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Photoreception is essential for the development of the visual system, shaping vision's first synapse to cortical development. Here, we find that the lighting environment controls developmental rod apoptosis via Opn4-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). Using genetics, sensory environment manipulations, and computational approaches, we establish a pathway where light-dependent glutamate released from ipRGCs is detected via a transiently expressed glutamate receptor (Grik3) on rod precursors within the inner retina. Communication between these cells is mediated by hybrid neurites on ipRGCs that sense light before eye opening. These structures span the ipRGC-rod precursor distance over development and contain the machinery for photoreception (Opn4) and neurotransmitter release (Vglut2 & Syp). Assessment of the human gestational retina identifies conserved hallmarks of an ipRGC-to-rod axis, including displaced rod precursors, transient GRIK3 expression, and ipRGCs with deep-projecting neurites. This analysis defines an adaptive retrograde pathway linking the sensory environment to rod precursors via ipRGCs prior to eye opening., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Papillary thyroid carcinoma (PTC) is the most common type of thyroid malignancy, with a rising incidence. Traditional treatments, such as thyroidectomy and radiotherapy, often lead to significant side effects, including impaired thyroid function. Therefore, there is an urgent need for non-invasive therapeutic approaches. This study aims to explore the potential of photobiomodulation therapy (PBMT), a non-invasive treatment using specific wavelengths of light, in the management of PTC., Methods: We investigated the effects of blue light PBMT on PTC cells, focusing on the Retinal-OPSIN 3 (OPN3) complex's role in mediating cellular responses. Blue light exposure was applied to PTC cells, and subsequent changes in cellular proliferation, cell cycle progression, and protein expression were analyzed. Statistical tests, including one-way ANOVA and t-tests, were used to evaluate the significance of the findings., Results: Blue light exposure led to the dissociation of 11-cis-retinal from OPN3, resulting in the accumulation of all-trans retinal. This accumulation disrupted cellular proliferation pathways and induced G0/G1 cell cycle arrest in PTC cells. The Retinal-OPN3 complex was found to be a key mediator in these processes, demonstrating that thyroid cells can respond to specific light wavelengths and utilize their photoreceptive potential for therapeutic purposes., Conclusions: Our findings suggest that PBMT, through the modulation of the Retinal-OPN3 complex, offers a promising non-invasive approach for treating PTC. This study highlights the therapeutic potential of light signal transduction in non-ocular tissues and opens new avenues for non-invasive cancer therapies., (© 2024. The Author(s).)

The ultrafast photochemical reaction mechanism, transient spectra, and transition kinetics of the human blue cone visual pigment have been recorded at room temperature. Ultrafast time-resolved absorption spectroscopy revealed the progressive formation and decay of several metastable photo-intermediates, corresponding to the Batho to Meta-II photo-intermediates previously observed with bovine rhodopsin and human green cone opsin, on the picosecond to millisecond timescales following pulsed excitation. The experimental data reveal several interesting similarities and differences between the photobleaching sequences of bovine rhodopsin, human green cone opsin, and human blue cone opsin. While Meta-II formation kinetics are comparable between bovine rhodopsin and blue cone opsin, the transition kinetics of earlier photo-intermediates and qualitative characteristics of the Meta-I to Meta-II transition are more similar for blue cone opsin and green cone opsin. Additionally, the blue cone photo-intermediate spectra exhibit a high degree of overlap with uniquely small spectral shifts. The observed variation in Meta-II formation kinetics between rod and cone visual pigments is explained based on key structural differences., Competing Interests: Competing interests statement:The authors declare no competing interest.

L-cone opsin expression by gene therapy is a promising treatment for blue cone monochromacy (BCM) caused by congenital lack of long- and middle-wavelength-sensitive (L/M) cone function. Eight patients with BCM and confirmed pathogenic variants at the OPN1LW/OPN1MW gene cluster participated. Optical coherence tomography (OCT), chromatic perimetry, chromatic microperimetry, chromatic visual acuity (VA), and chromaticity thresholds were performed with unmodified commercial equipment and/or methods available in the public domain. Adaptive optics scanning laser ophthalmoscope (AOSLO) imaging was performed in a subset of patients. Outer retinal changes were detectable by OCT with an age-related effect on the foveal disease stage. Rod and short-wavelength-sensitive (S) cone functions were relatively retained by perimetry, although likely impacted by age-related increases in the pre-retinal absorption of short-wavelength lights. The central macula showed a large loss of red sensitivity on dark-adapted microperimetry. Chromatic VAs with high-contrast red gratings on a blue background were not detectable. Color vision was severely deficient. AOSLO imaging showed reduced total cone density with majority of the population being non-waveguiding. This study developed and evaluated specialized outcomes that will be needed for the determination of efficacy and safety in human clinical trials. Dark-adapted microperimetry with a red stimulus sampling the central macula would be a key endpoint to evaluate the light sensitivity improvements. VA changes specific to L-opsin can be measured with red gratings on a bright blue background and should also be considered as outcome measures in future interventional trials.

There is growing interest in developing artificial lighting that stimulates intrinsically photosensitive retinal ganglion cells (ipRGCs) to entrain circadian rhythms to improve mood, sleep, and health. Efforts have focused on stimulating the intrinsic photopigment, melanopsin; however, specialized color vision circuits have been elucidated in the primate retina that transmit blue-yellow cone-opponent signals to ipRGCs. We designed a light that stimulates color-opponent inputs to ipRGCs by temporally alternating short- and long-wavelength components that strongly modulate short-wavelength sensitive (S) cones. Two-hour exposure to this S-cone modulating light produced an average circadian phase advance of 1 h and 20 min in 6 subjects (mean age = 30 years) compared to no phase advance for the subjects after exposure to a 500 lux white light equated for melanopsin effectiveness. These results are promising for developing artificial lighting that is highly effective in controlling circadian rhythms by invisibly modulating cone-opponent circuits., Competing Interests: Conflict of interest statementThe authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The University of Washington has filed U.S. Patent Application, entitled “LIGHTING DEVICES, SYSTEMS, METHODS FOR STIMULATING CIRCADIAN RHYTHMS” serial number 17/612,061 for which authors A.N., M.N., J.N., and J.A.K. receive licensing fees.

