ABSTRACT
This study characterizes a fluorescent Slc17a6 -tdTomato neuronal reporter mouse line offering strong labeling in axons throughout the optic nerve, dendrites and soma in 99% of retinal ganglion cells (RGCs). The model facilitates neuronal assessment ex vivo with wholemounts quantified to show neurodegeneration following optic nerve crush or elevated IOP as related to glaucoma, in vitro with robust Ca 2+ responses to P2X7 receptor stimulation in neuronal cultures, and in vivo using a confocal scanning laser ophthalmoscope (cSLO). While the tdTomato signal showed strong overlap with RGC markers, BRN3A and RBPMS, there was no cross-labeling of displaced amacrine cells in the ganglion cell layer. Controls indicated no impact of Slc17a6 -tdTomato expression on light-dependent neuronal function, as determined with a microelectrode array (MEA), or on structure, as measured with optical coherence tomography (OCT). In summary, this novel neuronal reporter mouse model offers an effective means to increase the efficiency for real-time, specific visualization of retinal ganglion cells. It holds substantial promise for enhancing our understanding of RGC pathology in glaucoma and other diseases of the optic nerve, and could facilitate the screening of targeted therapeutic interventions for neurodegeneration.
ABSTRACT
Piezo channels are associated with neuropathology in diseases like traumatic brain injury and glaucoma, but pathways linking tissue stretch to aberrant neural signaling remain unclear. The present study demonstrates that Piezo1 activation increases action potential frequency in response to light and the spontaneous dark signal from mouse retinal explants. Piezo1 stimulation was sufficient to increase cytoplasmic Ca 2+ in soma and neurites, while stretch increased spiking activity in current clamp recordings from of isolated retinal ganglion cells (RGCs). Axon-marker beta-tubulin III colocalized with both Piezo1 and Piezo2 protein in the mouse optic nerve head, while RGC nuclear marker BRN3A colocalized with Piezo channels in the soma. Piezo1 was also present on GFAP-positive regions in the optic nerve head and colocalized with glutamine synthetase in the nerve fiber layer, suggesting expression in optic nerve head astrocytes and Müller glia end feet, respectively. Human RGCs from induced pluripotent stem cells also expressed Piezo1 and Piezo2 in soma and axons, while staining patterns in rats resembled those in mice. mRNA message for Piezo1 was greatest in the RPE/choroid tissue, while Piezo2 levels were highest in the optic nerve, with both channels also expressed in the retina. Increased expression of Piezo1 and Piezo2 occurred both 1 and 10 days after a single stretch in vivo; this increase suggests a potential role in rising sensitivity to repeated nerve stretch. In summary, Piezo1 and Piezo2 were detected in the soma and axons of RGCs, and stimulation affected the light-dependent output of RGCs. The rise in RGCs excitability induced by Piezo stimulation may have parallels to the early disease progression in models of glaucoma and other retinal degenerations. Highlights: Activation of Piezo1 excites retinal ganglion cells, paralleling the early neurodegenerative progression in glaucoma mouse models and retinal degeneration.Piezo1 and Piezo2 were expressed in axons and soma of retinal ganglion cells in mice, rats, and human iPSC-RGCs.Functional assays confirmed Piezo1 in soma and neurites of neurons. Sustained elevation of Piezo1 and Piezo2 occurred after a single transient stretch may enhance damage from repeated traumatic nerve injury.
ABSTRACT
Glaucomatous neurodegeneration, a blinding disease affecting millions worldwide, has a need for the exploration of new and effective therapies. Previously, the glucagon-like peptide-1 receptor (GLP-1R) agonist NLY01 was shown to reduce microglia/macrophage activation, rescuing retinal ganglion cells after IOP elevation in an animal model of glaucoma. GLP-1R agonist use is also associated with a reduced risk for glaucoma in patients with diabetes. In this study, we demonstrate that several commercially available GLP-1R agonists, administered either systemically or topically, hold protective potential in a mouse model of hypertensive glaucoma. Further, the resulting neuroprotection likely occurs through the same pathways previously shown for NLY01. This work contributes to a growing body of evidence suggesting that GLP-1R agonists represent a viable therapeutic option for glaucoma.
