RESUMO
BACKGROUND: The primary pathological alterations of Pendred syndrome are endolymphatic pH acidification and luminal enlargement of the inner ear. However, the molecular contributions of specific cell types remain poorly characterized. Therefore, we aimed to identify pH regulators in pendrin-expressing cells that may contribute to the homeostasis of endolymph pH and define the cellular pathogenic mechanisms that contribute to the dysregulation of cochlear endolymph pH in Slc26a4-/- mice. METHODS: We used single-cell RNA sequencing to identify both Slc26a4-expressing cells and Kcnj10-expressing cells in wild-type (WT, Slc26a4+/+) and Slc26a4-/- mice. Bioinformatic analysis of expression data confirmed marker genes defining the different cell types of the stria vascularis. In addition, specific findings were confirmed at the protein level by immunofluorescence. RESULTS: We found that spindle cells, which express pendrin, contain extrinsic cellular components, a factor that enables cell-to-cell communication. In addition, the gene expression profile informed the pH of the spindle cells. Compared to WT, the transcriptional profiles in Slc26a4-/- mice showed downregulation of extracellular exosome-related genes in spindle cells. Immunofluorescence studies in spindle cells of Slc26a4-/- mice validated the increased expression of the exosome-related protein, annexin A1, and the clathrin-mediated endocytosis-related protein, adaptor protein 2. CONCLUSION: Overall, cell isolation of stria vascularis from WT and Slc26a4-/- samples combined with cell type-specific transcriptomic analyses revealed pH-dependent alternations in spindle cells and intermediate cells, inspiring further studies into the dysfunctional role of stria vascularis cells in SLC26A4-related hearing loss.
Assuntos
Surdez , Estria Vascular , Camundongos , Animais , Estria Vascular/metabolismo , Estria Vascular/patologia , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Cóclea/metabolismo , Cóclea/patologia , Surdez/genética , Transportadores de Sulfato/genética , RNA/metabolismoRESUMO
BACKGROUND: Neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD) display a strong male bias. Androgen exposure is profoundly increased in typical male development, but it also varies within the sexes, and previous work has sought to connect morphological proxies of androgen exposure, including digit ratio and facial morphology, to neurodevelopmental outcomes. The results of these studies have been mixed, and the relationships between androgen exposure and behavior remain unclear. METHODS: Here, we measured both digit ratio masculinity (DRM) and facial landmark masculinity (FLM) in the same neurodevelopmental cohort (N = 763) and compared these proxies of androgen exposure to clinical and parent-reported features as well as polygenic risk scores. RESULTS: We found that FLM was significantly associated with NDD diagnosis (ASD, ADHD, ID; all [Formula: see text]), while DRM was not. When testing for association with parent-reported problems, we found that both FLM and DRM were positively associated with concerns about social behavior ([Formula: see text], [Formula: see text]; [Formula: see text], [Formula: see text], respectively). Furthermore, we found evidence via polygenic risk scores (PRS) that DRM indexes masculinity via testosterone levels ([Formula: see text], [Formula: see text]), while FLM indexes masculinity through a negative relationship with sex hormone binding globulin (SHBG) levels ([Formula: see text], [Formula: see text]). Finally, using the SPARK cohort (N = 9419) we replicated the observed relationship between polygenic estimates of testosterone, SHBG, and social functioning ([Formula: see text], [Formula: see text], and [Formula: see text], [Formula: see text] for testosterone and SHBG, respectively). Remarkably, when considered over the extremes of each variable, these quantitative sex effects on social functioning were comparable to the effect of binary sex itself (binary male: [Formula: see text]; testosterone: [Formula: see text] from 0.1%-ile to 99.9%-ile; SHBG: [Formula: see text] from 0.1%-ile to 99.9%-ile). LIMITATIONS: In the devGenes and SPARK cohorts, our analyses rely on indirect, rather than direct measurement of androgens and related molecules. CONCLUSIONS: These findings and their replication in the large SPARK cohort lend support to the hypothesis that increasing net androgen exposure diminishes capacity for social functioning in both males and females.
