to, and encodes, visual information. Here, we address the critical question of which retinal photoreceptor is responsible for coordinating the release of this neuromodulator. Although all three photoreceptor classes-rods, cones, and melanopsin-containing retinal ganglion cells (mRGCs)-have been shown to provide electrophysiological inputs to dopaminergic amacrine cells (DACs), we show here that the release of dopamine is defined only by rod photoreceptors. Remarkably, this rod signal coordinates both a suppressive signal at low intensities and drives dopamine release at very bright light intensities. These data further reveal that dopamine release does not necessarily correlate with electrophysiological activity of DACs and add to a growing body of evidence that rods define aspects of retinal function at very bright light levels.

There is an urgent need for a more effective intervention to slow or prevent progression of age-related macular degeneration (AMD) from its early stages to vision-threatening late complications. Subthreshold nanosecond laser (SNL) treatment has shown promise in preclinical studies and a pilot study in intermediate AMD (iAMD) as a potential treatment. We aimed to evaluate the safety of SNL treatment in iAMD and its efficacy for slowing progression to late AMD.The Laser Intervention in Early Stages of Age-Related Macular Degeneration (LEAD) study is a 36-month, multicenter, randomized, sham-controlled trial.Two hundred ninety-two participants with bilateral large drusen and without OCT signs of atrophy.Participants were assigned randomly to receive Retinal Rejuvenation Therapy (2RTThe primary efficacy outcome was the time to development of late AMD defined by multimodal imaging (MMI). Safety was assessed by adverse events.Overall, progression to late AMD was not slowed significantly with SNL treatment compared with sham treatment (adjusted hazard ratio [HR], 0.61; 95% confidence interval [CI], 0.33-1.14; P = 0.122). However, a post hoc analysis showed evidence of effect modification based on the coexistence of reticular pseudodrusen (RPD; adjusted interaction P = 0.002), where progression was slowed for the 222 participants (76.0%) without coexistent RPD at baseline (adjusted HR, 0.23; 95% CI, 0.09-0.59; P = 0.002), whereas an increased progression rate (adjusted HR, 2.56; 95% CI, 0.80-8.18; P = 0.112) was observed for the 70 participants (24.0%) with RPD with SNL treatment. Differences between the groups in serious adverse events were not significant.In participants with iAMD without MMI-detected signs of late AMD, no significant difference in the overall progression rate to late AMD between those receiving SNL and sham treatment were observed. However, SNL treatment may have a role in slowing progression for those without coexistent RPD and may be inappropriate in those with RPD, warranting caution when considering treatment in clinical phenotypes with RPD. Our findings provide compelling evidence for further trials of the 2RT

Analysis of how retinal ganglion cells change in retinal degeneration is critical for evaluating the potential of photoreceptor restorative therapies. Immunocytochemistry in combination with image analysis provides a way for quantifying not only the density of ganglion cells during disease, but also information about their morphology and an evaluation of excitatory and inhibitory synaptic inputs. Here, we describe how indirect immunofluorescence can be used in retinal whole mounts to obtain information about ganglion cells in retinal degeneration.

Purpose Subthreshold, nanosecond pulsed laser treatment shows promise as a treatment for age-related macular degeneration (AMD); however, the safety profile needs to be robustly examined. The aim of this study was to investigate the effects of laser treatment in humans and mice. Methods Patients with AMD were treated with nanosecond pulsed laser at subthreshold (no visible retinal effect) energy doses (0.15-0.45 mJ) and retinal sensitivity was assessed with microperimetry. Adult C57BL6J mice were treated at subthreshold (0.065 mJ) and suprathreshold (photoreceptor loss, 0.5 mJ) energy settings. The retinal and vascular responses were analyzed by fundus imaging, histologic assessment, and quantitative PCR. Results Microperimetry analysis showed laser treatment had no effect on retinal sensitivity under treated areas in patients 6 months to 7 years after treatment. In mice, subthreshold laser treatment induced RPE loss at 5 hours, and by 7 days the RPE had retiled. Fundus imaging showed reduced RPE pigmentation but no change in retinal thickness up to 3 months. Electron microscopy revealed changes in melanosomes in the RPE, but Bruch's membrane was intact across the laser regions. Histologic analysis showed normal vasculature and no neovascularization. Suprathreshold laser treatment did not induce changes in angiogenic genes associated with neovascularization. Instead pigment epithelium-derived factor, an antiangiogenic factor, was upregulated. Conclusions In humans, low-energy, nanosecond pulsed laser treatment is not damaging to local retinal sensitivity. In mice, treatment does not damage Bruch's membrane or induce neovascularization, highlighting a reduced side effect profile of this nanosecond laser when used in a subthreshold manner.