The choroid embedded in between retina and sclera is essential for retinal photoreceptor nourishment, but is also a source of growth factors in the process of emmetropization that converts retinal visual signals into scleral growth signals. Still, the exact control mechanisms behind those functions are enigmatic while circadian rhythms are involved. These rhythms are attributed to daylight influences that are melanopsin (OPN4) driven. Recently, OPN4-mRNA has been detected in the choroid, and while its origin is unknown we here seek to identify the underlying structures using morphological methods. Human and chicken choroids were prepared for single- and double-immunohistochemistry of OPN4, vasoactive intestinal peptide (VIP), substance P (SP), CD68, and α-smooth muscle actin (ASMA). For documentation, light-, fluorescence-, and confocal laser scanning microscopy was applied. Retinal controls proved the reliability of the OPN4 antibody in both species. In humans, OPN4 immunoreactivity (OPN4-IR) was detected in nerve fibers of the choroid and adjacent ciliary nerve fibers. OPN4+ choroidal nerve fibers lacked VIP, but were co-localized with SP. OPN4-immunoreactivity was further detected in VIP+/SP + intrinsic choroidal neurons, in a hitherto unclassified CD68-negative choroidal cell population thus not representing macrophages, as well as in a subset of choroidal melanocytes. In chicken, choroidal nerve fibers were OPN4+, and further OPN4-IR was detected in clustered suprachoroidal structures that were not co-localized with ASMA and therefore do not represent non-vascular smooth-muscle cells. In the choroidal stroma, numerous cells displayed OPN4-IR, the majority of which was VIP-, while a few of those co-localized with VIP and were therefore classified as avian intrinsic choroidal neurons. OPN4-immunoreactivity was absent in choroidal blood vessels of both species. In summary, OPN4-IR was detected in both species in nerve fibers and cells, some of which could be identified (ICN, melanocytes in human), while others could not be classified yet. Nevertheless, the OPN4+ structures described here might be involved in developmental, light-, thermally-driven or nociceptive mechanisms, as known from other systems, but with respect to choroidal control this needs to be proven in upcoming studies., Competing Interests: Declaration of competing interest All authors confirm no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: This study investigates alterations in intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells (DACs) in lid suture myopia (LSM) rats., Methods: LSM was induced in rats by suturing the right eyes for 4 weeks. Double immunofluorescence staining of ipRGCs and DACs in whole-mount retinas was performed to analyze changes in the density and morphology of control, LSM, and fellow eyes. Real-time quantitative PCR and Western blotting were used to detect related genes and protein expression levels., Results: Significant myopia was induced in the lid-sutured eye, but the fellow eye was not different to control. Decreased ipRGC density with paradoxically increased overall melanopsin expression and enlarged dendritic beads was observed in both the LSM and fellow eyes of the LSM rat retinas. In contrast, DAC changes occurred only in the LSM eyes, with reduced DAC density and tyrosine hydroxylase (TH) expression, sparser dendritic processes, and fewer varicosities. Interestingly, contacts between ipRGCs and DACs in the inner plexiform layer (IPL) and the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and vesicular monoamine transporter protein 2 (VMAT2) mRNA were decreased in the LSM eyes., Conclusions: The ipRGCs and DACs in LSM rat retinas undergo multiple alterations in density, morphology, and related molecule expressions. However, the ipRGC changes alone appear not to be required for the development of myopia, given that myopia is only induced in the lid-sutured eye, and they are unlikely alone to drive the DAC changes. Reduced contacts between ipRGCs and DACs in the LSM eyes may be the structural foundation for the impaired signaling between them. PACAP and VMAT2, strongly associated with ipRGCs and DACs, may play important roles in LSM through complex mechanisms.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in several physiological light responses. In this study, we generate an improved Opn4 cre knockin allele (Opn4 cre(DSO) ), which faithfully reproduces endogenous Opn4 expression and improves compatibility with widely used reporters. We evaluated the efficacy and sensitivity of Opn4 cre(DSO) for labeling in retina and brain and provide an in-depth comparison with the extensively utilized Opn4 cre(Saha) line. Through this characterization, Opn4 cre(DSO) demonstrated higher specificity in labeling ipRGCs with minimal recombination escape. Leveraging a combination of electrophysiological, molecular, and morphological analyses, we confirmed its sensitivity in detecting all ipRGC types (M1-M6) and defined their unique topographical distribution across the retina. In the brain, the Opn4 cre(DSO) line labels ipRGC projections with minimal labeling of cell bodies. Overall, the Opn4 cre(DSO) mouse line represents an improved tool for studying ipRGC function and distribution, offering a means to selectively target these cells to study light-regulated behaviors and physiology., Competing Interests: Declaration of interests The authors declare no competing financial or non-financial interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The pupillary light reflex (PLR) adapts the amount of light reaching the retina, protecting it and improving image formation. Two PLR mechanisms have been described in vertebrates. First, the pretectum receives retinal inputs and projects to the Edinger-Westphal nucleus (EWN), which targets the ciliary ganglion through the oculomotor nerve (nIII). Postganglionic fibers enter the eye-globe, traveling to the iris sphincter muscle. Additionally, some vertebrates exhibit an iris-intrinsic PLR mechanism mediated by sphincter muscle cells that express melanopsin inducing muscle contraction. Given the high degree of conservation of the lamprey visual system, we investigated the mechanisms underlying the PLR to shed light onto their evolutionary origins. Recently, a PLR mediated by melanopsin was demonstrated in lampreys, suggested to be brain mediated. Remarkably, we found that PLR is instead mediated by direct retino-iridal cholinergic projections. This retina-mediated PLR acts synergistically with an iris-intrinsic mechanism that, as in other vertebrates, is mediated by melanopsin and has contribution of gap junctions between muscle fibers. In contrast, we show that lampreys lack the brain-mediated PLR. Our results suggest that two eye-intrinsic PLR mechanisms were present in early vertebrate evolution, whereas the brain-mediated PLR has a more recent origin., (© 2024. The Author(s).)

Light is fundamental for biological life, with most mammals possessing light-sensing photoreceptors in various organs. Opsin3 is highly expressed in adipose tissue which has extensive communication with other organs, particularly with the brain through the sympathetic nervous system (SNS). Our study reveals a new light-triggered crosstalk between adipose tissue and the hypothalamus. Direct blue-light exposure to subcutaneous white fat improves high-fat diet-induced metabolic abnormalities in an Opsin3-dependent manner. Metabolomic analysis shows that blue light increases circulating levels of histidine, which activates histaminergic neurons in the hypothalamus and stimulates brown adipose tissue (BAT) via SNS. Blocking central actions of histidine and denervating peripheral BAT blunts the effects of blue light. Human white adipocytes respond to direct blue light stimulation in a cell-autonomous manner, highlighting the translational relevance of this pathway. Together, these data demonstrate a light-responsive metabolic circuit involving adipose-hypothalamus communication, offering a potential strategy to alleviate obesity-induced metabolic abnormalities., (© 2024. The Author(s).)

Melanopsin is a photopigment belonging to the G Protein-Coupled Receptor (GPCR) family expressed in a subset of intrinsically photosensitive retinal ganglion cells (ipRGCs) and responsible for a variety of processes. The bistability and, thus, the possibility to function under low retinal availability would make melanopsin a powerful optogenetic tool. Here, we aim to utilize mouse melanopsin to trigger macrophage migration by its subcellular optical activation with localized blue light, while simultaneously imaging the migration with red light. To reduce melanopsin's red light sensitivity, we employ a combination of in silico structure prediction and automated quantum mechanics/molecular mechanics modeling to predict minimally invasive mutations to shift its absorption spectrum towards the shorter wavelength region of the visible spectrum without compromising the signaling efficiency. The results demonstrate that it is possible to achieve melanopsin mutants that resist red light-induced activation but are activated by blue light and display properties indicating preserved bistability. Using the A333T mutant, we show that the blue light-induced subcellular melanopsin activation triggers localized PIP3 generation and macrophage migration, which we imaged using red light, demonstrating the optogenetic utility of minimally engineered melanopsins., (© 2024. The Author(s).)

Migraine is a complex disorder characterized by episodes of moderate-to-severe, often unilateral headaches and generally accompanied by nausea, vomiting, and increased sensitivity to light (photophobia), sound (phonophobia), and smell (hyperosmia). Photophobia is considered the most bothersome symptom of migraine attacks. Although the underlying mechanism remains unclear, the intrinsically photosensitive retinal ganglion cells (ipRGCs) are considered to be involved in photophobia associated with migraine. In this study, we investigated the association between the sensitivity of ipRGCs and migraines and cortical spreading depression (CSD), which may trigger migraine attacks. The pupillary responses closely associated with the function of ipRGCs in patients with migraine who were irradiated with lights were evaluated. Blue (486 nm) light irradiation elicited a response from ipRGCs; however, red light (560 nm) had no such effect. Melanopsin, a photosensitive protein, phototransduces in ipRGCs following blue light stimulation. Hypersensitivity of ipRGCs was observed in patients with migraine. CSD was more easily induced with blue light than with incandescent light using a mouse CSD model. Moreover, CSD was suppressed, even in the presence of blue light, after injecting opsinamide, a melanopsin inhibitor. The hypersensitivity of ipRGCs in patients with migraine may induce CSD, resulting in migraine attacks.

Purpose: The purpose of this study was to evaluate pupillary light reflex (PLR) to chromatic flashes in patients with early-onset high-myopia (eoHM) without (myopic controls = M-CTRL) and with (female-limited myopia-26 = MYP-26) genetic mutations in the ARR3 gene encoding the cone arrestin., Methods: Participants were 26 female subjects divided into 3 groups: emmetropic controls (E-CTRL, N = 12, mean age = 28.6 ± 7.8 years) and 2 myopic (M-CTRL, N = 7, mean age = 25.7 ± 11.5 years and MYP-26, N = 7, mean age = 28.3 ± 15.4 years) groups. In addition, one hemizygous carrier and one control male subject were examined. Direct PLRs were recorded after 10-minute dark adaptation. Stimuli were 1-second red (peak wavelength = 621 nm) and blue (peak wavelength = 470 nm) flashes at photopic luminance of 250 cd/m². A 2-minute interval between the flashes was introduced. Baseline pupil diameter (BPD), peak pupil constriction (PPC), and postillumination pupillary response (PIPR) were extracted from the PLR. Group comparisons were performed with ANOVAs., Results: Dark-adapted BPD was comparable among the groups, whereas PPC to the red light was slightly reduced in patients with myopia (P = 0.02). PIPR at 6 seconds elicited by the blue flash was significantly weaker (P < 0.01) in female patients with MYP-26, whereas it was normal in the M-CTRL group and the asymptomatic male carrier., Conclusions: L/M-cone abnormalities due to ARR3 gene mutation is currently claimed to underlie the pathological eye growth in MYP-26. Our results suggest that malfunction of the melanopsin system of intrinsically photosensitive retinal ganglion cells (ipRGCs) is specific to patients with symptomatic MYP-26, and may therefore play an additional role in the pathological eye growth of MYP-26.

