RESUMO
Astrocytes release glutamate upon activation of various GPCRs to exert important roles in synaptic functions. However, the molecular mechanism of release has been controversial. Here, we report two kinetically distinct modes of nonvesicular, channel-mediated glutamate release. The fast mode requires activation of G(αi), dissociation of G(ßγ), and subsequent opening of glutamate-permeable, two-pore domain potassium channel TREK-1 through direct interaction between G(ßγ) and N terminus of TREK-1. The slow mode is Ca(2+) dependent and requires G(αq) activation and opening of glutamate-permeable, Ca(2+)-activated anion channel Best1. Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Diffusion modeling predicts that the fast mode can target neuronal mGluR with peak glutamate concentration of 100 µM, whereas slow mode targets neuronal NMDA receptors at around 1 µM. Our results reveal two distinct sources of astrocytic glutamate that can differentially influence neighboring neurons.
Assuntos
Astrócitos/metabolismo , Proteínas do Olho/metabolismo , Ácido Glutâmico/metabolismo , Canais Iônicos/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Bestrofinas , Células Cultivadas , Exocitose , Proteínas do Olho/genética , Células HEK293 , Humanos , Canais Iônicos/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Canais de Potássio de Domínios Poros em Tandem/genética , Alinhamento de Sequência , Transdução de SinaisRESUMO
PURPOSE: To examine the effects of autophagy deficiency induced by RPE-specific deletion of Atg5 or Atg7 in mice as a function of age. METHODS: Conditional knockout mice with a floxed allele of Atg5 or Atg7 were crossed with inducible VMD2-rtTA/Cre transgenic mice. VMD2-directed RPE-specific Cre recombinase expression was induced with doxycycline feeding in the resulting mice. Cre-mediated deletion of floxed Atg5 or Atg7 resulted in RPE-specific inactivation of the Atg5 or Atg7 gene. Plastic and thin retinal sections were analyzed with light and electron microscopy for histological changes. Photoreceptor outer segment (POS) thickness in plastic sections was measured using the Adobe Photoshop CS4 extended ruler tool. Autophagic adaptor p62/SQSTM1 and markers for oxidatively damaged lipids, proteins, and DNA were examined with immunofluorescence staining of cryosections. Fluorescence signals were quantified using Image J software. RESULTS: Accumulation of p62/SQSTM1 reflecting autophagy deficiency was observed in the RPE of the Atg5ΔRPE and Atg7ΔRPE mice. 3-nitrotyrosine, advanced glycation end products (AGEs), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), markers for oxidatively damaged proteins and DNA, were also found to accumulate in the RPE of these mice. We observed retinal degeneration in 35% of the Atg5ΔRPE mice and 45% of the Atg7ΔRPE mice at 8 to 24 months old. Degeneration severity and the number of mice with degeneration increased with age. The mean POS thickness of these mice was 25 µm at 8-12 months, 15 µm at 13-18 months, and 3 µm at 19-24 months, compared to 35 µm, 30 µm, and 24 µm in the wild-type mice, respectively. Early age-related macular degeneration (AMD)-like RPE defects were found in all the Atg5ΔRPE and Atg7ΔRPE mice 13 months old or older, including vacuoles, uneven RPE thickness, diminished basal infoldings, RPE hypertrophy/hypotrophy, pigmentary irregularities, and necrosis. The severity of the RPE defects increased with age and in the mice with retinal degeneration. RPE atrophy and choroidal neovascularization (CNV) were occasionally observed in the Atg5ΔRPE and Atg7ΔRPE mice with advanced age. CONCLUSIONS: Autophagy deficiency induced by RPE-specific deletion of Atg5 or Atg7 predisposes but does not necessarily drive the development of AMD-like phenotypes or retinal degeneration.
