RESUMO
Adeno-associated virus (AAV) has been studied as a safe delivery tool for gene therapy of retinal blinding diseases such as Leber's congenital amaurosis (LCA). The tropism of recombinant AAV (rAAV) including its specificity and efficiency in targeting retinal cell types has been studied with native or engineered capsids, along with specific promoters. However, one of the rAAV serotypes, rAAV2/6, has not been well-studied based on a report of low infection efficiency in the retina. We investigated the tropism of several rAAVs by subretinal injection in the adult mouse and found that rAAV2/6 predominantly infected cone photoreceptors including the main spectral type. Our data suggest that subretinal injection with rAAV2/6 may provide both an efficacious and specific means of gene delivery to cone photoreceptors in murine retinas.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Doenças Retinianas/terapia , Animais , Vetores Genéticos/administração & dosagem , Injeções , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/terapia , Camundongos da Linhagem 129 , Opsinas/genética , Opsinas/metabolismo , Retina/virologia , Células Fotorreceptoras Retinianas Cones/virologia , Doenças Retinianas/genética , Resultado do TratamentoRESUMO
With an increasing number of identified causative genes, the widespread use of gene therapy is quickly becoming feasible. Once a target gene is selected, it is important to have a cell delivery method that is both specific and efficient. Cell type specificity and high efficiency is particularly important for the treatment of retinal degeneration, since viruses are efficient gene delivery vehicles for the nervous system, but often bring with them non-specific infections. In this review, we focus on adeno-associated virus (AAV). Over the last few decades, AAV has become a leading choice for safe gene delivery, in part due to its replication deficiency in cells without a helper virus. Here, we summarize the tropism of recombinant AAV (rAAV) for various types of mammalian retinal neurons in relation to capsid serotype and administration method. We also include our recent findings on an AAV serotype that AAV was specifically infected mouse cone photoreceptors when delivered by subretinal administration.
Assuntos
Dependovirus , Marcação de Genes/métodos , Terapia Genética/métodos , Mamíferos , Degeneração Retiniana/terapia , Animais , Técnicas de Transferência de GenesRESUMO
An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.
Assuntos
Transdução de Sinal Luminoso/fisiologia , Isoformas de Proteínas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Células Bipolares da Retina/fisiologia , Canais de Cátion TRPM/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Eletrofisiologia , Humanos , Luz , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fases de Leitura Aberta , Isoformas de Proteínas/genética , Receptores de Glutamato Metabotrópico/genética , Células Bipolares da Retina/citologia , Canais de Cátion TRPM/genéticaRESUMO
(1) Background: The impairment of eye-hand coordination and smooth-pursuit eye movement caused by visual display terminal (VDT) operation is thought to impair daily living activities, for which no effective methods are currently known. On the other hand, various food ingredients, including astaxanthin, lutein, and zeaxanthin, are known to help improve the eye health of VDT operators. This study aimed to test the hypothesis that the combination of astaxanthin, lutein, and zeaxanthin can prevent the impairment of eye-hand coordination and smooth-pursuit eye movement caused by VDT operation. (2) Methods: We conducted a randomized, placebo-controlled, parallel-group clinical trial. Healthy subjects who regularly worked with VDTs were randomly assigned to the active and placebo groups. All of the subjects took soft capsules containing 6 mg of astaxanthin, 10 mg of lutein, and 2 mg of zeaxanthin or placebo soft capsules once daily for eight weeks. We evaluated the eye-hand coordination, smooth-pursuit eye movements, and macular pigment optical density (MPOD) at 0, two, four, and eight weeks after soft-capsule intake. (3) Results: The active group showed significantly improved eye-hand coordination after VDT operation at eight weeks. However, there was no clear improvement in the effect of the supplementation on smooth-pursuit eye movements. The active group also showed a significant increase in MPOD levels. (4) Conclusions: Consumption of a supplement containing astaxanthin, lutein, and zeaxanthin mitigates the decline of eye-hand coordination after VDT operation.
