Phototransduction Research Articles

Generation and characterization of a Stargardt’s disease-specific induced pluripotent stem cell line (LVPEIi008-A) with a homozygous nonsense mutation in exon 44 of ABCA4

The Stargardt’s Disease, Type 1 (STGD1) is associated with the loss of function mutations in ABCA4. This gene codes for a retina-specific, ATP-binding cassette (ABC) family transporter, involved in the transport of the key visual cycle intermediate, all-trans-retinaldehyde (atRAL), across the photoreceptor cell membranes. Here, we report the establishment of a patient-specific, iPSC line (LVPEIi008-A), that carries a homozygous nonsense mutation at (c.6088C > T) position, within exon 44 of ABCA4. The patient-specific skin fibroblasts were reprogrammed using episomal plasmids and the stably expanding iPSC line expressed the key stemness and pluripotency markers, maintained its chromosomal integrity and tested negative for mycoplasma.

Ablation of Fatty Acid Transport Protein-4 Enhances Cone Survival, M-cone Vision, and Synthesis of Cone-Tropic 9-cis-Retinal in rd12 Mouse Model of Leber Congenital Amaurosis.

The canonical visual cycle employing RPE65 as the retinoid isomerase regenerates 11-cis-retinal to support both rod- and cone-mediated vision. Mutations of RPE65 are associated with Leber congenital amaurosis that results in rod and cone photoreceptor degeneration and vision loss of affected patients at an early age. Dark-reared Rpe65-/- mouse has been known to form isorhodopsin that employs 9-cis-retinal as the photosensitive chromophore. The mechanism regulating 9-cis-retinal synthesis and the role of the endogenous 9-cis-retinal in cone survival and function remain largely unknown. In this study, we found that ablation of fatty acid transport protein-4 (FATP4), a negative regulator of 11-cis-retinol synthesis catalyzed by RPE65, increased the formation of 9-cis-retinal, but not 11-cis-retinal, in a light-independent mechanism in both sexes of RPE65-null rd12 mice. Both rd12 and rd12;Fatp4-/- mice contained a massive amount of all-trans-retinyl esters in the eyes, exhibiting comparable scotopic vision and rod degeneration. However, expression levels of M- and S-opsins as well as numbers of M- and S-cones surviving in the superior retinas of rd12;Fatp4-/ - mice were at least twofold greater than those in age-matched rd12 mice. Moreover, FATP4 deficiency significantly shortened photopic b-wave implicit time, improved M-cone visual function, and substantially deaccelerated the progression of cone degeneration in rd12 mice, whereas FATP4 deficiency in mice with wild-type Rpe65 alleles neither induced 9-cis-retinal formation nor influenced cone survival and function. These results identify FATP4 as a new regulator of synthesis of 9-cis-retinal, which is a "cone-tropic" chromophore supporting cone survival and function in the retinas with defective RPE65.

In search of the molecular mechanisms of adaptation memory in rods: basic activity of phosphodiesterase

Retinal rods, the photoreceptors responsible for twilight vision, are capable of adapting to a wide range of light levels. The molecular mechanisms of light adaptation have been well studied, but an interesting question is what changes occur in the phototransduction cascade after the adaptive light stimuli are eliminated. Previously, we showed the phenomenon of adaptation memory in amphibian rods: after background illumination photoreceptor sensitivity to light remained reduced for several minutes, while the dark current recovered within 20—30 s. This suggests the existence of additional, as yet unknown, regulatory mechanisms of the phototransduction cascade that act after the adaptive effect of light. In search of specific mechanisms that could explain the effect of adaptation memory, we performed electrophysiological experiments on isolated frog rods to evaluate the basal activity of the effector enzyme of the phototransduction cascade, the phosphodiesterase type 6, in the dark and after saturating background illumination. It was found that the post-adaptation state of rods was characterized by increased basal phosphodiesterase activity, which gradually decreased to the dark level within tens of seconds after turning off the adaptive illumination. These results also suggest that the components of the phototransduction cascade may undergo some unstudied changes after light adaptation.

