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Influence of the N-terminal segment and the PHY-tongue element on light-regulation in bacteriophytochromes
摘要： Photoreceptors enable the integration of ambient light stimuli to trigger lifestyle adaptations via modulation of central metabolite levels involved in diverse regulatory processes. Red light sensing bacteriophytochromes are attractive targets for the development of innovative optogenetic tools due to their natural modularity, diverse functionalities and the natural availability of the light-absorbing biliverdin in animal tissues. However, a rational design of such tools is complicated by the poor understanding of molecular mechanisms of light signal transduction over long distances – from the site of photon absorption to the active site of downstream enzymatic effectors. Here we show how swapping structural elements between two bacteriophytochrome homologs provides additional insights into light signal integration and effector regulation, involving a fine-tuned interplay of important structural elements of the sensor as well as the sensor-effector linker. Facilitated by the availability of structural information of inhibited and activated full-length structures of one of the two homologs (Idiomarina species A28L Phytochrome-activated diguanylyl Cyclase - IsPadC) and characteristic differences in photoresponses of the two homologs, we identify an important cross-talk between the N-terminal segment (NTS), containing the covalent attachment site of the chromophore, and the PHY-tongue region. Moreover, we highlight how these elements influence the dynamic range of photoactivation and how activation can be improved to light/dark ratios of ~800-fold by reducing basal dark-state activities at the same time as increasing photoconversion in the light-state. This will enable future optimization of optogenetic tools aiming at a direct allosteric regulation of enzymatic effectors.
关键词： phytochrome,diguanylate cyclase,photobiology,signal transduction,bilin,GGDEF,dimerization,photoreceptor
更新于2025-09-19 17:15:36
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Photoreceptor Progenitors Depend Upon Coordination of <i>gdf6a</i> , <i>thrβ</i> , and <i>tbx2b</i> to Generate Precise Populations of Cone Photoreceptor Subtypes
摘要： PURPOSE. Replacing cone photoreceptors, the units of the retina necessary for daytime vision, depends upon the successful production of a full variety of new cones from, for example, stem cells. Using genetic experiments in a model organism with high cone diversity, zebrafish, we map the intersecting effects of cone development factors gdf6a, tbx2b, and thrb. METHODS. We investigated these genes of interest by using genetic combinations of mutants, gene knockdown, and dominant negative gene expression, and then quantified cone subtype outcomes (which normally develop in tightly regulated ratios). RESULTS. Gdf6a mutants have reduced blue cones and, discovered here, reduced red cones. In combined gdf6a/tbx2b disruption, the loss of gdf6a in heterozygous tbx2b mutants reduced UV cones. Intriguingly, when we disrupted thrb in gdf6a mutants by using a thrb morpholino, their combined early disruption revealed a lamination phenotype. Disrupting thrb activity via expression of a dominant negative thrb (dnthrb) at either early or late retinal development had differential outcomes on red cones (reduced abundance), versus UV and blue cones (increased abundance). By using dnthrb in gdf6a mutants, we revealed that disrupting thrb activity did not change gdf6a mutant cone phenotypes. CONCLUSIONS. Gdf6a loss directly affects blue and red cones and indirectly affects UV cones by increasing sensitivity to additional disruption, such as reduced tbx2b, resulting in fewer UV cones. The effects of thrb change through photoreceptor development, first promoting red cones and restricting UV cones, and later restricting UV and blue cones. The effects of gdf6a on UV, blue, and red cone development overlap with, but likely supersede, those of thrb.
关键词： BMP signaling,progenitor,color vision,retinal lamination,thyroid signaling,zebrafish,retinal development,determination,regeneration,cone photoreceptor
更新于2025-09-19 17:15:36
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Mitochondrial Respiration in Outer Retina Contributes to Light-Evoked Increase in Hydration In Vivo
摘要： PURPOSE. To test the hypothesis that mitochondrial respiration contributes to local changes in hydration involved in phototransduction-driven expansion of outer retina, as measured by structural responses on optical coherence tomography (OCT) and diffusion magnetic resonance imaging (MRI). METHODS. Oxygen consumption rate and mitochondrial reserve capacity of freshly isolated C57BL/6 and 129S6/SvEvTac mouse retina were measured using a Seahorse Extracellular Flux Analyzer. Light-stimulated outer retina layer water content was determined by proton density MRI, structure and thickness by ultrahigh-resolution OCT, and water mobility by diffusion MRI. RESULTS. Compared with C57BL/6 mice, 129S6/SvEvTac retina demonstrated a less robust mitochondrial respiratory basal level, with a higher reserve capacity and lower oxygen consumption in the light, suggesting a relatively lower production of water. C57BL/6 mice showed a light-triggered surge in water content of outer retina in vivo as well as an increase in thickness, and water mobility. In contrast, light did not evoke augmented hydration in this region or an increase in hyporeflective bands or water mobility in the 129S6/SvEvTac outer retina. Nonetheless, we observed a significant but small increase in outer retinal thickness. CONCLUSIONS. These studies suggest that respiratory-controlled hydration in healthy retina is linked with a localized light-evoked expansion of the posterior retina in vivo and may serve as a useful biomarker of the function of photoreceptor/retinal pigment epithelium complex.
