Comprehensive Characterization of a Subfamily of Ca2+-Binding Proteins in Mouse and Human Retinal Neurons at Single-Cell Resolution.

Ca2+-binding proteins (CaBPs; CaBP1-5) are a subfamily of neuronal Ca2+ sensors with high homology to calmodulin. Notably, CaBP4, which is exclusively expressed in rod and cone photoreceptors, is crucial for maintaining normal retinal functions. However, the functional roles of CaBP1, CaBP2, and CaBP5 in the retina remain elusive, primarily due to limited understanding of their expression patterns within inner retinal neurons. In this study, we conducted a comprehensive transcript analysis using single-cell RNA sequencing datasets to investigate the gene expression profiles of CaBPs in mouse and human retinal neurons. Our findings revealed notable similarities in the overall expression patterns of CaBPs across both species. Specifically, nearly all amacrine cell, ganglion cell, and horizontal cell types exclusively expressed CaBP1. In contrast, the majority of bipolar cell types, including rod bipolar (RB) cells, expressed distinct combinations of CaBP1, CaBP2, and CaBP5, rather than a single CaBP as previously hypothesized. Remarkably, mouse rods and human cones exclusively expressed CaBP4, whereas mouse cones and human rods coexpressed both CaBP4 and CaBP5. Our single-cell reverse transcription polymerase chain reaction analysis confirmed the coexpression CaBP1 and CaBP5 in individual RBs from mice of either sex. Additionally, all three splice variants of CaBP1, primarily L-CaBP1, were detected in mouse RBs. Taken together, our study offers a comprehensive overview of the distribution of CaBPs in mouse and human retinal neurons, providing valuable insights into their roles in visual functions.

Analysis of Microtubule Polymerization During Axon Outgrowth Using Fluorescently Labeled Microtubule Plus-End Tracking Proteins.

The study of microtubules arrangements and dynamics during axon outgrowth and pathfinding has gained scientific interest during the last decade, and numerous technical resources for its visualization and analysis have been implemented. In this chapter, we describe the cell culture protocols of embryonic cortical and retinal neurons, the methods for transfecting them with fluorescent reporters of microtubule polymerization, and the procedures for time-lapse imaging and quantification in order to study microtubule dynamics during axon morphogenesis.

Artificial Retina Based on Organic Heterojunction Transistors for Mobile Recognition.

The flicker frequency of incident light constitutes a critical determinant in biology. Nevertheless, the exploration of methods to simulate external light stimuli with varying frequencies and develop artificial retinal neurons capable of responsive behavior remains an open question. This study presents an artificial neuron comprising organic phototransistors. The triggering properties of neurons are modulated by optical input, enabling them to execute rudimentary synaptic functions, emulating the biological characteristics of retinal neurons. The artificial retinal neuron exhibits varying responses to incoming light frequencies, allowing it to replicate the persistent visual behavior of the human eye and facilitating image discrimination. Additionally, through seamless integration with circuitry, it can execute motion recognition on a machine cart, preventing collisions with high-speed obstacles. The artificial retinal neuron offers a cost-effective and energy-efficient route for future mobile robot processors.

Genetic and epigenetic regulators of retinal Müller glial cell reprogramming.

Background: Retinal diseases characterized with irreversible loss of retinal nerve cells, such as optic atrophy and retinal degeneration, are the main causes of blindness. Current treatments for these diseases are very limited. An emerging treatment strategy is to induce the reprogramming of Müller glial cells to generate new retinal nerve cells, which could potentially restore vision. Main text: Müller glial cells are the predominant glial cells in retinae and play multiple roles to maintain retinal homeostasis. In lower vertebrates, such as in zebrafish, Müller glial cells can undergo cell reprogramming to regenerate new retinal neurons in response to various damage factors, while in mammals, this ability is limited. Interestingly, with proper treatments, Müller glial cells can display the potential for regeneration of retinal neurons in mammalian retinae. Recent studies have revealed that dozens of genetic and epigenetic regulators play a vital role in inducing the reprogramming of Müller glial cells in vivo. This review summarizes these critical regulators for Müller glial cell reprogramming and highlights their differences between zebrafish and mammals. Conclusions: A number of factors have been identified as the important regulators in Müller glial cell reprogramming. The early response of Müller glial cells upon acute retinal injury, such as the regulation in the exit from quiescent state, the initiation of reactive gliosis, and the re-entry of cell cycle of Müller glial cells, displays significant difference between mouse and zebrafish, which may be mediated by the diverse regulation of Notch and TGFß (transforming growth factor-ß) isoforms and different chromatin accessibility.

PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons.

PANoptosis is a newly identified type of regulated cell death that consists of pyroptosis, apoptosis, and necroptosis, which simultaneously occur during the pathophysiological process of infectious and inflammatory diseases. Although our previous literature mining study suggested that PANoptosis might occur in neuronal ischemia/reperfusion injury, little experimental research has been reported on the existence of PANoptosis. In this study, we used in vivo and in vitro retinal neuronal models of ischemia/reperfusion injury to investigate whether PANoptosis-like cell death (simultaneous occurrence of pyroptosis, apoptosis, and necroptosis) exists in retinal neuronal ischemia/reperfusion injury. Our results showed that ischemia/reperfusion injury induced changes in morphological features and protein levels that indicate PANoptosis-like cell death in retinal neurons both in vitro and in vivo. Ischemia/reperfusion injury also significantly upregulated caspase-1, caspase-8, and NLRP3 expression, which are important components of the PANoptosome. These results indicate the existence of PANoptosis-like cell death in ischemia/reperfusion injury of retinal neurons and provide preliminary experimental evidence for future study of this new type of regulated cell death.

Neural and Müller glial adaptation of the retina to photoreceptor degeneration.

The majority of inherited retinal degenerative diseases and dry age-related macular degeneration are characterized by decay of the outer retina and photoreceptors, which leads to progressive loss of vision. The inner retina, including second- and third-order retinal neurons, also shows aberrant structural changes at all stages of degeneration. Müller glia, the major glial cells maintain retinal homeostasis, activating and rearranging immediately in response to photoreceptor stress. These phenomena are collectively known as retinal remodeling and are anatomically well described, but their impact on visual function is less well characterized. Retinal remodeling has traditionally been considered a detrimental chain of events that decreases visual function. However, emerging evidence from functional assays suggests that remodeling could also be a part of a survival mechanism wherein the inner retina responds plastically to outer retinal degeneration. The visual system´s first synapses between the photoreceptors and bipolar cells undergo rewiring and functionally compensate to maintain normal signal output to the brain. Distinct classes of retinal ganglion cells remain even after the massive loss of photoreceptors. Müller glia possess the regenerative potential for retinal recovery and possibly exert adaptive transcriptional changes in response to neuronal loss. These types of homeostatic changes could potentially explain the well-maintained visual function observed in patients with inherited retinal degenerative diseases who display prominent anatomic retinal pathology. This review will focus on our current understanding of retinal neuronal and Müller glial adaptation for the potential preservation of retinal activity during photoreceptor degeneration. Targeting retinal self-compensatory responses could help generate universal strategies to delay sensory disease progression.

Omega-3/Omega-6 Long-Chain Fatty Acid Imbalance in Phase I Retinopathy of Prematurity.

There is a gap in understanding the effect of the essential ω-3 and ω-6 long-chain polyunsaturated fatty acids (LCPUFA) on Phase I retinopathy of prematurity (ROP), which precipitates proliferative ROP. Postnatal hyperglycemia contributes to Phase I ROP by delaying retinal vascularization. In mouse neonates with hyperglycemia-associated Phase I retinopathy, dietary ω-3 (vs. ω-6 LCPUFA) supplementation promoted retinal vessel development. However, ω-6 (vs. ω-3 LCPUFA) was also developmentally essential, promoting neuronal growth and metabolism as suggested by a strong metabolic shift in almost all types of retinal neuronal and glial cells identified with single-cell transcriptomics. Loss of adiponectin (APN) in mice (mimicking the low APN levels in Phase I ROP) decreased LCPUFA levels (including ω-3 and ω-6) in retinas under normoglycemic and hyperglycemic conditions. ω-3 (vs. ω-6) LCPUFA activated the APN pathway by increasing the circulating APN levels and inducing expression of the retinal APN receptor. Our findings suggested that both ω-3 and ω-6 LCPUFA are crucial in protecting against retinal neurovascular dysfunction in a Phase I ROP model; adequate ω-6 LCPUFA levels must be maintained in addition to ω-3 supplementation to prevent retinopathy. Activation of the APN pathway may further enhance the ω-3 and ω-6 LCPUFA's protection against ROP.

Photoelectric Dye, NK-5962, as a Potential Drug for Preventing Retinal Neurons from Apoptosis: Pharmacokinetic Studies Based on Review of the Evidence.