The pupil modulates the amount of light that reaches the retina. Not only luminance but also the spectral distribution defines the pupil size. Previous research has identified steady-state pupil size and melatonin attenuation to be predominantly driven by melanopsin, which is expressed by a unique subgroup of intrinsically photosensitive retinal ganglion cells (ipRGCs) that are sensitive to short-wavelength light (~480 nm). Here, we aimed to selectively target the melanopsin system during the evening, while measuring steady-state pupil size and melatonin concentrations under commonly experienced evening light levels (<90 lx). Therefore, we used a five-primary display prototype to generate light conditions that were matched in terms of L-, M-, and S-cone-opic irradiances, but with high and low melanopic irradiances (~3-fold difference). Seventy-two healthy, male participants completed a 2-week study protocol. The volunteers were assigned to one of the four groups that differed in luminance levels (27-285 cd/m 2 ). Within the four groups, each volunteer was exposed to a low melanopic (LM) and a high melanopic (HM) condition. The two 17-h study protocols comprised 3.5 h of light exposure starting 4 h before habitual bedtime. Median pupil size was significantly smaller during HM than LM in all four light intensity groups. In addition, we observed a significant correlation between melanopic weighted corneal illuminance (melanopic equivalent daylight illuminance [mEDI]) and pupil size, such that higher mEDI values were associated with smaller pupil size. Using pupil size to estimate retinal irradiance showed a qualitatively similar goodness of fit as mEDI for predicting melatonin suppression. Based on our results here, it remains appropriate to use melanopic irradiance measured at eye level when comparing light-dependent effects on evening melatonin concentrations in healthy young people at rather low light levels., Competing Interests: Conflict of interest statementI.S. and C.E. do not report any conflict of interest related to lighting. I.S. is an employee of Rodenstock GmbH. O.S. is listed as an inventor on the following patents: US8646939B2—Display system having circadian effect on humans; DE102010047207B4—Projection system and method for projecting image content; US8994292B2—Adaptive lighting system; WO2006013041A1—Projection device and filter therefor; and WO2016092112A1—Method for the selective adjustment of a desired brightness and/or color of a specific spatial area, and data processing device therefor. O.S. is a member of the Daylight Academy, Good Light Group, and Swiss Lighting Society. O.S. has had the following commercial interests in the last 2 years (2020-2023) related to lighting: Investigator-initiated research grants from SBB, Skyguide, Toshiba, Schneider, Lighting Europe, VW, VELUX, Porsche, and LightnTec. R.J.L. is a named inventor on patent applications relating to the design of multiprimary displays (“Improvements in image formation” and “Matching colors for a 5 primary display”); has received investigator-initiated research support from Philips Lighting and Signify; and received honoraria from Samsung Electronics. M.S. is named as an inventor on a patent application (“Determining metameric settings for a non-linear light source,” WO2020161499A1). C.C. has had the following commercial interests in the last 2 years (2020-2022) related to lighting: honoraria, travel, accommodation, and/or meals for invited keynote lectures; conference presentations; or teaching from Toshiba Materials, VELUX, Firalux, Lighting Europe, Electrosuisse, Novartis, Roche, Elite, Servier, and WIR Bank. C.C. is a member of the Daylight Academy.

Background: Neurodegenerative diseases share retinal abnormalities. Chromatic pupillometry allows in vivo assessment of photoreceptor functional integrity, including melanopsin-expressing retinal ganglion cells. This exploratory meta-analysis assesses retinal photoreceptor functionality in Alzheimer's vs. Parkinson's disease and conducts an in-depth review of applied pupillometric protocols., Methods: Literature reviews on PubMed and Scopus from 1991 to August 2023 identified chromatic pupillometry studies on Alzheimer's disease (AD; n = 42 patients from 2 studies) and Parkinson's disease (PD; n = 66 from 3 studies). Additionally, a pre-AD study (n = 10) and an isolated REM Sleep Behavior Disorder study (iRBD; n = 10) were found, but their results were not included in the meta-analysis statistics., Results: Melanopsin-mediated post-illumination pupil response to blue light was not significantly impaired in Alzheimer's (weighted mean difference = -1.54, 95% CI: 4.57 to 1.49, z = -1.00, p = 0.319) but was in Parkinson's (weighted mean difference = -9.14, 95% CI: 14.19 to -4.08, z = -3.54, p < 0.001). Other pupil light reflex metrics showed no significant differences compared to controls. Studies adhered to international standards of pupillometry with moderate to low bias. All studies used full-field stimulation. Alzheimer's studies used direct while Parkinson's studies used consensual measurement. Notably, studies did not control for circadian timing and Parkinson's patients were on dopaminergic treatment., Conclusion and Relevance: Results affirm chromatic pupillometry as a useful method to assess melanopsin-related retinal cell dysfunction in Parkinson's but not in Alzheimer's disease. While adhering to international standards, future studies may analyze the effects of local field stimulation, dopaminergic treatment, and longitudinal design to elucidate melanopsin dysfunction in Parkinson's disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The daily light-dark cycle is a key zeitgeber (time cue) for entraining an organism's biological clock, whereby light sensing by retinal photoreceptors, particularly intrinsically photosensitive retinal ganglion cells, stimulates the suprachiasmatic nucleus of the hypothalamus, a central pacemaker that in turn orchestrates the rhythm of peripheral metabolic activities. Non-rhythmic effects of light on metabolism have also been long known, and their transduction mechanisms are only beginning to unfold. Here, we summarize emerging evidence that, in mammals, light exposure or deprivation profoundly affects glucose homeostasis, thermogenesis and other metabolic activities in a clock-independent manner. Such light regulation could involve melanopsin-based, intrinsically photosensitive retinal ganglion cell-initiated brain circuits via the suprachiasmatic nucleus of the hypothalamus and other nuclei, or direct stimulation of opsins expressed in the hypothalamus, adipose tissue, blood vessels and skin to regulate sympathetic tone, lipolysis, glucose uptake, mitochondrial activation, thermogenesis, food intake, blood pressure and melanogenesis. These photic signalling events may coordinate with circadian-based mechanisms to maintain metabolic homeostasis, with dysregulation of this system underlying metabolic diseases caused by aberrant light exposure, such as environmental night light and shift work., (© 2024. Springer Nature Limited.)

The ambient daylight variation is coded by melanopsin photoreceptors and their luxotonic activity increases towards midday when colour temperatures are cooler, and irradiances are higher. Although melanopsin and cone photoresponses can be mediated via separate pathways, the connectivity of melanopsin cells across all levels of the retina enables them to modify cone signals. The downstream effects of melanopsin-cone interactions on human vision are however, incompletely understood. Here, we determined how the change in daytime melanopsin activation affects the human cone pathway signals in the visual cortex. A 5-primary silent-substitution method was developed to evaluate the dependence of cone-mediated signals on melanopsin activation by spectrally tuning the lights and stabilizing the rhodopsin activation under a constant cone photometric luminance. The retinal (white noise electroretinogram) and cortical responses (visual evoked potential) were simultaneously recorded with the photoreceptor-directed lights in 10 observers. By increasing the melanopsin activation, a reverse response pattern was observed with cone signals being supressed in the retina by 27% ( p = 0.03) and subsequently amplified by 16% ( p = 0.01) as they reach the cortex. We infer that melanopsin activity can amplify cone signals at sites distal to retinal bipolar cells to cause a decrease in the psychophysical Weber fraction for cone vision.

