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1.
bioRxiv ; 2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-39005302

RESUMEN

The retina is uniquely enriched in polyunsaturated fatty acids (PUFAs), which are primarily localized in cell membranes, where they govern membrane biophysical properties such as diffusion, permeability, domain formation, and curvature generation. During aging, alterations in lipid metabolism lead to reduced content of very long-chain PUFAs (VLC-PUFAs) in the retina, and this decline is associated with normal age-related visual decline and pathological age-related macular degeneration (AMD). ELOVL2 (Elongation of very-long-chain fatty acids-like 2) encodes a transmembrane protein that produces precursors to docosahexaenoic acid (DHA) and VLC-PUFAs, and methylation level of its promoter is currently the best predictor of chronological age. Here, we show that mice lacking ELOVL2-specific enzymatic activity (Elovl2 C234W ) have impaired contrast sensitivity and slower rod response recovery following bright light exposure. Intravitreal supplementation with the direct product of ELOVL2, 24:5n-3, in aged animals significantly improved visual function and reduced accumulation of ApoE, HTRA1 and complement proteins in sub-RPE deposits. At the molecular level, the gene expression pattern observed in retinas supplemented with 24:5n-3 exhibited a partial rejuvenation profile, including decreased expression of aging-related genes and a transcriptomic signature of younger retina. Finally, we present the first human genetic data showing significant association of several variants in the human ELOVL2 locus with the onset of intermediate AMD, underlying the translational significance of our findings. In sum, our study identifies novel therapeutic opportunities and defines ELOVL2 as a promising target for interventions aimed at preventing age-related vision loss.

2.
Nat Commun ; 15(1): 5943, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-39009597

RESUMEN

Inherited retinopathies are devastating diseases that in most cases lack treatment options. Disease-modifying therapies that mitigate pathophysiology regardless of the underlying genetic lesion are desirable due to the diversity of mutations found in such diseases. We tested a systems pharmacology-based strategy that suppresses intracellular cAMP and Ca2+ activity via G protein-coupled receptor (GPCR) modulation using tamsulosin, metoprolol, and bromocriptine coadministration. The treatment improves cone photoreceptor function and slows degeneration in Pde6ßrd10 and RhoP23H/WT retinitis pigmentosa mice. Cone degeneration is modestly mitigated after a 7-month-long drug infusion in PDE6A-/- dogs. The treatment also improves rod pathway function in an Rpe65-/- mouse model of Leber congenital amaurosis but does not protect from cone degeneration. RNA-sequencing analyses indicate improved metabolic function in drug-treated Rpe65-/- and rd10 mice. Our data show that catecholaminergic GPCR drug combinations that modify second messenger levels via multiple receptor actions provide a potential disease-modifying therapy against retinal degeneration.


Asunto(s)
Modelos Animales de Enfermedad , Reposicionamiento de Medicamentos , Retinitis Pigmentosa , Animales , Ratones , Perros , Retinitis Pigmentosa/tratamiento farmacológico , Retinitis Pigmentosa/genética , Mutación , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Ratones Noqueados , Amaurosis Congénita de Leber/tratamiento farmacológico , Amaurosis Congénita de Leber/genética , Bromocriptina/farmacología , Bromocriptina/uso terapéutico , cis-trans-Isomerasas/genética , cis-trans-Isomerasas/metabolismo , Humanos , Quimioterapia Combinada , Ratones Endogámicos C57BL , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Femenino , AMP Cíclico/metabolismo , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/genética , Masculino , Calcio/metabolismo
3.
Annu Rev Vis Sci ; 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38724080

RESUMEN

The continuous function of vertebrate photoreceptors requires regeneration of their visual pigment following its destruction upon activation by light (photobleaching). For rods, the chromophore required for the regeneration of rhodopsin is derived from the adjacent retinal pigmented epithelium (RPE) cells through a series of reactions collectively known as the RPE visual cycle. Mounting biochemical and functional evidence demonstrates that, for cones, pigment regeneration is supported by the parallel supply with chromophore by two pathways-the canonical RPE visual cycle and a second, cone-specific retina visual cycle that involves the Müller glial cells in the neural retina. In this article, we review historical information that led to the discovery of the retina visual cycle and discuss what is currently known about the reactions and molecular components of this pathway and its functional role in supporting cone-mediated vision.