NK-5962 is a key component of photoelectric dye-based retinal prosthesis (OUReP). In testing the safety and efficacy, NK-5962 was safe in all tests for the biological evaluation of medical devices (ISO 10993) and effective in preventing retinal cells from death even under dark conditions. The long-term implantation of the photoelectric dye-coupled polyethylene film in the subretinal space of hereditary retinal dystrophic (RCS) rats prevented neurons from apoptosis in the adjacent retinal tissue. The intravitreous injection of NK-5962 in the eyes of RCS rats, indeed, reduced the number of apoptotic cells in the retinal outer nuclear layer irrespective of light or dark conditions. In this study, we reviewed the in vitro and in vivo evidence of neuroprotective effect of NK-5962 and designed pharmacokinetic experiments. The in vitro IC50 of 1.7 µM, based on the protective effect on retinal cells in culture, could explain the in vivo EC50 of 3 µM that is calculated from concentrations of intravitreous injection to prevent retinal neurons from apoptosis. Pharmacokinetics of NK-5962 showed that intravenous administration, but not oral administration, led to the effective concentration in the eye of rats. NK-5962 would be a candidate drug for delaying the deterioration of retinal dystrophy, such as retinitis pigmentosa.

Nuclear PKR in retinal neurons in the early stage of diabetic retinopathy in streptozotocin­induced diabetic rats.

Retinal neuron apoptosis is a key component of diabetic retinopathy (DR), one of the most common complications of diabetes. Stress due to persistent hyperglycaemia and corresponding glucotoxicity represents one of the primary pathogenic mechanisms of diabetes and its complications. Apoptosis of retinal neurons serves a critical role in the pathogenesis of DR observed in patients with diabetes and streptozotocin (STZ)­induced diabetic rats. Retinal neuron apoptosis occurs one month after STZ injection, which is considered the early stage of DR. The molecular mechanism involved in the suppression of retinal neuron apoptosis during the early stage of DR remains unclear. RNA­dependent protein kinase (PKR) is a stress­sensitive pro­apoptotic kinase. Our previous study indicated that PKR­associated protein X, a stress­sensitive activator of PKR, is upregulated in the early stage of STZ­induced diabetes. In order to assess the role of PKR in DR prior to apoptosis of retinal neurons, immunofluorescence and western blotting were performed to investigate the cellular localization and expression of PKR in the retina in the early stage of STZ­induced diabetes in rats. PKR activity was indirectly assessed by expression levels of phosphorylated eukaryotic translation initiation factor 2α (p­eIF2­α) and the presence of apoptotic cells in the retina was investigated by TUNEL assay. The findings revealed that PKR was localized in the nucleus of retinal ganglion and inner nuclear layer cells from normal and diabetic rats. To the best of our knowledge, the present study is the first to demonstrate nuclear localization of PKR in retinal neurons. Immunofluorescence analysis demonstrated that PKR was expressed in the nuclei of retinal neurons at 3 and 6 days and its expression was decreased at 15 days after STZ treatment. In addition, p­eIF2­α expression and cellular localization followed the trend of PKR, suggesting that this pro­apoptotic kinase was active in the nuclei of retinal neurons. These findings are consistent with the hypothesis that nuclear translocation of PKR may be a mechanism to sequester active PKR, thus preventing upregulation of cytosolic signalling pathways that induce apoptosis in retinal neurons. Apoptotic cells were not detected in the retina in the early stage of DR. A model was proposed to explain the mechanism by which apoptosis of retinal neurons by PKR is suppressed in the early stage of DR. The possible role of mitochondrial RNA (mtRNA) and Alu RNA in this phenomenon is also discussed since it was demonstrated that the cellular stress due to prolonged hyperglycaemia induces the release of mtRNA and transcription of Alu RNA. Moreover, it mtRNA activates PKR, whereas Alu RNA inhibits the activation of this protein kinase.

Bioinspired Photoresponsive Single Transistor Neuron for a Neuromorphic Visual System.

Realizing a neuromorphic-based artificial visual system with low-cost hardware requires a neuromorphic device that can react to light stimuli. This study introduces a photoresponsive neuron device composed of a single transistor, developed by engineering an artificial neuron that responds to light, just like retinal neurons. Neuron firing is activated primarily by electrical stimuli such as current via a well-known single transistor latch phenomenon. Its firing characteristics, represented by spiking frequency and amplitude, are additionally modulated by optical stimuli such as photons. When light is illuminated onto the neuron transistor, electron-hole pairs are generated, and they allow the neuron transistor to fire at lower firing threshold voltage. Different photoresponsive properties can be modulated by the intensity and wavelength of the light, analogous to the behavior of retinal neurons. The artificial visual system can be miniaturized because a photoresponsive neuronal function is realized without bulky components such as image sensors and extra circuits.