Assuntos
Degeneração Macular , Pigmento Macular , Humanos , Luteína , Zeaxantinas , Movimentos Oculares , Voluntários Saudáveis , Degeneração Macular/tratamento farmacológico , Acuidade Visual , Suplementos Nutricionais , Método Duplo-CegoRESUMO
Expression of channels to specific neuronal sites can critically impact their function and regulation. Currently, the molecular mechanisms underlying this targeting and intracellular trafficking of transient receptor potential (TRP) channels remain poorly understood, and identifying proteins involved in these processes will provide insight into underlying mechanisms. Vision is dependent on the normal function of retinal depolarizing bipolar cells (DBCs), which couple a metabotropic glutamate receptor 6 to the TRP melastatin 1 (TRPM1) channel to transmit signals from photoreceptors. We report that the extracellular membrane-attached protein nyctalopin is required for the normal expression of TRPM1 on the dendrites of DBCs in mus musculus. Biochemical and genetic data indicate that nyctalopin and TRPM1 interact directly, suggesting that nyctalopin is acting as an accessory TRP channel subunit critical for proper channel localization to the synapse.
Assuntos
Dendritos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas/metabolismo , Retina/citologia , Células Bipolares da Retina/citologia , Canais de Cátion TRPM/metabolismo , Aminoácidos/farmacologia , Animais , Proteínas de Bactérias/genética , Biofísica , Linhagem Celular Transformada , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Técnicas In Vitro , Proteínas de Repetições Ricas em Leucina , Proteínas Luminescentes/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Técnicas de Patch-Clamp , Proteínas/genética , Proteoglicanas/genética , Proteoglicanas/metabolismo , Retina/crescimento & desenvolvimento , Canais de Cátion TRPM/deficiência , Transfecção/métodos , Xantenos/farmacologiaRESUMO
In darkness, glutamate released from photoreceptors activates the metabotropic glutamate receptor 6 (mGluR6) on retinal ON bipolar cells. This activates the G protein G(o), which then closes transient receptor potential melastatin 1 (TRPM1) channels, leading to cells' hyperpolarization. It has been generally assumed that deleting mGluR6 would render the cascade inactive and the ON bipolar cells constitutively depolarized. Here we show that the rod bipolar cells in mGluR6-null mice were hyperpolarized. The slope conductance of the current-voltage curves and the current noise were smaller than in wild type. Furthermore, while in wild-type rod bipolar cells, TRPM1 could be activated by local application of capsaicin; in null cells, it did not. These results suggest that the TRPM1 channel in mGluR6-null rod bipolar cells is inactive. To explore the reason for this lack of activity, we tested if mGluR6 deletion affected expression of cascade components. Immunostaining for G protein subunit candidates Gα(o), Gß(3), and Gγ(13) showed no significant changes in their expression or distribution. Immunostaining for TRPM1 in the dendritic tips was greatly reduced, but the channel was still present in the soma and primary dendrites of mGluR6-null bipolar cells, where a certain fraction of TRPM1 appears to localize to the plasma membrane. Consequently, the lack of TRPM1 activity in the null retina is unlikely to be due to failure of the channels to localize to the plasma membrane. We speculate that, to be constitutively active, TRPM1 channels in ON bipolar cells have to be in a complex, or perhaps require an unidentified factor.