4.
Cell Rep ; 43(5): 114143, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38676924

RESUMEN

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.


Asunto(s)
Proteínas Portadoras , Ratones Noqueados , Células Fotorreceptoras Retinianas Conos , Epitelio Pigmentado de la Retina , Animales , Ratones , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Células Ependimogliales/metabolismo , Ratones Endogámicos C57BL , Células Fotorreceptoras Retinianas Conos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Pigmentos Retinianos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Masculino , Femenino
5.
Curr Biol ; 34(7): 1492-1505.e6, 2024 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-38508186

RESUMEN

Vision under dim light relies on primary cilia elaborated by rod photoreceptors in the retina. This specialized sensory structure, called the rod outer segment (ROS), comprises hundreds of stacked, membranous discs containing the light-sensitive protein rhodopsin, and the incorporation of new discs into the ROS is essential for maintaining the rod's health and function. ROS renewal appears to be primarily regulated by extrinsic factors (light); however, results vary depending on different model organisms. We generated two independent transgenic mouse lines where rhodopsin's fate is tracked by a fluorescently labeled rhodopsin fusion protein (Rho-Timer) and show that rhodopsin incorporation into nascent ROS discs appears to be regulated by both external lighting cues and autonomous retinal clocks. Live-cell imaging of the ROS isolated from mice exposed to six unique lighting conditions demonstrates that ROS formation occurs in a periodic manner in cyclic light, constant darkness, and artificial light/dark cycles. This alternating bright/weak banding of Rho-Timer along the length of the ROS relates to inhomogeneities in rhodopsin density and potential points of structural weakness. In addition, we reveal that prolonged dim ambient light exposure impacts not only the rhodopsin content of new discs but also that of older discs, suggesting a dynamic interchange of material between new and old discs. Furthermore, we show that rhodopsin incorporation into the ROS is greatly altered in two autosomal recessive retinitis pigmentosa mouse models, potentially contributing to the pathogenesis. Our findings provide insights into how extrinsic (light) and intrinsic (retinal clocks and genetic mutation) factors dynamically regulate mammalian ROS renewal.


Asunto(s)
Células Fotorreceptoras Retinianas Bastones , Rodopsina , Animales , Ratones , Luz , Ratones Transgénicos , Especies Reactivas de Oxígeno/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Segmento Externo de la Célula en Bastón/metabolismo
6.
Proc Natl Acad Sci U S A ; 121(11): e2316118121, 2024 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-38442152

RESUMEN

Retinitis pigmentosa (RP) is a common form of retinal dystrophy that can be caused by mutations in any one of dozens of rod photoreceptor genes. The genetic heterogeneity of RP represents a significant challenge for the development of effective therapies. Here, we present evidence for a potential gene-independent therapeutic strategy based on targeting Nr2e3, a transcription factor required for the normal differentiation of rod photoreceptors. Nr2e3 knockout results in hybrid rod photoreceptors that express the full complement of rod genes, but also a subset of cone genes. We show that germline deletion of Nr2e3 potently protects rods in three mechanistically diverse mouse models of retinal degeneration caused by bright-light exposure (light damage), structural deficiency (rhodopsin-deficient Rho-/- mice), or abnormal phototransduction (phosphodiesterase-deficient rd10 mice). Nr2e3 knockout confers strong neuroprotective effects on rods without adverse effects on their gene expression, structure, or function. Furthermore, in all three degeneration models, prolongation of rod survival by Nr2e3 knockout leads to lasting preservation of cone morphology and function. These findings raise the possibility that upregulation of one or more cone genes in Nr2e3-deficient rods may be responsible for the neuroprotective effects we observe.