Assuntos
Androgênios , Transtorno do Espectro Autista , Estudos de Coortes , Feminino , Humanos , Masculino , Herança Multifatorial , TestosteronaRESUMO
The locus coeruleus (LC) is a nucleus within the brainstem that consists of norepinephrine-releasing neurons. It is involved in broad processes including cognitive and emotional functions. Understanding the mechanisms that control the excitability of LC neurons is important because they innervate widespread brain regions. One of the key regulators is cytosolic calcium concentration ([Ca2+]c), the increases in which can be amplified by calcium-induced calcium release (CICR) from intracellular calcium stores. Although the electrical activities of LC neurons are regulated by changes in [Ca2+]c, the extent of CICR involvement in this regulation has remained unclear. Here we show that CICR hyperpolarizes acutely dissociated LC neurons of the rat and demonstrate the underlying pathway. When CICR was activated by extracellular application of 10 mM caffeine, LC neurons were hyperpolarized in the current-clamp mode of patch-clamp recording, and the majority of neurons showed an outward current in the voltage-clamp mode. This outward current was accompanied by increased membrane conductance, and its reversal potential was close to the K+ equilibrium potential, indicating that it is mediated by opening of K+ channels. The outward current was generated in the absence of extracellular calcium and was blocked when the calcium stores were inhibited by applying ryanodine. Pharmacological blockers indicated that it was mediated by Ca2+-activated K+ channels of the non-small conductance type. The application of caffeine increased [Ca2+]c, as visualized by fluorescence microscopy. These findings show CICR suppresses LC neuronal activity, and indicate its dynamic role in modulating the LC-mediated noradrenergic tone in the brain.
Assuntos
Neurônios Adrenérgicos/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Locus Cerúleo/metabolismo , Neurônios Adrenérgicos/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/farmacologia , Locus Cerúleo/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Single-cell RNA sequencing is a powerful tool by which to characterize the transcriptional profile of low-abundance cell types, but its application to the inner ear has been hampered by the bony labyrinth, tissue sparsity, and difficulty dissociating the ultra-rare cells of the membranous cochlea. Herein, we present a method to isolate individual inner hair cells (IHCs), outer hair cells (OHCs), and Deiters' cells (DCs) from the murine cochlea at any post-natal time point. We harvested more than 200 murine IHCs, OHCs, and DCs from post-natal days 15 (p15) to 228 (p228) and leveraged both short- and long-read single-cell RNA sequencing to profile transcript abundance and structure. Our results provide insights into the expression profiles of these cells and document an unappreciated complexity in isoform variety in deafness-associated genes. This refined view of transcription in the organ of Corti improves our understanding of the biology of hearing and deafness.
Assuntos
Surdez/genética , Órgão Espiral/metabolismo , Transcriptoma , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Camundongos , Órgão Espiral/crescimento & desenvolvimento , Análise de Célula ÚnicaRESUMO
Autosomal-dominant, early-onset DYT1 dystonia is associated with an in-frame deletion of a glutamic acid codon (ΔE) in the TOR1A gene. The gene product, torsinA, is an evolutionarily conserved AAA+ ATPase. The fact that constitutive secretion from patient fibroblasts is suppressed indicates that the ΔE-torsinA protein influences the cellular secretory machinery. However, which component is affected remains unclear. Prompted by recent reports that abnormal protein trafficking through the Golgi apparatus, the major protein-sorting center of the secretory pathway, is sometimes associated with a morphological change in the Golgi, we evaluated the influence of ΔE-torsinA on this organelle. Specifically, we examined its structure by confocal microscopy, in cultures of striatal, cerebral cortical and hippocampal neurons obtained from wild-type, heterozygous and homozygous ΔE-torsinA knock-in mice. In live neurons, the Golgi was assessed following uptake of a fluorescent ceramide analog, and in fixed neurons it was analyzed by immuno-fluorescence staining for the Golgi-marker GM130. Neither staining method indicated genotype-specific differences in the size, staining intensity, shape or localization of the Golgi. Moreover, no genotype-specific difference was observed as the neurons matured in vitro. These results were supported by a lack of genotype-specific differences in GM130 expression levels, as assessed by Western blotting. The Golgi was also disrupted by treatment with brefeldin A, but no genotype-specific differences were found in the immuno-fluorescence staining intensity of GM130. Overall, our results demonstrate that the ΔE-torsinA protein does not drastically influence Golgi morphology in neurons, irrespective of genotype, brain region (among those tested), or maturation stage in culture. While it remains possible that functional changes in the Golgi exist, our findings imply that any such changes are not severe enough to influence its morphology to a degree detectable by light microscopy.