Dominant role for pigment epithelial CRALBP in supplying visual chromophore to photoreceptors

Cellular retinaldehyde-binding protein (CRALBP) supports production of 11-cis-retinaldehyde and its delivery to photoreceptors. It is found in the retinal pigment epithelium (RPE) and Müller glia (MG), but the relative functional importance of these two cellular pools is debated. Here, we report RPE- and MG-specific CRALBP knockout (KO) mice and examine their photoreceptor and visual cycle function. Bulk visual chromophore regeneration in RPE-KO mice is 15-fold slower than in controls, accounting for their delayed rod dark adaptation and protection against retinal phototoxicity, whereas MG-KO mice have normal bulk visual chromophore regeneration and retinal light damage susceptibility. Cone pigment regeneration is significantly impaired in RPE-KO mice but mildly affected in MG-KO mice, disclosing an unexpectedly strong reliance of cone photoreceptors on the RPE-based visual cycle. These data reveal a dominant role for RPE-CRALBP in supporting rod and cone function and highlight the importance of RPE cell targeting for CRALBP gene therapies.

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina

Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation. We found that photoreceptor fluorescence lifetimes increase and decrease in sync with light and dark exposure, respectively. This is likely due to changes in all-trans-retinol and all-trans-retinal levels in the outer segments, mediated by phototransduction and visual cycle activity. During light exposure, RPE fluorescence lifetime was observed to increase steadily over time, as a result of all-trans-retinol accumulation during the visual cycle and decreasing metabolism caused by the lack of normal perfusion of the sample. Our system can measure the fluorescence lifetime of intrinsic retinal fluorophores on a cellular scale, revealing differences in lifetime between retinal cell classes under different conditions of light and dark exposure.

DisCrit Mothering As A Radical Act

Influenced by revolutionary mothering, activists of color, and radical feminists, this article explores Neurodivergent families through the lens of Disability Critical Race Theory. As a parent of an Autistic child, I embark on redefining my role by integrating art and design into community advocacy for Autistic communities. I challenge unjust stigmas by breaking visual cycles within social and clinical practices. This journey culminated in the creation of “KnoxRoxs,” a photo zine dedicated to my Autistic son, aimed at increasing visibility for children of color in Neurodiverse communities. I emphasize the multifaceted nature of Neurodivergent mothering, involving caregiving, community engagement, and creative resistance.

The mouse retinal pigment epithelium mounts an innate immune defense response following retinal detachment

The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNA-Seq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes (Bmp4, Crim1, Degs1, Gja1, Itgav, Mfap3l, Pdpn, Ptgds, Rbp1, Rnf13, Rpe65, Slc4a2, Sulf1 and Ttr) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd) were defined as mRNA with a significant (padj≤0.05) fold change (FC) of 0.67 ≥ FC ≥ 1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 (Lcn2) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, and tight junctions were downregulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b. Validation studies confirmed RNA-Seq results. Thus, the RPE quickly downregulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space.

Exposure to flutolanil at environmentally relevant concentrations can induce image and non-image-forming failure of zebrafish larvae through neuro and visual disruptions

Numerous pesticides pose a threat to aquatic ecosystems, jeopardizing aquatic animal species and impacting human health. While the contamination of aquatic environment by flutolanil and its adverse effects on animal in the treatment of rich sheath blight have been reported, the neuro-visual effects of flutolanil at environmentally relevant concentrations remain unknown. In this study, we administered flutolanil to zebrafish embryos (0, 0.125, 0.50 and 2.0 mg/L) for 4 days to investigate its impact on the neuro and visual system. The results revealed that flutolanil induced abnormal behavior in larvae, affecting locomotor activity, stimuli response and phototactic response. Additionally, it led to defective brain and ocular development and differentiation. The disruption extended to the neurological system and visual phototransduction of larvae, evidenced by significant disturbances in genes and proteins related to neurodevelopment, neurotransmission, eye development, and visual function. Untargeted metabolomics analysis revealed that the GABAergic signaling pathway and increased levels of glutamine, glutamate, andγ-aminobutyric acid were implicated in the response to neuro and visual system injury induced by flutolanil, contributing to aberrant development, behavioral issues, and endocrine disruption. This study highlights the neuro-visual injury caused by flutolanil in aquatic environment, offering fresh insights into the mechanisms underlying image and non-image effects.