Assuntos
Proteína 5 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/genética , Autofagia , Deleção de Genes , Degeneração Macular/genética , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/patologia , Alelos , Animais , Biomarcadores/metabolismo , Eletrorretinografia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Degeneração Retiniana/patologia , Epitélio Pigmentado da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Adenylyl cyclases (ACs) are a family of enzymes which convert ATP to cAMP, an essential intermediate in many signal transduction pathways. Of the 10 AC genes in man, 9 fall into the category of transmembrane ACs (tmACs), which associate with G-protein coupled receptors (GPCRs) and are activated by forskolin. The 10th AC, termed soluble AC (sAC) is neither activated by forskolin nor does it interact with GPCRs. Rather, sAC can be found in many compartments within the cell and is activated by bicarbonate. As such, sAC is considered a major sensor of bicarbonate in many tissues. The pathways involving sAC vary in different tissues and organ systems, and are as diverse as facilitating sperm capacitation and regulating pressure in the eye. The role of sAC in the eye has only recently begun to receive significant attention. Here we summarize what is known about the roles of sAC in the eye. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.
Assuntos
Adenilil Ciclases/metabolismo , Olho/enzimologia , Animais , HumanosRESUMO
Mutations in bestrophin-1 (Best1) cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited retinal degenerative disease. Best1 is a homo-oligomeric anion channel localized to the basolateral surface of retinal pigment epithelial (RPE) cells. A number of Best1 mutants mislocalize in Madin-Darby canine kidney (MDCK) cells. However, many proteins traffic differently in MDCK and RPE cells, and MDCK cells do not express endogenous Best1. Thus, effects of Best1 mutations on localization in MDCK cells may not translate to RPE cells. To determine whether BVMD causing mutations affect Best1 localization, we compared localization and oligomerization of Best1 with Best1 mutants V9M, W93C, and R218C. In MDCK cells, Best1 and Best1(R218C) were basolaterally localized. Best1(W93C) and Best1(V9M) accumulated in cells. In cultured fetal human retinal pigment epithelium cells (fhRPE) expressing endogenous Best1, Best1(R218C) and Best1(W93C) were basolateral. Best1(V9M) was intracellular. All three mutants exhibited similar fluorescence resonance energy transfer (FRET) efficiencies to, and co-immunoprecipitated with Best1, indicating unimpaired oligomerization. When human Best1 was expressed in RPE in mouse eyes it was basolaterally localized. However, Best1(V9M) accumulated in intracellular compartments in mouse RPE. Co-expression of Best1 and Best1(W93C) in MDCK cells resulted in basolateral localization of both Best1 and Best1(W93C), but co-expression of Best1 with Best1(V9M) resulted in mislocalization of both proteins. We conclude that different mutations in Best1 cause differential effects on its localization and that this effect varies with the presence or absence of wild-type (WT) Best1. Furthermore, MDCK cells can substitute for RPE when examining the effects of BVMD causing mutations on Best1 localization if co-expressed with WT Best1.
Assuntos
Canais de Cloreto/metabolismo , Proteínas do Olho/metabolismo , Canais Iônicos/metabolismo , Distrofia Macular Viteliforme/patologia , Animais , Bestrofinas , Sinalização do Cálcio , Membrana Celular/metabolismo , Células Cultivadas , Canais de Cloreto/genética , Olho/metabolismo , Proteínas do Olho/genética , Regulação da Expressão Gênica , Humanos , Canais Iônicos/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Transporte Proteico/genética , Distrofia Macular Viteliforme/genéticaRESUMO
PURPOSE: Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1. METHODS: Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp. RESULTS: Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control monolayers that was greater than that observed in those expressing Best1(W93C). Examination of [Ca2+]i following ATP stimulation demonstrated that the expression of Best1W93C impaired intracellular Ca2+ signaling. CONCLUSIONS: These data indicate that Best1 activity strongly influences electrophysiology and Ca2+ signaling in RPE cells, and that a common BVMD mutation disrupts both of these parameters. Our findings support the hypothesis that Best1 functions as an anion channel in human RPE.