Because the selective estrogen receptor modulator tamoxifen was shown to be retina-protective in the light damage and rd10 models of retinal degeneration, the purpose of this study was to test whether tamoxifen is retina-protective in a model where retinal pigment epithelium (RPE) toxicity appears to be the primary insult: the sodium iodate (NaIO 3 ) model. C57Bl/6J mice were given oral tamoxifen (in the diet) or the same diet lacking tamoxifen, then given an intraperitoneal injection of NaIO 3 at 25 mg/kg. The mice were imaged a week later using optical coherence tomography (OCT). ImageJ with a custom macro was utilized to measure retinal thicknesses in OCT images. Electroretinography (ERG) was used to measure retinal function one week post-injection. After euthanasia, quantitative real-time PCR (qRT-PCR) was performed. Tamoxifen administration partially protected photoreceptors. There was less photoreceptor layer thinning in OCT images of tamoxifen-treated mice. qRT-PCR revealed, in the tamoxifen-treated group, less upregulation of antioxidant and complement factor 3 mRNAs, and less reduction in the rhodopsin and short-wave cone opsin mRNAs. Furthermore, ERG results demonstrated preservation of photoreceptor function for the tamoxifen-treated group. Cone function was better protected than rods. These results indicate that tamoxifen provided structural and functional protection to photoreceptors against NaIO 3 . RPE cells were not protected. These neuroprotective effects suggest that estrogen-receptor modulation may be retina-protective. The fact that cones are particularly protected is intriguing given their importance for human visual function and their survival until the late stages of retinitis pigmentosa. Further investigation of this protective pathway could lead to new photoreceptor-protective therapeutics., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The principal eyes of jumping spiders (Salticidae) integrate a dual-lens system, a tiered retinal matrix with multiple photoreceptor classes and muscular control of retinal movements to form high resolution images, extract color information, and dynamically evaluate visual scenes. While much work has been done to characterize these more complex principal anterior eyes, little work has investigated the three other pairs of simpler secondary eyes: the anterior lateral eye pair and two posterior (lateral and median) pairs of eyes. We investigated the opsin protein component of visual pigments in the eyes of three species of salticid using transcriptomics and immunohistochemistry. Based on characterization and localization of a set of three conserved opsins (Rh1 - green sensitive, Rh2 - blue sensitive, and Rh3 - ultraviolet sensitive) we have identified potential photoreceptors for blue light detection in the eyes of two out of three species: Menemerus bivittatus (Chrysillini) and Habrocestum africanum (Hasarinii). Additionally, the photoreceptor diversity of the secondary eyes exhibits more variation than previous estimates, particularly for the small, posterior median eyes previously considered vestigial in some species. In all three species investigated the lateral eyes were dominated by green-sensitive visual pigments (RH1 opsins), while the posterior median retinas were dominated by opsins forming short-wavelength sensitive visual pigments (e.g. RH2 and/or RH3/RH4). There was also variation among secondary eye types and among species in the distribution of opsins in retinal photoreceptors, particularly for the putatively blue-sensitive visual pigment formed from RH2. Our findings suggest secondary eyes have the potential for color vision, with observed differences between species likely associated with different ecologies and visual tasks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

It is unclear whether normal human skin tissue or abnormal scarring are photoreceptive. Therefore, this study investigated photosensitivity in normal skin tissue and hypertrophic scars. The expression of opsins, which are photoreceptor proteins, in normal dermal fibroblasts (NDFs) and hypertrophic scar fibroblasts (HSFs) was examined. After exposure to blue light (BL), changes in the expression levels of αSMA and clock-related genes, specifically PER2 and BMAL1 , were examined in both fibroblast types. Opsins were expressed in both fibroblast types, with OPN3 exhibiting the highest expression levels. After peripheral circadian rhythm disruption, BL induced rhythm formation in NDFs. In contrast, although HSFs showed changes in clock-related gene expression levels, no distinct rhythm formation was observed. The expression level of αSMA was significantly higher in HSFs and decreased to the same level as that in NDFs upon BL exposure. When OPN3 knocked-down HSFs were exposed to BL, the reduction in αSMA expression was inhibited. This study showed that BL exposure directly triggers peripheral circadian synchronization in NDFs but not in HSFs. OPN3-mediated BL exposure inhibited HSFs. Although the current results did not elucidate the relationship between peripheral circadian rhythms and hypertrophic scars, they show that BL can be applied for the prevention and treatment of hypertrophic scars and keloids.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are specialized retinal output neurons that mediate behavioral, neuroendocrine, and developmental responses to environmental light. There are diverse molecular strategies for marking ipRGCs, especially in mice, making them among the best characterized retinal ganglion cells (RGCs). With the development of more sensitive reporters, new subtypes of ipRGCs have emerged. We therefore tested high-sensitivity reporter systems to see whether we could reveal yet more. Substantial confusion remains about which of the available methods, if any, label all and only ipRGCs. Here, we compared many different methods for labeling of ipRGCs, including anti-melanopsin immunofluorescence, Opn4-GFP BAC transgenic mice, and Opn4 cre mice crossed with three different Cre-specific reporters (Z/EG, Ai9, and Ai14) or injected with Cre-dependent (DIO) AAV2. We show that Opn4 cre mice, when crossed with sensitive Cre-reporter mice, label numerous ganglion cell types that lack intrinsic photosensitivity. Though other methods label ipRGCs specifically, they do not label the entire population of ipRGCs. We conclude that no existing method labels all and only ipRGCs. We assess the appropriateness of each reporter for particular applications and integrate findings across reporters to estimate that the overall abundance of ipRGCs among mouse RGCs may approach 11%., (© 2024 Wiley Periodicals LLC.)

Background: To compare the expression levels of long non-coding RNA (lncRNA) and messenger RNA (mRNA) in pre-receptive endometrium between patients with Polycystic Ovary Syndrome (PCOS)and normal ovulation undergoing in vitro fertilization-embryo transfer (IVF-ET)., Methods: Endometrial tissues were collected with endometrial vacuum curette in pre-receptive phase (3 days after oocytes retrieval) from PCOS and control groups. LncRNAs and mRNAs of endometrium were identified via RNA sequencing and alignments. A subset of 9 differentially expressed lncRNAs and 11 mRNAs were validated by quantitative reverse transcription polymerase chain reaction(qRT-PCR)in 22 PCOS patients and 18 ovulation patients. The function of mRNAs with differential expression patterns were explored using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)., Results: We found out 687 up-regulated and 680 down-regulated mRNAs, as well as 345 up-regulated and 63 down-regulated lncRNAs in the PCOS patients in contrast to normal ovulation patients. qRT-PCR was used to detect the expression of 11 mRNAs, and validated that the expression of these 6 mRNAs CXCR4, RABL6, OPN3, SYBU, IDH1, NOP10 were significantly elevated among PCOS patients, and the expression of ZEB1 was significantly decreased. qRT-PCR was performed to detect the expression of 9 lncRNAs, and validated that the expression of these 7 lncRNAs IDH1-AS1, PCAT14, FTX, DANCR, PRKCQ-AS1, SNHG8, TPT1-AS1 were significantly enhanced among PCOS patients. Bioinformatics analysis showed that differentially expressed genes (DEGs) involved KEGG pathway were tyrosine metabolism, PI3K-Akt pathway, metabolic pathway, Jak-STAT pathway, pyruvate metabolism, protein processing in endoplasmic reticulum, oxidative phosphorylation and proteasome. The up-regulation of GO classification was involved in ATP metabolic process, oxidative phosphorylation, RNA catabolic process, and down-regulation of GO classification was response to corticosteroid, steroid hormone, and T cell activation., Conclusion: Our results determined the characteristics and expression profile of endometrial lncRNAs and mRNAs in PCOS patients in pre-receptive phase, which is the day 3 after oocytes retrival. The possible pathways and related genes of endometrial receptivity disorders were found, and those lncRNAs may be developed as a predictive biomarker of endometrium in pre-receptive phase., (© 2024. The Author(s).)

