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Biallelic mutations in interphotoreceptor matrix proteoglycan 2 (IMPG2) in humans cause retinitis pigmentosa (RP) with early macular involvement, albeit the disease progression varies widely due to genetic heterogeneity and IMPG2 mutation type. There are currently no treatments for IMPG2-RP. To aid preclinical studies toward eventual treatments, there is a need to better understand the progression of disease pathology in appropriate animal models. Toward this goal, we developed mouse models with patient mimicking homozygous frameshift (T807Ter) or missense (Y250C) Impg2 mutations, as well as mice with a homozygous frameshift mutation (Q244Ter) designed to completely prevent IMPG2 protein expression, and characterized the trajectory of their retinal pathologies across postnatal development until late adulthood. We found that the Impg2T807Ter/T807Ter and Impg2Q244Ter/Q244Ter mice exhibited early onset gliosis, impaired photoreceptor outer segment maintenance, appearance of subretinal deposits near the optic disc, disruption of the outer retina, and neurosensorial detachment, whereas the Impg2Y250C/Y250C mice exhibited minimal retinal pathology. These results demonstrate the importance of mutation type in disease progression in IMPG2-RP and provide a toolkit and preclinical data for advancing therapeutic approaches.
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Proteoglicanas , Retinose Pigmentar , Humanos , Animais , Camundongos , Adulto , Proteoglicanas/genética , Retina , Mutação , Retinose Pigmentar/genética , Progressão da DoençaRESUMO
Nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) is required for nuclear nicotinamide adenine mononucleotide (NAD+) biosynthesis in all nucleated cells, and despite its functional ubiquity, mutations in this gene lead to an isolated retinal degeneration. The mechanisms underlying how mutant NMNAT1 causes disease are not well understood, nor is the reason why the pathology is confined to the retina. Using a mouse model of NMNAT1-associated retinal degeneration that harbors the p.Val9Met mutation, we tested the hypothesis that decreased function of mutant NMNAT1 has a greater effect on the levels of NAD+ in the retina than elsewhere in the body. Measurements by liquid chromatography with tandem mass spectrometry showed an early and sustained decrease of NAD+ in mutant retinas that was not observed in other tissues. To understand how consumers of nuclear NAD+ are affected by the reduced availability of NAD+ in mutant retinas, poly(ADP-ribose) polymerase (PARP) and nuclear sirtuin activity were evaluated. PARP activity was elevated during disease progression, as evidenced by overproduction of poly(ADP-ribose) (PAR) in photoreceptors, whereas histone deacetylation activity of nuclear sirtuins was not altered. We hypothesized that PARP could be activated because of elevated levels of oxidative stress; however, we did not observe oxidative DNA damage, lipid peroxidation, or a low glutathione to oxidized glutathione ratio. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed that photoreceptors appear to ultimately die by apoptosis, although the low NAD+ levels and overproduction of PAR suggest that cell death may include aspects of the parthanatos cell death pathway.
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Modelos Animais de Doenças , Mutação , NAD/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Poli Adenosina Difosfato Ribose/metabolismo , Retina/metabolismo , Degeneração Retiniana/genética , Animais , Apoptose/genética , Cromatografia Líquida , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Estresse Oxidativo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Degeneração Retiniana/metabolismo , Sirtuínas/metabolismo , Espectrometria de Massas em TandemRESUMO
Intraflagellar transport (IFT) is a bidirectional transport process that occurs along primary cilia and specialized sensory cilia, such as photoreceptor outersegments. Genes coding for various IFT components are associated with ciliopathies. Mutations in IFT172 lead to diseases ranging from isolated retinal degeneration to severe syndromic ciliopathies. In this study, we created a mouse model of IFT172-associated retinal degeneration to investigate the ocular disease mechanism. We found that depletion of IFT172 in rod photoreceptors leads to a rapid degeneration of the retina, with severely reduced electroretinography (ERG) responses by 1 month and complete outer-nuclear layer (ONL) degeneration by 2 months. We investigated molecular mechanisms of degeneration and show that IFT172 protein reduction leads to mislocalization of specific photoreceptor outersegment (OS) proteins (RHO, RP1, IFT139), aberrant light-driven translocation of alpha transducin and altered localization of glioma-associated oncogene family member 1 (GLI1). This mouse model exhibits key features of the retinal phenotype observed in patients with IFT172-associated blindness and can be used for in vivo testing of ciliopathy therapies.