Assuntos
Distonia/genética , Deleção de Genes , Complexo de Golgi/metabolismo , Chaperonas Moleculares/genética , Animais , Autoantígenos/metabolismo , Brefeldina A/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ceramidas/metabolismo , Dendritos/efeitos dos fármacos , Dendritos/metabolismo , Fluorescência , Técnicas de Introdução de Genes , Complexo de Golgi/efeitos dos fármacos , Hipocampo/patologia , Processamento de Imagem Assistida por Computador , Proteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/metabolismo , Neostriado/patologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
BACKGROUND: NPAS3 has been established as a robust genetic risk factor in major mental illness. In mice, loss of neuronal PAS domain protein 3 (NPAS3) impairs postnatal hippocampal neurogenesis, while loss of the related protein NPAS1 promotes it. These and other findings suggest a critical role for NPAS proteins in neuropsychiatric functioning, prompting interest in the molecular pathways under their control. METHODS: We used RNA sequencing coupled with chromatin immunoprecipitation sequencing to identify genes directly regulated by NPAS1 and NPAS3 in the hippocampus of wild-type, Npas1-/-, and Npas3-/- mice. Computational integration with human genetic and expression data revealed the disease relevance of NPAS-regulated genes and pathways. Specific findings were confirmed at the protein level by Western blot. RESULTS: This is the first in vivo, transcriptome-scale investigation of genes regulated by NPAS1 and NPAS3. These transcription factors control an ensemble of genes that are themselves also major regulators of neuropsychiatric function. Specifically, Fmr1 (fragile X syndrome) and Ube3a (Angelman syndrome) are transcriptionally regulated by NPAS3, as is the neurogenesis regulator Notch. Dysregulation of these pathways was confirmed at the protein level. Furthermore, NPAS1/3 targets show increased human genetic burden for schizophrenia and intellectual disability. CONCLUSIONS: Together, these data provide a clear, unbiased view of the full spectrum of genes regulated by NPAS1 and NPAS3 and show that these transcription factors are master regulators of neuropsychiatric function. These findings expose the molecular pathophysiology of NPAS1/3 mutations and provide a striking example of the shared, combinatorial nature of molecular pathways that underlie diagnostically distinct neuropsychiatric conditions.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transtornos Mentais/genética , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Feminino , Regulação da Expressão Gênica/fisiologia , Predisposição Genética para Doença , Hipocampo/metabolismo , Humanos , Masculino , Transtornos Mentais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Transcrição Gênica , TranscriptomaRESUMO
High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.
Assuntos
Encéfalo/citologia , Técnicas de Cultura de Células/métodos , Técnicas de Genotipagem/métodos , Neurônios/citologia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Distúrbios Distônicos/genética , Camundongos , Chaperonas Moleculares/genética , RatosRESUMO
Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.
Assuntos
Pareamento Cromossômico/fisiologia , Corantes Fluorescentes/química , Compostos de Piridínio/química , Compostos de Amônio Quaternário/química , Vesículas Sinápticas/fisiologia , Animais , Camundongos , Fotodegradação , Terminações Pré-Sinápticas/química , Terminações Pré-Sinápticas/fisiologia , Ratos , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/químicaRESUMO
BACKGROUND: Images in biomedical imaging research are often affected by non-specific background noise. This poses a serious problem when the noise overlaps with specific signals to be quantified, e.g. for their number and intensity. A simple and effective means of removing background noise is to prepare a filtered image that closely reflects background noise and to subtract it from the original unfiltered image. This approach is in common use, but its effectiveness in identifying and quantifying synaptic puncta has not been characterized in detail. NEW ANALYSIS: We report on our assessment of the effectiveness of isolating punctate signals from diffusely distributed background noise using one variant of this approach, "Difference of Gaussian(s) (DoG)" which is based on a Gaussian filter. RESULTS: We evaluated immunocytochemically stained, cultured mouse hippocampal neurons as an example, and provided the rationale for choosing specific parameter values for individual steps in detecting glutamatergic nerve terminals. The intensity and width of the detected puncta were proportional to those obtained by manual fitting of two-dimensional Gaussian functions to the local information in the original image. COMPARISON WITH EXISTING METHODS: DoG was compared with the rolling-ball method, using biological data and numerical simulations. Both methods removed background noise, but differed slightly with respect to their efficiency in discriminating neighboring peaks, as well as their susceptibility to high-frequency noise and variability in object size. CONCLUSIONS: DoG will be useful in detecting punctate signals, once its characteristics are examined quantitatively by experimenters.