ABSTRACT
Lack of non-muscle ß -actin gene (Actb) leads to early embryonic lethality in mice, however mice with ß - to γ -actin replacement develop normally and show no detectable phenotypes at young age. Here we investigated the effect of this replacement in the retina. During aging, these mice have accelerated de-generation of retinal structure and function, including elongated microvilli and defective mitochondria of retinal pigment epithelium (RPE), abnormally bulging photoreceptor outer segments (OS) accompanied by reduced transducin concentration and light sensitivity, and accumulation of autofluorescent microglia cells in the subretinal space between RPE and OS. These defects are accompanied by changes in the F-actin binding of several key actin interacting partners, including ezrin, myosin, talin, and vinculin known to play central roles in modulating actin cytoskeleton and cell adhesion and mediating the phagocytosis of OS. Our data show that ß -actin protein is essential for maintaining normal retinal structure and function.
ABSTRACT
Photopharmacological compounds such as azobenzene-based photoswitches have been shown to control the conductivity of ionic channels in a light-dependent manner and are considered a potential strategy to restore vision in patients with end-stage photoreceptor degeneration. Here, we report the effects of DENAQ, a second-generation azobenzene-based photoswitch on retinal ganglion cells (RGC) in canine retinas using multi-electrode array (MEA) recordings (from nine degenerated and six WT retinas). DENAQ treatment conferred increased light sensitivity to RGCs in degenerated canine retinas. RGC light responses were observed in degenerated retinas following ex vivo application of 1 mM DENAQ (n = 6) or after in vivo DENAQ injection (n = 3, 150 µL, 3-10 mM) using 455 nm light at intensities as low as 0.2 mW/cm2. The number of light-sensitive cells and the per cell response amplitude increased with light intensity up to the maximum tested intensity of 85 mW/cm2. Application of DENAQ to degenerated retinas with partially preserved cone function caused appearance of DENAQ-driven responses both in cone-driven and previously non-responsive RGCs, and disappearance of cone-driven responses. Repeated stimulation slowed activation and accelerated recovery of the DENAQ-driven responses. The latter is likely responsible for the delayed appearance of a response to 4 Hz flicker stimulation. Limited aqueous solubility of DENAQ results in focal drug aggregates associated with ocular toxicity. While this limits the therapeutic potential of DENAQ, more potent third-generation photoswitches may be more promising, especially when delivered in a slow-release formulation that prevents drug aggregation.
ABSTRACT
Glaucoma is an optic neuropathy characterized by permanent visual field loss caused by the death of retinal ganglion cells (RGCs) and it is the leading cause of irreversible blindness worldwide. Consequently, there is an unmet need for the development of new strategies for its treatment. We investigated RGC replacement therapy as a treatment for ganglion cell loss. Human-induced pluripotent stem cells (hiPSCs) were differentiated into mature, functional RGCs in vitro, labeled with AAV2.7m8-SNCG-eGFP, and transplanted intravitreally in wild-type 4-month-old C57BL/6J mice. Survival of the transplanted hiPSC-RGCs was assessed by color fundus photography and histological studies confirmed the localization of the transplanted hiPSC-RGCs within the retina. Two-photon live imaging of retinal explants and electrophysiological studies confirmed that the morphology and function of the transplanted hiPSC-RGCs were similar to native RGCs. These experiments will provide key strategies to enhance the efficiency of stem cell replacement therapy for neurodegenerative diseases, including glaucoma.