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Membrana Celular/metabolismo , Canais de Cloreto/metabolismo , Células Epiteliais/metabolismo , Proteínas do Olho/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Trifosfato de Adenosina/farmacologia , Adenovírus Humanos/genética , Bestrofinas , Membrana Celular/efeitos dos fármacos , Canais de Cloreto/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Proteínas do Olho/genética , Feto , Expressão Gênica , Vetores Genéticos , Células HEK293 , Humanos , Transporte de Íons/efeitos dos fármacos , Ionomicina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mutação , Ácido Niflúmico/farmacologia , Técnicas de Patch-Clamp , Cultura Primária de Células , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Transfecção , Distrofia Macular Viteliforme/genética , Distrofia Macular Viteliforme/metabolismo , Distrofia Macular Viteliforme/patologiaRESUMO
Body temperature is maintained in a narrow range in mammals, primarily controlled by sweating. In humans, the dynamic thermoregulatory organ, comprised of 2-4 million sweat glands distributed over the body, can secrete up to 4 L of sweat per day, thereby making it possible to withstand high temperatures and endure prolonged physical stress (e.g., long-distance running). The genetic basis for sweat gland function, however, is largely unknown. We find that the forkhead transcription factor, FoxA1, is required to generate mouse sweating capacity. Despite continued sweat gland morphogenesis, ablation of FoxA1 in mice results in absolute anihidrosis (lack of sweating). This inability to sweat is accompanied by down-regulation of the Na-K-Cl cotransporter 1 (Nkcc1) and the Ca(2+)-activated anion channel Bestrophin 2 (Best2), as well as glycoprotein accumulation in gland lumens and ducts. Furthermore, Best2-deficient mice display comparable anhidrosis and glycoprotein accumulation. These findings link earlier observations that both sodium/potassium/chloride exchange and Ca(2+) are required for sweat production. FoxA1 is inferred to regulate two corresponding features of sweat secretion. One feature, via Best2, catalyzes a bicarbonate gradient that could help to drive calcium-associated ionic transport; the other, requiring Nkcc1, facilitates monovalent ion exchange into sweat. These mechanistic components can be pharmaceutical targets to defend against hyperthermia and alleviate defective thermoregulation in the elderly, and may provide a model relevant to more complex secretory processes.
Assuntos
Regulação da Temperatura Corporal/fisiologia , Canais de Cloreto/metabolismo , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/fisiologia , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Sudorese/fisiologia , Análise de Variância , Animais , Bestrofinas , Western Blotting , Cruzamentos Genéticos , Primers do DNA/genética , Imunofluorescência , Galactosídeos , Perfilação da Expressão Gênica , Genótipo , Fator 3-alfa Nuclear de Hepatócito/genética , Indóis , Camundongos , Modelos Biológicos , Reação em Cadeia da Polimerase em Tempo Real , Membro 2 da Família 12 de Carreador de Soluto , Sudorese/genéticaRESUMO
BEST1 encodes Bestrophin-1 (Best1), a homo-oligomeric, integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium. Mutations in BEST1 cause five distinct retinal degenerative diseases, including adult vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). The mechanisms underlying these diseases and why mutations cause one disease over another are, for the most part, unknown. To gain insights into these four diseases, we expressed 28 Best1 mutants fused to YFP in polarized MDCK monolayers and, via confocal microscopy and immunofluorescence, live-cell FRET, and reciprocal co-immunoprecipitation experiments, screened these mutants for defects in localization and oligomerization. All 28 mutants exhibited comparable FRET efficiencies to and co-immunoprecipitated with WT Best1, indicating unimpaired oligomerization. RP- and ADVIRC-associated mutants were properly localized to the basolateral plasma membrane of cells, while two AVMD and most ARB mutants were mislocalized. When co-expressed, all mislocalized mutants caused mislocalization of WT Best1 to intracellular compartments. Our current and past results indicate that mislocalization of Best1 is not an absolute feature of any individual bestrophinopathy, occurring in AVMD, BVMD, and ARB. Furthermore, some ARB mutants that do not also cause dominant disease cause mislocalization of Best1, indicating that mislocalization is not a cause of disease, and that absence of Best1 activity from the plasma membrane is tolerated. Lastly, we find that the ARB truncation mutants L174Qfs*57 and R200X can form oligomers with WT Best1, indicating that the first â¼174 amino acids of Best1 are sufficient for oligomerization to occur.
