RESUMEN
Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins.
Asunto(s)
Diabetes Insípida Neurogénica , Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Humanos , Ratones , Animales , Arginina Vasopresina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Vasopresinas/farmacología , Vasopresinas/metabolismo , Diabetes Insípida Neurogénica/metabolismo , Neurofisinas/genética , Proteínas Mutantes/metabolismo , MutaciónRESUMEN
Pituitary organoids are promising graft sources for transplantation in treatment of hypopituitarism. Building on development of self-organizing culture to generate pituitary-hypothalamic organoids (PHOs) using human pluripotent stem cells (hPSCs), we established techniques to generate PHOs using feeder-free hPSCs and to purify pituitary cells. The PHOs were uniformly and reliably generated through preconditioning of undifferentiated hPSCs and modulation of Wnt and TGF-ß signaling after differentiation. Cell sorting using EpCAM, a pituitary cell-surface marker, successfully purified pituitary cells, reducing off-target cell numbers. EpCAM-expressing purified pituitary cells reaggregated to form three-dimensional pituitary spheres (3D-pituitaries). These exhibited high adrenocorticotropic hormone (ACTH) secretory capacity and responded to both positive and negative regulators. When transplanted into hypopituitary mice, the 3D-pituitaries engrafted, improved ACTH levels, and responded to in vivo stimuli. This method of generating purified pituitary tissue opens new avenues of research for pituitary regenerative medicine.
Asunto(s)
Hormona Adrenocorticotrópica , Células Madre Pluripotentes , Ratones , Animales , Humanos , Molécula de Adhesión Celular Epitelial , Técnicas de Cultivo de Célula/métodos , Diferenciación CelularRESUMEN
Introduction: The pituitary gland, regulating various hormones, is central in the endocrine system. As spontaneous recovery from hypopituitarism is rare, and exogenous-hormone substitution is clumsy, pituitary replacement via regenerative medicine, using pluripotent stem cells, is desirable. We have developed a differentiation method that in mice yields pituitary organoids (POs) derived from human embryonic stem cells (hESC). Efficacy of these POs, transplanted subcutaneously into hypopituitary mice, in reversing hypopituitarism was studied. Methods: hESC-derived POs were transplanted into inguinal subcutaneous white adipose tissue (ISWAT) and beneath dorsal skin, a relatively avascular region (AR), of hypophysectomized severe combined immunodeficient (SCID) mice. Pituitary function was evaluated thereafter for ¾ 6mo, assaying basal plasma ACTH and ACTH response to corticotropin-releasing hormone (CRH) stimulation. Histopathologic examination of organoids 150d after transplantation assessed engraftment. Some mice received an inhibitor of vascular endothelial growth factor (VEGF) to permit assessment of how angiogenesis contributed to subcutaneous engraftment. Results: During follow-up, both basal and CRH-stimulated plasma ACTH levels were significantly higher in the ISWAT group (p < 0.001 - 0.05 and 0.001 - 0.005, respectively) than in a sham-operated group. ACTH secretion also was higher in the ISWAT group than in the AR group. Histopathologic study found ACTH-producing human pituitary-cell clusters in both groups of allografts, which had acquired a microvasculature. POs qPCR showed expression of angiogenetic factors. Plasma ACTH levels decreased with VEGF-inhibitor administration. Conclusions: Subcutaneous transplantation of hESC-derived POs into hypopituitary SCID mice efficaciously renders recipients ACTH-sufficient.
Asunto(s)
Células Madre Embrionarias Humanas , Hipopituitarismo , Enfermedades de la Hipófisis , Humanos , Ratones , Animales , Células Madre Embrionarias Humanas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Hormona Adrenocorticotrópica/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Ratones SCID , Hipófisis/metabolismo , Enfermedades de la Hipófisis/metabolismo , Hipopituitarismo/metabolismoRESUMEN
When damaged, restoring the function of the hypothalamus is currently impossible. It is unclear whether neural stem cells exist in the hypothalamus. Studies have reported that adult rodent tanycytes around the third ventricle function as hypothalamic neural stem cell-like cells. However, it is currently impossible to collect periventricular cells from humans. We attempted to generate hypothalamic neural stem cell-like cells from human embryonic stem cells (ESCs). We focused on retina and anterior neural fold homeobox (RAX) because its expression is gradually restricted to tanycytes during the late embryonic stage. We differentiated RAX::VENUS knockin human ESCs (hESCs) into hypothalamic organoids and sorted RAX+ cells from mature organoids. The isolated RAX+ cells formed neurospheres and exhibited self-renewal and multipotency. Neurogenesis was observed when neurospheres were transplanted into the mouse hypothalamus. We isolated RAX+ hypothalamic neural stem cell-like cells from wild-type human ES organoids. This is the first study to differentiate human hypothalamic neural stem cell-like cells from pluripotent stem cells.