Asunto(s)
Fármacos Neuroprotectores , Distrofias Retinianas , Retinitis Pigmentosa , Animales , Ratones , Células Fotorreceptoras Retinianas Conos , Retinitis Pigmentosa/genética , Modelos Animales de Enfermedad , Células Germinativas , Receptores Nucleares Huérfanos
7.
bioRxiv ; 2023 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-37961274

RESUMEN

Vision is initiated by the reception of light by photoreceptors and subsequent processing via parallel retinal circuits. Proper circuit organization depends on the multi-functional tissue polarity protein FAT3, which is required for amacrine cell connectivity and retinal lamination. Here we investigated the retinal function of Fat3 mutant mice and found decreases in physiological and perceptual responses to high frequency flashes. These defects did not correlate with abnormal amacrine cell wiring, pointing instead to a role in bipolar cell subtypes that also express FAT3. Indeed, similar deficits were observed in mice lacking the bipolar cell glutamate receptors GRIK1 (OFF-bipolar cells) and GRM6 (ON-bipolar cells). Mechanistically, FAT3 binds to the synaptic protein PTPσ and is required to localize GRIK1 to OFF-cone bipolar cell synapses with cone photoreceptors. How FAT3 impacts ON-cone bipolar cell function at high temporal frequency remains to be uncovered. These findings expand the repertoire of FAT3's functions and reveal the importance of both ON- and OFF-bipolar cells for high frequency light response.

8.
Cell Rep ; 42(8): 112982, 2023 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-37585292

RESUMEN

In daylight, demand for visual chromophore (11-cis-retinal) exceeds supply by the classical visual cycle. This shortfall is compensated, in part, by the retinal G-protein-coupled receptor (RGR) photoisomerase, which is expressed in both the retinal pigment epithelium (RPE) and in Müller cells. The relative contributions of these two cellular pools of RGR to the maintenance of photoreceptor light responses are not known. Here, we use a cell-specific gene reactivation approach to elucidate the kinetics of RGR-mediated recovery of photoreceptor responses following light exposure. Electroretinographic measurements in mice with RGR expression limited to either cell type reveal that the RPE and a specialized subset of Müller glia contribute both to scotopic and photopic function. We demonstrate that 11-cis-retinal formed through photoisomerization is rapidly hydrolyzed, consistent with its role in a rapid visual pigment regeneration process. Our study shows that RGR provides a pan-retinal sink for all-trans-retinal released under sustained light conditions and supports rapid chromophore regeneration through the photic visual cycle.


Asunto(s)
Epitelio Pigmentado de la Retina , Retinaldehído , Animales , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Retinaldehído/metabolismo , Pigmentos Retinianos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neuroglía/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo
9.
Hum Mol Genet ; 32(17): 2735-2750, 2023 08 26.
Artículo en Inglés | MEDLINE | ID: mdl-37384398

RESUMEN

Phosphodiesterase-6 (PDE6) is the key phototransduction effector enzyme residing in the outer segment (OS) of photoreceptors. Cone PDE6 is a tetrameric protein consisting of two inhibitory subunits (γ') and two catalytic subunits (α'). The catalytic subunit of cone PDE6 contains a C-terminus prenylation motif. Deletion of PDE6α' C-terminal prenylation motif is linked to achromatopsia (ACHM), a type of color blindness in humans. However, mechanisms behind the disease and roles for lipidation of cone PDE6 in vision are unknown. In this study, we generated two knock-in mouse models expressing mutant variants of cone PDE6α' lacking the prenylation motif (PDE6α'∆C). We find that the C-terminal prenylation motif is the primary determinant for the association of cone PDE6 protein with membranes. Cones from PDE6α'∆C homozygous mice are less sensitive to light, and their response to light is delayed, whereas cone function in heterozygous PDE6α'∆C/+ mice is unaffected. Surprisingly, the expression level and assembly of cone PDE6 protein were unaltered in the absence of prenylation. Unprenylated assembled cone PDE6 in PDE6α'∆C homozygous animals is mislocalized and enriched in the cone inner segment and synaptic terminal. Interestingly, the disk density and the overall length of cone OS in PDE6α'∆C homozygous mutants are altered, highlighting a novel structural role for PDE6 in maintaining cone OS length and morphology. The survival of cones in the ACHM model generated in this study bodes well for gene therapy as a treatment option for restoring vision in patients with similar mutations in the PDE6C gene.


Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Células Fotorreceptoras Retinianas Conos , Humanos , Ratones , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Fototransducción , Prenilación
10.
Proc Natl Acad Sci U S A ; 120(23): e2217885120, 2023 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-37252956

RESUMEN

Retinitis pigmentosa (RP) is an ocular disease characterized by the loss of night vision, followed by the loss of daylight vision. Daylight vision is initiated in the retina by cone photoreceptors, which are gradually lost in RP, often as bystanders in a disease process that initiates in their neighboring rod photoreceptors. Using physiological assays, we investigated the timing of cone electroretinogram (ERG) decline in RP mouse models. A correlation between the time of loss of the cone ERG and the loss of rods was found. To investigate a potential role of the visual chromophore supply in this loss, mouse mutants with alterations in the regeneration of the retinal chromophore, 11-cis retinal, were examined. Reducing chromophore supply via mutations in Rlbp1 or Rpe65 resulted in greater cone function and survival in a RP mouse model. Conversely, overexpression of Rpe65 and Lrat, genes that can drive the regeneration of the chromophore, led to greater cone degeneration. These data suggest that abnormally high chromophore supply to cones upon the loss of rods is toxic to cones, and that a potential therapy in at least some forms of RP is to slow the turnover and/or reduce the level of visual chromophore in the retina.


Asunto(s)
Visión de Colores , Retinitis Pigmentosa , Ratones , Animales , Retina , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Retinitis Pigmentosa/genética , Modelos Animales de Enfermedad
11.
eNeuro ; 10(3)2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36823167

RESUMEN

Rhodopsin is the critical receptor molecule which enables vertebrate rod photoreceptor cells to detect a single photon of light and initiate a cascade of molecular events leading to visual perception. Recently, it has been suggested that the F45L mutation in the transmembrane helix of rhodopsin disrupts its dimerization in vitro To determine whether this mutation of rhodopsin affects its signaling properties in vivo, we generated knock-in mice expressing the rhodopsin F45L mutant. We then examined the function of rods in the mutant mice versus wild-type controls, using in vivo electroretinography and transretinal and single cell suction recordings, combined with morphologic analysis and spectrophotometry. Although we did not evaluate the effect of the F45L mutation on the state of dimerization of the rhodopsin in vivo, our results revealed that F45L-mutant mice exhibit normal retinal morphology, normal rod responses as measured both in vivo and ex vivo, and normal rod dark adaptation. We conclude that the F45L mutation does not affect the signaling properties of rhodopsin in its natural setting.


Asunto(s)
Células Fotorreceptoras Retinianas Bastones , Rodopsina , Ratones , Animales , Rodopsina/genética , Retina , Mutación/genética , Adaptación a la Oscuridad/genética
12.
PLoS One ; 17(8): e0272506, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35939447

RESUMEN

Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and identical posttranslational farnesylation of their Gγ subunits, rods and cones rely on unique Gγ1 (Gngt1) and Gγc (Gngt2) isoforms, respectively. The only other farnesylated G-protein γ-subunit, Gγ11 (Gng11), is expressed in multiple tissues but not retina. To determine whether Gγ1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11 in Gγ1-deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Gγ in rod cells and restoration of Gαt1 expression, which is greatly reduced in Gγ1-deficient rods. Electroretinography showed restoration of visual function in all three transgenic Gγ1-deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Gγ1-deficient rods was also fully restored. In all dark-adapted transgenic lines, Gαt1 was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1 translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1 translocation in Gγ11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11 transgenic mice but not in Gγ1 controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11 affects the ability of rods to adapt to background light.