Assuntos
Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Oftalmopatias Hereditárias/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Mutação de Sentido Incorreto , Multimerização Proteica/fisiologia , Doenças Retinianas/genética , Adenoviridae/genética , Animais , Proteínas de Bactérias/metabolismo , Bestrofinas , Western Blotting , Doenças da Coroide/genética , Doenças da Coroide/metabolismo , Cães , Eletrofisiologia , Oftalmopatias Hereditárias/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/metabolismo , Células Madin Darby de Rim Canino/metabolismo , Microscopia Confocal , Técnicas de Patch-Clamp , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Doenças Retinianas/metabolismo , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismo , Transfecção , Distrofia Macular Viteliforme/genética , Distrofia Macular Viteliforme/metabolismoRESUMO
Purpose: No large-mammal surgical models exist for geographic atrophy (GA), choroidal neovascularization (CNV), and pachychoroidal vascular remodeling. Our goal was to develop a porcine RPE debridement model of advanced macular degeneration to study photoreceptor cell loss and choroidal remodeling. Methods: Seven 2-month-old female domestic pigs were used for this study. After 25G vitrectomy, the area centralis was detached via subretinal bleb. A nitinol wire (Finesse Flex Loop) was used to debride RPE cells across a 3- to 5-mm diameter region. Fluid-air exchange was performed, and 20% SF6 gas injected. Animals underwent fundus photography, fluorescein angiography, optical coherence tomography (OCT), and OCT-angiography (OCTA) at 2 weeks, 1 month, 2 months, 3 months, and 6 months postoperatively. Retinal histology was obtained at euthanasia, 2 months (n = 3), 3 months (n = 2), or 6 months (n = 2) after surgery. Results: RPE debridement resulted in GA with rapid loss of choriocapillaris, progressive loss of photoreceptors, and pachychoroidal changes in Sattler's and Haller's layers in all seven eyes undergoing debridement within 2 months. OCT and histological findings included subretinal disciform scar with overlying outer retinal atrophy; outer retinal tubulations and subretinal hyper-reflective material. OCTA revealed type 2 CNV (n = 4) at the edges of the debridement zone by 2 months, but there was no significant exudation noted at any time point. Conclusions: Surgical debridement of the RPE results in GA, CNV, and pachychoroid and reproduced all forms of advanced macular degeneration. This surgical model may be useful in examining the role of RPE and other cell replacement in treating advanced macular disease.
Assuntos
Neovascularização de Coroide , Atrofia Geográfica , Degeneração Macular , Feminino , Suínos , Animais , Desbridamento , Degeneração Macular/diagnóstico , Degeneração Macular/cirurgia , Atrofia Geográfica/diagnóstico , Sus scrofa , Retina , Corioide , Neovascularização de Coroide/diagnóstico , Neovascularização de Coroide/cirurgiaRESUMO
Purpose: We investigated whether a clinically used carbonic anhydrase inhibitor (CAIs) can modulate intraocular pressure (IOP) through soluble adenylyl cyclase (sAC) signaling. Methods: IOP was measured 1 h after topical treatment with brinzolamide, a topically applied and clinically used CAIs, using direct cannulation of the anterior chamber in sAC knockout (KO) mice or C57BL/6J mice in the presence or absence of the sAC inhibitor (TDI-10229). Results: Mice treated with the sAC inhibitor TDI-10229 had elevated IOP. CAIs treatment significantly decreased increased intraocular pressure (IOP) in wild-type, sAC KO mice, as well as TDI-10229-treated mice. Conclusions: Inhibiting carbonic anhydrase reduces IOP independently from sAC in mice. Our studies suggest that the signaling cascade by which brinzolamide regulates IOP does not involve sAC.