Human amniotic epithelial stem cells (hAESCs) are regarded as potential alternatives to keratinocytes (KCs) used for skin wound healing. Light is an alternative approach for inducing stem cell differentiation. Opsins (OPNs), a family of light-sensitive, G protein-coupled receptors, play a multitude of light-dependent and light-independent functions in extraocular tissues. However, it remains unclear whether the light sensitivity and function of OPNs are involved in light-induced differentiation of hAESCs to KCs. Herein, we determine the role of OPNs in differentiation of hAESCs into KCs through cell and molecular biology approaches in vitro. It is shown that mRNA expression of OPN3 in the amniotic membrane and hAESCs was higher than the other four primary OPNs by RT-qPCR analysis. Changes in OPN3 gene expression had a significant impact on cell proliferation, stemness and differentiation capability of hAESCs. Furthermore, we found a significant upregulation of OPN3, KRT5 and KRT14 with hAESCs treated at 3 × 33 J/cm 2 irradiation from blue-light LED. Taken together, these results suggest that OPN3 acts as a positive regulator of differentiation of hAESCs into KCs. This study provides a novel insight into photosensitive OPNs associated with photobiomodulation(PBM)-induced differentiation in stem cells., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Intrinsically photosensitive retinal ganglion cells (ipRGCs) serve as primary photoceptors by expressing the photopigment, melanopsin, and also as retinal relay neurons for rod and cone signals en route to the brain, in both cases for the purpose of non-image vision as well as aspects of image vision. So far, six subtypes of ipRGCs (M1 through M6) have been characterized. Regarding their phototransduction mechanisms, we have previously found that, unconventionally, rhabdomeric (microvillous) and ciliary signaling motifs co-exist within a given M1-, M2-, and M4-ipRGC, with the first mechanism involving PLCβ4 and TRPC6,7 channels and the second involving cAMP and HCN channels. We have now examined M3-, M5-, and M6-cells and found that each cell likewise uses both signaling pathways for phototransduction, despite differences in the percentage representation by each pathway in a given ipRGC subtype for bright-flash responses (and saturated except for M6-cells). Generally, M3- and M5-cells show responses quite similar in kinetics to M2-responses, and M6-cell responses resemble broadly those of M1-cells although much lower in absolute sensitivity and amplitude. Therefore, similar to rod and cone subtypes in image vision, ipRGC subtypes possess the same phototransduction mechanism(s) even though they do not show microvilli or cilia morphologically., Competing Interests: Competing interests statement:The authors declare no competing interest.

Animal opsins are light activated G-protein-coupled receptors, capable of optogenetic control of G-protein signalling for research or therapeutic applications. Animal opsins offer excellent photosensitivity, but their temporal resolution can be limited by long photoresponse duration when expressed outside their native cellular environment. Here, we explore methods for addressing this limitation for a prototypical animal opsin (human rod opsin) in HEK293T cells. We find that the application of the canonical rhodopsin kinase (GRK1)/visual arrestin signal termination mechanism to this problem is complicated by a generalised suppressive effect of GRK1 expression. This attenuation can be overcome using phosphorylation-independent mutants of arrestin, especially when these are tethered to the opsin protein. We further show that point mutations targeting the Schiff base stability of the opsin can also reduce signalling lifetime. Finally, we apply one such mutation (E122Q) to improve the temporal fidelity of restored visual responses following ectopic opsin expression in the inner retina of a mouse model of retinal degeneration (rd1). Our results reveal that these two strategies (targeting either arrestin binding or Schiff-base hydrolysis) can produce more time-delimited opsin signalling under heterologous expression and establish the potential of this approach to improve optogenetic performance., (© 2023. The Author(s).)

Light-emitting diode (LED) irradiation has been used in the differentiation of mesenchymal stem cells into a variety of cell types. This study investigated the effect of green LED (GLED) irradiation on the differentiation of adipocyte-derived mesenchymal cells into hepatocyte-like cells (HLCs) and the mechanism of its action. HLCs in the hepatocyte maturation phase were irradiated with GLED (520 nm, 21 W/m 2 , 5 min/day for 10 days). The cells were then assessed for expression of hepatocyte maturity genes and opsin 3 (OPN3), hepatocyte function, viability, apoptosis, and levels of reactive oxygen species (ROS), intracellular adenosine triphosphate (ATP) and calcium ions (Ca 2+ ). GLED irradiation increased Alpha-1 antitrypsin and Ornithine transcarbamylase gene expression, promoted Cytochrome P450 3A4 activity and urea synthesis, and elevated intracellular ROS, ATP and Ca 2+ levels. OPN3 expression was significantly more upregulated in GLED-irradiated HLCs than in the non-irradiated HLCs. No significant difference in cell viability or apoptosis was observed between GLED-irradiated and non-irradiated HLCs. GLED irradiation can promote hepatocyte maturation and functions through OPN3. GLED irradiation also stimulated mitochondrial function via Ca 2+ /ATP/ROS activation. GLED irradiation has potential to support cell-based transplantation in patients., (© 2023. The Author(s).)

Melanopsin signaling within intrinsically photosensitive retinal ganglion cell (ipRGC) subtypes impacts a broad range of behaviors from circadian photoentrainment to conscious visual perception. Yet, how melanopsin phototransduction within M1-M6 ipRGC subtypes impacts cellular signaling to drive diverse behaviors is still largely unresolved. The identity of the phototransduction channels in each subtype is key to understanding this central question but has remained controversial. In this study, we resolve two opposing models of M4 phototransduction, demonstrating that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dispensable for this process and providing support for a pathway involving melanopsin-dependent potassium channel closure and canonical transient receptor potential (TRPC) channel opening. Surprisingly, we find that HCN channels are likewise dispensable for M2 phototransduction, contradicting the current model. We instead show that M2 phototransduction requires TRPC channels in conjunction with T-type voltage-gated calcium channels, identifying a novel melanopsin phototransduction target. Collectively, this work resolves key discrepancies in our understanding of ipRGC phototransduction pathways in multiple subtypes and adds to mounting evidence that ipRGC subtypes employ diverse phototransduction cascades to fine-tune cellular responses for downstream behaviors., Competing Interests: EC, JB, TS, LB, TS No competing interests declared

Melanopsin (OPN4) is a light-sensitive protein that plays a vital role in the regulation of circadian rhythms and other nonvisual functions. Current research on OPN4 has focused on mammals; more evidence is needed from non-mammalian vertebrates to fully assess the significance of the non-visual photosensitization of OPN4 for circadian rhythm regulation. There are species differences in the regulatory mechanisms of OPN4 for vertebrate circadian rhythms, which may be due to the differences in the cutting variants, tissue localization, and photosensitive activation pathway of OPN4. We here summarize the distribution of OPN4 in mammals, birds, and teleost fish, and the classical excitation mode for the non-visual photosensitive function of OPN4 in mammals is discussed. In addition, the role of OPN4-expressing cells in regulating circadian rhythm in different vertebrates is highlighted, and the potential rhythmic regulatory effects of various neuropeptides or neurotransmitters expressed in mammalian OPN4-expressing ganglion cells are summarized among them., (© 2023. Springer Nature Limited.)