Assuntos
Receptores de Glutamato Metabotrópico/fisiologia , Células Bipolares da Retina/fisiologia , Canais de Cátion TRPM/fisiologia , Animais , Capsaicina/farmacologia , Dendritos/química , Dendritos/fisiologia , Deleção de Genes , Proteínas Heterotriméricas de Ligação ao GTP/análise , Proteínas Heterotriméricas de Ligação ao GTP/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Glutamato Metabotrópico/genética , Células Bipolares da Retina/efeitos dos fármacos , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPM/análiseRESUMO
Mutations in TRPM1 are found in humans with an autosomal recessive form of complete congenital stationary night blindness (cCSNB). The Trpm1(-/-) mouse has been an important animal model for this condition. Here we report a new mouse mutant, tvrm27, identified in a chemical mutagenesis screen. Genetic mapping of the no b-wave electroretinogram (ERG) phenotype of tvrm27 localized the mutation to a chromosomal region that included Trpm1. Complementation testing with Trpm1(-/-) mice confirmed a mutation in Trpm1. Sequencing identified a nucleotide change in exon 23, converting a highly conserved alanine within the pore domain to threonine (p.A1068T). Consistent with prior studies of Trpm1(-/-) mice, no anatomical changes were noted in the Trpm1(tvrm27/tvrm27) retina. The Trpm1(tvrm27/tvrm27) phenotype is distinguished from that of Trpm1(-/-) by the retention of TRPM1 expression on the dendritic tips of depolarizing bipolar cells (DBCs). While ERG b-wave amplitudes of Trpm1(+/-) heterozygotes are comparable to wild type, those of Trpm1(+/tvrm27) mice are reduced by 32%. A similar reduction in the response of Trpm1(+/tvrm27) DBCs to LY341495 or capsaicin is evident in whole cell recordings. These data indicate that the p.A1068T mutant TRPM1 acts as a dominant negative with respect to TRPM1 channel function. Furthermore, these data indicate that the number of functional TRPM1 channels at the DBC dendritic tips is a key factor in defining DBC response amplitude. The Trpm1(tvrm27/tvrm27) mutant will be useful for elucidating the role of TRPM1 in DBC signal transduction, for determining how Trpm1 mutations impact central visual processing, and for evaluating experimental therapies for cCSNB.
Assuntos
Mutação Puntual , Células Bipolares da Retina/fisiologia , Canais de Cátion TRPM/genética , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Sequência de Aminoácidos , Aminoácidos/farmacologia , Animais , Capsaicina/farmacologia , Mapeamento Cromossômico , Cromossomos de Mamíferos/genética , Dendritos/fisiologia , Modelos Animais de Doenças , Éxons , Oftalmopatias Hereditárias/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Heterozigoto , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Miopia/genética , Cegueira Noturna/genética , Retina/patologia , Retina/fisiologia , Análise de Sequência de DNA , Canais de Cátion TRPM/metabolismo , Treonina/genética , Xantenos/farmacologiaRESUMO
Retinal photoreceptor cells, rods and cones, convert photons of light into chemical and electrical signals as the first step of the visual transduction cascade. Although the chemical processes in the phototransduction system are very similar to each other in these photoreceptors, the light sensitivity and time resolution of the photoresponse in rods are functionally different than those in the photoresponses of cones. To systematically investigate how photoresponses are divergently regulated in rods and cones, we have developed a detailed mathematical model on the basis of the Hamer model. The current model successfully reconstructed light intensity-, ATP- and GTP-dependent changes in concentrations of phosphorylated visual pigments (VPs), activated transducins (Tr*s) and phosphodiesterases (PDEs) in rods and cones. In comparison to rods, the lower light sensitivity of cones was attributed not only to the lower affinity of activated VPs for Trs but also to the faster desensitization of the VPs. The assumption of an intermediate inactive state, MIIi, in the thermal decay of activated VPs was essential for inducing faster inactivation of VPs in rods, and possibly also in cones.
