Phase Separation of Phytochrome B in HEK293T Cells.

In this method, we employed HEK293T cells to express the plant photoreceptor phytochrome B (phyB). Through the application of various treatments such as phycocyanobilin (PCB) supplementation, red light exposure, and temperature adjustments, the phyB proteins exhibited liquid-liquid phase separation, leading to the formation of biomolecular condensates. Here, we present a comprehensive description of the protein expression, cell treatment, and imaging capture procedures. This detailed guide provides step-by-step instructions on how to induce phase separation of phyB proteins in HEK293T cells. By utilizing this approach, researchers can investigate the physicochemical characteristics and dynamic formation process of phyB photobodies with precision.

Isolation of Phytochrome B Photobodies.

Phytochrome B (phyB), a plant photoreceptor, forms a membraneless organelle known as a photobody. Here, we present a protocol for the isolation of phyB photobodies through fluorescence-activated particle sorting from mature transgenic Arabidopsis leaves expressing phyB-GFP. This protocol involves the isolation of nuclei from frozen ground leaves using sucrose gradient centrifugation, the disruption of nuclear envelopes by sonication, and the subsequent isolation of phyB photobodies through fluorescence-activated particle sorting. We include experimental tips and notes for each step.

The Logistical Backbone of Photoreceptor Cell Function: Complementary Mechanisms of Dietary Vitamin A Receptors and Rhodopsin Transporters.

In this review, we outline our current understanding of the mechanisms involved in the absorption, storage, and transport of dietary vitamin A to the eye, and the trafficking of rhodopsin protein to the photoreceptor outer segments, which encompasses the logistical backbone required for photoreceptor cell function. Two key mechanisms of this process are emphasized in this manuscript: ocular and systemic vitamin A membrane transporters, and rhodopsin transporters. Understanding the complementary mechanisms responsible for the generation and proper transport of the retinylidene protein to the photoreceptor outer segment will eventually shed light on the importance of genes encoded by these proteins, and their relationship on normal visual function and in the pathophysiology of retinal degenerative diseases.

MIR96 Has Good Potential to Differentiate Human Bone Marrow-Derived Mesenchymal Stem Cells into Photoreceptor-Like Cells.

OBJECTIVES: MicroRNAs play an important role in the development and function of neuron cells. Among these, the miRNA known as MIR96 is abundantly expressed in mammalian retina and significantly affects differentiation, maturation, and survival of human photoreceptor cells. In this study, a mimic to miRNA-96 was transfected into human bone marrowderived mesenchymal stem cells to explore the biological functions of MIR96 at differentiation processing. MATERIALS AND METHODS: A mimic to miRNA-96 and a competitive control were transfected into human bone marrow-derived mesenchymal stem cells using Lipofectamine. After 24 and 48 hours, we evaluated changes in expression levels of genes associated with neural progenitor and photoreceptor differentiation (OTX2, NRL, protein kinase C, SLC1A1, and recoverin) by real-time polymerase chain reaction. In addition, we measured expression of mRNA and protein of the CRX gene (neuroretinal progenitor cell marker) and the RHO gene (terminal differentiation marker) using real-time polymerase chain reaction and immunocytochemistry, respectively. RESULTS: Real-time polymerase chain reaction results showed increased levels of RHO and recoverin mRNA after 24 hours in transfected cells. In addition, mRNA levels of OTX2, CRX, NRL, RHO, recoverin, and protein kinase C increased after 48 hours in transfected cells. Immunocytochemistry results confirmed these findings by demonstrating RHO and CRX at both 24 and 48 hours in transfected cells. CONCLUSIONS: Control of the expression of MIR96 can be a good strategy to promote cell differentiation and can be used in cell therapy for retinal degeneration. Our results showed that human bone marrow-derived mesenchymal stem cells can differentiate into photoreceptor cells after transfection with MIR96. These results support therapeutic use of MIR96 in retinal degeneration and suggest human bone marrowderived mesenchymal stem cells as a promising tool for interventions.

Phytochrome B photobody components.