Analysis of how retinal ganglion cells change in retinal degeneration is critical for evaluating the potential of photoreceptor restorative therapies. Immunocytochemistry in combination with image analysis provides a way for quantifying not only the density of ganglion cells during disease, but also information about their morphology and an evaluation of excitatory and inhibitory synaptic inputs. Here, we describe how indirect immunofluorescence can be used in retinal whole mounts to obtain information about ganglion cells in retinal degeneration.

Retinal dopamine is released by a specialised subset of amacrine cells in response to light and has a potent influence on how the retina responds to, and encodes, visual information. Here we address the critical question of which retinal photoreceptor is responsible for coordinating the release of this neuromodulator. While all three photoreceptor classes: rods, cones and melanopsin-containing retinal ganglion cells (mRGCs) have been shown to provide electrophysiological inputs to dopaminergic amacrine cells (DACs), we show here that the release of dopamine is defined only by rod photoreceptors. Remarkably, this rod signal coordinates both a suppressive signal at low intensities, and drives dopamine release at very bright light intensities. These data further reveal that dopamine release does not necessarily correlate with electrophysiological activity of dopaminergic amacrine cells and add to a growing body of evidence that rods define aspects of retinal function at very bright light levels.

To consider whether a circumlimbal suture can be used to chronically elevate intraocular pressure (IOP) in mice and to assess its effect on retinal structure, function and gene expression of stretch sensitive channels. Anesthetized adult C57BL6/J mice had a circumlimbal suture (10/0) applied around the equator of one eye. In treated eyes (n = 23) the suture was left in place for 12 weeks whilst in sham control eyes the suture was removed at day two (n = 17). Contralateral eyes served as untreated controls. IOP was measured after surgery and once a week thereafter. After 12 weeks, electroretinography (ERG) was performed to assess photoreceptor, bipolar cell and retinal ganglion cell (RGC) function. Retinal structure was evaluated using optical coherence tomography. Retinae were processed for counts of ganglion cell density or for quantitative RT-PCR to quantify purinergic (P2x7, Adora3, Entpd1) or stretch sensitive channel (Panx1, Trpv4) gene expression. Immediately after suture application, IOP spiked to 33 ± 3 mmHg. After 1 day, IOP had recovered to 27 ± 3 mmHg. Between weeks 2 and 12, IOP remained elevated above baseline (control 14 ± 1 mmHg, ocular hypertensive 19 ± 1 mmHg). Suture removal at day 2 (Sham) restored IOP to baseline levels, where it remained through to week 12. ERG analysis showed that 12 weeks of IOP elevation reduced photoreceptor (−15 ± 4%), bipolar cell (−15 ± 4%) and ganglion cell responses (−19 ± 6%) compared to sham controls and respective contralateral eyes (untreated). The retinal nerve fiber layer was thinned in the presence of normal total retinal thickness. Ganglion cell density was reduced across all quadrants (superior −12 ± 5%; temporal, −7% ± 2%; inferior −9 ± 4%; nasal −8 ± 5%). Quantitative RT-PCR revealed a significant increase in Entpd1 gene expression (+11 ± 4%), whilst other genes were not significantly altered (P2x7, Adora3, Trpv4, Panx1). Our results show that circumlimbal ligation produces mild chronic ocular hypertension and retinal dysfunction in mice. Consistent with a sustained change to purinergic signaling we found an up-regulation of Entpd1.

Age-related macular degeneration (AMD) is a leading cause of irreversible, severe vision loss in Western countries. Recently, we identified a novel pathway involving P2X7 receptor scavenger function expressed on ocular immune cells as a risk factor for advanced AMD. In this study, we investigate the effect of loss of P2X7 receptor function on retinal structure and function during aging. P2X7-null and wild-type C57bl6J mice were investigated at 4, 12, and 18 months of age for macrophage phagocytosis activity, ocular histological changes, and retinal function. Phagocytosis activity of blood-borne macrophages decreased with age at 18 months in the wild-type mouse. Lack of P2X7 receptor function reduced phagocytosis at all ages compared to wild-type mice. At 12 months of age, P2X7-null mice had thickening of Bruchs membrane and retinal pigment epithelium dysfunction. By 18 months of age, P2X7-null mice displayed phenotypic characteristics consistent with early AMD, including Bruchs membrane thickening, retinal pigment epithelium cell loss, retinal functional deficits, and signs of subretinal inflammation. Our present study shows that loss of function of the P2X7 receptor in mice induces retinal changes representing characteristics of early AMD, providing a valuable model for investigating the role of scavenger receptor function and the immune system in the development of this age-related disease.