Assuntos
Fotofobia , Células Fotorreceptoras Retinianas Bastonetes , Humanos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Transdução de Sinal Luminoso/fisiologia , Transducina/metabolismoRESUMO
Purpose: C1q/TNF-related protein (CTRP) 9 is one of the adiponectin paralogs, and a genetic ablation of its receptor, AdipoR1, is known to cause retinal degeneration. The purpose of this study was to determine the role played by CTRP9 in the retina. Methods: The retinas of Ctrp9 gene knockout (KO) and wild type (WT) mice were examined by electroretinography (ERG), histology, RNA sequencing, and quantitative real-time PCR. Results: The amplitude of the photopic ERG elicited by the maximum stimulus intensity was smaller by 40% in the Ctrp9 KO mice than in WT mice at 8 weeks of age. However, the photopic ERGs was not reduced from 8 weeks to 6 months of age. The amplitudes of the scotopic ERGs were not reduced in the Ctrp9 KO mice at 8 weeks and 6 months of age. No distinct histological abnormalities were found in the retinal sections but the density of peanut agglutinin-stained cells in the retinal flat mount of KO mice was reduced to about 70% of that of WT mice. Genomewide RNA sequencing of the retina revealed the absence of the expression of CTRP9 in both KO and WT mice. RNA sequencing and quantitative real-time PCR analysis showed that the expressions of the transcripts of genes expressed in cones, Opn1sw, Opn1mw, Gnat2, and Cnga3, were reduced in the KO mice retina, however, the degree of expression of the transcripts in rods was not significantly reduced. Conclusions: CTRP9 is released ectopically from other tissues, and it regulates the number of cones in the mouse retinas.
Assuntos
Adiponectina , Glicoproteínas , Células Fotorreceptoras Retinianas Cones , Adiponectina/genética , Adiponectina/metabolismo , Animais , Eletrorretinografia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Adiponectina/genética , Receptores de Adiponectina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/fisiologiaRESUMO
The 15q13.3 microdeletion syndrome is a genetic disorder characterized by a wide spectrum of psychiatric disorders that is caused by the deletion of a region containing 7 genes on chromosome 15 (MTMR10, FAN1, TRPM1, MIR211, KLF13, OTUD7A, and CHRNA7). The contribution of each gene in this syndrome has been studied using mutant mouse models, but no single mouse model recapitulates the whole spectrum of human 15q13.3 microdeletion syndrome. The behavior of Trpm1-/- mice has not been investigated in relation to 15q13.3 microdeletion syndrome due to the visual impairment in these mice, which may confound the results of behavioral tests involving vision. We were able to perform a comprehensive behavioral test battery using Trpm1 null mutant mice to investigate the role of Trpm1, which is thought to be expressed solely in the retina, in the central nervous system and to examine the relationship between TRPM1 and 15q13.3 microdeletion syndrome. Our data demonstrate that Trpm1-/- mice exhibit abnormal behaviors that may explain some phenotypes of 15q13.3 microdeletion syndrome, including reduced anxiety-like behavior, abnormal social interaction, attenuated fear memory, and the most prominent phenotype of Trpm1 mutant mice, hyperactivity. While the ON visual transduction pathway is impaired in Trpm1-/- mice, we did not detect compensatory high sensitivities for other sensory modalities. The pathway for visual impairment is the same between Trpm1-/- mice and mGluR6-/- mice, but hyperlocomotor activity has not been reported in mGluR6-/- mice. These data suggest that the phenotype of Trpm1-/- mice extends beyond that expected from visual impairment alone. Here, we provide the first evidence associating TRPM1 with impairment of cognitive function similar to that observed in phenotypes of 15q13.3 microdeletion syndrome.