In vivo retinoid metabolic and visual cycle pathways assessment of carotenoid-rich refined red palm-pressed mesocarp olein

The Palm-pressed mesocarp is a by-product from the palm oil mill and is comprised of 5–7 % of residual oil, which could be further refined into red palm-pressed mesocarp olein (PPMO). This study aimed to obtain the chemical profile of PPMO and assess its eye health effects through retinoid metabolic and visual cycle pathways regulation. PPMO contains exceptionally high vitamin E (1819.0 ppm) and carotenoid (1113.8 ppm) contents with a similar fatty acid composition to palm olein. The rats fed with 2 g/kg PPMO daily for 84 days through oral gavage up-regulated the retinol and retinyl palmitate levels, as well as the mRNA and protein expression levels of RBP4 in blood, CRBP1 and LRAT in the liver, RPE65 and RDH5 in the eye. The outcomes showed that PPMO showed comparable regulation effects with β-carotene in the retinoid metabolic and visual cycle pathways, revealing it as a phytonutrient-rich edible oil with beneficial eye health effects.

A CASE REPORT: EXPLORING STARGARDT DISEASE - CLINICAL FEATURES AND GENETIC INSIGHTS

Stargardt disease (SD) stands as a prominent inherited macular dystrophy affecting both adults and children, characterized by bilateral and symmetrical maculopathy with autosomal recessive transmission linked to the ABCA4 gene. We aim to present a comprehensive exploration of SD, delving into its clinical manifestations, genetic intricacies, and potential therapeutic interventions. This case report highlights a 46-year-old north African male with late-onset STGD with gradual bilateral vision decline, central macular atrophy, and yellow deposits in the perimacular region. the spectrum of SD is diverse, ranging from childhood onset to adult manifestation, often mimicking age-related maculopathy. Stargardt diseases genetic landscape is intricately governed by the ABCA4 gene, marked by over 900 variants across its 50 exons. This genetic diversity extends to dominant genes such as STGD4, ELOVL4, and PRPH2, contributing to a spectrum of retinal disorders. Therapeutic approaches for Stargardt disease involve drugs targeting the visual cycle, a modified vitamin A, and ongoing gene replacement and stem cell therapy trials This comprehensive review amalgamates clinical observations, genetic considerations, and emerging therapeutic avenues.

Unraveling the mystery of ocular retinoid turnover: Insights from albino mice and the role of STRA6

A delicate balance between photon absorption for vision and protection of photoreceptors from light damage is pivotal for ocular health. This equilibrium is governed by the light-absorbing 11-cis-retinylidene chromophore of visual pigments, which, upon bleaching, transforms into all-trans-retinal and undergoes regeneration through an enzymatic pathway, named the visual cycle. Chemical side reactions of retinaldehydes during the recycling process can generate byproducts that may result in a depletion of retinoids. In our study, we have clarified the crucial roles played by melanin pigmentation and the retinoid transporter STRA6 in preventing this loss and preserving the integrity of the visual cycle. Our experiments initially confirmed that consecutive green and blue light bleaching of isolated bovine rhodopsin produced 9-cis and 13-cis retinal. The same unusual retinoids were found in the retinas of mice exposed to intense light, with elevated concentrations observed in albino mice. Examining the metabolic fate of these visual cycle byproducts revealed that 9-cis-retinal, but not 13-cis-retinal, was recycled back to all-trans-retinal through an intermediate called isorhodopsin. However, investigations in Stra6 knockout mice unveiled that the generation of these visual cycle byproducts correlated with a light-induced loss of ocular retinoids and visual impairment. Collectively, our findings uncover important novel aspects of visual cycle dynamics, with implications for ocular health and photoreceptor integrity.