Asunto(s)
Subunidades gamma de la Proteína de Unión al GTP , Células Fotorreceptoras Retinianas Bastones , Animales , Electrorretinografía , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Ratones , Ratones Transgénicos , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Transducina/genética , Transducina/metabolismo
13.
Invest Ophthalmol Vis Sci ; 63(8): 18, 2022 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-35861670

RESUMEN

Purpose: Light detection in retinal rod photoreceptors is initiated by activation of the visual pigment rhodopsin. A critical, yet often-overlooked, step enabling efficient perception of light is rhodopsin dephosphorylation mediated by protein phosphatase 2A (PP2A). PP2A deficiency has been reported to impair rhodopsin regeneration after phosphorylation by G protein receptor kinase 1 (GRK1) and binding of arrestin (Arr1), thereby delaying rod dark adaptation. However, its effects on the viability of photoreceptors in the absence of GRK1 and Arr1 remain unclear. Here, we investigated the effects of PP2A deficiency in the absence of GRK1 or Arr1, both of which have been implicated in Oguchi disease, a form of night blindness. Methods: Rod-specific mice lacking the predominant catalytic Cα-subunit of PP2A were crossed with the Grk1-/- or Arr1-/- strains to obtain double knockout lines. Rod photoreceptor viability was analyzed in histological cross-sections of the retina stained with hematoxylin and eosin, and rod function was evaluated by ex vivo electroretinography. Results: PP2A deficiency alone did not impair photoreceptor viability up to 12 months of age. Retinal degeneration was more pronounced in rods lacking GRK1 compared to rods lacking Arr1, and degeneration was accelerated in both Grk1-/- or Arr1-/- strains where PP2A was also deleted. In Arr1-/- mice, rod maximal photoresponse amplitudes were reduced by 80% at 3 months, and this diminution was enhanced further with concomitant PP2A deficiency. Conclusions: These results suggest that although PP2A is not required for the survival of rods, its deletion accelerates the degeneration induced by the absence of either GRK1 or Arr1.


Asunto(s)
Arrestina , Degeneración Retiniana , Animales , Arrestina/metabolismo , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Ratones , Ratones Noqueados , Proteína Fosfatasa 2 , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Células Fotorreceptoras Retinianas Bastones/fisiología , Rodopsina/metabolismo
14.
FASEB J ; 36(7): e22390, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35665537

RESUMEN

The daylight and color vision of diurnal vertebrates depends on cone photoreceptors. The capability of cones to operate and respond to changes in light brightness even under high illumination is attributed to their fast rate of recovery to the ground photosensitive state. This process requires the rapid replenishing of photoisomerized visual chromophore (11-cis-retinal) to regenerate cone visual pigments. Recently, several gene candidates have been proposed to contribute to the cone-specific retinoid metabolism, including acyl-CoA wax alcohol acyltransferase 2 (AWAT2, aka MFAT). Here, we evaluated the role of AWAT2 in the regeneration of visual chromophore by the phenotypic characterization of Awat2-/- mice. The global absence of AWAT2 enzymatic activity did not affect gross retinal morphology or the rate of visual chromophore regeneration by the canonical RPE65-dependent visual cycle. Analysis of Awat2 expression indicated the presence of the enzyme throughout the murine retina, including the retinal pigment epithelium (RPE) and Müller cells. Electrophysiological recordings revealed reduced maximal rod and cone dark-adapted responses in AWAT2-deficient mice compared to control mice. While rod dark adaptation was not affected by the lack of AWAT2, M-cone dark adaptation both in isolated retina and in vivo was significantly suppressed. Altogether, these results indicate that while AWAT2 is not required for the normal operation of the canonical visual cycle, it is a functional component of the cone-specific visual chromophore regenerative pathway.