关键词： mitochondria,water content,functional imaging,genetics,photoreceptor
更新于2025-09-19 17:15:36
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Phosphorylation at Serine 21 in G protein‐coupled receptor kinase 1 (GRK1) is required for normal kinetics of dark adaption in rod but not cone photoreceptors
摘要： Timely recovery of the light response in photoreceptors requires efficient inactivation of photoactivated rhodopsin. This process is initiated by phosphorylation of its carboxyl terminus by G protein-coupled receptor kinase 1 (GRK1). Previously, we showed that GRK1 is phosphorylated in the dark at Ser21 in a cAMP-dependent manner and dephosphorylated in the light. Results in vitro indicate that dephosphorylation of Ser21 increases GRK1 activity, leading to increased phosphorylation of rhodopsin. This creates the possibility of light-dependent regulation of GRK1 activity and its efficiency in inactivating the visual pigment. To address the functional role of GRK1 phosphorylation in rods and cones in vivo, we generated mutant mice in which Ser21 is substituted with alanine (GRK1-S21A), preventing dark-dependent phosphorylation of GRK1. GRK1-S21A mice had normal retinal morphology, without evidence of degeneration. The function of dark-adapted GRK1-S21A rods and cones was also unaffected, as demonstrated by the normal amplitude and kinetics of their responses obtained by ex vivo and in vivo ERG recordings. In contrast, rod dark adaptation following exposure to bright bleaching light was significantly delayed in GRK1-S21A mice, suggesting that the higher activity of this kinase results in enhanced rhodopsin phosphorylation and therefore delays its regeneration. In contrast, dark adaptation of cones was unaffected by the S21A mutation. Taken together, these data suggest that rhodopsin phosphorylation/dephosphorylation modulates the recovery of rhodopsin to the ground state and rod dark adaptation. They also reveal a novel role for cAMP-dependent phosphorylation of GRK1 in regulating the dark adaptation of rod but not cone photoreceptors.
关键词： vision,phototransduction,photoreceptor,protein phosphorylation,cAMP
更新于2025-09-16 10:30:52
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<i>ATF6</i> Is Mutated in Early Onset Photoreceptor Degeneration With Macular Involvement
摘要： PURPOSE. Photoreceptor degeneration (PRD) is a genetically heterogeneous retinal disorder. Although a number of genes involved in PRD have been identi?ed, their genetic basis remains unknown in a signi?cant number of patients. In this study, we aimed to identify novel disease-causing genes of PRD. METHODS. Comprehensive ocular examinations were performed in a 2-year-old patient diagnosed with early onset PRD. Retinal capture sequencing was performed to screen causative mutations in known retinal disease-causing loci. Whole-exome sequencing (WES) and a series of variant-?ltering strategies were applied for identifying novel disease-causing genes. Retina ATF6 expression was con?rmed by immunohistochemistry. RT-PCR was performed to identify ATF6 mRNA in the patient. RESULTS. The patient showed typical PRD features, with macular involvement and ellipsoid zone irregularities. Results of retinal capture sequencing were negative. WES data led to identi?cation of biallelic loss-of-function mutations in the ATF6 gene. The ?rst variant generates a premature stop codon (NCBI accession no. NM_007348: c.1126C>T, p.R376*) and the second variant affects a splicing donor site (NM_007348: c.1533t1G>C). Sanger sequencing con?rmed the 2 alleles are from 1 parent each. Both of the variants are extremely rare in the population. The splicing variant causes either intron inclusion or exon skipping in the patient, thus severely disrupting ATF6 functional domains. ATF6 is expressed in three neuronal cell layers of mouse retina. CONCLUSIONS. Our results support ATF6 as a novel disease-causing gene for PRD and suggest that disrupted protein quality control mechanisms may be a novel pathological mechanism underlying human retinal degeneration.