Peripheral blood-derived monocytes show neuronal properties and integration in immune-deficient rd1 mouse model upon phenotypic differentiation and induction with retinal growth factors.

BACKGROUND: Cell therapy is one of the most promising therapeutic interventions for retinitis pigmentosa. In the current study, we aimed to assess if peripheral blood-derived monocytes which are highly abundant and accessible could be utilized as a potential candidate for phenotypic differentiation into neuron-like cells. METHODS: The peripheral blood-derived monocytes were reconditioned phenotypically using extrinsic growth factors to induce pluripotency and proliferation. The reconditioned monocytes (RM) were further incubated with a cocktail of growth factors involved in retinal development and growth to induce retinal neuron-like properties. These cells, termed as retinal neuron-like cells (RNLCs) were characterized for their morphological, molecular and functional behaviour in vitro and in vivo. RESULTS: The monocytes de-differentiated in vitro and acquired pluripotency with the expression of prominent stem cell markers. Treatment of RM with retinal growth factors led to an upregulation of neuronal and retinal lineage markers and downregulation of myeloid markers. These cells show morphological alterations resembling retinal neuron-like cells and expressed photoreceptor (PR) markers. The induced RNLCs also exhibited relative membrane potential change upon light exposure suggesting that they have gained some neuronal characteristics. Further studies showed that RNLCs could also integrate in an immune-deficient retinitis pigmentosa mouse model NOD.SCID-rd1 upon sub-retinal transplantation. The RNLCs engrafted in the inner nuclear layer (INL) and ganglion cell layer (GCL) of the RP afflicted retina. Mice transplanted with RNLCs showed improvement in depth perception, exploratory behaviour and the optokinetic response. CONCLUSIONS: This proof-of-concept study demonstrates that reconditioned monocytes can be induced to acquire retinal neuron-like properties through differentiation using a defined growth media and can be a potential candidate for cell therapy-based interventions and disease modelling for ocular diseases.

Interaction between SDF1 and CXCR4 Promotes Photoreceptor Differentiation via Upregulation of NFκB Pathway Signaling Activity in Pax6 Gene-Transfected Photoreceptor Precursors.

BACKGROUND: CCL2 (also known as monocyte chemoattractant protein 1) and CX3CR1 (also known as Fractalkine receptor)-deficient mice have damaged photoreceptors. OBJECTIVES: We examined the interaction of SDF1 and CXCR4 on the differentiation of retinal progenitors into rhodopsin-positive photoreceptors. METHODS: Cloned retinal progenitors were obtained by Pax6 gene transfection of mouse iPS cells followed by serial dilution. Clones were selected by expression of nestin, Musashi1, Six3, and Chx10 mRNA. Cell surface protein expression was analyzed by flow cytometry. The levels of mRNA and intracellular protein were examined by real-time PCR and immunochemistry, respectively. Transient transfection experiments of retinal progenitors were conducted using a human rhodopsin promoter luciferase plasmid. RESULTS: We selected 10 clones that expressed Six3, Chx10, Crx, Rx1, Nrl, CD73, and rhodopsin mRNA, which, except for rhodopsin, are photoreceptor precursor markers. Clones expressed both CD73 and CXCR4 on the cell surface and differentiated into rhodopsin-positive photoreceptors, which was reinforced by the addition of exogenous SDF1. A CXCR4 inhibitor AMD3100 blocked SDF1-mediated differentiation of progenitors into photoreceptors. SDF1 enhanced human rhodopsin promoter transcription activity, possibly via the NFκB pathway. Addition of SDF1 to the cell culture induced nuclear translocation of NFκB on retinal progenitor cell clones. Neonatal and newborn mouse retinas expressed SDF1 and CXCR4. Cells in the outer nuclear layer where photoreceptors are located expressed CXCR4 at P14 and P56. Cells in the inner nuclear layer expressed SDF1. CONCLUSIONS: These findings suggest that retinal progenitor cell differentiation was at least partly regulated by SDF1 and CXCR4 via upregulation of NFκB activity.

Cellular localization of melatonin receptor Mel1b in pigeon retina.

Melatonin, an important neuromodulator involved in circadian rhythms, modulates a series of physiological processes via activating its specific receptors, namely Mel1a (MT1), Mel1b (MT2) and Mel1c receptors. In this work, the localization of Mel1b receptor was studied in pigeon retina using double immunohistochemistry staining and confocal scanning microscopy. Our results showed that Mel1b receptor widely existed in the outer segment of photoreceptors and in the somata of dopaminergic amacrine cells, cholinergic amacrine cells, glycinergic AII amacrine cells, conventional ganglion cells and intrinsically photosensitive retinal ganglion cells, while horizontal cells, bipolar cells and Müller glial cells seemed to lack immunoreactivity of Mel1b receptor. That multiple types of retinal cells expressing Mel1b receptor suggests melatonin may directly modulate the activities of retina via activating Mel1b receptor.