Asunto(s)
Células-Madre Neurales , Células Madre Pluripotentes , Ratones , Animales , Humanos , Diferenciación Celular/fisiología , Neurogénesis/fisiología , Hipotálamo/metabolismoRESUMEN
Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) differentiate into AVP neurons, whereas human ESCs/iPSCs die. Human ESCs/iPSCs are generally more similar to mouse epiblast stem cells (mEpiSCs) compared to mouse ESCs. In this study, we converted human FNDI-specific iPSCs by the naive conversion kit. Although the conversion was partial, we found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.
Asunto(s)
Diabetes Insípida Neurogénica , Células Madre Pluripotentes Inducidas , Animales , Diferenciación Celular , Humanos , Hipotálamo/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Neuronas/metabolismo , Vasopresinas/metabolismoRESUMEN
Human stem cell-derived organoid culture enables the in vitro analysis of the cellular function in three-dimensional aggregates mimicking native organs, and also provides a valuable source of specific cell types in the human body. We previously established organoid models of the hypothalamic-pituitary (HP) complex using human pluripotent stem cells. Although the models are suitable for investigating developmental and functional HP interactions, we consider that isolated pituitary cells are also useful for basic and translational research on the pituitary gland, such as stem cell biology and regenerative medicine. To develop a method for the purification of pituitary cells in HP organoids, we performed surface marker profiling of organoid cells derived from human induced pluripotent stem cells (iPSCs). Screening of 332 human cell surface markers and a subsequent immunohistochemical analysis identified epithelial cell adhesion molecule (EpCAM) as a surface marker of anterior pituitary cells, as well as their ectodermal precursors. EpCAM was not expressed on hypothalamic lineages; thus, anterior pituitary cells were successfully enriched by magnetic separation of EpCAM+ cells from iPSC-derived HP organoids. The enriched pituitary population contained functional corticotrophs and their progenitors; the former responded normally to a corticotropin-releasing hormone stimulus. Our findings would extend the applicability of organoid culture as a novel source of human anterior pituitary cells, including stem/progenitor cells and their endocrine descendants.
Asunto(s)
Células Madre Pluripotentes Inducidas , Hormonas Adenohipofisarias , Células Madre Pluripotentes , Biomarcadores/metabolismo , Molécula de Adhesión Celular Epitelial/metabolismo , Humanos , Organoides/metabolismo , Hipófisis/metabolismo , Hormonas Adenohipofisarias/metabolismoRESUMEN
Prolactin (PRL), a hormone involved in lactation, is mainly produced and secreted by the lactotrophs of the anterior pituitary (AP) gland. We previously reported a method to generate functional adrenocorticotropic hormone-producing cells by differentiating the AP and hypothalamus simultaneously from human induced pluripotent stem cells (iPSCs). However, PRL-producing cells in the induced AP have not been investigated. Here, we confirmed the presence of PRL-producing cells and evaluated their endocrine functions. We differentiated pituitary cells from human iPSCs using serum-free floating culture of embryoid-like aggregates with quick reaggregation (SFEB-q) method and evaluated the appearance and function of PRL-producing cells. Secretion of PRL from the differentiated aggregates was confirmed, which increased with further culture. Fluorescence immunostaining and immunoelectron microscopy revealed PRL-producing cells and PRL-positive secretory granules, respectively. PRL secretion was promoted by various prolactin secretagogues such as thyrotropin-releasing hormone, vasoactive intestinal peptide, and prolactin-releasing peptide, and inhibited by bromocriptine. Moreover, the presence of tyrosine hydroxylase-positive dopaminergic nerves in the hypothalamic tissue area around the center of the aggregates connecting to PRL-producing cells indicated the possibility of recapitulating PRL regulatory mechanisms through the hypothalamus. In conclusion, we generated pituitary lactotrophs from human iPSCs; these displayed similar secretory responsiveness as human pituitary cells in vivo. In the future, this is expected to be used as a model of human PRL-producing cells for various studies, such as drug discovery, prediction of side effects, and elucidation of tumorigenic mechanisms using disease-specific iPSCs. Furthermore, it may help to develop regenerative medicine for the pituitary gland.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/fisiología , Lactotrofos/fisiología , Adenohipófisis/citología , Prolactina/biosíntesis , Técnicas de Cultivo de Célula , Línea Celular , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Lactotrofos/efectos de los fármacos , Hormona Liberadora de Prolactina/farmacología , Hormona Liberadora de Tirotropina/farmacología , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