Asunto(s)
Células Fotorreceptoras Retinianas Conos , Células Fotorreceptoras Retinianas Bastones , Acilcoenzima A/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Animales , Ratones , Retina/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Retinaldehído/metabolismo
15.
Sci Rep ; 12(1): 2897, 2022 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-35190581

RESUMEN

Calcium regulates the response sensitivity, kinetics and adaptation in photoreceptors. In striped bass cones, this calcium feedback includes direct modulation of the transduction cyclic nucleotide-gated (CNG) channels by the calcium-binding protein CNG-modulin. However, the possible role of EML1, the mammalian homolog of CNG-modulin, in modulating phototransduction in mammalian photoreceptors has not been examined. Here, we used mice expressing mutant Eml1 to investigate its role in the development and function of mouse photoreceptors using immunostaining, in-vivo and ex-vivo retinal recordings, and single-cell suction recordings. We found that the mutation of Eml1 causes significant changes in the mouse retinal structure characterized by mislocalization of rods and cones in the inner retina. Consistent with the fraction of mislocalized photoreceptors, rod and cone-driven retina responses were reduced in the mutants. However, the Eml1 mutation had no effect on the dark-adapted responses of rods in the outer nuclear layer. Notably, we observed no changes in the cone sensitivity in the Eml1 mutant animals, either in darkness or during light adaptation, ruling out a role for EML1 in modulating cone CNG channels. Together, our results suggest that EML1 plays an important role in retina development but does not modulate phototransduction in mammalian rods and cones.


Asunto(s)
Movimiento Celular/genética , Supervivencia Celular/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Células Fotorreceptoras Retinianas Bastones/fisiología , Animales , Calcio/fisiología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Retina/patología , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Bastones/patología , Visión Ocular/genética
16.
Hum Mol Genet ; 31(7): 1035-1050, 2022 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-34652420

RESUMEN

Heteromeric Kv2.1/Kv8.2 channels are voltage-gated potassium channels localized to the photoreceptor inner segment. They carry IKx, which is largely responsible for setting the photoreceptor resting membrane potential. Mutations in Kv8.2 result in childhood-onset cone dystrophy with supernormal rod response (CDSRR). We generated a Kv8.2 knockout (KO) mouse and examined retinal signaling and photoreceptor degeneration to gain deeper insight into the complex phenotypes of this disease. Using electroretinograms, we show that there were delayed or reduced signaling from rods depending on the intensity of the light stimulus, consistent with reduced capacity for light-evoked changes in membrane potential. The delayed response was not seen ex vivo where extracellular potassium levels were controlled by the perfusion buffer, so we propose the in vivo alteration is influenced by genotype-associated ionic imbalance. We observed mild retinal degeneration. Signaling from cones was reduced but there was no loss of cone density. Loss of Kv8.2 altered responses to flickering light with responses attenuated at high frequencies and altered in shape at low frequencies. The Kv8.2 KO line on an all-cone retina background had reduced cone-driven ERG b wave amplitudes and underwent degeneration. Altogether, we provide insight into how a deficit in the dark current affects the health and function of photoreceptors.


Asunto(s)
Canales de Potasio con Entrada de Voltaje , Degeneración Retiniana , Enfermedades de la Retina , Animales , Electrorretinografía , Ratones , Canales de Potasio con Entrada de Voltaje/genética , Retina/fisiología , Células Fotorreceptoras Retinianas Conos/fisiología , Degeneración Retiniana/genética
17.
Front Cell Neurosci ; 15: 761416, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34690705

RESUMEN

The past decades have seen tremendous progress in our understanding of the function of photoreceptors and olfactory sensory neurons, uncovering the mechanisms that determine their properties and, ultimately, our ability to see and smell. This progress has been driven to a large degree by the powerful combination of physiological experimental tools and genetic manipulations, which has enabled us to identify the main molecular players in the transduction cascades of these sensory neurons, how their properties affect the detection and discrimination of stimuli, and how diseases affect our senses of vision and smell. This review summarizes some of the common and unique features of photoreceptors and olfactory sensory neurons that make these cells so exciting to study.