Assuntos
Glaucoma , Pressão Intraocular , Animais , Camundongos , Inibidores da Anidrase Carbônica , Adenilil Ciclases/uso terapêutico , Camundongos Endogâmicos C57BL , Glaucoma/tratamento farmacológicoRESUMO
To investigate downstream molecular changes caused by mitogen-activated protein kinase (MEK) inhibitor treatment and further explore the impact of direct knockdown of early growth response-1 ( EGR1 ) in melanoma cell culture. RNA-sequencing (RNA-Seq) was performed to determine gene expression changes with MEK inhibitor treatment. Treatment with MEK inhibitor (trametinib) was then assessed in two cutaneous (MEL888, MEL624) and one conjunctival (YUARGE 13-3064) melanoma cell line. Direct knockdown of EGR1 was accomplished using lentiviral vectors containing shRNA. Cell viability was measured using PrestoBlueHS Cell Viability Reagent. Total RNA and protein were assessed by qPCR and SimpleWestern. RNA-Seq demonstrated a profound reduction in EGR1 with MEK inhibitor treatment, prompting further study of melanoma cell lines. Following trametinib treatment of melanoma cells, viability was reduced in both cutaneous (MEL888 26%, P â <â 0.01; MEL624 27%, P â <â 0.001) and conjunctival (YUARGE 13-3064 33%, P â <â 0.01) melanoma compared with DMSO control, with confirmed EGR1 knockdown to 0.04-, 0.01-, and 0.16-fold DMSO-treated levels (all P â <â 0.05) in MEL888, MEL624, and YUARGE 13-3064, respectively. Targeted EGR1 knockdown using shRNA reduced viability in both cutaneous (MEL624 78%, P â =â 0.05) and conjunctival melanoma (YUARGE-13-3064 67%, P â =â 0.02). RNA-Sequencing in MEK inhibitor-treated cells identified EGR1 as a candidate effector molecule of interest. In a malignant melanoma cell population, MEK inhibition reduced viability in both cutaneous and conjunctival melanoma with a profound downstream reduction in EGR1 expression. Targeted knockdown of EGR1 reduced both cutaneous and conjunctival melanoma cell viability independent of MEK inhibition, suggesting a key role for EGR1 in melanoma pathobiology.
Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Mitógenos , Dimetil Sulfóxido , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno , RNA Interferente Pequeno , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
Glaucoma is a leading cause of blindness affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). IOP results from the resistance to drainage of aqueous humor (AH) produced by the ciliary body in a process requiring bicarbonate. Once secreted into the anterior chamber, AH drains from the eye via two pathways: uveoscleral and pressure-dependent or conventional outflow (C(t)). Modulation of "inflow" and "outflow" pathways is thought to occur via distinct, local mechanisms. Mice deficient in the bicarbonate channel bestrophin-2 (Best2), however, exhibit a lower IOP despite an increase in AH production. Best2 is expressed uniquely in nonpigmented ciliary epithelial (NPE) cells providing evidence for a bicarbonate-dependent communicative pathway linking inflow and outflow. Here, we show that bicarbonate-sensitive soluble adenylyl cyclase (sAC) is highly expressed in the ciliary body in NPE cells, but appears to be absent from drainage tissues. Pharmacologic inhibition of sAC in mice causes a significant increase in IOP due to a decrease in C(t) with no effect on inflow. In mice deficient in sAC IOP is elevated, and C(t) is decreased relative to wild-type mice. Pharmacologic inhibition of sAC did not alter IOP or C(t) in sAC-deficient mice. Based on these data we propose that the ciliary body can regulate C(t) and that sAC serves as a critical sensor of bicarbonate in the ciliary body regulating the secretion of substances into the AH that govern outflow facility independent of pressure.