For pituitary regenerative medicine, the creation of a hypophyseal model in monkeys is necessary to conduct future preclinical studies; however, previous studies reported that hypophysectomy in monkeys is not always safe or satisfactory. This study aimed to create a hypophyseal dysfunction model in a cynomolgus monkey using a safer surgical technique and establish the protocol of pituitary hormone replacement therapy for this model. Surgical resection of the pituitary gland of a 7.8-year-old healthy adult cynomolgus male monkey weighing 5.45 kg was performed to create a hypophyseal dysfunction model for future regenerative studies. Endoscopic transoral transsphenoidal surgery was used to perform hypophysectomy under navigation support. These procedures were useful for confirming total removal of the pituitary gland without additional bone removal and preventing complications such as cerebrospinal fluid leakage. Total removal was confirmed by pathological examination and computed tomography. Hypopituitarism was verified with endocrinological examinations including stimulation tests. Postoperatively, the monkey's general condition of hypopituitarism was treated with hormone replacement therapy, resulting in long-term survival. The success of a minimally invasive and safe surgical method and long-term survival indicate the creation of a hypophyseal dysfunction model in a cynomolgus monkey; hence, this protocol can be employed in the future.
Asunto(s)
Hipofisectomía/efectos adversos , Hipopituitarismo/diagnóstico por imagen , Hipopituitarismo/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Terapia de Reemplazo de Hormonas , Humanos , Hipopituitarismo/etiología , Macaca fascicularis , Masculino , Procedimientos Quirúrgicos Mínimamente Invasivos , Medicina Regenerativa , Tomografía Computarizada por Rayos X , Resultado del TratamientoRESUMEN
The pituitary is a major hormone center that secretes systemic hormones responding to hypothalamus-derived-releasing hormones. Previously, we reported the independent pituitary induction and hypothalamic differentiation of human embryonic stem cells (ESCs). Here, a functional hypothalamic-pituitary unit is generated using human induced pluripotent stem (iPS) cells in vitro. The adrenocorticotropic hormone (ACTH) secretion capacity of the induced pituitary reached a comparable level to that of adult mouse pituitary because of the simultaneous maturation with hypothalamic neurons within the same aggregates. Corticotropin-releasing hormone (CRH) from the hypothalamic area regulates ACTH cells similarly to our hypothalamic-pituitary axis. Our induced hypothalamic-pituitary units respond to environmental hypoglycemic condition in vitro, which mimics a life-threatening situation in vivo, through the CRH-ACTH pathway, and succeed in increasing ACTH secretion. Thus, we generated powerful hybrid organoids by recapitulating hypothalamic-pituitary development, showing autonomous maturation on the basis of interactions between developing tissues.
Asunto(s)
Hipotálamo/fisiología , Células Madre Pluripotentes Inducidas/citología , Hipófisis/fisiología , Hormona Adrenocorticotrópica/metabolismo , Envejecimiento/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Corticotrofos/citología , Corticotrofos/ultraestructura , Humanos , Células Madre Pluripotentes Inducidas/ultraestructura , Ratones , Neuronas/citología , Organoides/citologíaRESUMEN
High differentiation efficiency is one of the most important factors in developing an in vitro model from pluripotent stem cells. In this report, we improved the handling technique applied to mouse-induced pluripotent stem (iPS) cells, resulting in better differentiation into hypothalamic vasopressin (AVP) neurons. We modified the culture procedure to make the maintenance of iPS cells in an undifferentiated state much easier. Three-dimensional floating culture was demonstrated to be effective for mouse iPS cells. We also improved the differentiation method with regards to embryology, resulting in a greater number of bigger colonies of AVP neurons differentiating from mouse iPS cells. Fgf8, which was not used in the original differentiation method, increased iPS differentiation into AVP neurons. These refinements will be useful as a valuable tool for the modeling of degenerative disease in AVP neurons in vitro using disease-specific iPS cells in future studies.