Photoreception and phototransduction in human skin

Light allows us to see. Seeing begins with photoreceptors in the retina where light is absorbed, converted into an electrical response and transmitted to the brain. Photoreception is highly conserved, and in animals, almost exclusively based on a single class of proteins, the opsins. Image-forming information goes to the lateral geniculate nucleus and eventual processing into visual images, sight. What may not be immediately apparent is the transmission of non-image information. However, cells in the skin contain the full set of opsins and their phototransduction cascades which are active, cover the whole solar spectrum and appear to participate in the skin’s protective mechanisms. This is obviously related to collection of non-image information. Why is this system of photoreception and transmission duplicated in the skin and what is its function?

De novo pigmentation of amelanotic retinal pigment epithelium – An ex vivo model

Purpose: Retinal Pigment Epithelial (RPE) cells perform critical functions in the visual cycle. Their melanin pigmentation, which is organized into specialized compartments ‐ melanosomes, is highly critical for proper vision. We hereby show an ex vivo model for de novo pigmentation of RPE cells.Methods: A chemical method to induce pigmentation in a non‐pigment model of ARPE‐19 cells was applied using L‐DOPA as a repurposed drug from the current treatment of Parkinson's disease.Results: L‐DOPA was optimized for its toxic effect on ARPE‐19 cells along with pigmentation development. We found 1000 μM L‐DOPA to start inducing pigmentation of RPEs by Day 3, and achieve full pigmentation by Day 5. The gene expression of RPE‐specific markers (Tyrosinase, CRALBP, PEDF) was significantly higher in L‐DOPA‐treated ARPE‐19 compared to non‐treated ones. Positive expression for Tyrosinase was confirmed by ICC at both Day 3 and Day 5 of L‐DOPA treatment. Transmission electron microscopy showed the de novo melanosome formation with ultrastructural features of various stages of maturity (Stage I to IV), apical‐basal polarity, and melanosomes localization on the apical side of the RPEs in the L‐DOPA treated cells.Conclusions: Our study showed that L‐DOPA treatment could induce de novo melanosome formation in amelanotic RPE cells.Keywords: RPE, Melanosomes, L‐DOPA, Pigmentation.

Phenotypic high-throughput screening identifies aryl hydrocarbon receptor agonism as common inhibitor of toxin-induced retinal pigment epithelium cell death.

The retinal pigment epithelium (RPE) is essential to maintain retinal function, and RPE cell death represents a key pathogenic stage in the progression of several blinding ocular diseases, including age-related macular degeneration (AMD). To identify pathways and compounds able to prevent RPE cell death, we developed a phenotypic screening pipeline utilizing a compound library and high-throughput screening compatible assays on the human RPE cell line, ARPE-19, in response to different disease relevant cytotoxic stimuli. We show that the metabolic by-product of the visual cycle all-trans-retinal (atRAL) induces RPE apoptosis, while the lipid peroxidation by-product 4-hydroxynonenal (4-HNE) promotes necrotic cell death. Using these distinct stimuli for screening, we identified agonists of the aryl hydrocarbon receptor (AhR) as a consensus target able to prevent both atRAL mediated apoptosis and 4-HNE-induced necrotic cell death. This works serves as a framework for future studies dedicated to screening for inhibitors of cell death, as well as support for the discussion of AhR agonism in RPE pathology.