ABSTRACT
Purpose: Optogenetic gene therapy to render remaining retinal cells light-sensitive in end-stage retinal degeneration is a promising strategy for treatment of individuals blind because of a variety of different inherited retinal degenerations. The clinical trials currently in progress focus on delivery of optogenetic genes to ganglion cells. Delivery of optogenetic molecules to cells in the outer neural retina is predicted to be even more advantageous because it harnesses more of the retinal circuitry. However, this approach has not yet been tested in large animal models. For this reason, we evaluated the safety and efficacy of optogenetic therapy targeting remaining diseased cone photoreceptors in the Rcd1 dog model of retinitis pigmentosa. Methods: Imaging and measures of retinal function and functional vision were carried out, as well as terminal studies evaluating multi-electrode array recordings and histology. Results: Animals remained healthy and active throughout the study and showed improved retinal and visual function as assessed by electroretinography and visual-evoked potentials, improved navigational vision, and improved function of cone photoreceptors and the downstream retinal circuitry. Conclusions: The findings demonstrate that an optogenetic approach targeting the outer retina in a blind large animal model can partially restore vision. Translational Relevance: This work has translational relevance because the approach could potentially be extrapolated to treat humans who are totally blind because of retinal degenerative disease.
Subject(s)
Dependovirus , Retinal Degeneration , Animals , Dependovirus/genetics , Dogs , Optogenetics/methods , Retina , Retinal Cone Photoreceptor Cells/pathology , Retinal Degeneration/genetics , Retinal Degeneration/pathology , Retinal Degeneration/therapy , Vision, OcularABSTRACT
Regulator of G-protein signaling 7 (RGS7) is predominately present in the nervous system and is essential for neuronal signaling involving G-proteins. Prior studies in cultured cells showed that RGS7 is regulated via proteasomal degradation, however no protein is known to facilitate proteasomal degradation of RGS7 and it has not been shown whether this regulation affects G-protein signaling in neurons. Here we used a knockout mouse model with conditional deletion of arginyltransferase (Ate1) in the nervous system and found that in retinal ON bipolar cells, where RGS7 modulates a G-protein to signal light increments, deletion of Ate1 raised the level of RGS7. Electroretinographs revealed that lack of Ate1 leads to increased light-evoked response sensitivities of ON-bipolar cells, as well as their downstream neurons. In cultured mouse embryonic fibroblasts (MEF), RGS7 was rapidly degraded via proteasome pathway and this degradation was abolished in Ate1 knockout MEF. Our results indicate that Ate1 regulates RGS7 protein level by facilitating proteasomal degradation of RGS7 and thus affects G-protein signaling in neurons.
Subject(s)
Aminoacyltransferases/physiology , Fibroblasts/metabolism , Light , Nervous System/metabolism , RGS Proteins/metabolism , Retinal Bipolar Cells/metabolism , Animals , Female , Fibroblasts/pathology , Fibroblasts/radiation effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nervous System/pathology , Nervous System/radiation effects , RGS Proteins/genetics , Retinal Bipolar Cells/pathology , Retinal Bipolar Cells/radiation effects , Signal TransductionABSTRACT
Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). The loss of RGCs manifests as characteristic cupping or optic nerve degeneration, resulting in visual field loss in patients with Glaucoma. Published studies on in vitro RGC differentiation from stem cells utilized classical RGC signaling pathways mimicking retinal development in vivo. Although many strategies allowed for the generation of RGCs, increased variability between experiments and lower yield hampered the cross comparison between individual lines and between experiments. To address this critical need, we developed a reproducible chemically defined in vitro methodology for generating retinal progenitor cell (RPC) populations from iPSCs, that are efficiently directed towards RGC lineage. Using this method, we reproducibly differentiated iPSCs into RGCs with greater than 80% purity, without any genetic modifications. We used small molecules and peptide modulators to inhibit BMP, TGF-ß (SMAD), and canonical Wnt pathways that reduced variability between iPSC lines and yielded functional and mature iPSC-RGCs. Using CD90.2 antibody and Magnetic Activated Cell Sorter (MACS) technique, we successfully purified Thy-1 positive RGCs with nearly 95% purity.