Phytochrome B (phyB) is a red and far-red photoreceptor that promotes light responses. Upon photoactivation, phyB enters the nucleus and forms a molecular condensate called a photobody through liquid-liquid phase separation. Phytochrome B photobody comprises phyB, the main scaffold molecule, and at least 37 client proteins. These clients belong to diverse functional categories enriched with transcription regulators, encompassing both positive and negative light signaling factors, with the functional bias toward the negative factors. The functionally diverse clients suggest that phyB photobody acts either as a trap to capture proteins, including negatively acting transcription regulators, for processes such as sequestration, modification, or degradation or as a hub where proteins are brought into close proximity for interaction in a light-dependent manner.

Photoreceptor calyceal processes accompany the developing outer segment, adopting a stable length despite a dynamic core.

Vertebrate photoreceptors detect light through a large cilium-based outer segment, which is filled with photopigment-laden membranous discs. Surrounding the base of the outer segment are microvilli-like calyceal processes (CPs). Although CP disruption has been associated with altered outer segment morphology and photoreceptor degeneration, the role of the CPs remains elusive. Here, we used zebrafish as a model to characterize CPs. We quantified CP parameters and report a strong disparity in outer segment coverage between photoreceptor subtypes. CP length is stable across light and dark conditions, yet heat-shock inducible expression of tagged actin revealed rapid turnover of the CP actin core. Detailed imaging of the embryonic retina uncovered substantial remodeling of the developing photoreceptor apical surface, including a transition from dynamic tangential processes to vertically oriented CPs immediately prior to outer segment formation. Remarkably, we also found a direct connection between apical extensions of the Müller glia and retinal pigment epithelium, arranged as bundles around the ultraviolet sensitive cones. In summary, our data characterize the structure, development and surrounding environment of photoreceptor microvilli in the zebrafish retina.

Cell adhesion and actin dynamics factors promote axonal extension and synapse formation in transplanted Drosophila photoreceptor cells.

Vision is formed by the transmission of light stimuli to the brain through axons extending from photoreceptor cells. Damage to these axons leads to loss of vision. Despite research on neural circuit regeneration through transplantation, achieving precise axon projection remains challenging. To achieve optic nerve regeneration by transplantation, we employed the Drosophila visual system. We previously established a transplantation method for Drosophila utilizing photoreceptor precursor cells extracted from the eye disc. However, little axonal elongation of transplanted cells into the brain, the lamina, was observed. We verified axonal elongation to the lamina by modifying the selection process for transplanted cells. Moreover, we focused on N-cadherin (Ncad), a cell adhesion factor, and Twinstar (Tsr), which has been shown to promote actin reorganization and induce axon elongation in damaged nerves. Overexpression of Ncad and tsr promoted axon elongation to the lamina, along with presynaptic structure formation in the elongating axons. Furthermore, overexpression of Neurexin-1 (Nrx-1), encoding a protein identified as a synaptic organizer, was found to not only promote presynapse formation but also enhance axon elongation. By introducing Ncad, tsr, and Nrx-1, we not only successfully achieved axonal projection of transplanted cells to the brain beyond the retina, but also confirmed the projection of transplanted cells into a deeper ganglion, the medulla. The present study offers valuable insights to realize regeneration through transplantation in a more complex nervous system.

T-type voltage-gated channels, Na+/Ca2+-exchanger, and calpain-2 promote photoreceptor cell death in inherited retinal degeneration.