Assuntos
Ansiedade/genética , Cromossomos Humanos Par 15/genética , Hipercinese/genética , Canais de Cátion TRPM/genética , Animais , Monoaminas Biogênicas/análise , Química Encefálica , Comportamento Exploratório , Estudos de Associação Genética , Humanos , Masculino , Aprendizagem em Labirinto/fisiologia , Metilfenidato/farmacologia , Camundongos , Camundongos Knockout , Teste de Campo Aberto , Reflexo de Sobressalto , Teste de Desempenho do Rota-Rod , Deleção de Sequência , Interação Social , Memória Espacial , Natação , Canais de Cátion TRPM/deficiência , Transtornos da Visão/genéticaRESUMO
PURPOSE: To identify human transient receptor potential cation channel, subfamily M, member 1 (TRPM1) gene mutations in patients with congenital stationary night blindness (CSNB). METHODS: We analyzed four different Japanese patients with complete CSNB in whom previous molecular examination revealed no mutation in either nyctalopin (NYX) or glutamate receptor, metabotropic 6 (GRM6). The ophthalmologic examination included best-corrected visual acuity, refraction, biomicroscopy, ophthalmoscopy, fundus photography, Goldmann kinetic perimetry, color vision tests, and electroretinography (ERG). Exons 2 through 27 and the exon-intron junction regions of human TRPM1 were sequenced. RESULTS: Five different mutations in human TRPM1 were identified. Mutations were present in three unrelated patients with complete CSNB. All three patients were compound heterozygotes. Fundus examination revealed no abnormalities other than myopic changes, and the single bright-flash, mixed rod-cone ERG showed a "negative-type" configuration with a reduced normal a-wave and a significantly reduced b-wave amplitude. Our biochemical and cell biologic analyses suggest that the two identified IVS mutations lead to abnormal TRPM1 protein production, and imply that the two identified missense mutations lead to the mislocalization of the TRPM1 protein in bipolar cells (BCs). CONCLUSIONS: Human TRPM1 mutations are associated with the complete form of CSNB in Japanese patients, suggesting that TRPM1 plays an essential role in mediating the photoresponse in ON BCs in humans as well as in mice.
Assuntos
Predisposição Genética para Doença , Mutação/genética , Cegueira Noturna/congênito , Cegueira Noturna/genética , Canais de Cátion TRPM/genética , Adulto , Animais , Sequência de Bases , Linhagem Celular , Criança , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Heterozigoto , Humanos , Espaço Intracelular/metabolismo , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Linhagem , Transporte Proteico , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/metabolismo , Adulto JovemRESUMO
The critical flicker-fusion frequency (CFF), defined as the frequency at which a flickering light is indistinguishable from a continuous light, is a useful measure of visual temporal resolution. The mouse CFF has been studied by electrophysiological approaches such as recordings of the electroretinogram (ERG) and the visually evoked potential (VEP), but it has not been measured behaviorally. Here we estimated the mouse CFF by using a touchscreen based operant system. The test with ascending series of frequencies and that with randomized frequencies resulted in about 17 and 14 Hz, respectively, as the frequency which could not be distinguished from steady lights. Since the ascending method of limits tend to overestimate the threshold than the descending method, we estimated the mouse CFF to be about 14 Hz. Our results highlight usefulness of the operant conditioning method in measurement of the mouse visual temporal resolution.
Assuntos
Discriminação Psicológica , Percepção Visual , Animais , Condicionamento Operante , Potenciais Evocados Visuais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Understanding the molecular mechanisms by which distinct cell fate is determined during organogenesis is a central issue in development and disease. Here, using conditional gene ablation in mice, we show that the transcription factor Otx2 is essential for retinal photoreceptor cell fate determination and development of the pineal gland. Otx2-deficiency converted differentiating photoreceptor cells to amacrine-like neurons and led to a total lack of pinealocytes in the pineal gland. We also found that Otx2 transactivates the cone-rod homeobox gene Crx, which is required for terminal differentiation and maintenance of photoreceptor cells. Furthermore, retroviral gene transfer of Otx2 steers retinal progenitor cells toward becoming photoreceptors. Thus, Otx2 is a key regulatory gene for the cell fate determination of retinal photoreceptor cells. Our results reveal the key molecular steps required for photoreceptor cell-fate determination and pinealocyte development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Células Fotorreceptoras/citologia , Células Fotorreceptoras/metabolismo , Glândula Pineal/metabolismo , Transativadores/genética , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/fisiologia , Fatores de Transcrição Otx , Células Fotorreceptoras/embriologia , Glândula Pineal/citologia , Glândula Pineal/embriologia , Transativadores/biossíntese , Transativadores/deficiência , Transativadores/fisiologiaRESUMO
ãRhythmic neural activities are observed in many brain regions, and these are considered to play an important role in neural information processing. On the other hand, distinct rhythmic neural activities emerge under several pathological conditions, suggesting that rhythmic neural activity has a close relation to brain function and dysfunction. In many pathological cases, the intrinsic property of unusual rhythm generation in a neuron or a neuronal network is prevented under normal conditions, but released by the pathological condition. Therefore, it may be useful to explore which conditions determine rhythm generation in order to understand the mechanisms of brain function/dysfunction. The pathological retina in retinal degeneration exhibits rhythmic neural activity not observed in the healthy retina. In this review, we first provide a brief introduction to the possible mechanisms of rhythm generation in a neural system. Then we introduce experimental evidence of rhythm generation in the pathological retina, as well as two hypotheses regarding this mechanism. Finally, we raise several issues to be solved for the further understanding of pathological rhythm generation.