关键词： unfolded protein response,next-generation sequencing,ATF6,photoreceptor degeneration
更新于2025-09-11 14:15:04
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Molecular Diagnosis of Inherited Retinal Diseases in Indigenous African Populations by Whole-Exome Sequencing
摘要： PURPOSE. A majority of genes associated with inherited retinal diseases (IRDs) have been identi?ed in patients of European origin. Indigenous African populations exhibit rich genomic diversity, and evaluation of reported genetic mutations has yielded low returns so far. Our goal was to perform whole-exome sequencing (WES) to examine variants in known IRD genes in underrepresented African cohorts. METHODS. Whole-exome sequencing was performed on 56 samples from 16 families with diverse IRD phenotypes that had remained undiagnosed after screening for known mutations using genotyping-based microarrays (Asper Ophthalmics). Variants in reported IRD genes were identi?ed using WES and validated by Sanger sequencing. Custom TaqMan assays were used to screen for identi?ed mutations in 193 unrelated indigenous Africans with IRDs. RESULTS. A total of 3494 variants were identi?ed in 217 known IRD genes, leading to the identi?cation of seven different mutations (including six novel) in six genes (RHO, PRPF3, PRPF31, ABCA4, CERKL, and PDE6B) in six distinct families. TaqMan screening in additional probands revealed identical homozygous CERKL and PDE6B variants in four more patients. CONCLUSIONS. This is the ?rst report of WES of patients with IRDs in indigenous African populations. Our study identi?ed genetic defects in almost 40% of the families analyzed, signi?cantly enhancing the molecular diagnosis of IRD in South Africa. Thus, WES of understudied cohorts seems to present an effective strategy for determining novel mutations in heterogeneous retinal diseases.
关键词： genetic testing,vision loss,inherited blindness,South Africa,retinal degeneration,next generation sequencing,photoreceptor dysfunction,clinical genetics
更新于2025-09-11 14:15:04
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The 3-Phosphoinositide-Dependent Protein Kinase 1 Inhibits Rod Photoreceptor Development
摘要： The transition of rod precursor cells to post-mitotic rod photoreceptors can be promoted by extrinsic factors such as insulin-like growth factor 1 (IGF-1), which regulates phosphatidylinositide concentration, and consequently the 3-phosphoinositide-dependent protein kinase-1 (PDPK-1). PDPK-1 is a 63 kDa cytoplasmic kinase that controls cell proliferation and differentiation. In the mouse retina, PDPK-1 and its phosphorylated derivative p-PDPK-1 (Ser241), showed peak expression during the first postnatal (PN) day with a substantial decline by PN7 and in the adult retina. Though initially widely distributed among cell types, PDPK-1 expression decreased first in the inner retina and later in the outer retina. When PDPK-1 is inhibited in neonatal retinal explants by BX795, there is a robust increase in rod photoreceptor numbers. The increase in rods depended on the activity of PKC, as BX795 had no effect when PKC is inhibited. Inhibition of PDPK-1-dependent kinases, such as P70-S6K, but not others, such as mTORC-1, stimulated rod development. The P70-S6K-dependent increase in rods appears to be correlated with phosphorylation of Thr252 and not at Thr389, a substrate of mTORC-1. This pathway is also inactive while PKC activity is inhibited. We also found that inhibition of the kinase mTORC-2, also stimulated by insulin activity, similarly increased rod formation, and this effect appears to be independent intracellular signaling pathway that also stimulates photoreceptor development. Consistent with previous studies, stimulation of STAT3 activity is sufficient to prevent any PDPK-1, P70-S6K, or mTORC2-dependent increase in rods. Together the data indicate that PDPK-1 and other intrinsic kinases downstream of IGF-1 are key regulators of rod photoreceptor formation.
关键词： development,PDPK-1,retina,mTORC2,photoreceptor,PKC,STAT3,IGF-1
更新于2025-09-10 09:29:36
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Otx2 enhances transdifferentiation of Müller cells-derived retinal stem cells into photoreceptor-like cells
摘要： Retinal Müller glial cells have the potential of neurogenic retinal progenitor cells, and could reprogram into retinal‐specific cell types such as photoreceptor cells. How to promote the differentiation of Müller cells into photoreceptor cells represents a promising therapy strategy for retinal degeneration diseases. This study aimed to enhance the transdifferentiation of rat Müller cells‐derived retinal stem cells (MC‐RSCs) into photoreceptor‐like cells and explore the signalling mechanism. We dedifferentiated rat Müller cells into MC‐RSCs which were infected with Otx2 overexpression lentivirus or control. The positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus was significantly higher compared to control. Furthermore, pre‐treatment with Crx siRNA, Nrl siRNA, or GSK‐3 inhibitor SB‐216763 reduced the positive rate of photoreceptor‐like cells among MC‐RSCs treated with Otx2 overexpression lentivirus. Finally, Otx2 induced photoreceptor precursor cells were injected into subretinal space of N‐methyl‐N‐nitrosourea induced rat model of retinal degeneration and partially recovered retinal degeneration in the rats. In conclusion, Otx2 enhances transdifferentiation of MC‐RSCs into photoreceptor‐like cells and this is associated with the inhibition of Wnt signalling. Otx2 is a potential target for gene therapy of retinal degenerative diseases.