Assuntos
Adenilil Ciclases/metabolismo , Bicarbonatos/metabolismo , Corpo Ciliar/enzimologia , Glaucoma/enzimologia , Pressão Intraocular , Adenilil Ciclases/genética , Animais , Humor Aquoso/enzimologia , Bestrofinas , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glaucoma/epidemiologia , Glaucoma/genética , Humanos , Camundongos , Camundongos Knockout , SuínosRESUMO
Mutations in BEST1, encoding bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited macular degeneration characterized by a diminished electrooculogram light peak (LP), lipofuscin in retinal pigment epithelial cells (RPE), and fluid- and debris-filled retinal detachments. To understand the pathogenesis of BVMD we generated knock-in mice carrying the BVMD-causing mutation W93C in Best1. Both Best1(+/W93C)and Best1(W93C/W93C) mice had normal ERG a- and b-waves, but exhibited an altered LP luminance response reminiscent of that observed in BVMD patients. Morphological analysis identified fluid- and debris-filled retinal detachments in mice as young as 6 months of age. By 18-24 months of age Best1(+/W93C)and Best1(W93C/W93C) mice exhibited enhanced accumulation of lipofuscin in the RPE, and a significant deposition of debris composed of unphagocytosed photoreceptor outer segments and lipofuscin granules in the subretinal space. Although Best1 is thought to function as a Ca(2+)-activated Cl(-) channel, RPE cells from Best1(W93C) mice exhibited normal Cl(-) conductances. We have previously shown that Best1(-/-) mice exhibit increased [Ca(2+)](i) in response to ATP stimulation. However, ATP-stimulated changes in [Ca(2+)](i) in RPE cells from Best1(+/W93C) and Best1(W93C/W93C) mice were suppressed relative to Best1(+/+) littermates. Based on these data we conclude that mice carrying the Best1(W93C) mutation are a valid model for BVMD. Furthermore, these data suggest that BVMD is not because of Best1 deficiency, as the phenotypes of Best1(+/W93C) and Best1(W93C/W93C) mice are distinct from that of Best1(-/-) mice with regard to lipofuscin accumulation, and changes in the LP and ATP Ca(2+) responses.
Assuntos
Sinalização do Cálcio , Modelos Animais de Doenças , Degeneração Macular/metabolismo , Trifosfato de Adenosina/farmacologia , Substituição de Aminoácidos/genética , Animais , Bestrofinas , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos da radiação , Cloretos/metabolismo , Eletroculografia , Eletrorretinografia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Técnicas de Introdução de Genes , Genótipo , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/efeitos da radiação , Canais Iônicos , Luz , Degeneração Macular/genética , Degeneração Macular/patologia , Degeneração Macular/fisiopatologia , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/patologia , Epitélio Pigmentado Ocular/efeitos da radiação , Epitélio Pigmentado Ocular/ultraestruturaRESUMO
Purpose: To improve outcomes for subretinal implantation surgery in pigs. Methods: Analysis of variables affecting the success of subretinal implantation surgery was performed on videos of 37 surgeries. Ex vivo experiments were conducted to measure intraocular pressure (IOP) and test various prototyped implanters for effectiveness at maintaining IOP. Results: A video analysis revealed a prolonged sclerotomy open time owing to a combination of uncontrolled bleeding and excessive fluid outflow often resulting in retinal prolapse. Precauterization of the choroid before full-thickness sclerotomy (n = 10) resulted in a reduced incidence of uncontrolled bleeding from 39.1% (9/23) versus 0% (0/10) (P = 0.005) and improved implantation success from 73% to 90%. An ex vivo analysis of the IOP revealed a mean decrease in the IOP from 30.2 ± 3.0 mm Hg to 5.0 ± 2.1 mm Hg after a fully penetrating sclerotomy. To address this situation, we produced a series of plugs that integrated with a custom implant insertion device to seal the sclerotomy during implantation. The use of the plugs was cumbersome, however, and so we opted instead to increase the width of the inserter tip to fill the open sclerotomy. This improved device restored and maintained IOP during implantation (27.1 ± 1.9 mm Hg). Combined with precauterization the improved inserter resulted in 100% successful implantation (n = 4). Conclusions: For subretinal implantation in pigs, a modified procedure to precauterize the choroid before sclerotomy combined with an instrument that better fills the scleral opening decreases bleeding, hypotony, and open sclerotomy time, improving the success rate. Translational Relevance: Better management of IOP and bleeding from a sclerotomy will improve implant-based therapies.