Assuntos
Imunofluorescência , Interpretação de Imagem Assistida por Computador , Neurônios/citologia , Distribuição Normal , Sinapses/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Hipocampo/citologia , Camundongos , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
BACKGROUND: Although recent developments in methodologies for light microscopy have enabled imaging of fine biological structures, such imaging is often accompanied by two types of problems. One is a tilting of the specimen with respect to the x-y plane (i.e. rotation around the x- or y-axis) such that the sample is not perpendicular to the optical z-axis, and the other is rotation around the z-axis that precludes optimal orientations for imaging and experimentation. These rotation problems can cause optical aberrations and hamper imaging experiments, even when the angular difference from the ideal position is small. NEW METHOD: In order to correct for these practical issues, we have developed a specimen holder with 3-axis (x-y-z) rotation for an inverted light microscope. This allows for full-range rotations of 2-4° for x-, y-axes, ~24° for z-axis, and a small-angle control of <0.1° for either axis. RESULTS: Using this device, we observed the cultured hippocampal neurons stained by immunofluorescence for a dendritic marker, or the sub-resolution fluorescent beads plated on a glass coverslip. The rotations and associated problems could be manipulated, while viewing the specimens by laser-scanning confocal microscopy. COMPARISON WITH EXISTING METHODS: This tilting/rotation device is easily manufactured and installed on a conventional microscope stage without requiring changes to the existing optical components. Similar devices with full capability have not been available. CONCLUSIONS: It will be useful for imaging experiments with biomedical applications.
Assuntos
Hipocampo/citologia , Microscopia Confocal/instrumentação , Microscopia/instrumentação , Neurônios/citologia , Animais , Imunofluorescência , CamundongosRESUMO
DYT1 dystonia is the most common hereditary form of primary torsion dystonia. This autosomal-dominant disorder is characterized by involuntary muscle contractions that cause sustained twisting and repetitive movements. It is caused by an in-frame deletion in the TOR1A gene, leading to the deletion of a glutamic acid residue in the torsinA protein. Heterozygous knock-in mice, which reproduce the genetic mutation in human patients, have abnormalities in synaptic transmission at the principal GABAergic neurons in the striatum, a brain structure that is involved in the execution and modulation of motor activity. However, whether this mutation affects the excitability of striatal GABAergic neurons has not been investigated in this animal model. Here, we examined the excitability of cultured striatal neurons obtained from heterozygous knock-in mice, using calcium imaging as indirect readout. Immunofluorescence revealed that more than 97% of these neurons are positive for a marker of GABAergic neurons, and that more than 92% are also positive for a marker of medium spiny neurons, indicating that these are mixed cultures of mostly medium spiny neurons and a few (~5%) GABAergic interneurons. When these neurons were depolarized by field stimulation, the calcium concentration in the dendrites increased rapidly and then decayed slowly. The amplitudes of calcium transients were larger in heterozygous neurons than in wild-type neurons, resulting in ~15% increase in cumulative calcium transients during a train of stimuli. However, there was no change in other parameters of calcium dynamics. Given that calcium dynamics reflect neuronal excitability, these results suggest that the mutation only slightly increases the excitability of striatal GABAergic neurons in DYT1 dystonia.