Asunto(s)
Diferenciación Celular , Línea Celular/citología , Hipotálamo/citología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/citología , Animales , Línea Celular/metabolismo , Células Cultivadas , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Hipotálamo/metabolismo , Células Madre Pluripotentes Inducidas/citología , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Vasopresinas/metabolismoRESUMEN
Tanycytes have recently been accepted as neural stem/progenitor cells in the postnatal hypothalamus. Persistent retina and anterior neural fold homeobox (Rax) expression is characteristic of tanycytes in contrast to its transient expression of whole hypothalamic precursors. In this study, we found that Rax+ residual cells in the maturation phase of hypothalamic differentiation in mouse embryonic stem cell (mESC) cultures had similar characteristics to ventral tanycytes. They expressed typical neural stem/progenitor cell markers, including Sox2, vimentin, and nestin, and differentiated into mature neurons and glial cells. Quantitative RT-PCR analysis showed that Rax+ residual cells expressed Fgf-10, Fgf-18, and Lhx2, which are expressed by ventral tanycytes. They highly expressed tanycyte-specific genes Dio2 and Gpr50 compared with Rax+ early hypothalamic progenitor cells. Therefore, Rax+ residual cells in the maturation phase of hypothalamic differentiation were considered to be more differentiated and similar to late progenitor cells and tanycytes. They self-renewed and formed neurospheres when cultured with exogenous FGF-2. Additionally, these Rax+ neurospheres differentiated into three neuronal lineages (neurons, astrocytes, and oligodendrocytes), including neuropeptide Y+ neuron, that are reported to be differentiated from ventral tanycytes toward the arcuate nuclei. Thus, Rax+ residual cells were multipotent neural stem/progenitor cells. Rax+ neurospheres were stably passaged and retained high Sox2 expression even after multiple passages. These results suggest the successful induction of Rax+ tanycyte-like cells from mESCs [induced tanycyte-like (iTan) cells]. These hypothalamic neural stem/progenitor cells may have potential in regenerative medicine and as a research tool.
Asunto(s)
Linaje de la Célula/fisiología , Células Madre Embrionarias/metabolismo , Células Ependimogliales/metabolismo , Hipotálamo/metabolismo , Células-Madre Neurales/metabolismo , Animales , Células Cultivadas , Células Madre Embrionarias/citología , Células Ependimogliales/citología , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Hipotálamo/citología , Proteínas con Homeodominio LIM/metabolismo , Ratones , Células-Madre Neurales/citología , Factores de Transcripción/metabolismoRESUMEN
Arginine-vasopressin (AVP) neurons exist in the hypothalamus, a major region of the diencephalon, and play an essential role in water balance. Here, we established the differentiation method for AVP-secreting neurons from human embryonic stem cells (hESCs) by recapitulating in vitro the in vivo embryonic developmental processes of AVP neurons. At first, the differentiation efficiency was improved. That was achieved through the optimization of the culture condition for obtaining dorsal hypothalamic progenitors. Secondly, the induced AVP neurons were identified by immunohistochemistry and these neurons secreted AVP after potassium chloride stimulation. Additionally, other hypothalamic neuropeptides were also detected, such as oxytocin, corticotropin-releasing hormone, thyrotropin-releasing hormone, pro-opiomelanocortin, agouti-related peptide, orexin, and melanin-concentrating hormone. This is the first report describing the generation of secretory AVP neurons derived from hESCs. This method will be applicable to research using disease models and, potentially, for regenerative medicine of the hypothalamus.