Inherited retinal degenerations (IRDs) are a group of untreatable and commonly blinding diseases characterized by progressive photoreceptor loss. IRD pathology has been linked to an excessive activation of cyclic nucleotide-gated channels (CNGC) leading to Na+- and Ca2+-influx, subsequent activation of voltage-gated Ca2+-channels (VGCC), and further Ca2+ influx. However, a connection between excessive Ca2+ influx and photoreceptor loss has yet to be proven.Here, we used whole-retina and single-cell RNA-sequencing to compare gene expression between the rd1 mouse model for IRD and wild-type (wt) mice. Differentially expressed genes indicated links to several Ca2+-signalling related pathways. To explore these, rd1 and wt organotypic retinal explant cultures were treated with the intracellular Ca2+-chelator BAPTA-AM or inhibitors of different Ca2+-permeable channels, including CNGC, L-type VGCC, T-type VGCC, Ca2+-release-activated channel (CRAC), and Na+/Ca2+ exchanger (NCX). Moreover, we employed the novel compound NA-184 to selectively inhibit the Ca2+-dependent protease calpain-2. Effects on the retinal activity of poly(ADP-ribose) polymerase (PARP), sirtuin-type histone-deacetylase, calpains, as well as on activation of calpain-1, and - 2 were monitored, cell death was assessed via the TUNEL assay.While rd1 photoreceptor cell death was reduced by BAPTA-AM, Ca2+-channel blockers had divergent effects: While inhibition of T-type VGCC and NCX promoted survival, blocking CNGCs and CRACs did not. The treatment-related activity patterns of calpains and PARPs corresponded to the extent of cell death. Remarkably, sirtuin activity and calpain-1 activation were linked to photoreceptor protection, while calpain-2 activity was related to degeneration. In support of this finding, the calpain-2 inhibitor NA-184 protected rd1 photoreceptors.These results suggest that Ca2+ overload in rd1 photoreceptors may be triggered by T-type VGCCs and NCX. High Ca2+-levels likely suppress protective activity of calpain-1 and promote retinal degeneration via activation of calpain-2. Overall, our study details the complexity of Ca2+-signalling in photoreceptors and emphasizes the importance of targeting degenerative processes specifically to achieve a therapeutic benefit for IRDs. Video Abstract.

Aggregation of rhodopsin mutants in mouse models of autosomal dominant retinitis pigmentosa.

Mutations in rhodopsin can cause it to misfold and lead to retinal degeneration. A distinguishing feature of these mutants in vitro is that they mislocalize and aggregate. It is unclear whether or not these features contribute to retinal degeneration observed in vivo. The effect of P23H and G188R misfolding mutations were examined in a heterologous expression system and knockin mouse models, including a mouse model generated here expressing the G188R rhodopsin mutant. In vitro characterizations demonstrate that both mutants aggregate, with the G188R mutant exhibiting a more severe aggregation profile compared to the P23H mutant. The potential for rhodopsin mutants to aggregate in vivo was assessed by PROTEOSTAT, a dye that labels aggregated proteins. Both mutants mislocalize in photoreceptor cells and PROTEOSTAT staining was detected surrounding the nuclei of photoreceptor cells. The G188R mutant promotes a more severe retinal degeneration phenotype and greater PROTEOSTAT staining compared to that promoted by the P23H mutant. Here, we show that the level of PROTEOSTAT positive cells mirrors the progression and level of photoreceptor cell death, which suggests a potential role for rhodopsin aggregation in retinal degeneration.

Neurexin-1-dependent circuit activity is required for the maintenance of photoreceptor subtype identity in Drosophila.

In the human and Drosophila color vision system, each photoreceptor neuron (cone cell in humans and R7/R8 photoreceptor cell in Drosophila) makes a stochastic decision to express a single photopigment of the same family with the exclusion of the others. While recent studies have begun to reveal the mechanisms that specify the generation of cone subtypes during development in mammals, nothing is known about how the mosaic of mutually exclusive cone subtypes is maintained in the mammalian retina. In Drosophila, recent work has led to the identification of several intrinsic factors that maintain the identity of R8 photoreceptor subtypes in adults. Whether and how extrinsic mechanisms are involved, however, remain unknown. In this study, we present evidence that supports that the Drosophila transsynaptic adhesion molecule Neurexin 1 (Dnrx-1) is required non-cell autonomously in R8p subtypes for the maintenance of R8y subtype identity. Silencing the activity of R8p subtypes caused a phenotype identical to that in dnrx-1 mutants. These results support a novel role for Nrx-1-dependent circuit activity in mediating the communication between R8 photoreceptor subtypes for maintaining the subtype identity in the retina.