First results of long-term follow-up of children in Russia after gene therapy for hereditary retinal dystrophies associated with biallelic mutations in the RPE65 gene

Purpose: to evaluate the results of gene therapy by the recombinant adeno-associated viral vector voretigene neparvovec (VN) in children with follow-up periods of 1, 3, 6, and 12 months.Material and methods. The study included 6 children (12 eyes) aged 5 to 15 with a confirmed biallelic mutation in the RPE65 gene, treated with VN (Luxturna, USA). Treatment efficacy was assessed by surveying which included questions on spatial orientation under various types of illumination, Goldman perimetry, microperimetry, best corrected visual acuity (BCVA), electroretinogram (ERG), and visual evoked potentials (VEP). To assess the structure of the retina, the central retinal thickness (CRT) was evaluated by optical coherence tomography.Results. All children showed subjective changes in visual perception, including improved orientation in the dark and twilight, and improved contrast. In one case, the child with initially low visual acuity showed improved visual fixation. In 4 patients out of 6 (8 eyes), an expansion of the visual fields was noted, including 2 cases who displayed significant expansion thereof. In 2 patients (4 eyes), the visual fields were not narrowed and remained so throughout the entire observation period. Mean light sensitivity of the retina in 3 patients and fixation indices in 1 patient improved significantly as shown by microperimetry. BCVA remained stable throughout the study or changed insignificantly. Initially, ERG could not be detected in 8 eyes, but after an VN injection, 6 eyes demonstrated a partial recovery at different times — from 1 to 12 months. An increase in the amplitude of the P1 component to pattern VEP and P2 component to flash VEP was observed in all patients, which indicates an enhanced activity in the projection of the visual cortex after the restoration of the visual cycle. No significant changes were revealed in CRT (p = 0.9). Complications and adverse events were noted in 9 eyes (75 %): chorioretinal dystrophy at the injection site in 3 patients (5 eyes), multifocal nummular dystrophy in 2 patients (4 eyes), local episcleritis in 1 eye, transient increase in intraocular pressure in 2 patients (3 eyes).Conclusion. The results of a one-year post VN treatment follow-up of Russian patients with RPE65-associated inherited retinal disease demonstrate stabilization and improvement of visual functions, which is especially important for otherwise incurable patients with a progressive course of the disease.

Scavenging of Cation Radicals of the Visual Cycle Retinoids by Lutein, Zeaxanthin, Taurine, and Melanin.

In the retina, retinoids involved in vision are under constant threat of oxidation, and their oxidation products exhibit deleterious properties. Using pulse radiolysis, this study determined that the bimolecular rate constants of scavenging cation radicals of retinoids by taurine are smaller than 2 × 107 M-1s-1 whereas lutein scavenges cation radicals of all three retinoids with the bimolecular rate constants approach the diffusion-controlled limits, while zeaxanthin is only 1.4-1.6-fold less effective. Despite that lutein exhibits greater scavenging rate constants of retinoid cation radicals than other antioxidants, the greater concentrations of ascorbate in the retina suggest that ascorbate may be the main protectant of all visual cycle retinoids from oxidative degradation, while α-tocopherol may play a substantial role in the protection of retinaldehyde but is relatively inefficient in the protection of retinol or retinyl palmitate. While the protection of retinoids by lutein and zeaxanthin appears inefficient in the retinal periphery, it can be quite substantial in the macula. Although the determined rate constants of scavenging the cation radicals of retinol and retinaldehyde by dopa-melanin are relatively small, the high concentration of melanin in the RPE melanosomes suggests they can be scavenged if they are in proximity to melanin-containing pigment granules.

Iron overload and chelation modulates bisretinoid levels in the retina.