Assuntos
Cálcio/metabolismo , Corpo Estriado/metabolismo , Citoplasma/metabolismo , Distonia Muscular Deformante/genética , Distúrbios Distônicos/genética , Neurônios GABAérgicos/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Neurônios Colinérgicos/metabolismo , Modelos Animais de Doenças , Heterozigoto , Interneurônios/metabolismo , Camundongos , Camundongos Transgênicos , MutaçãoRESUMO
Increased activities of cytoplasmic calcium and the excitatory neurotransmitter glutamate have been independently implicated in dystonia pathophysiology. However, cellular-level evidence linking these two features is not available. Here we show that glutamate-dependent changes in neuronal calcium dynamics occur in a knock-in mouse model of DYT1 dystonia, the most common hereditary form of this disorder. Fluorescence-based analysis of the dynamics of cytoplasmic calcium concentration ([Ca(2+)]c) in cultured hippocampal neurons shows that electrical stimulation depolarizes the neurons and increases the dendritic [Ca(2+)]c, which then decays slowly to the pre-stimulus level. Whereas the peak amplitude of [Ca(2+)]c was not affected, the decay period was prolonged in neurons of heterozygous mice whose genotype reflects the human condition. We found that this effect was blocked by the antagonists of ionotropic glutamate receptors, and confirmed that glutamate receptors are present in these neurons. As the [Ca(2+)]c is readout and regulator of neuronal excitability, its abnormality represents an important cellular phenotype of dystonia.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Citoplasma/metabolismo , Distonia/metabolismo , Hipocampo , Neurônios/metabolismo , Receptores de Glutamato/metabolismo , Animais , Células Cultivadas , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos KnockoutRESUMO
TorsinA is an evolutionarily conserved AAA+ ATPase, and human patients with an in-frame deletion of a single glutamate (ΔE) codon from the encoding gene suffer from autosomal-dominant, early-onset generalized DYT1 dystonia. Although only 30-40% of carriers of the mutation show overt motor symptoms, most experience enhanced excitability of the central nervous system. The cellular mechanism responsible for this change in excitability is not well understood. Here we show the effects of the ΔE-torsinA mutation on miniature neurotransmitter release from neurons. Neurotransmitter release was characterized in cultured hippocampal neurons obtained from wild-type, heterozygous, and homozygous ΔE-torsinA knock-in mice using two approaches. In the first approach, patch-clamp electrophysiology was used to record glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in the presence of the Na⺠channel blocker tetrodotoxin (TTX) and absence of GABA(A) receptor antagonists. The intervals between mEPSC events were significantly shorter in neurons obtained from the mutant mice than in those obtained from wild-type mice. In the second approach, the miniature exocytosis of synaptic vesicles was detected by imaging the unstimulated release of FM dye from the nerve terminals in the presence of TTX. Cumulative FM dye release was higher in neurons obtained from the mutant mice than in those obtained from wild-type mice. The number of glutamatergic nerve terminals was also assessed, and we found that this number was unchanged in heterozygous relative to wild-type neurons, but slightly increased in homozygous neurons. Notably, in both heterozygous and homozygous neurons, the unitary synaptic charge during each mEPSC event was unchanged. Overall, our results suggest more frequent miniature glutamate release in neurons with ΔE-torsinA mutations. This change may be one of the underlying mechanisms by which the excitability of the central nervous system is enhanced in the context of DYT1 dystonia. Moreover, qualitative differences between heterozygous and homozygous neurons with respect to certain synaptic properties indicate that the abnormalities observed in homozygotes may reflect more than a simple gene dosage effect.
Assuntos
Exocitose/fisiologia , Ácido Glutâmico/metabolismo , Hipocampo/fisiologia , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Chaperonas Moleculares/genética , Neurônios/fisiologia , Animais , Modelos Animais de Doenças , Distonia Muscular Deformante/genética , Potenciais Pós-Sinápticos Excitadores/fisiologia , Heterozigoto , Hipocampo/citologia , Homozigoto , Potenciais Pós-Sinápticos Inibidores/fisiologia , Camundongos , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Mutação , Receptores de GABA-A/metabolismo , Canais de Sódio/efeitos dos fármacos , Canais de Sódio/metabolismo , Vesículas Sinápticas/metabolismo , Tetrodotoxina/farmacologia , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
Early-onset generalized dystonia, DYT1, is caused by a mutation in the gene encoding the evolutionarily conserved AAA+ ATPase torsinA. Synaptic abnormalities have been implicated in DYT1 dystonia, but the details of the synaptic pathophysiology are only partially understood. Here, we demonstrate a novel role for torsinA in synaptic vesicle recycling, using cultured hippocampal neurons from a knock-in mouse model of DYT1 dystonia (ΔE-torsinA) and live-cell imaging with styryl FM dyes. Neurons from heterozygous ΔE-torsinA mice released a larger fraction of the total recycling pool (TRP) during a single round of electrical stimulation than did wild-type neurons. Moreover, when the neurons were subjected to prior high activity, the time course of release was shortened. In neurons from homozygous mice, these enhanced exocytosis phenotypes were similar, but in addition the size of the TRP was reduced. Notably, when release was triggered by applying a calcium ionophore rather than electrical stimuli, neither a single nor two ΔE-torsinA alleles affected the time course of release. Thus, the site of action of ΔE-torsinA is at or upstream of the rise in calcium concentration in nerve terminals. Our results suggest that torsinA regulates synaptic vesicle recycling in central neurons. They also indicate that this regulation is influenced by neuronal activity, further supporting the idea that synaptic abnormalities contribute to the pathophysiology of DYT1 dystonia.