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Proteínas de Transporte/genética , Cílios/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Degeneração Retiniana/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Cílios/patologia , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Eletrorretinografia , Humanos , Camundongos , Camundongos Knockout , Mutação , Retina/diagnóstico por imagem , Retina/patologia , Degeneração Retiniana/diagnóstico por imagem , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Proteína GLI1 em Dedos de Zinco/genéticaRESUMO
Parthanatos is a programmed cell death pathway mediated by the effects of pathogenically high levels of poly(ADP-ribose) polymerase 1 (PARP1) activity. This process underlies a broad range of diseases affecting many tissues and organs across the body, including the retina. This chapter reviews mechanisms that are currently understood to drive parthanatos in the context of retinal diseases associated with this form of cell death. Toxicity of upregulated poly(ADP-ribose) (PAR) content, NAD+ and ATP depletion, translocation of apoptosis-inducing factor (AIF) to the nucleus, and loss of glycolytic function are discussed. Since therapies that preserve vulnerable cells remain elusive for the vast majority of retinal diseases, pharmacologically blocking parthanatos may be an effective treatment strategy for cases in which this process contributes to pathogenesis.
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Parthanatos , Doenças Retinianas/patologia , Fator de Indução de Apoptose , Humanos , Poli(ADP-Ribose) Polimerase-1/genética , Poli Adenosina Difosfato Ribose , Transporte ProteicoRESUMO
The nicotinamide nucleotide adenylyltransferase 1 (NMNAT1) enzyme is essential for regenerating the nuclear pool of NAD(+) in all nucleated cells in the body, and mounting evidence also suggests that it has a separate role in neuroprotection. Recently, mutations in the NMNAT1 gene were associated with Leber congenital amaurosis, a severe retinal degenerative disease that causes blindness during infancy. Availability of a reliable mammalian model of NMNAT1-Leber congenital amaurosis would assist in determining the mechanisms through which disruptions in NMNAT1 lead to retinal cell degeneration and would provide a resource for testing treatment options. To this end, we identified two separate N-ethyl-N-nitrosourea-generated mouse lines that harbor either a p.V9M or a p.D243G mutation. Both mouse models recapitulate key aspects of the human disease and confirm the pathogenicity of mutant NMNAT1. Homozygous Nmnat1 mutant mice develop a rapidly progressing chorioretinal disease that begins with photoreceptor degeneration and includes attenuation of the retinal vasculature, optic atrophy, and retinal pigment epithelium loss. Retinal function deteriorates in both mouse lines, and, in the more rapidly progressing homozygous Nmnat1(V9M) mutant mice, the electroretinogram becomes undetectable and the pupillary light response weakens. These mouse models offer an opportunity for investigating the cellular mechanisms underlying disease pathogenesis, evaluating potential therapies for NMNAT1-Leber congenital amaurosis, and conducting in situ studies on NMNAT1 function and NAD(+) metabolism.