Melatonin protects photoreceptor cells against ferroptosis in dry AMD disorder by inhibiting GSK-3B/Fyn-dependent Nrf2 nuclear translocation.

BACKGROUND: Ferroptosis is a type of non-apoptotic cell death that relies on iron ions and reactive oxygen species to induce lipid peroxidation. This study aimed to determine whether ferroptosis exists in the pathogenesis of dry age-related macular degeneration (AMD) and to confirm that melatonin (MLT) suppresses the photoreceptor cell ferroptosis signaling pathway. METHODS: We exposed 661W cells to sodium iodate (NaIO3) in vitro and treated them with different concentrations of MLT. In vivo, C57BL/6 mice were given a single caudal vein injection of NaIO3, followed by an intraperitoneal injection of MLT, and eyeballs were taken for subsequent trials. RESULTS: We found that NaIO3 could induce photoreceptor cell death and lipid peroxide accumulation, and result in changes in the expression of ferroptosis-related factors and iron maintenance proteins, which were treated by MLT. We further demonstrated that MLT can block Fyn-dependent Nrf2 nuclear translocation by suppressing the GSK-3ß signaling pathway. In addition, the therapeutic effect of MLT was significantly inhibited when Nrf2 was silenced. CONCLUSIONS: Our findings provide a novel insight that NaIO3 induces photoreceptor cell ferroptosis in dry AMD and suggest that MLT has therapeutic effects by suppressing GSK-3ß/Fyn-dependent Nrf2 nuclear translocation.

Peripheral monocytes and neutrophils promote photoreceptor cell death in an experimental retinal detachment model.

Photoreceptor cell death and immune cell infiltration are two major events that contribute to retinal degeneration. However, the relationship between these two events has not been well delineated, primarily because of an inadequate understanding of the immunological processes involved in photoreceptor degeneration, especially that of peripheral leukocytes that infiltrate the subretinal space and retinal tissues. In this work, we characterized the role of leukocyte infiltration within the detached retina. We observed that CD45+ CD11b+ Ly6G+ neutrophils and CD45+ CD11b+ Ly6G- Ly6C+ monocytes are the predominant peripheral immune cell populations that infiltrate the retinal and subretinal space after detachment. Selective depletion of monocytes or neutrophils using cell-specific targeting is neuroprotective for photoreceptors. These results indicate that peripheral innate immune cells contribute to photoreceptor degeneration, and targeting these immune cell populations could be therapeutic during retinal detachment.

ROM1 is redundant to PRPH2 as a molecular building block of photoreceptor disc rims.

Visual signal transduction takes place within a stack of flattened membranous 'discs' enclosed within the light-sensitive photoreceptor outer segment. The highly curved rims of these discs, formed in the process of disc enclosure, are fortified by large hetero-oligomeric complexes of two homologous tetraspanin proteins, PRPH2 (a.k.a. peripherin-2 or rds) and ROM1. While mutations in PRPH2 affect the formation of disc rims, the role of ROM1 remains poorly understood. In this study, we found that the knockout of ROM1 causes a compensatory increase in the disc content of PRPH2. Despite this increase, discs of ROM1 knockout mice displayed a delay in disc enclosure associated with a large diameter and lack of incisures in mature discs. Strikingly, further increasing the level of PRPH2 rescued these morphological defects. We next showed that disc rims are still formed in a knockin mouse in which the tetraspanin body of PRPH2 was replaced with that of ROM1. Together, these results demonstrate that, despite its contribution to the formation of disc rims, ROM1 can be replaced by an excess of PRPH2 for timely enclosure of newly forming discs and establishing normal outer segment structure.

CCR1 mediates Müller cell activation and photoreceptor cell death in macular and retinal degeneration.