Assuntos
Implantes para Drenagem de Glaucoma , Glaucoma de Ângulo Aberto , Animais , Glaucoma de Ângulo Aberto/cirurgia , Pressão Intraocular , Suínos , Tonometria Ocular , Resultado do TratamentoRESUMO
Activation of an apical Ca(2+)-dependent Cl(-) channel (CaCC) is the rate-limiting step for fluid secretion in many exocrine tissues. Here, we compared the properties of native CaCC in mouse submandibular salivary gland acinar cells to the Ca(2+)-gated Cl(-) currents generated by Tmem16A and Best2, members from two distinct families of Ca(2+)-activated Cl(-) channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca(2+)-activated Cl(-) currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca(2+)-activated Cl(-) current found in native salivary acinar cells. Best2(-/-) and Tmem16A(-/-) mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca(2+)-activated Cl(-) current in submandibular gland acinar cells from Best2-deficient mice were the same as in wild type cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable with that in wild type mice. In contrast, submandibular gland acinar cells from Tmem16A(-/-) mice lacked a Ca(2+)-activated Cl(-) current and a Ca(2+)-mobilizing agonist failed to stimulate Cl(-) efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wild type and Best2(-/-) mice. Our results demonstrate that both Tmem16A and Best2 generate Ca(2+)-activated Cl(-) current in vitro with similar properties to those expressed in native cells, yet only Tmem16A appears to be a critical component of the acinar Ca(2+)-activated Cl(-) channel complex that is essential for saliva production by the submandibular gland.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Glândula Submandibular/metabolismo , Animais , Anoctamina-1 , Canais de Cloreto/genética , Regulação da Expressão Gênica/fisiologia , Transporte de Íons/fisiologia , Camundongos , Camundongos Knockout , Especificidade de Órgãos/fisiologia , Glândula Submandibular/citologiaRESUMO
Fibrin is a degradable biopolymer with an excellent clinical safety profile. Use of higher mechanical strength fibrin hydrogels is limited by the rapid rate of fibrin polymerization. We recently demonstrated the use of higher mechanical strength (fibrinogen concentrations >30 mg/ml) fibrin scaffolds for surgical implantation of cells. The rapid polymerization of fibrin at fibrinogen concentrations impaired our ability to scale production of these fibrin scaffolds. We serendipitously discovered that the azo dye Trypan blue (TB) slowed fibrin gelation kinetics allowing for more uniform mixing of fibrinogen and thrombin at high concentrations. A screen of closely related compounds identified similar activity for Evans blue (EB), an isomer of TB. Both TB and EB exhibited a concentration dependent increase in clot time, though EB had a larger effect. While gelation time was increased by TB or EB, overall polymerization time was unaffected. Scanning electron microscopy showed similar surface topography, but transmission electron microscopy showed a higher cross-linking density for gels formed with TB or EB versus controls. Based on these data we conclude that addition of TB or EB during thrombin mediated fibrin polymerization slows the initial gelation time permitting generation of larger more uniform fibrin hydrogels with high-mechanical strength.
Assuntos
Compostos Azo/química , Fibrina/química , Hidrogéis/química , Modelos Químicos , CinéticaRESUMO
PURPOSE: To report a case of adult-onset vitelliform macular dystrophy in a patient who was found to have a previously unreported variant of the IMPG2 gene. METHODS: Case report. RESULTS: A 65-year-old white woman with no significant medical or ocular history presented with a complaint of persistent wavy vision for 10 months. On funduscopic examination, bilateral vitelliform lesions of approximately 1 mm in the right eye and 0.5 mm in the left eye were evident, with no choroidal neovascularization in either eye. The patient was diagnosed with adult-onset vitelliform macular dystrophy. Genetic testing revealed a single likely pathogenic variant of the IMPG2 gene that may explain the examination findings. CONCLUSION: Adult-onset vitelliform macular dystrophy is a common and relatively benign condition occurring in approximately 1 in 8,000 individuals. Although vitelliform lesions can be a manifestation of systemic diseases or be idiopathic, in a minority of patients, genetic predisposition may play a role. Mutations in four particular genes BEST1, PRPH2, IMPG1, and IMPG2 have been associated with some cases of adult-onset vitelliform macular dystrophy, with this particular gene variant of IMPG2 being previously unreported.