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Modelos Animais de Doenças , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/fisiopatologia , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Animais , Genótipo , Humanos , Camundongos , Camundongos Mutantes , Reação em Cadeia da PolimeraseRESUMO
Specific variants of human long-wavelength (L) and middle-wavelength (M) cone opsin genes have recently been associated with a variety of vision disorders caused by cone malfunction, including red-green color vision deficiency, blue cone monochromacy, myopia, and cone dystrophy. Strikingly, unlike disease-causing mutations in rhodopsin, most of the cone opsin alleles that are associated with vision disorders do not have deleterious point mutations. Instead, specific combinations of normal polymorphisms that arose by genetic recombination between the genes encoding L and M opsins appear to cause disease. Knockout/knock-in mice promise to make it possible to study how these deleterious cone opsin variants affect the structure, function, and viability of the cone photoreceptors. Ideally, we would like to evaluate different variants that cause vision disorders in humans against a control pigment that is not associated with vision disorders, and each variant should be expressed as the sole photopigment in each mouse cone, as is the case in humans. To evaluate the feasibility of this approach, we created a line of mice to serve as the control in the analysis of disease-causing mutations by replacing exon 2 through 6 of the mouse M-opsin gene with the corresponding cDNA for a human L-opsin variant that is associated with normal vision. Experiments reported here establish that the resulting pigment, which differs from the endogenous mouse M opsin at 35 amino acid positions, functions normally in mouse cones. This pigment was evaluated in mice with and without coexpression of the mouse short wavelength (S) opsin. Here, the creation and validation of two lines of genetically engineered mice that can be used to study disease-causing variants of human L/M-opsins, in vivo, are described.
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Modelos Animais , Opsinas/genética , Sequência de Aminoácidos , Animais , Eletrorretinografia , Técnicas de Genotipagem , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Opsinas/química , RNA Mensageiro/isolamento & purificação , Proteínas Recombinantes , Transcrição GênicaRESUMO
The electroretinogram (ERG) provides information about outer retina function in both clinical and research applications. ERG components elicited by light increments and decrements can be separated using a long-flash paradigm in which periods of light ON and OFF are alternated. Here, the ON-OFF ERG is combined with a silent substitution technique to elicit responses from individual cone photoreceptor classes by modulating the intensities of three color lights between the two periods. The results focus on the short wavelength (S) cone pathways since they are vulnerable to disease and because there are many unanswered questions about S-cone ON and OFF circuitry.
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Percepção de Cores/fisiologia , Eletrorretinografia/métodos , Células Fotorreceptoras Retinianas Cones/citologia , Transdução de Sinais , Animais , Humanos , PapioRESUMO
Importance: Approximately 70% of crew members who complete long-duration missions to the International Space Station develop signs of optic disc edema, a hallmark finding of spaceflight-associated neuro-ocular syndrome. The onset and magnitude of edema differ across individuals, and the reason for this variability remains unknown. Identifying risk factors for spaceflight-induced disc edema is important because this condition may become more severe during extended-duration missions to the moon and Mars and could be associated with irreversible vision loss. Objective: To assess whether preflight indicators of crowded optic nerve head morphology, other ocular measures (such as choroid thickness and axial length), body weight, body mass index, sex, age, and previous flight experience are associated with optic disc edema development. Design, Setting, and Participants: This cohort study analyzed ocular, body weight, and demographic data collected from 31 US and international crew members before, during, and after spaceflight at the NASA Johnson Space Center and International Space Station. Ocular factors assessed included preflight and in-flight peripapillary total retinal thickness, minimum rim width, optic cup volume, mean cup depth, mean cup width, cup-disc ratio, Bruch membrane opening area, retinal nerve fiber layer thickness, choroid thickness, axial length, and refractive error. In addition, body weight, body mass index, sex, age, and previous spaceflight experience were assessed for associations with optic disc edema development. The data were analyzed from August 2021 to June 2022. Exposure: Approximately 6 to 12 months of spaceflight. Main Outcomes and Measures: In-flight increases in peripapillary total retinal thickness. Linear mixed models were used to assess for associations between a wide range of risk factors and in-flight increases in peripapillary total retinal thickness, which is a sensitive objective measure for detecting optic disc edema. Results: This study included 31 International Space Station crew members with a mean (SD) age of 46.9 (6.0) years (25 men [80.6%]). During spaceflight, mean (SE) peripapillary total retinal thickness increased from 392.0 (5.8) µm to 430.2 (9.6) µm (P < .001), and greater individual changes were associated with smaller preflight cup volume (slope [SE], -62.8 [18.9]; P = .002), shallower preflight cup depth (slope [SE], -0.11 [0.03]; P < .001), and narrower preflight cup width (slope [SE], -0.03 [0.01]; P = .03). No associations were observed between changes in peripapillary total retinal thickness and any other variable evaluated. Conclusions and Relevance: Findings of this cohort study suggest that smaller optic cup morphology may be associated with optic disc edema development during spaceflight. Crew members with this cup profile may benefit from enhanced ophthalmic monitoring during spaceflight and use of countermeasures against spaceflight-associated neuro-ocular syndrome.