The purpose was to quantify ocular dopamine in rabbits after stimulation of the optic nerve head with short-wavelength (blue) light to activate melanopsin expressed in the axons of intrinsically photosensitive retinal ganglion cells (ipRGCs). Dopamine levels in tears, aqueous humor, vitreous body, and retina (including choroid) were quantified after blue light stimulation of the optic nerve head of 15 rabbits with an optical fiber for 1 min, 10 min, or no stimulation (n = 5, each group). The left eye of all rabbits was operated on to introduce the optical fiber and stimulate the optic nerve, while the contralateral eye served as internal control. One minute of blue light stimulation significantly increased dopamine concentration in the vitreous body of the treated eyes compared to the contralateral ones (P = 0.015). Stimulation for 10 min significantly increased dopamine concentration in the vitreous body, as well as the aqueous humor (P < 0.05). Therefore, using an optical fiber approach to stimulate the optic nerve head with blue light significantly increased dopamine concentration in the aqueous humor and the vitreous body. This likely reflects an upregulation of retinal dopamine synthesis that could be attributed to ipRGC activation. However, the data provided in this study fell short of establishing a definitive link between dopamine release and ipRGC activation, mainly due to the lack of evidence supporting the expression of the melanopsin photopigment in the optic nerve., Competing Interests: Declaration of competing interest Hamed Bahmani and Tim Schilling are employees of Dopavision GmbH. Hamed Bahmani is also named as co-inventor for a patent application owned by Dopavision GmbH (WO2018/224671) which relates to a system and method to stimulate the optic nerve head. The remaining authors have no financial or intellectual property interest in any material or method mentioned., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mammalian type opsin 5 (Opn5m), a UV-sensitive G protein-coupled receptor opsin highly conserved in vertebrates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we examined Opn5m from diverse species using an aequorin luminescence assay and Gα-KO cell line. Beyond the commonly studied major Gα classes, Gαq, Gα11, Gα14, and Gα15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type Gα deletion and rescued by cotransfection with mouse and medaka Gq-type Gα proteins. Opn5m preferentially activated Gα14 and close relatives. Mutational analysis implicated specific regions, including α3-β5 and αG-α4 loops, αG and α4 helices, and the extreme C terminus, in the preferential activation of Gα14 by Opn5m. FISH revealed co-expression of genes encoding Opn5m and Gα14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of Gα14 by Opn5m is relevant for UV sensing in specific cell types., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the content of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Melanopsin is a photopigment that plays a role in non-visual, light-driven, cellular processes such as modulation of circadian rhythms, retinal vascular development, and the pupillary light reflex (PLR). In this study, computational methods were used to understand which chromophore is harbored by melanopsin in red-eared slider turtles (Trachemys scripta elegans). In mammals, the vitamin A derivative 11-cis-retinal (A1) is the chromophore, which provides functionality for melanopsin. However, in red-eared slider turtles, a member of the reptilian class, the identity of the chromophore remains unclear. Red-eared slider turtles, similar to other freshwater vertebrates, possess visual pigments that harbor a different vitamin A derivative, 11-cis-3,4-didehydroretinal (A2), making their pigments more sensitive to red-light than blue-light, therefore, suggesting the chromophore to be the A2 derivative instead of the A1. To help resolve the chromophore identity, in this work, computational homology models of melanopsin in red-eared slider turtles were first constructed. Next, quantum mechanics/molecular mechanics (QM/MM) calculations were carried out to compare how A1 and A2 derivatives bind to melanopsin. Time dependent density functional theory (TDDFT) calculations were then used to determine the excitation energy of the pigments. Lastly, calculated excitation energies were compared to experimental spectral sensitivity data from responses by the irises of red-eared sliders. Contrary to what was expected, our results suggest that melanopsin in red-eared slider turtles is more likely to harbor the A1 chromophore than the A2. Furthermore, a glutamine (Q62 2.56 ) and tyrosine (Y85 3.28 ) residue in the chromophore binding pocket are shown to play a role in the spectral tuning of the chromophore., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The intrinsically photosensitive retinal ganglion cells (ipRGCs) are known to serve non-image-forming functions, such as photoentrainment of the circadian rhythm and pupillary light reflex. However, how they affect human spatial vision is largely unknown. The spatial contrast sensitivity function (CSF), which measures contrast sensitivity as a function of spatial frequency, was used in the current study to investigate the function of ipRGCs in pattern vision. To compare the effects of different background lights on the CSF, we utilized the silent substitution technique. We manipulated the stimulation level of melanopsin (i.e., the visual pigment of ipRGCs) from the background light while keeping the cone stimulations constant, or vice versa. We conducted four experiments to measure the CSFs at various spatial frequencies, eccentricities, and levels of background luminance. Results showed that melanopsin stimulation from the background light enhances spatial contrast sensitivity across different eccentricities and luminance levels. Our finding that melanopsin contributes to CSF, combined with the receptive field analysis, suggests a role for the magnocellular pathway and challenges the conventional view that ipRGCs are primarily responsible for non-visual functions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Langerhans cell histiocytosis (LCH) is a type of -histiocytic disorder characterized by aberrant function, differentiation or proliferation of mononuclear phagocyte system cells, however, the pathogenesis is not fully understood. Opsin 3 (OPN3) plays an important role in regulating cell function., Objectives: We aimed to investigate OPN3 expression in LCH and Langerhans cells and evaluate its possible regulation of cellular function in a Langerhans cell-like cell line (ELD-1)., Materials & Methods: Expression of OPN3 in LCH and paired adjacent healthy skin tissue was determined using microscopic tools (immunohistochemical and immunofluorescence staining) and RNA scope. OPN3 protein and mRNA levels in primary dendritic cells and ELD-1 were measured by real-time quantitative PCR and western blotting, respectively. The effects of reduced or over-expressed OPN3 mRNA level, via a lentiviral vector, were examined on ELD-1 proliferation, migration, cell cycle and apoptosis using the Cell Counting Kit 8, EdU-594 kit, Transwell assays and Cell Cycle Analysis Kit and Annexin V-PE apoptosis kit, respectively. Lastly, the signalling pathway mediating these functions was investigated via RNA sequencing and western blotting., Results: OPN3 was highly expressed in human LCH tissue compared to healthy tissue, and was expressed in primary dendritic cells and ELD-1. Knockdown of OPN3 in ELD-1 inhibited cell proliferation, the cell cycle, and cell migration, while over-expression reversed these processes. These functions correlated with induction of the MAPK (p38/JNK/ERK) signalling pathway., Conclusion: Our results provide insight into the role of OPN3 in LCH which may become a molecular target for the clinical treatment of LCH.

Blue cone monochromacy (BCM) is a rare X-linked retinal disease characterized by the absence of L- and M-opsin in cone photoreceptors, considered a potential gene therapy candidate. However, most experimental ocular gene therapies utilize subretinal vector injection which would pose a risk to the fragile central retinal structure of BCM patients. Here we describe the use of ADVM-062, a vector optimized for cone-specific expression of human L-opsin and administered using a single intravitreal (IVT) injection. Pharmacological activity of ADVM-062 was established in gerbils, whose cone-rich retina naturally lacks L-opsin. A single IVT administration dose of ADVM-062 effectively transduced gerbil cone photoreceptors and produced a de novo response to long-wavelength stimuli. To identify potential first-in-human doses we evaluated ADVM-062 in non-human primates. Cone-specific expression of ADVM-062 in primates was confirmed using ADVM-062.myc, a vector engineered with the same regulatory elements as ADVM-062. Enumeration of human OPN1LW.myc-positive cones demonstrated that doses ≥3 × 10 10 vg/eye resulted in transduction of 18%-85% of foveal cones. A Good Laboratory Practice (GLP) toxicology study established that IVT administration of ADVM-062 was well tolerated at doses that could potentially achieve clinically meaningful effect, thus supporting the potential of ADVM-062 as a one-time IVT gene therapy for BCM., Competing Interests: Declaration of interests K.H., D.C., J.N., C.D., P.S., K.O.B., M.R., B.S., C.M.G., B.E.R., and R.N.G. are current or previous employees of Adverum Biotechnologies and hold stock grants. J.V.H. is a paid consultant of Adverum Biotechnologies., (Copyright © 2023 Adverum Biotechnologies Inc. Published by Elsevier Inc. All rights reserved.)

Animal opsins, light-sensitive G protein-coupled receptors, have been used for optogenetic tools to control G protein-dependent signaling pathways. Upon G protein activation, the Gα and Gβγ subunits drive different intracellular signaling pathways, leading to complex cellular responses. For some purposes, Gα- and Gβγ-dependent signaling needs to be separately modulated, but these responses are simultaneously evoked due to the 1:1 stoichiometry of Gα and Gβγ Nevertheless, we show temporal activation of G protein using a self-inactivating invertebrate opsin, Platynereis c-opsin1, drives biased signaling for Gβγ-dependent GIRK channel activation in a light-dependent manner by utilizing the kinetic difference between Gβγ-dependent and Gα-dependent responses. The opsin-induced transient Gi/o activation preferentially causes activation of the kinetically fast Gβγ-dependent GIRK channels rather than slower Gi/oα-dependent adenylyl cyclase inhibition. Although similar Gβγ-biased signaling properties were observed in a self-inactivating vertebrate visual pigment, Platynereis c-opsin1 requires fewer retinal molecules to evoke cellular responses. Furthermore, the Gβγ-biased signaling properties of Platynereis c-opsin1 are enhanced by genetically fusing with RGS8 protein, which accelerates G protein inactivation. The self-inactivating invertebrate opsin and its RGS8-fusion protein can function as optical control tools biased for Gβγ-dependent ion channel modulation.