Subject(s)
Cell Differentiation/drug effects , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/metabolism , Smad Proteins/antagonists & inhibitors , Wnt Proteins/antagonists & inhibitors , Computational Biology , Gene Expression Profiling , Humans , Immunohistochemistry , Immunophenotyping , Neurogenesis , Retina/cytology , Signal TransductionABSTRACT
Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an â¼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.
Subject(s)
Blindness/metabolism , Blindness/therapy , Dependovirus/genetics , Genetic Therapy/methods , Animals , Electroretinography , Eye Proteins/genetics , Eye Proteins/metabolism , Female , Humans , Leber Congenital Amaurosis/metabolism , Leber Congenital Amaurosis/therapy , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolismABSTRACT
There has been marked progress in recent years in developing gene delivery approaches for the treatment of inherited blinding diseases. Many of the proof-of-concept studies have utilized rodent models of retinal degeneration. In those models, tests of visual function include a modified water maze swim test, optokinetic nystagmus, and light-dark activity assays. Test paradigms used in rodents can be difficult to replicate in large animals due to their size and awareness of non-visual aspects of the test system. Two types of visual behavior assays have been utilized in canines: an obstacle avoidance course and a forced choice Y maze. Given the progress in developing cell and gene therapies in large animals, such tests will become more and more valuable. This study provides guidelines for carrying out such tests and assesses the challenges and benefits associated with each test.
ABSTRACT
Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0(460)). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0(460) was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0(460) was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0(460) in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0(460) in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0(460) was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.
Subject(s)
Ion Channel Gating , Ion Channels/radiation effects , Light , Retinal Ganglion Cells/radiation effects , Vision, Ocular , Animals , Blindness/physiopathology , Ion Channels/physiology , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Retinal Ganglion Cells/physiologyABSTRACT
Mammalian cones respond to light by closing a cGMP-gated channel via a cascade that includes a heterotrimeric G-protein, cone transducin, comprising Gαt2, Gß3 and Gγt2 subunits. The function of Gßγ in this cascade has not been examined. Here, we investigate the role of Gß3 by assessing cone structure and function in Gß3-null mouse (Gnb3(-/-)). We found that Gß3 is required for the normal expression of its partners, because in the Gnb3(-/-) cone outer segments, the levels of Gαt2 and Gγt2 are reduced by fourfold to sixfold, whereas other components of the cascade remain unaltered. Surprisingly, Gnb3(-/-) cones produce stable responses with normal kinetics and saturating response amplitudes similar to that of the wild-type, suggesting that cone phototransduction can function efficiently without a Gß subunit. However, light sensitivity was reduced by approximately fourfold in the knock-out cones. Because the reduction in sensitivity was similar in magnitude to the reduction in Gαt2 level in the cone outer segment, we conclude that activation of Gαt2 in Gnb3(-/-) cones proceeds at a rate approximately proportional to its outer segment concentration, and that activation of phosphodiesterase and downstream cascade components is normal. These results suggest that the main role of Gß3 in cones is to establish optimal levels of transducin heteromer in the outer segment, thereby indirectly contributing to robust response properties.
Subject(s)
Heterotrimeric GTP-Binding Proteins/genetics , Retinal Cone Photoreceptor Cells/physiology , Transducin/genetics , Vision, Ocular/physiology , Animals , Color , Female , GABA Plasma Membrane Transport Proteins/genetics , Green Fluorescent Proteins/genetics , Heterotrimeric GTP-Binding Proteins/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Neurological , Photic Stimulation , Retinal Photoreceptor Cell Outer Segment/physiology , Transducin/physiology , Ultraviolet RaysABSTRACT
In absence of their natural ligand, 11-cis-retinal, cone opsin G-protein-coupled receptors fail to traffic normally, a condition associated with photoreceptor degeneration and blindness. We created a mouse with a point mutation (F81Y) in cone S-opsin. As expected, cones with this knock-in mutation respond to light with maximal sensitivity red-shifted from 360 to 420 nm, consistent with an altered interaction between the apoprotein and ligand, 11-cis-retinal. However, cones expressing F81Y S-opsin showed an â¼3-fold reduced absolute sensitivity that was associated with a corresponding reduction in S-opsin protein expression. The reduced S-opsin expression did not arise from decreased S-opsin mRNA or cone degeneration, but rather from enhanced endoplasmic reticulum (ER)-associated degradation of the nascent protein. Exogenously increased 11-cis-retinal restored F81Y S-opsin protein expression to normal levels, suggesting that ligand binding in the ER facilitates proper folding. Immunohistochemistry and electron microscopy of normal retinas showed that Mueller cells, which synthesize a precursor of 11-cis-retinal, are closely adjoined to the cone ER, so they could deliver the ligand to the site of opsin synthesis. Together, these results suggest that the binding of 11-cis-retinal in the ER is important for normal folding during cone opsin biosynthesis.