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Papiledema , Voo Espacial , Masculino , Humanos , Pessoa de Meia-Idade , Papiledema/diagnóstico , Papiledema/etiologia , Estudos de Coortes , Edema , Peso CorporalRESUMO
Importance: Countermeasures that reverse the headward fluid shift experienced in weightlessness have the potential to mitigate spaceflight-associated neuro-ocular syndrome. This study investigated whether use of the countermeasure lower-body negative pressure during spaceflight was associated with changes in ocular structure. Objective: To determine whether changes to the optic nerve head and retina during spaceflight can be mitigated by brief in-flight application of 25-mm Hg lower-body negative pressure. Design, Setting, and Participants: In the National Aeronautics and Space Administration's "Fluid Shifts Study," a prospective cohort study, optical coherence tomography scans of the optic nerve head and macula were obtained from US and international crew members before flight, in-flight, and up to 180 days after return to Earth. In-flight scans were obtained both under normal weightless conditions and 10 to 20 minutes into lower-body negative pressure exposure. Preflight and postflight data were collected in the seated, supine, and head-down tilt postures. Crew members completed 6- to 12-month missions that took place on the International Space Station. Data were analyzed from 2016 to 2021. Interventions or Exposures: Spaceflight and lower-body negative pressure. Main Outcomes and Measures: Changes in minimum rim width, optic cup volume, Bruch membrane opening height, peripapillary total retinal thickness, and macular thickness. Results: Mean (SD) flight duration for the 14 crew members (mean [SD] age, 45 [6] years; 11 male crew members [79%]) was 214 (72) days. Ocular changes on flight day 150, as compared with preflight seated, included an increase in minimum rim width (33.8 µm; 95% CI, 27.9-39.7 µm; P < .001), decrease in cup volume (0.038 mm3; 95% CI, 0.030-0.046 mm3; P < .001), posterior displacement of Bruch membrane opening (-9.0 µm; 95% CI, -15.7 to -2.2 µm; P = .009), and decrease in macular thickness (fovea to 500 µm, 5.1 µm; 95% CI, 3.5-6.8 µm; P < .001). Brief exposure to lower-body negative pressure did not affect these parameters. Conclusions and Relevance: Results of this cohort study suggest that peripapillary tissue thickening, decreased cup volume, and mild central macular thinning were associated with long-duration spaceflight. Acute exposure to 25-mm Hg lower-body negative pressure did not alter optic nerve head or retinal morphology, suggesting that longer durations of a fluid shift reversal may be needed to mitigate spaceflight-induced changes and/or other factors are involved.
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Disco Óptico , Voo Espacial , Estudos de Coortes , Deslocamentos de Líquidos Corporais/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Retina/diagnóstico por imagem , Voo Espacial/métodosRESUMO
Spaceflight associated neuro-ocular syndrome (SANS) is hypothesized to develop as a consequence of the chronic headward fluid shift that occurs in sustained weightlessness. We exposed healthy subjects (n = 24) to strict 6° head-down tilt bed rest (HDTBR), an analog of weightlessness that generates a sustained headward fluid shift, and we monitored for ocular changes similar to findings that develop in SANS. Two-thirds of the subjects received a daily 30-min exposure to artificial gravity (AG, 1 g at center of mass, ~0.3 g at eye level) during HDTBR by either continuous (cAG, n = 8) or intermittent (iAG, n = 8) short-arm centrifugation to investigate whether this intervention would attenuate headward fluid shift-induced ocular changes. Optical coherence tomography images were acquired to quantify changes in peripapillary total retinal thickness (TRT), retinal nerve fiber layer thickness, and choroidal thickness, and to detect chorioretinal folds. Intraocular pressure (IOP), optical biometry, and standard automated perimetry data were collected. TRT increased by 35.9 µm (95% CI, 19.9-51.9 µm, p < 0.0001), 36.5 µm (95% CI, 4.7-68.2 µm, p = 0.01), and 27.6 µm (95% CI, 8.8-46.3 µm, p = 0.0005) at HDTBR day 58 in the control, cAG, and iAG groups, respectively. Chorioretinal folds developed in six subjects across the groups, despite small increases in IOP. Visual function outcomes did not change. These findings validate strict HDTBR without elevated ambient CO2 as a model for investigating SANS and suggest that a fluid shift reversal of longer duration and/or greater magnitude at the eye may be required to prevent or mitigate SANS.