The presence of melanopsin (OPN4) has been shown in cultured murine melanocytes and was associated with ultraviolet A radiation (UVA) reception. Here we demonstrated the protective role of OPN4 in skin physiology and the increased UVA-induced damage in its absence. Histological analysis showed a thicker dermis and thinner hypodermal white adipose tissue layer in Opn4 -/- (KO) mice than in wild-type (WT) animals. Proteomics analyses revealed molecular signatures associated with proteolysis, remodeling chromatin, DNA damage response (DDR), immune response, and oxidative stress coupled with antioxidant responses in the skin of Opn4 KO mice compared to WT. Skin protein variants were found in Opn4 KO mice and Opn2, Opn3, and Opn5 gene expressions were increased in the genotype. We investigated each genotype response to UVA stimulus (100 kJ/m 2 ). We found an increase of Opn4 gene expression following stimulus on the skin of WT mice suggesting melanopsin as a UVA sensor. Proteomics findings suggest that UVA decreases DDR pathways associated with ROS accumulation and lipid peroxidation in the skin of Opn4 KO mice. Relative changes in methylation (H3-K79) and acetylation sites of histone between genotypes and differentially modulated by UVA stimulus were also observed. We also identified alterations of molecular traits of the central hypothalamus-pituitary- adrenal (HPA) and the skin HPA-like axes in the absence of OPN4. Higher skin corticosterone levels were detected in UVA-stimulated Opn4 KO compared to irradiated WT mice. Taken altogether, functional proteomics associated with gene expression experiments allowed a high-throughput evaluation that suggests an important protective role of OPN4 in regulating skin physiology in the presence and absence of UVA radiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

State-of-the-art all-optical systems promise unprecedented access to neural activity in vivo , using multiphoton optogenetics to allow simultaneous imaging and control of activity in selected neurons at cellular resolution. However, to achieve wide use of all-optical stimulation and imaging, simple strategies are needed to robustly and stably express opsins and indicators in the same cells. Here, we describe a bicistronic adeno-associated virus (AAV) that expresses both the fast and bright calcium indicator jGCaMP8s, and a soma-targeted (st) and two-photon-activatable opsin, ChrimsonR. With this method, stChrimsonR stimulation with two-photon holography in the visual cortex of mice drives robust spiking in targeted cells, and neural responses to visual sensory stimuli and spontaneous activity are strong and stable. Cells expressing this bicistronic construct show responses to both photostimulation and visual stimulation that are similar to responses measured from cells expressing the same opsin and indicator via separate viruses. This approach is a simple and robust way to prepare neurons in vivo for two-photon holography and imaging., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 LaFosse et al.)

This study investigated the effect of low-intensity blue light on the albino Wistar rat retina, including intrinsically photosensitive retinal ganglion cells (ipRGCs). Three groups of nine albino Wistar rats were used. One group was continuously exposed to blue light (150 lx) for 2 d (STE); one was exposed to 12 h of blue light and 12 h of darkness for 10 d (LTE); one was maintained in 12 h of white light (150 lx) and 12 h of darkness for 10 d (control). Melanopsin (Opn4) was immunolabelled on retinal whole-mounts. To count and measure Opn4-positive ipRGC somas and dendrites (including Sholl profiles), Neuron J was used. Retinal cryosections were immunolabeled for glial fibrillary acid protein (GFAP) and with terminal deoxynucleotidyl transferase dUTP nick-end labelling for apoptosis detection. LTE reduced the length of Opn4-positive ipRGC dendrites ( p = 0.03) and decreased Opn4-immunoreactivity in ipRGC outer stratifying dendrites. LTE and STE decreased the complexity of dendritic arborization (Sholl profile; p < 0.001, p = 0.03, respectively), increased retinal GFAP immunoreactivity ( p < 0.001, p = 0.002, respectively), and caused outer segment vesiculation and outer nuclear layer apoptosis. Ultrastructural analysis showed that LTE damaged mitochondria in retinal ganglion cells and in the inner plexiform layer. Thus, LTE to low-intensity blue light harms the retinas of albino Wistar rats.

The retinas of fishes often have single and double cone photoreceptors that are organized in lattice-like mosaics. In flatfishes experiencing eye migration (i.e., the metamorphic process whereby one eye migrates to the other side of the head), the hexagonal lattice of single cones present in the larva undergoes major restructuring resulting in a dominant square mosaic postmetamorphosis consisting of four double cones surrounding each single cone. The expression of different opsin types during eye migration has not been examined despite its importance in understanding photoreceptor plasticity and whether cell fate (in terms of spectral phenotype) could influence square mosaic formation. Here, we probed the retina of Atlantic halibut undergoing eye migration for opsin expression using two antibodies, AHblue and AB5407, that labeled short wavelength sensitive 2 (SWS2) opsin and longer wavelength (predominantly middle wavelength sensitive, RH2) opsins, respectively. Throughout the retina, double and triple cones labeled with AB5407 exclusively, whereas the vast majority of single cones labeled with AHblue. A minority (<5%) of single cones in the square mosaic of the centroventral retina labeled with AB5407. In regions of mosaic transition and near peripheral growth zones, some single cones co-expressed at least two opsins as they labeled with both antibodies. Short wavelength (SWS2 expressing, or S) cones formed a nonrandom mosaic gradient from central to dorsal retina in a region dominated by the larval single cone mosaic. Our results demonstrate the expression of at least two opsins throughout the postmetamorphic retina and suggest opsin switching as a mechanism to create new cone spectral phenotypes. In addition, the S cone gradient at the onset of eye migration may underlie a plastic, cell induction mechanism by which a cone's phenotype determines that of its neighbors and the formation of the square mosaic., (© 2022 Wiley Periodicals LLC.)

Opsins, light-sensitive G protein-coupled receptors, have been identified in corals but their properties are largely unknown. Here, we identified six opsin genes (acropsins 1-6) from a coral species Acropora millepora, including three novel opsins (acropsins 4-6), and successfully characterized the properties of four out of the six acropsins. Acropsins 1 and 6 exhibited light-dependent cAMP increases in cultured cells, suggesting that the acropsins could light-dependently activate Gs-type G protein like the box jellyfish opsin from the same opsin group. Spectral sensitivity curves having the maximum sensitivities at ~ 472 nm and ~ 476 nm were estimated for acropsins 1 and 6, respectively, based on the light wavelength-dependent cAMP increases in these opsins-expressing cells (heterologous action spectroscopy). Acropsin 2 belonging to the same group as acropsins 1 and 6 did not induce light-dependent cAMP or Ca 2+ changes. We then successfully estimated the acropsin 2 spectral sensitivity curve having its maximum value at ~ 471 nm with its chimera mutant which possessed the third cytoplasmic loop of the Gs-coupled jellyfish opsin. Acropsin 4 categorized as another group light-dependently induced intracellular Ca 2+ increases but not cAMP changes. Our results uncovered that the Acropora coral possesses multiple opsins coupling two distinct cascades, cyclic nucleotide and Ca 2+ signaling light-dependently., (© 2023. The Author(s).)

The function of a hormone receptor requires mechanisms to control precisely where, when, and at what level the receptor gene is expressed. An intriguing case concerns the selective induction of thyroid hormone receptor β2 (TRβ2), encoded by Thrb, in the pituitary and also in cone photoreceptors, in which it critically regulates expression of the opsin photopigments that mediate color vision. Here, we investigate the physiological significance of a candidate enhancer for induction of TRβ2 by mutagenesis of a conserved intron region in its natural context in the endogenous Thrb gene in mice. Mutation of e-box sites for bHLH (basic-helix-loop-helix) transcription factors preferentially impairs TRβ2 expression in cones whereas mutation of nearby sequences preferentially impairs expression in pituitary. A deletion encompassing all sites impairs expression in both tissues, indicating bifunctional activity. In cones, the e-box mutations disrupt chromatin acetylation, blunt the developmental induction of TRβ2, and ultimately impair cone opsin expression and sensitivity to longer wavelengths of light. These results demonstrate the necessity of studying an enhancer in its natural chromosomal context for defining biological relevance and reveal surprisingly critical nuances of level and timing of enhancer function. Our findings illustrate the influence of noncoding sequences over thyroid hormone functions., (Published by Oxford University Press on behalf of the Endocrine Society 2023.)