Subject(s)
Opsins/biosynthesis , Opsins/genetics , Retinal Cone Photoreceptor Cells/metabolism , Retinaldehyde/physiology , Algorithms , Animals , Animals, Genetically Modified , Blotting, Western , Electrophysiological Phenomena , Endoplasmic Reticulum/metabolism , Fluorescent Antibody Technique , Immunohistochemistry , Immunoprecipitation , Light , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron , Mutation/physiology , Real-Time Polymerase Chain Reaction , Receptors, G-Protein-Coupled/metabolism , Retinal Rod Photoreceptor Cells/metabolismABSTRACT
Decoding the wiring diagram of the retina requires simultaneous observation of activity in identified neuron populations. Available recording methods are limited in their scope: electrodes can access only a small fraction of neurons at once, whereas synthetic fluorescent indicator dyes label tissue indiscriminately. Here, we describe a method for studying retinal circuitry at cellular and subcellular levels combining two-photon microscopy and a genetically encoded calcium indicator. Using specific viral and promoter constructs to drive expression of GCaMP3, we labeled all five major neuron classes in the adult mouse retina. Stimulus-evoked GCaMP3 responses as imaged by two-photon microscopy permitted functional cell type annotation. Fluorescence responses were similar to those measured with the small molecule dye OGB-1. Fluorescence intensity correlated linearly with spike rates >10 spikes/s, and a significant change in fluorescence always reflected a significant change in spike firing rate. GCaMP3 expression had no apparent effect on neuronal function. Imaging at subcellular resolution showed compartment-specific calcium dynamics in multiple identified cell types.
Subject(s)
Action Potentials/physiology , Neurons/physiology , Retina/physiology , Vision, Ocular/physiology , Voltage-Sensitive Dye Imaging/methods , Animals , Female , Male , Mice , Mice, Inbred C57BL , Neurons/classification , Neurons/cytology , Organ Culture Techniques , Retina/cytologyABSTRACT
Mouse cone photoreceptors, like those of most mammals including humans, express cone opsins derived from two ancient families: S-opsin (gene Opn1sw) and M-opsin (gene Opn1mw). Most C57Bl/6 mouse cones co-express both opsins, but in dorso-ventral counter-gradients, with M-opsin dominant in the dorsal retina and S-opsin in the ventral retina, and S-opsin 4-fold greater overall. We created a mouse lacking S-opsin expression by the insertion of a Neomycin selection cassette between the third and fourth exons of the Opn1sw gene (Opn1sw(Neo/Neo)). In strong contrast to published results characterizing mice lacking rhodopsin (Rhoâ»/â») in which retinal rods undergo cell death by 2.5 months, cones of the Opn1sw(Neo/Neo) mouse remain viable for at least 1.5 yrs, even though many ventral cones do not form outer segments, as revealed by high resolution immunohistochemistry and electron microscopy. Suction pipette recordings revealed that functional ventral cones of the Opn1sw(Neo/Neo) mouse not only phototransduce light with normal kinetics, but are more sensitive to mid-wavelength light than their WT counterparts. Quantitative Western blot analysis revealed the basis of the heightened sensitivity to be increased M-opsin expression. Because S- and M-opsin transcripts must compete for the same translational machinery in cones where they are co-expressed, elimination of S-opsin mRNA in ventral Opn1sw(Neo/Neo) cones likely increases M-opsin expression by relieving competition for translational machinery, revealing an important consequence of eliminating a dominant transcript. Overall, our results reveal a striking capacity for cone photoreceptors to function with much reduced opsin expression, and to remain viable in the absence of an outer segment.