Assuntos
Mutação , Proteoglicanas , Distrofia Macular Viteliforme , Idade de Início , Idoso , Feminino , Predisposição Genética para Doença , Testes Genéticos , Humanos , Proteoglicanas/genética , Distrofia Macular Viteliforme/genéticaRESUMO
We investigated the molecular determinants of Ca(2+)-activated chloride current (CaCC) expressed in adult sensory neurons after a nerve injury. Dorsal root ganglia express the transcripts of three gene families known to induce CaCCs in heterologous systems: bestrophin, tweety, and TMEM16. We found with quantitative transcriptional analysis and in situ hybridization that nerve injury induced upregulation of solely bestrophin-1 transcripts in sensory neurons. Gene screening with RNA interference in single neurons demonstrated that mouse Best1 is required for the expression of CaCC in injured sensory neurons. Transfecting injured sensory neurons with bestrophin-1 mutants inhibited endogenous CaCC. Exogenous expression of the fusion protein green fluorescent protein-Bestrophin-1 in naive neurons demonstrated a plasma membrane localization of the protein that generates a CaCC with biophysical and pharmacological properties similar to endogenous CaCC. Our data suggest that Best1 belongs to a group of genes upregulated by nerve injury and supports functional CaCC expression in injured sensory neurons.
Assuntos
Cálcio/metabolismo , Cloretos/metabolismo , Proteínas do Olho/metabolismo , Gânglios Espinais/fisiologia , Nervo Isquiático/lesões , Células Receptoras Sensoriais/fisiologia , Animais , Bestrofinas , Membrana Celular/metabolismo , Proteínas do Olho/genética , Técnicas de Silenciamento de Genes , Proteínas de Fluorescência Verde/genética , Canais Iônicos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Técnicas de Patch-Clamp , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Macular degenerations (MD), age-related or inherited, interfere with the ability to read, drive and recognize faces. Understanding this class of diseases has been challenging because the mouse, the mammal most amenable to genetic manipulation, lacks a macula. Here we discuss whether we can model MD in the mouse, present criteria for an 'ideal' mouse model of MD and discuss how mouse models have contributed to our knowledge of MD by contrasting how well they meet the 'ideal' criteria with how informative they have actually been. By modeling MD in mice, we can learn about aspects of MD that an animal with a macula would be unable to teach us.
Assuntos
Modelos Animais de Doenças , Degeneração Macular/genética , Envelhecimento/patologia , Animais , Proteínas da Matriz Extracelular/genética , Proteínas do Olho/genética , Previsões , Proteínas de Filamentos Intermediários/genética , Degeneração Macular/patologia , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Periferinas , Proteínas Recombinantes/genética , Degeneração Retiniana/genética , Degeneração Retiniana/patologiaRESUMO
PURPOSE: Mice in which bestrophin 2 (Best2) is disrupted exhibit changes in aqueous flow and drainage, resulting in a reduction in intraocular pressure in comparison to wild-type mice. Best2 encodes a putative anion channel localized uniquely to the basolateral plasma membrane of non-pigmented epithelium cells in mice. In this study, we examine the localization of Best2 in the human eye. METHODS: Rabbit polyclonal antibodies recognizing human Best2 (hBest2) were generated and characterized for use in western blot, immunoprecipitation, and immunofluorescence assays. The expression of hBest2 using these antibodies was examined using human donor eye tissues. RESULTS: We could not detect hBest2 in human ciliary bodies or other ocular tissues by western blot. However, when enriched by immunoprecipitation, hBest2 was identified in ciliary bodies, but not in the retinal pigment epithelium. Using immunofluorescence, we located hBest2 in the basolateral plasma membrane of non-pigmented epithelial cells. CONCLUSIONS: We found expression of hBest2 similar to mice only in NPE cells. These data suggest that Best2 may play a functional role in the regulation of aqueous flow and drainage in humans. We conclude that Best2 represents a new potential target for glaucoma therapy.