Assuntos
Fenômenos Eletrofisiológicos/fisiologia , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Células Amácrinas/fisiologia , Animais , Encéfalo/fisiologia , Humanos , Rede Nervosa/fisiologia , Estimulação Luminosa , Células Fotorreceptoras de Vertebrados/fisiologiaRESUMO
ãThe vertebrate retina is one of the most sophisticated parts of the nervous system. It comprises five classes of neurons and one glial type cell. During development, but prior to a vertebrate's eyes opening, retinal circuits are refined by endogenous neural activity. Characteristic patterns of activity, including oscillatory activity, occur in the normal retina, whereas distinctive alternative patterns occur in abnormal retinas. In this paper, we first describe the electrophysiological and spike sorting methods used to study retinal oscillations. Next, we describe the mechanisms and functions of oscillation in the normal retina. Finally, we characterize the distinctive oscillations and abnormal spontaneous activities in the degenerative retina.
Assuntos
Eletrofisiologia/métodos , Retina/fisiologia , Retina/fisiopatologia , Degeneração Retiniana/fisiopatologia , Potenciais de Ação , Células Amácrinas/fisiologia , Animais , Neuroglia/fisiologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Retina/citologia , Retina/crescimento & desenvolvimento , Células Ganglionares da Retina/fisiologiaRESUMO
TRPM1, the first member of the melanoma-related transient receptor potential (TRPM) subfamily, is the visual transduction channel downstream of metabotropic glutamate receptor 6 (mGluR6) on retinal ON bipolar cells (BCs). Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB). In both TRPM1 and mGluR6 KO mouse retinas, OFF but not ON BCs respond to light stimulation. Here we report an unexpected difference between TRPM1 knockout (KO) and mGluR6 KO mouse retinas. We used a multielectrode array (MEA) to record spiking in retinal ganglion cells (RGCs). We found spontaneous oscillations in TRPM1 KO retinas, but not in mGluR6 KO retinas. We performed a structural analysis on the synaptic terminals of rod ON BCs. Intriguingly, rod ON BC terminals were significantly smaller in TRPM1 KO retinas than in mGluR6 KO retinas. These data suggest that a deficiency of TRPM1, but not of mGluR6, in rod ON bipolar cells may affect synaptic terminal maturation. We speculate that impaired signaling between rod BCs and AII amacrine cells (ACs) leads to spontaneous oscillations. TRPM1 and mGluR6 are both essential components in the signaling pathway from photoreceptors to ON BC dendrites, yet they differ in their effects on the BC terminal and postsynaptic circuitry.