Nonimage-forming vision in mammals is mediated primarily by melanopsin (OPN4)-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs). In mouse M1-ipRGCs, melanopsin predominantly activates, via Gα q,11,14 , phospholipase C-β4 to open transient receptor 6 (TRPC6) and TRPC7 channels. In M2- and M4-ipRGCs, however, a prominent phototransduction mechanism involves the opening of hyperpolarization- and cyclic nucleotide-gated channels via cyclic nucleotide, although the upstream steps remain uncertain. We report here experiments, primarily on M4-ipRGCs, with photo-uncaging of cyclic nucleotides and virally expressed CNGA2 channels to conclude that the second messenger is cyclic adenosine monophosphate (cAMP) - very surprising considering that cyclic guanosine monophosphate (cGMP) is used in almost all cyclic nucleotide-mediated phototransduction mechanisms across the animal kingdom. We further found that the upstream G protein is likewise G q , which via its Gβγ subunits directly activates adenylyl cyclase (AC). Our findings are a demonstration in a native cell of a cross-motif GPCR signaling pathway from G q directly to AC with a specific function.

Opsin 3 (OPN3), a member of the light-sensitive, retinal-dependent opsin family, is widely expressed in a variety of human tissues and plays a multitude of light-dependent and light-independent roles. We recently identified five missense variants of OPN3, including p. I51T, p. V134A, p. V183I, p. M256I and p. C331Y, in human melanocytic tumours. However, it remains unclear how these OPN3 variants affect OPN3 protein structure and function. Herein, we conducted structural and functional studies of these variant proteins in OPN3 by molecular docking and molecular dynamics simulations. Moreover, we performed in vitro fluorescence calcium imaging to assess the functional properties of five single-nucleotide variant (SNV) proteins using a site-directed mutagenesis method. Notably, the p. I51T variant was not able to effectively dock with 11-cis-retinal. Additionally, in vitro, the p. I51T SNVs failed to induce any detectable changes in intracellular Ca 2+ concentration at room temperature. Taken together, these results reveal that five SNVs in the OPN3 gene have deleterious effects on protein structure and function, suggesting that these mutations, especially the p. I51T variant, significantly disrupt the canonical function of the OPN3 protein. Our findings provide new insight into the role of OPN3 variants in the loss of protein function., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Zebrafish retinal cone signals shift in spectral shape through larval, juvenile, and adult development as expression patterns of eight cone-opsin genes change. An algorithm extracting signal amplitudes for the component cone spectral types is developed and tested on two thyroxin receptor β2 (trβ2) gain-of-function lines crx:mYFP-2A-trβ2 and gnat2:mYFP-2A-trβ2 , allowing correlation between opsin signaling and opsin immunoreactivity in lines with different developmental timing and cell-type expression of this red-opsin-promoting transgene. Both adult transgenics became complete, or nearly complete, "red-cone dichromats," with disproportionately large long-wavelength-sensitive (LWS)1 opsin amplitudes as compared with controls, where LWS1 and LWS2 amplitudes were about equal, and significant signals from SWS1, SWS2, and Rh2 opsins were detected. But in transgenic larvae and juveniles of both lines it was LWS2 amplitudes that increased, with LWS1 cone signals rarely encountered. In gnat2:mYFP-2A-trβ2 embryos at 5 d postfertilization (dpf), red-opsin immunoreactive cone density doubled, but red-opsin amplitudes (LWS2) increased <10%, and green-opsin, blue-opsin, and UV-opsin signals were unchanged, despite co-expressed red opsins, and the finding that an sws1 UV-opsin reporter gene was shut down by the gnat2:mYFP-2A-trβ2 transgene. By contrast both LWS2 red-cone amplitudes and the density of red-cone immunoreactivity more than doubled in 5-dpf crx:mYFP-2A-trβ2 embryos, while UV-cone amplitudes were reduced 90%. Embryonic cones with trβ2 gain-of-function transgenes were morphologically distinct from control red, blue or UV cones, with wider inner segments and shorter axons than red cones, suggesting cone spectral specification, opsin immunoreactivity and shape are influenced by the abundance and developmental timing of trβ2 expression., Competing Interests: The authors declare no competing financial interests., (Copyright © 2022 Nelson et al.)

N-terminal P23H opsin mutation accounts for most of retinitis pigmentosa (RP) cases. P23H functions and folding can be rescued by small chaperone ligands, which contributes to validate mutant opsin as a suitable target for pharmacological treatment of RP. However, the lack of structural details on P23H mutant opsin strongly impairs drug design, and new chemotypes of effective chaperones of P23H opsin are in high demand. Here, a computational-boosted workflow combining homology modeling with molecular dynamics (MD) simulations and virtual screening was used to select putative P23H opsin chaperones among different libraries through a structure-based approach. In vitro studies corroborated the reliability of the structural model generated in this work and identified a number of novel chemotypes of safe and effective chaperones able to promote P23H opsin trafficking to the outer cell membrane.

The common final pathway to blindness in many forms of retinal degeneration is the death of the light-sensitive primary retinal neurons. However, the normally light-insensitive second- and third-order neurons persist optogenetic gene therapy aims to restore sight by rendering such neurons light-sensitive. Here, we investigate whether bReaChES, a newly described high sensitivity Type I opsin with peak sensitivity to long-wavelength visible light, can restore vision in a murine model of severe early-onset retinal degeneration. Intravitreal injection of an adeno-associated viral vector carrying the sequence for bReaChES downstream of the calcium calmodulin kinase IIα promoter resulted in sustained retinal expression of bReaChES. Retinal ganglion cells (RGCs) expressing bReaChES generated action potentials at light levels consistent with bright indoor lighting (from 13.6 log photons cm -2  s -1 ). They could also detect flicker at up to 50 Hz, which approaches the upper temporal limit of human photopic vision. Topological response maps of bReaChES-expressing RGCs suggest that optogenetically activated RGCs may demonstrate similar topographical responses to RGCs stimulated by photoreceptor activation. Furthermore, treated dystrophic mice displayed restored cortical neuronal activity in response to light and rescued behavioral responses to a looming stimulus that simulated an aerial predator. Finally, human surgical retinal explants exposed to the bReaChES treatment vector demonstrated transduction. Together, these findings suggest that intravitreal gene therapy to deliver bReaChES to the retina may restore vision in human retinal degeneration in vivo at ecologically relevant light levels with spectral and temporal response characteristics approaching those of normal human photopic vision., (© 2022. The Author(s).)

Opsin-3 ( Opn3 , encephalopsin) was the first nonvisual opsin gene discovered in mammals. Since then, several Opn3 functions have been described, and in two cases (adipose tissue, smooth muscle) light sensing activity is implicated. In addition to peripheral tissues, Opn3 is robustly expressed within the central nervous system, for which it derives its name. Despite this expression, no studies have investigated developmental or adult CNS consequences of Opn3 loss-of-function. Here, the behavioral consequences of mice deficient in Opn3 were investigated. Opn3 -deficient mice perform comparably to wild-type mice in measures of motor coordination, socialization, anxiety-like behavior, and various aspects of learning and memory. However, Opn3 -deficient mice have an attenuated acoustic startle reflex (ASR) relative to littermates. This deficit is not because of changes in hearing sensitivity, although Opn3 was shown to be expressed in auditory and vestibular structures, including cochlear outer hair cells. Interestingly, the ASR was not acutely light-dependent and did not vary between daytime and nighttime trials, despite known functions of Opn3 in photoreception and circadian gene amplitude. Together, these results demonstrate the first role of Opn3 on behavior, although the role of this opsin in the CNS remains largely elusive., (Copyright © 2022 Upton et al.)

During development, melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) become light sensitive much earlier than rods and cones. IpRGCs project to many subcortical areas, whereas physiological functions of these projections are yet to be fully elucidated. Here, we found that ipRGC-mediated light sensation promotes synaptogenesis of pyramidal neurons in various cortices and the hippocampus. This phenomenon depends on activation of ipRGCs and is mediated by the release of oxytocin from the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) into cerebral-spinal fluid. We further characterized a direct connection between ipRGCs and oxytocin neurons in the SON and mutual projections between oxytocin neurons in the SON and PVN. Moreover, we showed that the lack of ipRGC-mediated, light-promoted early cortical synaptogenesis compromised learning ability in adult mice. Our results highlight the importance of light sensation early in life on the development of learning ability and therefore call attention to suitable light environment for infant care., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)