Subject(s)
Retina/physiology , Retinal Cone Photoreceptor Cells/physiology , Rod Opsins/deficiency , Rod Opsins/metabolism , Animals , Blotting, Western , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA, Messenger/metabolism , Retina/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Rod Opsins/immunologyABSTRACT
Maturation of the mammalian nervous system requires adequate provision of thyroid hormone and mechanisms that enhance tissue responses to the hormone. Here, we report that the development of cones, the photoreceptors for daylight and color vision, requires protection from thyroid hormone by type 3 deiodinase, a thyroid hormone-inactivating enzyme. Type 3 deiodinase, encoded by Dio3, is expressed in the immature mouse retina. In Dio3(-/-) mice, approximately 80% of cones are lost through neonatal cell death. Cones that express opsin photopigments for response to both short (S) and medium-long (M) wavelength light are lost. Rod photoreceptors, which mediate dim light vision, remain essentially intact. Excessive thyroid hormone in wild-type pups also eliminates cones. Cone loss is mediated by cone-specific thyroid hormone receptor beta2 (TRbeta2) as deletion of TRbeta2 rescues cones in Dio3(-/-) mice. However, rescued cones respond to short but not longer wavelength light because TRbeta2 under moderate hormonal stimulation normally induces M opsin and controls the patterning of M and S opsins over the retina. The results suggest that type 3 deiodinase limits hormonal exposure of the cone to levels that safeguard both cone survival and the patterning of opsins that is required for cone function.
Subject(s)
Iodide Peroxidase/genetics , Retina/enzymology , Retina/growth & development , Retinal Cone Photoreceptor Cells/enzymology , Thyroid Hormones/metabolism , Animals , Cell Death/genetics , Cell Differentiation/genetics , Cell Survival/genetics , Female , Gene Expression Regulation, Developmental/genetics , Light , Male , Mice , Mice, Knockout , Opsins/metabolism , Photic Stimulation , Retina/cytology , Retinal Cone Photoreceptor Cells/cytology , Retinal Cone Photoreceptor Cells/radiation effects , Thyroid Hormone Receptors beta/metabolism , Vision, Ocular/geneticsABSTRACT
Arrestins are proteins that arrest the activity of G protein-coupled receptors (GPCRs). While it is well established that normal inactivation of photoexcited rhodopsin, the GPCR of rod phototransduction, requires arrestin (Arr1), it has been controversial whether the same requirement holds for cone opsin inactivation. Mouse cone photoreceptors express two distinct visual arrestins: Arr1 and Arr4. By means of recordings from cones of mice with one or both arrestins knocked out, this investigation establishes that a visual arrestin is required for normal cone inactivation. Arrestin-independent inactivation is 70-fold more rapid in cones than in rods, however. Dual arrestin expression in cones could be a holdover from ancient genome duplication events that led to multiple isoforms of arrestin, allowing evolutionary specialization of one form while the other maintains the basic function.