Assuntos
Receptores de Glutamato Metabotrópico/metabolismo , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Canais de Cátion TRPM/metabolismo , Células Amácrinas/metabolismo , Animais , Dendritos/metabolismo , Oftalmopatias Hereditárias/metabolismo , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Camundongos , Camundongos Knockout , Miopia/metabolismo , Cegueira Noturna/metabolismo , Células Bipolares da Retina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
The photoreceptor is a highly polarized neuron and also has epithelial characteristics such as adherens junctions. To investigate the mechanisms of polarity formation of the photoreceptor cells, we conditionally knocked out atypical protein kinase Clambda (aPKClambda), which has been proposed to play a critical role in the establishment of epithelial and neuronal polarity, in differentiating photoreceptor cells using the Cre-loxP system. In aPKClambda conditional knock-out (CKO) mice, the photoreceptor cells displayed morphological defects and failed to form ribbon synapses. Intriguingly, lack of aPKClambda in differentiating photoreceptors led to severe laminar disorganization not only in the photoreceptor layer but also in the entire retina. Cell fate determination was not affected by total laminar disorganization. After Cre recombinase began to be expressed in the developing photoreceptors at embryonic day 12.5, both the immature photoreceptors and mitotic progenitors were dispersed throughout the CKO retina. We detected that adherens junction formation between the immature photoreceptors and the progenitors was lost in the CKO retina, whereas it was maintained between the progenitors themselves. These results indicate that the expression of aPKClambda in differentiating photoreceptors is required for total retinal lamination. Our data suggest that properly polarized photoreceptors anchor progenitors at the apical edge of the neural retina, which may be essential for building correct laminar organization of the retina.
Assuntos
Diferenciação Celular/fisiologia , Isoenzimas/fisiologia , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/enzimologia , Proteína Quinase C/fisiologia , Animais , Animais Recém-Nascidos , Polaridade Celular/genética , Polaridade Celular/fisiologia , Isoenzimas/deficiência , Isoenzimas/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Retina/citologia , Retina/enzimologia , Sinapses/genética , Sinapses/patologiaRESUMO
BACKGROUND: Sterile alpha motif (SAM) domains are approximately 70 residues long and have been reported as common protein-protein interaction modules. This domain is found in a large number of proteins, including Polycomb group (PcG) proteins and ETS family transcription factors. In this work, we report the cloning and functional characterization of a novel SAM domain-containing protein, which is predominantly expressed in retinal photoreceptors and the pineal gland and is designated mouse mr-s (major retinal SAM domain protein). RESULTS: mr-s is evolutionarily conserved from zebrafish through human, organisms through which the mechanism of photoreceptor development is also highly conserved. Phylogenetic analysis suggests that the SAM domain of mr-s is most closely related to a mouse polyhomeotic (ph) ortholog, Mph1/Rae28, which is known as an epigenetic molecule involved in chromatin modifications. These findings provide the possibility that mr-s may play a critical role by regulating gene expression in photoreceptor development. mr-s is preferentially expressed in the photoreceptors at postnatal day 3-6 (P3-6), when photoreceptors undergo terminal differentiation, and in the adult pineal gland. Transcription of mr-s is directly regulated by the cone-rod homeodomain protein Crx. Immunoprecipitation assay showed that the mr-s protein self-associates mainly through the SAM domain-containing region as well as ph. The mr-s protein localizes mainly in the nucleus, when mr-s is overexpressed in HEK293T cells. Moreover, in the luciferase assays, we found that mr-s protein fused to GAL4 DNA-binding domain functions as a transcriptional repressor. We revealed that the repression activity of mr-s is not due to a homophilic interaction through its SAM domain but to the C-terminal region. CONCLUSION: We identified a novel gene, mr-s, which is predominantly expressed in retinal photoreceptors and pineal gland. Based on its expression pattern and biochemical analysis, we predict that mr-s may function as a transcriptional repressor in photoreceptor cells and in pinealocytes of the pineal gland.