Mononuclear cells are involved in the pathogenesis of retinal diseases, including age-related macular degeneration (AMD). Here, we examined the mechanisms that underlie macrophage-driven retinal cell death. Monocytes were extracted from patients with AMD and differentiated into macrophages (hMdɸs), which were characterized based on proteomics, gene expression, and ex vivo and in vivo properties. Using bioinformatics, we identified the signaling pathway involved in macrophage-driven retinal cell death, and we assessed the therapeutic potential of targeting this pathway. We found that M2a hMdɸs were associated with retinal cell death in retinal explants and following adoptive transfer in a photic injury model. Moreover, M2a hMdɸs express several CCRI (C-C chemokine receptor type 1) ligands. Importantly, CCR1 was upregulated in Müller cells in models of retinal injury and aging, and CCR1 expression was correlated with retinal damage. Lastly, inhibiting CCR1 reduced photic-induced retinal damage, photoreceptor cell apoptosis, and retinal inflammation. These data suggest that hMdɸs, CCR1, and Müller cells work together to drive retinal and macular degeneration, suggesting that CCR1 may serve as a target for treating these sight-threatening conditions.

The PKG Inhibitor CN238 Affords Functional Protection of Photoreceptors and Ganglion Cells against Retinal Degeneration.

Hereditary retinal degeneration (RD) is often associated with excessive cGMP signalling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can reduce photoreceptor loss in two different RD animal models. In this study, we identified a PKG inhibitor, the cGMP analogue CN238, which preserved photoreceptor viability and functionality in rd1 and rd10 mutant mice. Surprisingly, in explanted retinae, CN238 also protected retinal ganglion cells from axotomy-induced retrograde degeneration and preserved their functionality. Furthermore, kinase activity-dependent protein phosphorylation of the PKG target Kv1.6 was reduced in CN238-treated rd10 retinal explants. Ca2+-imaging on rd10 acute retinal explants revealed delayed retinal ganglion cell repolarization with CN238 treatment, suggesting a PKG-dependent modulation of Kv1-channels. Together, these results highlight the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, illustrating their broad potential for the treatment of retinal diseases and possibly neurodegenerative diseases in general.

Atonal homolog 7 (ATOH7) confers neuroprotection for photoreceptor cells in glaucoma via inhibition of the notch pathway.

Single-cell RNA sequencing (scRNA-seq) technologies enable the profiling and analysis of the transcriptomes of single cells and hold promise for clarifying gene mechanisms at single-cell resolution. We based this study on scRNA-seq data to reveal glaucoma-related genes and downstream pathways with neuroprotection effects. The scRNA-seq datasets related to glaucoma of retinal tissue samples of human beings and Atonal Homolog 7 (ATOH7)-null mice were obtained from the GEO database. The 74 top marker genes and 20 cell clusters were obtained in human retinal tissue samples. The key gene ATOH7 was found after the intersection with genes from GeneCards data. In the ATOH7-null mouse retinal tissue samples, pseudotime inference demonstrated significant changes in cell differentiation. Moreover, mouse retinal photoreceptor cells (PRCs) were cultured and treated with lentivirus carrying oe-ATOH7 alone or in combination with Notch signaling pathway activator Jagged-1/FC, after which cell biological functions were determined. The involvement of ATOH7 in glaucoma was identified through regulating PRCs. Furthermore, ATOH7 conferred neuroprotection in PRCs in glaucoma by mediating the Notch signaling pathway. In vitro data confirmed that ATOH7 overexpression promoted the differentiation of PRCs and inhibited their apoptosis by suppressing the Notch signaling pathway. The evidence provided by our study highlighted the involvement of ATOH7 in the blockade of the Notch signaling pathway, resulting in the neuroprotection for PRCs in glaucoma.

Primary versus Secondary Elevations in Fundus Autofluorescence.