Subject(s)
Arrestin/metabolism , Retinal Cone Photoreceptor Cells/physiology , Vision, Ocular/physiology , Analysis of Variance , Animals , Arrestin/classification , Arrestin/deficiency , Electrophysiology , Light , Membrane Potentials/genetics , Membrane Potentials/radiation effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Reaction Time/physiology , Retina/cytology , Rod Opsins/pharmacology , Vision, Ocular/radiation effectsABSTRACT
Cone cells constitute only 3% of the photoreceptors of the wild-type (WT) mouse. While mouse rods have been thoroughly investigated with suction pipette recordings of their outer segment membrane currents, to date no recordings from WT cones have been published, likely because of the rarity of cones and the fragility of their outer segments. Recently, we characterized the photoreceptors of Nrl(-/-) mice, using suction pipette recordings from their "inner segments" (perinuclear region), and found them to be cones. Here we report the use of this same method to record for the first time the responses of single cones of WT mice, and of mice lacking the alpha-subunit of the G-protein transducin (G(t)alpha(-/-)), a loss that renders them functionally rodless. Most cones were found to functionally co-express both S- (lambda(max) = 360 nm) and M- (lambda(max) = 508 nm) cone opsins and to be maximally sensitive at 360 nm ("S-cones"); nonetheless, all cones from the dorsal retina were found to be maximally sensitive at 508 nm ("M-cones"). The dim-flash response kinetics and absolute sensitivity of S- and M-cones were very similar and not dependent on which of the coexpressed cone opsins drove transduction; the time to peak of the dim-flash response was approximately 70 ms, and approximately 0.2% of the circulating current was suppressed per photoisomerization. Amplification in WT cones (A approximately 4 s(-2)) was found to be about twofold lower than in rods (A approximately 8 s(-2)). Mouse M-cones maintained their circulating current at very nearly the dark adapted level even when >90% of their M-opsin was bleached. S-cones were less tolerant to bleached S-opsin than M-cones to bleached M-opsin, but still far more tolerant than mouse rods to bleached rhodopsin, which exhibit persistent suppression of nearly 50% of their circulating current following a 20% bleach. Thus, the three types of mouse opsin appear distinctive in the degree to which their bleached, unregenerated opsins generate "dark light."
Subject(s)
Retina/physiology , Retinal Cone Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/physiology , Rod Opsins/physiology , Vision, Ocular , Animals , Basic-Leucine Zipper Transcription Factors/genetics , Dark Adaptation , Electroretinography , Eye Proteins/genetics , Gene Expression Regulation , Light , Mice , Mice, Inbred C57BL , Mice, Knockout , Transducin/geneticsABSTRACT
PURPOSE: To test the hypothesis that Nrl(-)(/)(-) photoreceptors are cones, by comparing them with WT rods and cones using morphological, molecular, histochemical, and electrophysiological criteria. METHODS: The photoreceptor layer of fixed retinal tissue of 4- to 6-week-old mice was examined in plastic sections by electron microscopy, and by confocal microscopy in frozen sections immunolabeled for the mouse UV-cone pigment and colabeled with PNA. Quantitative immunoblot analysis was used to determine the levels of expression of key cone-specific proteins. Single- and paired-flash methods were used to extract the spectral sensitivity, kinetics, and amplification of the a-wave of the ERG. RESULTS: Outer segments of Nrl(-/-) photoreceptors ( approximately 7 mum) are shorter than those of wild-type (WT) rods ( approximately 25 mum) and cones ( approximately 15 mum); but, like WT cones, they have 25 or more basal discs open to the extracellular space, extracellular matrix sheaths stained by PNA, chromatin "clumping" in their nuclei, and mitochondria two times shorter than rods. Nrl(-/-) photoreceptors express the mouse UV cone pigment, cone transducin, and cone arrestin in amounts expected, given the relative size and density of cones in the two retinas. The ERG a-wave was used to assay the properties of the photocurrent response. The sensitivity of the Nrl(-/-) a-wave is at its maximum at 360 nm, with a secondary mode at 510 nm having approximately one-tenth the maximum sensitivity. These wavelengths are the lambda(max) of the two mouse cone pigments. The time to peak of the dim-flash photocurrent response was approximately 50 ms, more than two times faster than that of rods. CONCLUSIONS: Many morphological, molecular, and electrophysiological features of the Nrl(-/-) photoreceptors are cone-like, and strongly distinguish these cells from rods. This retina provides a model for the investigation of cone function and cone-specific genetic disease.