Iron dysregulation in conjunction with other disease processes may exacerbate retinal degeneration. We employed models of iron overload and iron chelation to explore the interactions between iron-catalyzed oxidation and photoreactive bisretinoid lipofuscin. The mice were injected intravitreally with ferric ammonium citrate (FAC) or were treated using the iron chelator deferiprone (DFP) from birth to 2months of age. Short-wavelength fundus autofluorescence (SW-AF) and spectral-domain optical coherence tomography (SD-OCT) scans were acquired. The bisretinoid levels were quantified using ultra performance liquid chromatography (UPLC) and in vivo through quantitative fundus autofluorescence (qAF). In histologic sections, the photoreceptor cell viability was assessed by measuring the thickness of the outer nuclear layer (ONL). The levels of bisretinoids, all-trans-retinal dimers, and A2PE were significantly increased in the FAC-injected eyes of C57BL/6J mice. Seven days after FAC injection, hyperautofluorescent foci were visible in fundus autofluorescence (488nm) images, and in SD-OCT scans, aberrant hyperreflectivity was present in the outer retina and ONL thinning was observed. In FAC-injected Abca4-/- mice with pronounced RPE bisretinoid lipofuscin accumulation, the hyperautofluorescent puncta were more abundant than in the wild-type mice, and the extent of ONL thinning was greater. Conversely, the intravitreal injection of FAC in Mertk-/- mice led to a more modest increase in A2PE after 2 days. In contrast to the effect of iron accumulation, chelation with DFP resulted in significantly increased levels of A2E and A2-GPE and qAF due to the reduced iron-catalyzed oxidation of bisretinoids. In Mertk-/- mice, the A2E level was significantly lower and the ONL area was smaller than in DFP-treated mice. DFP chelation did not impair the visual cycle in BALB/cJ mice. Iron accumulation was associated with progressive impairment in photoreceptor cells that was associated with the increased formation of a bisretinoid species known to form in photoreceptor outer segments as a precursor to A2E. Additionally, disease features such as the development of hyperautofluorescence puncta in fundus AF images, hyperreflectivity in the outer retina of SD-OCT scans, and ONL thinning were more pronounced when iron was delivered to Abca4-/- mice with a greater propensity for bisretinoid formation. Higher bisretinoid levels and enhanced qAF are indicative of lesser bisretinoid loss due to oxidation.

Establishing Fully-Automated Fundus-Controlled Dark Adaptometry: A Validation and Retest-Reliability Study.

The purpose of this study was to establish and validate a novel fundus-controlled dark-adaptometry method. We developed a custom dark-adaptometry software for the S-MAIA device using the open-perimetry-interface. In the validation-substudy, participants underwent dark-adaptometry testing with a comparator device (MonCvONE, 59% rhodopsin bleach, cyan and red stimuli centered at 2 degrees, 4 degrees, and 6 degrees eccentricity). Following a brief break (approximately 5 minutes), the participants were bleached again and underwent dark-adaptometry testing with the S-MAIA device (same loci). In the retest reliability-substudy, participants were tested twice with the S-MAIA device (same loci as above). Nonlinear curve fitting was applied to extract dark-adaptation curve parameters. Validity and repeatability were summarized in terms of the mean bias and 95% limits of agreement (LoAs). In the validation-substudy (N = 20 participants, median age interquartile range [IQR] 31.5 years [IQR = 25.8, 62.0]), measures of rod-mediated dark-adaptation showed little to no between method differences for the cone-rod-break-time (bias 95% confidence interval [95% CI] of +0.1 minutes [95% CI = -0.6 to 0.8]), rod-intercept-time (-0.23 minutes [95% CI = -1.38 to 0.93]), and S2 slope (-0.01 LogUnits/minutes [95% CI = -0.02 to -0.01]). In the retest reliability-substudy (N = 10 participants, 32.0 years [95% CI = 27.0, 57.5]), the corresponding LoAs were (cone-rod-break-time) -3.94 to 2.78 minutes, (rod-intercept-time) -4.55 to 3.11 minutes, and (S2 slope [rate-limited component of rod recovery]) -0.03 to 0.03 LogUnits/minutes. The LoAs for the steady-state cone and rod thresholds were -0.28 to 0.33 LogUnits and -0.34 to 0.28 LogUnits. The devised fundus-controlled dark-adaptometry method yields valid and reliable results. Fundus-controlled dark-adaptometry solves the critical need for localized testing of the visual cycle and retinoid transfer in eyes with unstable fixation.

Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function

The sixth family phosphodiesterases (PDE6) are principal effector enzymes of the phototransduction cascade in rods and cones. Maturation of nascent PDE6 protein into a functional enzyme relies on a coordinated action of ubiquitous chaperone HSP90, its specialized cochaperone aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and the regulatory Pγ-subunit of PDE6. Deficits in PDE6 maturation and function underlie severe visual disorders and blindness. Here, to elucidate the roles of HSP90, AIPL1, and Pγ in the maturation process, we developed the heterologous expression system of human cone PDE6C in insect cells allowing characterization of the purified enzyme. We demonstrate that in the absence of Pγ, HSP90, and AIPL1 convert the inactive and aggregating PDE6C species into dimeric PDE6C that is predominantly misassembled. Nonetheless, a small fraction of PDE6C is properly assembled and fully functional. From the analysis of mutant mice that lack both rod Pγ and PDE6C, we conclude that, in contrast to the cone enzyme, no maturation of rod PDE6AB occurs in the absence of Pγ. Co-expression of PDE6C with AIPL1 and Pγ in insect cells leads to a fully mature enzyme that is equivalent to retinal PDE6. Lastly, using immature PDE6C and purified chaperone components, we reconstituted the process of the client maturation invitro. Based on this analysis we propose a scheme for the PDE6 maturation process.

Crosstalk of CGMP and CAMP in the vertebrate phototransduction cascade

The conventional phototransduction cascade suggests that cyclic guanosine monophosphate (cGMP) functions as the chief secondary messenger while intracellular calcium concentration dominates as the central feedback modulator. Countless years and several scientific institutions have led to this conclusion, and it stands as the most extensively researched and explicit among all sensory transduction schemes. However, experimental evidence suggests that our understanding of the phototransduction cascade mechanisms remains significantly incomplete [1]. According to the canonical cascade scheme, all transients should be completed within a second once the light stimulus is no longer present. However, our data indicates that there are long-lasting changes in cell sensitivity and dark current parameters after the stimulus, which can last for more than 10 s. Phenomena that deviate from the standard phototransduction cascade behavior can potentially be clarified by an alternative regulatory mechanism that is based on cyclic adenosine monophosphate (cAMP). Previous research has provided convincing evidence that the intracellular levels of cAMP can significantly impact the functioning of the phototransduction cascade on both slow (day) [2] and relatively fast (minutes) [3] time scales. Additionally, there is phenomenological evidence indicating the existence of other regulatory signaling pathways in the phototransduction cascade without a corresponding mechanism in the classical phototransduction scheme, including inositol triphosphate (IP3) and diacylglycerol (DAG). We investigate whether cAMP, IP3, and DAG regulate the phototransduction cascade during photoresponse. For this regulatory effect to occur, there must be a change in the signaling molecule concentration during the process. These processes occur in less than a second, and it is crucial for the presence of the regulatory effect. Given that traditional fluorescence methods cannot measure the concentration of any signaling molecule in the retina, a hardware-software setup has been developed that allows cryofixation of retinal samples at the required speed. The setup permits fixing up to six samples in a series with a delay of no more than 80 milliseconds after light stimulation. The concentration of signal molecules is assessed using high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry. The results demonstrate a 4.5-fold elevation in cAMP concentration 1.1 s after switching on a light with an intensity close to saturation. The concentration of cAMP is directly proportional to the intensity of the stimulating light; there is no increase in cAMP at lower light intensities. No noteworthy changes in IP3 and DAG concentration were detected in response to light stimulation. The findings align with existing literature on the kinetics of light-triggered protein kinase A (PKA) activity [3], which indicated an initial decrease followed by an increase in PKA activity. These results could potentially inform the revision and expansion of the phototransduction cascade model.