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Repouso em Cama/efeitos adversos , Doenças da Coroide/patologia , Decúbito Inclinado com Rebaixamento da Cabeça/efeitos adversos , Papiledema/patologia , Doenças Retinianas/patologia , Simulação de Ausência de Peso/efeitos adversos , Adulto , Estudos de Casos e Controles , Doenças da Coroide/etiologia , Feminino , Humanos , Masculino , Papiledema/etiologia , Doenças Retinianas/etiologiaRESUMO
Head-to-foot gravitationally induced hydrostatic pressure gradients in the upright posture on Earth are absent in weightlessness. This results in a relative headward fluid shift in the vascular and cerebrospinal fluid compartments and may underlie multiple physiological consequences of spaceflight, including the spaceflight-associated neuro-ocular syndrome. Here, we tested three mechanical countermeasures [lower body negative pressure (LBNP), venoconstrictive thigh cuffs (VTC), and impedance threshold device (ITD) resistive inspiratory breathing] individually and in combination to reduce a posture-induced headward fluid shift as a ground-based spaceflight analog. Ten healthy subjects (5 male) underwent baseline measures (seated and supine postures) followed by countermeasure exposure in the supine posture. Noninvasive measurements included ultrasound [internal jugular veins (IJV) cross-sectional area, cardiac stroke volume, optic nerve sheath diameter, noninvasive IJV pressure], transient evoked otoacoustic emissions (OAE; intracranial pressure index), intraocular pressure, choroidal thickness from optical coherence tomography imaging, and brachial blood pressure. Compared with the supine posture, IJV area decreased 48% with application of LBNP [mean ratio: 0.52, 95% confidence interval (CI): 0.44-0.60, P < 0.001], 31% with VTC (mean ratio: 0.69, 95% CI: 0.55-0.87, P < 0.001), and 56% with ITD (mean ratio: 0.44, 95% CI: 0.12-1.70, P = 0.46), measured at end-inspiration. LBNP was the only individual countermeasure to decrease the OAE phase angle (Δ -12.9 degrees, 95% CI: -25 to -0.9, P = 0.027), and use of combined countermeasures did not result in greater effects. Thus, LBNP, and to a lesser extent VTC and ITD, represents promising headward fluid shift countermeasures but will require future testing in analog and spaceflight environments.NEW & NOTEWORTHY As a weightlessness-induced headward fluid shift is hypothesized to be a primary factor underlying several physiological consequences of spaceflight, countermeasures aimed at reversing the fluid shift will likely be crucial during exploration-class spaceflight missions. Here, we tested three mechanical countermeasures individually and in various combinations to reduce a posture-induced headward fluid shift as a ground-based spaceflight analog.