Assuntos
Distúrbios Distônicos/metabolismo , Chaperonas Moleculares/metabolismo , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Células Cultivadas , Distúrbios Distônicos/genética , Endocitose/fisiologia , Exocitose/fisiologia , Técnicas de Introdução de Genes , Heterozigoto , Camundongos , Modelos Animais , Chaperonas Moleculares/genética , Vesículas Sinápticas/genéticaRESUMO
Transient receptor potential V1 (TRPV1) has been suggested to play an important role in detecting decreases in extracellular pH (pH(o)). Results from recent in vivo studies, however, have suggested that TRPV1 channels play less of a role in sensing a moderately acidic pH(o) (6.0 < pH < 7.0) than predicted from the in vitro experiments. A clear explanation for this discrepancy between the in vitro and in vivo data has not yet been provided. We report here that intracellular acidification induced by a moderately low pH(o) (6.4) almost completely inhibited the effect of extracellular acidosis on TRPV1 activity. In our experiments, sodium acetate (20 mm), which was used to induce intracellular acidosis, attenuated the capsaicin-evoked TRPV1 current (I(CAP)) in a reversible manner in whole-cell patch-clamp mode and shifted the concentration-response curve to the right. Likewise, the concentration-response curve was significantly shifted to the right by lowering the pH of the pipette solution from 7.2 to 6.5. In addition, application of an acidic bath solution (pH 6.4) to the intracellular side also significantly suppressed I(CAP) in inside-out patch mode. In cell-attached patch mode, the single-channel activity of i(CAP) was significantly attenuated by intracellular acidosis that was induced by a decrease in pH(o) (6.4). These results suggested that intracellular acidification induced by a low pH(o) inhibited TRPV1 activity. When studied in perforated patch mode or by acidifying the intracellular pipette solution, potentiation or activation of TRPV1 by extracellular acidosis (pH 6.4) at 37 °C was almost completely inhibited. Likewise, enhancement of neuronal excitability by a moderately acidic pH(o) (6.4) at a physiological temperature (37 °C) was attenuated by lowering the pH of the pipette solution to 6.5 or using perforated patch mode. Taken together, these results suggest that extracellular acidosis of moderate intensity may not significantly modulate TRPV1 activity in physiological conditions at which intracellular pH can be readily affected by pH(o), and this phenomenon is due to attenuation of TRPV1 channel activity by low-pH(o)-induced intracellular acidification.
Assuntos
Acidose/fisiopatologia , Gânglios Espinais/fisiologia , Canais de Cátion TRPV/fisiologia , Animais , Capsaicina/farmacologia , Células Cultivadas , Concentração de Íons de Hidrogênio , Masculino , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/antagonistas & inibidoresRESUMO
The steroid receptor-type transcription factor Nurr1 has a crucial role in the development of the mesencephalic dopamine (DA) neurons. Although ectopic expression of Nurr1 in cultured neural precursor cells is sufficient in establishing the DA phenotype, Nurr1-induced DA cells are morphologically and functionally immature, suggesting the necessity of additional factor(s) for full neuronal differentiation. In this study, we demonstrate that neurogenic basic helix-loop-helix (bHLH) factors Mash1, neurogenins (Ngns) and NeuroD play contrasting roles in Nurr1-induced DA neuronal differentiation. Mash1, but not Ngn2, spatially and temporally colocalized with aldehyde dehydrogenase 2 (AHD2), a specific midbrain DA neuronal progenitor marker, in the early embryonic ventral mesencephalon. Forced expression of Mash1 caused immature Nurr1-induced DA cells to differentiate into mature and functional DA neurons as judged by electrophysiological characteristics, release of DA, and expression of presynaptic DA neuronal markers. By contrast, atonal-related bHLHs, represented by Ngn1, Ngn2 and NeuroD, repressed Nurr1-induced expression of DA neuronal markers. Domain-swapping experiments with Mash1 and NeuroD indicated that the helix-loop-helix domain, responsible for mediating dimerization of bHLH transcription factors, imparts the distinct effect. Finally, transient co-transfection of the atonal-related bHLHs with Nurr1 resulted in an E-box-independent repression of Nurr1-induced transcriptional activation of a reporter containing Nurr1-binding element (NL3) as well as a reporter driven by the native tyrosine hydroxylase gene promoter. Taken together, these findings suggest that Mash1 contributes to the generation of DA neurons in cooperation with Nurr1 in the developing midbrain whereas atonal-related bHLH genes inhibit the process.