Assuntos
Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Fatores de Transcrição Otx/genética , Células Fotorreceptoras/fisiologia , Proteínas Repressoras/genética , Retina/fisiologia , Transativadores/genética , Animais , Linhagem Celular , Clonagem Molecular , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos , Fatores de Transcrição Otx/metabolismo , Glândula Pineal/fisiologia , Proteínas do Grupo Polycomb , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismoRESUMO
Quaking (QKI), which belongs to the STAR family of KH domain-containing RNA-binding proteins, functions in pre-mRNA splicing, microRNA regulation, and formation of circular RNA. QKI plays critical roles in myelinogenesis in the central and peripheral nervous systems and has been implicated neuron-glia fate decision in the brain; however, neither the expression nor function of QKI in the neural retina is known. Here we report the expression of QKI RNA-binding protein in the developing and mature mouse retina. QKI was strongly expressed by Müller glial cells in both the developing and adult retina. Intriguingly, during development, QKI was expressed in early differentiating neurons, such as the horizontal and amacrine cells, and subsequently in later differentiating bipolar cells, but not in photoreceptors. Neuronal expression was uniformly weak in the adult. Among QKI isoforms (5, 6, and 7), QKI-5 was the predominantly expressed isoform in the adult retina. To study the function of QKI in the mouse retina, we examined quakingviable(qkv) mice, which have a dysmyelination phenotype that results from deficiency of QKI expression and reduced numbers of mature oligodendrocytes. In homozygous qkv mutant mice (qkv/qkv), the optic nerve expression levels of QKI-6 and 7, but not QKI-5 were reduced. In the retina of the mutant homozygote, QKI-5 levels were unchanged, and QKI-6 and 7 levels, already low, were also unaffected. We conclude that QKI is expressed in developing and adult Müller glia. QKI is additionally expressed in progenitors and in differentiating neurons during retinal development, but expression weakened or diminished during maturation. Among QKI isoforms, we found that QKI-5 predominated in the adult mouse retina. Since Müller glial cells are thought to share properties with retinal progenitor cells, our data suggest that QKI may contribute to maintaining retinal progenitors prior to differentiation into neurons. On the other hand, the expression of QKI in different retinal neurons may suggest a role in neuronal cell type specific fate determination and maturation. The data raises the possibility that QKI may function in retinal cell fate determination and maturation in both glia and neurons.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a RNA/metabolismo , Retina/metabolismo , Animais , Células Ependimogliais/metabolismo , Camundongos , Nervo Óptico/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/genética , Retina/citologia , Retina/crescimento & desenvolvimento , Neurônios Retinianos/metabolismoRESUMO
Crx, an Otx-like homeobox gene, is expressed primarily in the photoreceptors of the retina and in the pinealocytes of the pineal gland. The CRX homeodomain protein is a transactivator of many photoreceptor/pineal-specific genes in vivo, such as rhodopsin and the cone opsins. Mutations in Crx are associated with the retinal diseases, cone-rod dystrophy-2, retinitis pigmentosa, and Leber's congenital amaurosis, which lead to loss of vision. We have generated transgenic mice, using 5'- and/or 3'-flanking sequences from the mouse Crx homeobox gene fused to the beta-galactosidase (lacZ) reporter gene, and we have investigated the promoter function of the cell-specific and developmentally regulated expression of Crx. All of the independent transgenic lines commonly showed lacZ expression in the photoreceptor cells of the retina and in the pinealocytes of the pineal gland. We characterized the transgenic lines in detail for cell-specific lacZ expression patterns by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining and lacZ immunostaining. The lacZ expression was observed in developing and developed photoreceptor cells. This observation was confirmed by coimmunostaining of dissociated retinal cells with the lacZ and opsin antibodies. The ontogeny analysis indicated that the lacZ expression completely agrees with a temporal expression pattern of Crx during retinal development. This study demonstrates that the mouse Crx 5'-upstream genomic sequence is capable of directing a cell-specific and developmentally regulated expression of Crx in photoreceptor cells.