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Voo Espacial , Ausência de Peso , Deslocamentos de Líquidos Corporais , Humanos , Pressão Intracraniana , Pressão Negativa da Região Corporal Inferior , Masculino , Ausência de Peso/efeitos adversosRESUMO
Spaceflight-associated neuro-ocular syndrome (SANS) develops during long-duration (>1 mo) spaceflight presumably because of chronic exposure to a headward fluid shift that occurs in weightlessness. We aimed to determine whether reversing this headward fluid shift with acute application of lower body negative pressure (LBNP) can influence outcome measures at the eye. Intraocular pressure (IOP) and subfoveal choroidal thickness were therefore evaluated by tonometry and optical coherence tomography (OCT), respectively, in 14 International Space Station crewmembers before flight in the seated, supine, and 15° head-down tilt (HDT) postures and during spaceflight, without and with application of 25 mmHg LBNP. IOP in the preflight seated posture was 14.4 mmHg (95% CI, 13.5-15.2 mmHg), and spaceflight elevated this value by 1.3 mmHg (95% CI, 0.7-1.8 mmHg, P < 0.001). Acute exposure to LBNP during spaceflight reduced IOP to 14.2 mmHg (95% CI, 13.4-15.0 mmHg), which was equivalent to that of the seated posture (P > 0.99), indicating that venous fluid redistribution by LBNP can influence ocular outcome variables during spaceflight. Choroidal thickness during spaceflight (374 µm, 95% CI, 325-423 µm) increased by 35 µm (95% CI, 25-45 µm, P < 0.001), compared with the preflight seated posture (339 µm, 95% CI, 289-388 µm). Acute use of LBNP during spaceflight did not affect choroidal thickness (381 µm, 95% CI, 331-430 µm, P = 0.99). The finding that transmission of reduced venous pressure by LBNP did not decrease choroidal thickness suggests that engorgement of this tissue during spaceflight may reflect changes that are secondary to the chronic cerebral venous congestion associated with spaceflight.NEW & NOTEWORTHY Spaceflight induces a chronic headward fluid shift that is believed to underlie ocular changes observed in astronauts. The present study demonstrates, for the first time, that reversing this headward fluid shift via application of lower body negative pressure (LBNP) during spaceflight may alter the ocular venous system, as evidenced by a decrease in intraocular pressure. This finding indicates that LBNP has the potential to be an effective countermeasure against the headward fluid shift during spaceflight, which may then be beneficial in preventing or reversing associated ocular changes.
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Voo Espacial , Ausência de Peso , Corioide , Humanos , Pressão Intraocular , Pressão Negativa da Região Corporal Inferior , Tonometria Ocular , Ausência de Peso/efeitos adversosRESUMO
No treatment is available for nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1)-associated retinal degeneration, an inherited disease that leads to severe vision loss early in life. Although the causative gene, NMNAT1, plays an essential role in nuclear nicotinamide adenine dinucleotide (NAD)+ metabolism in tissues throughout the body, NMNAT1-associated disease is isolated to the retina. Since this condition is recessive, supplementing the retina with a normal copy of NMNAT1 should protect vulnerable cells from disease progression. We tested this hypothesis in a mouse model that harbors the p.Val9Met mutation in Nmnat1 and consequently develops a retinal degenerative phenotype that recapitulates key features of the human disease. Gene augmentation therapy, delivered by subretinal injection of adeno-associated virus (AAV) carrying a normal human copy of NMNAT1, rescued retinal structure and function. Due to the early-onset profile of the phenotype, a rapidly activating self-complementary AAV was required to initiate transgene expression during the narrow therapeutic window. These data represent the first proof of concept for a therapy to treat patients with NMNAT1-associated disease.
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Treatment strategies for dominantly inherited disorders typically involve silencing or ablating the pathogenic allele. CRISPR-Cas nucleases have shown promise in allele-specific knockout approaches when the dominant allele creates unique protospacer adjacent motifs that can lead to allele-restricted targeting. Here, we present a spacer-mediated allele-specific knockout approach that utilizes both SpCas9 variants and truncated single-guide RNAs to achieve efficient discrimination of a single-nucleotide mutation in rhodopsin (Rho)-P23H mice, a model of dominant retinitis pigmentosa. We found that approximately 45% of the mutant P23H allele was edited at the DNA level and that the relative RNA expression of wild-type Rho was about 2.8 times more than that of mutant Rho in treated retinas. Furthermore, the progression of photoreceptor cell degeneration in outer nuclear layer was significantly delayed in treated regions of the Rho-P23H retinas at 5 weeks of age. Our proof-of-concept study therefore outlines a general strategy that could potentially be expanded to examine the therapeutic benefit of allele-specific gene editing approach to treat human P23H patients. Our study also extends allele-specific editing strategies beyond discrimination within the protospacer adjacent motif sites, with potentially broad applicability to other dominant diseases.