The method of quantitative fundus autofluorescence (qAF) can be used to assess the levels of bisretinoids in retinal pigment epithelium (RPE) cells so as to aid the interpretation and management of a variety of retinal conditions. In this review, we focused on seven retinal diseases to highlight the possible pathways to increased fundus autofluorescence. ABCA4- and RDH12-associated diseases benefit from known mechanisms whereby gene malfunctioning leads to elevated bisretinoid levels in RPE cells. On the other hand, peripherin2/RDS-associated disease (PRPH2/RDS), retinitis pigmentosa (RP), central serous chorioretinopathy (CSC), acute zonal occult outer retinopathy (AZOOR), and ceramide kinase like (CERKL)-associated retinal degeneration all express abnormally high fundus autofluorescence levels without a demonstrated pathophysiological pathway for bisretinoid elevation. We suggest that, while a known link from gene mutation to increased production of bisretinoids (as in ABCA4- and RDH12-associated diseases) causes primary elevation in fundus autofluorescence, a secondary autofluorescence elevation also exists, where an impairment and degeneration of photoreceptor cells by various causes leads to an increase in bisretinoid levels in RPE cells.

HDAC inhibition delays photoreceptor loss in Pde6b mutant mice of retinitis pigmentosa: insights from scRNA-seq and CUT&Tag.

Purpose: This research aimed to ascertain the neuroprotective effect of histone deacetylase (HDAC) inhibition on retinal photoreceptors in Pde6brd1 mice, a model of retinitis pigmentosa (RP). Methods: Single-cell RNA-sequencing (scRNA-seq) explored HDAC and poly (ADP-ribose) polymerase (PARP)-related gene expression in both Pde6b-mutant rd1 and wild-type (WT) mice. The CUT&Tag method was employed to examine the functions of HDAC in rd1 mice. Organotypic retinal explant cultures from WT and rd1 mice were exposed to the HDAC inhibitor SAHA (suberoylanilide hydroxamic acid) postnatally, from day 5 to day 11. The terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay was applied to quantify the percentage of photoreceptor loss in the outer nuclear layer (ONL). HDAC activity was confirmed to be inhibited by SAHA through an HDAC activity assay. Moreover, the study evaluated PARP activity, a key driver of the initial response to DNA damage during photoreceptor degeneration, following HDAC inhibition. Results: The scRNA-seq revealed that diverse roles of HDAC and PARP isoforms in photoreceptor cell death. HDAC-related genes appeared to regulate cell death and primary immunodeficiency. Alterations in HDAC activity were consistent with the TUNEL-positive cells in the ONL at different time points. Notably, SAHA significantly postponed photoreceptor loss and decreased HDAC and PARP activity, thereby implicating both in the same degenerative pathway. Conclusions: This study highlights that the interaction between HDAC inhibition and PARP can delay photoreceptor cell death, proposing a promising therapeutic approach for RP.

Structure of the photoreceptor synaptic assembly of the extracellular matrix protein pikachurin with the orphan receptor GPR179.

Precise synapse formation is essential for normal functioning of the nervous system. Retinal photoreceptors establish selective contacts with bipolar cells, aligning the neurotransmitter release apparatus with postsynaptic signaling cascades. This involves transsynaptic assembly between the dystroglycan-dystrophin complex on the photoreceptor and the orphan receptor GPR179 on the bipolar cell, which is mediated by the extracellular matrix protein pikachurin (also known as EGFLAM). This complex plays a critical role in the synaptic organization of photoreceptors and signal transmission, and mutations affecting its components cause blinding disorders in humans. Here, we investigated the structural organization and molecular mechanisms by which pikachurin orchestrates transsynaptic assembly and solved structures of the human pikachurin domains by x-ray crystallography and of the GPR179-pikachurin complex by single-particle, cryo-electron microscopy. The structures reveal molecular recognition principles of pikachurin by the Cache domains of GPR179 and show how the interaction is involved in the transsynaptic alignment of the signaling machinery. Together, these data provide a structural basis for understanding the synaptic organization of photoreceptors and ocular pathology.

Photoreceptor Ion Channels in Signaling and Disease.

Photoreceptors (PRs) in the neural retina convert photon capture into an electrical signal that is communicated across a chemical synapse to second-order neurons in the retina and on through the rest of the visual pathway. This information is decoded in the visual cortex to create images. The activity of PRs depends on the concerted action of several voltage-gated ion channels that will be discussed in this chapter.