18.
Polymers (Basel) ; 13(19)2021 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-34641139

RESUMEN

The ocular immune privilege is a phenomenon brought about by anatomical and physiological barriers to shield the eye from immune and inflammation responses. While this phenomenon is beneficial for eyes protection, it is, at the same time, a hindrance for drug delivery to the posterior segment of the eye to treat retinal diseases. Some ocular barriers can be bypassed by intravitreal injections, but these are associated with several side effects and patient noncompliance, especially when frequent injections are required. As an alternative, applying drugs as an eye drop is preferred due to the safety and ease. This study investigated the possible use of topically-applied hyaluronic acid-coated gold nanoparticles as drug delivery vehicles to the back of the eye. The coated gold nanoparticles were topically applied to mouse eyes, and results were compared to topically applied uncoated gold nanoparticles and phosphate-buffered saline (PBS) solution. Retina sections from these mice were then analyzed using fluorescence microscopy, inductively coupled plasma mass spectrometry (ICP-MS), and transmission electron microscopy (TEM). All characterization techniques used in this study suggest that hyaluronic acid-coated gold nanoparticles have higher distribution in the posterior segment of the eye than uncoated gold nanoparticles. Electroretinogram (ERG) analysis revealed that the visual function of mice receiving the coated gold nanoparticles was not affected, and these nanoparticles can, therefore, be applied safely. Together, our results suggest that hyaluronic acid-coated gold nanoparticles constitute potential drug delivery vehicles to the retina when applied noninvasively as an eye drop.

19.
ACS Omega ; 6(9): 6172-6184, 2021 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-33718708

RESUMEN

Delivering therapeutics to the posterior segment of the eye is challenging due to various anatomical and physical barriers. While significant improvements have been realized by introducing direct injections to diseased sites, these approaches come with potential side effects that can range from simple inflammation to severe retinal damage. The topical instillation of drugs remains a safer and preferred alternative for patients' compliance. Here, we report the synthesis of penetratin-complexed, redox-responsive hyaluronic acid-based nanogels for the triggered release and delivery of therapeutics to the posterior part of the eye via topical application. The synthesized nanogels were shown to release their load only when exposed to a reducing environment, similar to the cytoplasm. As a model drug, visual chromophore analog, 9-cis-retinal, was loaded into nanogels and efficiently delivered to the mouse retina's photoreceptors when applied topically. Electroretinogram measurements showed a partial recovery of photoreceptor function in all treated eyes versus untreated controls. To the best of our knowledge, this report constitutes the first attempt to use a topically applied triggered-release drug delivery system to target the pigmented layer of the retina, in addition to the first attempt to deliver the visual chromophore topically.

20.
J Gen Physiol ; 153(1)2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33216847

RESUMEN

Cone photoreceptors mediate daytime vision in vertebrates. The rapid and efficient regeneration of their visual pigments following photoactivation is critical for the cones to remain photoresponsive in bright and rapidly changing light conditions. Cone pigment regeneration depends on the recycling of visual chromophore, which takes place via the canonical visual cycle in the retinal pigment epithelium (RPE) and the Müller cell-driven intraretinal visual cycle. The molecular mechanisms that enable the neural retina to regenerate visual chromophore for cones have not been fully elucidated. However, one known component of the two visual cycles is the cellular retinaldehyde-binding protein (CRALBP), which is expressed both in the RPE and in Müller cells. To understand the significance of CRALBP in cone pigment regeneration, we examined the function of cones in mice heterozygous for Rlbp1, the gene encoding CRALBP. We found that CRALBP expression was reduced by ∼50% in both the RPE and retina of Rlbp1+/- mice. Electroretinography (ERG) showed that the dark adaptation of rods and cones is unaltered in Rlbp1+/- mice, indicating a normal RPE visual cycle. However, pharmacologic blockade of the RPE visual cycle revealed suppressed cone dark adaptation in Rlbp1+/- mice in comparison with controls. We conclude that the expression level of CRALPB specifically in the Müller cells modulates the efficiency of the retina visual cycle. Finally, blocking the RPE visual cycle also suppressed further cone dark adaptation in Rlbp1-/- mice, revealing a shunt in the classical RPE visual cycle that bypasses CRALBP and allows partial but unexpectedly rapid cone dark adaptation.


Asunto(s)
Retina , Células Fotorreceptoras Retinianas Conos , Animales , Adaptación a la Oscuridad , Ratones , Pigmentos Retinianos , Células Fotorreceptoras Retinianas Bastones
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