Retinal Neuron Is More Sensitive to Blue Light-Induced Damage than Glia Cell Due to DNA Double-Strand Breaks.

Blue light is a major component of visible light and digital displays. Over-exposure to blue light could cause retinal damage. However, the mechanism of its damage is not well defined. Here, we demonstrate that blue light (900 lux) impairs cell viability and induces cell apoptosis in retinal neurocytes in vitro. A DNA electrophoresis assay shows severe DNA damage in retinal neurocytes at 2 h after blue light treatment. γ-H2AX foci, a specific marker of DNA double-strand breaks (DSBs), is mainly located in the Map2-posotive neuron other than the glia cell. After assaying the expression level of proteins related to DNA repair, Mre11, Ligase IV and Ku80, we find that Ku80 is up-regulated in retinal neurocytes after blue light treatment. Interestingly, Ku80 is mainly expressed in glia fibrillary acidic protein (GFAP)-positive glia cells. Moreover, following blue light exposure in vivo, DNA DSBs are shown in the ganglion cell layer and only observed in Map2-positive cells. Furthermore, long-term blue light exposure significantly thinned the retina in vivo. Our findings demonstrate that blue light induces DNA DSBs in retinal neurons, and the damage is more pronounced compared to glia cells. Thus, this study provides new insights into the mechanisms of the effect of blue light on the retina.

Expression of Sirtuins in the Retinal Neurons of Mice, Rats, and Humans.

Sirtuins are a class of histone deacetylases (HDACs) that have been shown to regulate a range of pathophysiological processes such as cellular aging, inflammation, metabolism, and cell proliferation. There are seven mammalian Sirtuins (SIRT1-7) that play important roles in stress response, aging, and neurodegenerative diseases. However, the location and function of Sirtuins in neurons are not well defined. This study assessed the retinal expression of Sirtuins in mice, rats, and humans and measured the expression of Sirtuins in aged and injured retinas. Expression of all 7 Sirtuins was confirmed by Western blot and Real-Time PCR analysis in all three species. SIRT1 is highly expressed in mouse, rat, and human retinas, whereas SIRT2-7 expression was relatively lower in human retinas. Immunofluorescence was also used to examine the expression and localization of Sirtuins in rat retinal neurons. Importantly, we demonstrate a marked reduction of SIRT1 expression in aged retinal neurons as well as retinas injured by acute ischemia-reperfusion. On the other hand, none of the other Sirtuins exhibit any significant age-related changes in expression except for SIRT5, which was significantly higher in the retinas of adults compared to both young and aged rats. Our work presents the first composite analysis of Sirtuins in the retinal neurons of mice, rats, and humans, and suggests that increasing the expression and activity of SIRT1 may be beneficial for the treatment of glaucoma and other age-related eye dysfunction.

Structural and functional changes in retinal vasculature induced by retinal ischemia-reperfusion in rats.

Recent studies have shown retinal blood vessel damage in experimental models of retinal degeneration. The present study aimed to provide a detailed description of the structural and functional changes in retinal vasculature induced by retinal ischemia-reperfusion (I/R) in rats. Retinal ischemia was induced for 60 min by raising the intraocular pressure to 130 mmHg. Morphological changes in vascular components (endothelial cells, pericytes, and basement membranes), the patency and perfusion of blood vessels, and expression of vascular endothelial growth factor (VEGF) were assessed in the retinas at 2, 7, and 14 days after I/R. Significant reductions in vascular densities were observed at 7 and 14 days after I/R. Pericyte loss occurred after the appearance of endothelial cell degeneration, whereas the vascular basement membranes remained unchanged. Some vessels showed no perfusion in damaged retina. A decrease in the immunoreactivity of VEGF in the region extending from the ganglion cell layer to the outer plexiform layer was evident 2 days after I/R. In retinal I/R model, retinal ganglion cells are rapidly (<2 day) damaged following reperfusion, therefore, the current results suggest that neuronal cell damage precedes capillary degeneration, and neuronal cells may play an important role in maintaining vascular structure and function through the production and release of endothelial cell survival factors, including VEGF. Neuronal cell damage could be an additional cause of progression of ischemic retinal damage by reducing blood supply to the retinal neurons due to the destruction of the blood vessel network.