关键词： Müller cells,photoreceptor cells,stem cells,Wnt signalling,Otx2
更新于2025-09-10 09:29:36
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Repeatability of Cone Spacing Measures in Eyes With Inherited Retinal Degenerations
摘要： PURPOSE. To determine short-term variability of adaptive optics scanning laser ophthalmoscopy (AOSLO)–derived cone spacing measures in eyes with inherited retinal degenerations (IRD) and in normal eyes. METHODS. Twenty IRD patients and 10 visually normal subjects underwent AOSLO imaging at two visits separated by no more than 1 month (NCT00254605). Cone spacing was measured in multiple macular regions in each image by three independent graders. Variability of cone spacing measures between visits, between graders, and between eyes was determined and correlated with standard clinical measures. RESULTS. Cone spacing was measured in 2905 regions. Interobserver agreement was high both in normal eyes and eyes with IRD (mean intraclass correlation coef?cient [ICC] ? 0.838 for normal and 0.892 for eyes with IRD). Cone spacing measures were closely correlated between visits (ICC > 0.869 for both study groups). Mean relative intervisit spacing difference (absolute difference in measures divided by the mean at each region) was 4.0% for normal eyes and 4.9% for eyes with IRD. Cone spacing measures from fellow eyes of the same subject showed strong agreement for all subjects (ICC > 0.85 for both study groups). CONCLUSIONS. Adaptive optics scanning laser ophthalmoscopy–derived macular cone spacing measures were correlated between observers, visits, and fellow eyes of the same subject in normal eyes and in eyes with IRD. This information may help establish the role of cone spacing measures derived from images of the cone mosaic obtained with AOSLO as a sensitive biomarker for longitudinal tracking of photoreceptor loss during disease progression and in response to treatment. (ClinicalTrials.gov number, NCT00254605.)
关键词： retinal,adaptive optics,photoreceptor,imaging,retinitis pigmentosa,retinal degeneration
更新于2025-09-10 09:29:36
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Quantification of Oxygen Consumption in Retina Ex Vivo Demonstrates Limited Reserve Capacity of Photoreceptor Mitochondria
摘要： PURPOSE. Cell death in neurodegeneration occurs at the convergence of diverse metabolic pathways. In the retina, a common underlying mechanism involves mitochondrial dysfunction since photoreceptor homeostasis and survival are highly susceptible to altered aerobic energy metabolism. We sought to develop an assay to directly measure oxygen consumption in intact retina with the goal of identifying alterations in respiration during photoreceptor dysfunction and degeneration. METHODS. Circular punches of freshly isolated mouse retina, adjacent to the optic nerve head, were used in the microplate-based Seahorse Extracellular Flux Analyzer to measure oxygen consumption. Tissue integrity was evaluated by propidium iodide staining and live imaging. Different substrates were tested for mitochondrial respiration. Basal and maximal respiration were expressed as oxygen consumption rate (OCR) and respectively measured in Ames’ medium before and after the addition of mitochondrial uncoupler, BAM15. RESULTS. We show that glucose is an essential substrate for retinal mitochondria. At baseline, mitochondria respiration in the intact wild-type retina was close to maximal, with limited reserve capacity. Similar OCR and limited mitochondrial reserve capacity was also observed in cone-only Nrl(cid:2)/(cid:2) retina. However, the retina of Pde6brd1/rd1, Cep290rd16/rd16 and Rpgrip1(cid:2)/(cid:2) mice, all with dysfunctional or no photoreceptors, had reduced OCR and higher mitochondrial reserve capacity. CONCLUSIONS. We have optimized a method to directly measure oxygen consumption in acutely isolated, ex vivo mouse retina and demonstrate that photoreceptors have low mitochondrial reserve capacity. Our data provide a plausible explanation for the high vulnerability of photoreceptors to altered energy homeostasis caused by mutations or metabolic challenges.
关键词： photoreceptor homeostasis,neurodegeneration,mitochondrial function,retinal disease,oxidative stress
更新于2025-09-10 09:29:36