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PURPOSE: Human long (L) and middle (M) wavelength cone opsin genes are highly variable due to intermixing. Two L/M cone opsin interchange mutants, designated LIAVA and LVAVA, are associated with clinical diagnoses, including red-green color vision deficiency, blue cone monochromacy, cone degeneration, myopia, and Bornholm Eye Disease. Because the protein and splicing codes are carried by the same nucleotides, intermixing L and M genes can cause disease by affecting protein structure and splicing. METHODS: Genetically engineered mice were created to allow investigation of the consequences of altered protein structure alone, and the effects on cone morphology were examined using immunohistochemistry. In humans and mice, cone function was evaluated using the electroretinogram (ERG) under L/M- or short (S) wavelength cone isolating conditions. Effects of LIAVA and LVAVA genes on splicing were evaluated using a minigene assay. RESULTS: ERGs and histology in mice revealed protein toxicity for the LVAVA but not for the LIAVA opsin. Minigene assays showed that the dominant messenger RNA (mRNA) was aberrantly spliced for both variants; however, the LVAVA gene produced a small but significant amount of full-length mRNA and LVAVA subjects had correspondingly reduced ERG amplitudes. In contrast, the LIAVA subject had no L/M cone ERG. CONCLUSIONS: Dramatic differences in phenotype can result from seemingly minor differences in genotype through divergent effects on the dual amino acid and splicing codes. TRANSLATIONAL RELEVANCE: The mechanism by which individual mutations contribute to clinical phenotypes provides valuable information for diagnosis and prognosis of vision disorders associated with L/M interchange mutations, and it informs strategies for developing therapies.
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PURPOSE: With the goal of eventually restoring functional vision in patients with retinal degenerative diseases, USC/Second Sight Medical Products, Inc. chronically implanted blind human subjects with a prototype epiretinal prosthesis consisting of a 4 x 4 array of 16 stimulating electrodes. To accurately represent a visual scene, a visual prosthesis must convey luminance information across a range of brightness levels. To achieve this, the brightness of phosphenes produced by an individual electrode should scale appropriately with luminance, and the same luminance should produce equivalently bright phosphenes across the entire electrode array. The goal was to examine how apparent brightness changes as a function of stimulation intensity across electrodes. METHODS: As described in previous studies, electrical stimulation of intact cells of the neural retina using this prosthetic device reliably elicits visual percepts in human subjects blinded by retinitis pigmentosa. Here, apparent brightness for a range of electrical amplitudes was measured using both subjective magnitude rating and brightness-matching procedures in chronically implanted human subjects. RESULTS: It was found that apparent brightness can be described as a power function of stimulation intensity. The same model can also predict brightness matching across electrodes. CONCLUSIONS: These results suggest that a relatively simple model for scaling current across electrodes may be capable of producing equivalently bright phosphenes across an entire array. (ClinicalTrials.gov number, NCT00279500.).
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Eletrodos Implantados , Luz , Fosfenos/fisiologia , Implantação de Prótese , Retina/fisiologia , Degeneração Retiniana/fisiopatologia , Percepção Visual/fisiologia , Cegueira/reabilitação , Estimulação Elétrica , Humanos , Microeletrodos , Modelos Biológicos , Limiar Sensorial/fisiologiaRESUMO
PURPOSE: With the long-term goal of restoring functional vision in patients with retinal degenerative diseases, the eyes of blind human subjects were implanted chronically with epiretinal prostheses consisting of two-dimensional electrode arrays that directly stimulated cells of the neural retina. METHODS: Psychophysical techniques were used to measure the brightness of electrically generated percepts on single electrodes using a variety of electrical stimulation patterns. RESULTS: It was possible to predict the sensitivity of the human visual system to a wide variety of retinal electrical stimulation patterns using a simple and biologically plausible model. CONCLUSIONS: This is the first study to demonstrate that, on the single-electrode level, retinal electrical stimulation in humans can produce visual qualia that are predictable using a quantitative model, a prerequisite for a successful retinal prosthesis. (ClinicalTrials.gov number, NCT00279500.).