Asunto(s)
Arginina Vasopresina/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteína Relacionada con Agouti/metabolismo , Hormona Liberadora de Corticotropina/metabolismo , Humanos , Hormonas Hipotalámicas/metabolismo , Hipotálamo/citología , Hipotálamo/metabolismo , Inmunohistoquímica , Melaninas/metabolismo , Neurofisinas/metabolismo , Orexinas/metabolismo , Oxitocina/metabolismo , Hormonas Hipofisarias/metabolismo , Precursores de Proteínas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Vasopresinas/metabolismoRESUMEN
During embryonic development, oral ectoderm differentiates into the adenohypophysis, dental epithelia, salivary glands, and nasal pit. Few reports exist concerning the induction of oral ectoderm from embryonic stem (ES) cells. Generally, any lot differences in fetal bovine serum (FBS) and serum replacer may affect the induction of ES cell-differentiation. Using a previously established culture strategy for differentiation, the proportion of cell aggregates containing Pitx1+ oral ectoderm varied widely between 9-36% when several different lots of FBS or serum replacer were used. We therefore tried to enhance the differentiation method. We found that bone morphogenetic protein (BMP) 4 and fibroblast growth factor (FGF) treatments improved oral ectoderm induction. Such treatment also improved the differentiation of oral ectoderm into the adenohypophysis. Furthermore, increased BMP4 treatment induced dental epithelium and mesenchyme. Such differentiation suggests that the Pitx1+ layer displays similar properties to oral ectoderm, as found in vivo. Differentiation of ES cells into oral ectoderm using different lots of FBS and serum replacer increased 78-90% after treatment with BMP4 and FGF. In summary, we have established a robust strategy for the induction of oral ectoderm differentiation from mouse ES cells.
Asunto(s)
Proteína Morfogenética Ósea 4/farmacología , Diferenciación Celular/efectos de los fármacos , Factores de Crecimiento de Fibroblastos/farmacología , Animales , Ectodermo/citología , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción Paired Box/genética , Factores de Transcripción Paired Box/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factor de Transcripción Pit-1/genética , Factor de Transcripción Pit-1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína del Homeodomínio PITX2RESUMEN
Cell migration is a critical cellular process that determines embryonic development and the progression of human diseases. Therefore, cell- or context-specific mechanisms by which multiple promigratory proteins differentially regulate cell migration must be analyzed in detail. Girdin (girders of actin filaments) (also termed GIV, Gα-interacting vesicle associated protein) is an actin-binding protein that regulates migration of various cells such as endothelial cells, smooth muscle cells, neuroblasts, and cancer cells. Here we show that Girdin regulates the establishment of cell polarity, the deregulation of which may result in the disruption of directional cell migration. We found that Girdin interacts with Par-3, a scaffolding protein that is a component of the Par protein complex that has an established role in determining cell polarity. RNA interference-mediated depletion of Girdin leads to impaired polarization of fibroblasts and mammary epithelial cells in a way similar to that observed in Par-3-depleted cells. Accordingly, the expression of Par-3 mutants unable to interact with Girdin abrogates cell polarization in fibroblasts. Further biochemical analysis suggests that Girdin is present in the Par protein complex that includes Par-3, Par-6, and atypical protein kinase C. Considering previous reports showing the role of Girdin in the directional migration of neuroblasts, network formation of endothelial cells, and cancer invasion, these data may provide a specific mechanism by which Girdin regulates cell movement in biological contexts that require directional cell movement.
Asunto(s)
Movimiento Celular , Polaridad Celular , Proteínas de Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , División Celular , Línea Celular , Citoplasma/metabolismo , Regulación hacia Abajo , Fibroblastos/citología , Técnicas de Inactivación de Genes , Humanos , Glándulas Mamarias Humanas/citología , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Neuronas/citología , Estructura Terciaria de Proteína , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/deficiencia , Proteínas de Transporte Vesicular/genéticaRESUMEN
Disrupted-In-Schizophrenia 1 (DISC1) is a promising candidate gene for susceptibility to psychiatric disorders, including schizophrenia. DISC1 appears to be involved in neurogenesis, neuronal migration, axon/dendrite formation and synapse formation; during these processes, DISC1 acts as a scaffold protein by interacting with various partners. However, the lack of Disc1 knockout mice and a well-characterized antibody to DISC1 has made it difficult to determine the exact role of DISC1 in vivo. In this study, we generated mice lacking exons 2 and 3 of the Disc1 gene and prepared specific antibodies to the N- and C-termini of DISC1. The Disc1 mutant mice are viable and fertile, and no gross phenotypes, such as disorganization of the brain's cytoarchitecture, were observed. Western blot analysis revealed that the DISC1-specific antibodies recognize a protein with an apparent molecular mass of ~100 kDa in brain extracts from wild-type mice but not in brain extracts from DISC1 mutant mice. Immunochemical studies demonstrated that DISC1 is mainly localized to the vicinity of the Golgi apparatus in hippocampal neurons and astrocytes. A deficiency of full-length Disc1 induced a threshold shift in the induction of long-term potentiation in the dentate gyrus. The Disc1 mutant mice displayed abnormal emotional behavior as assessed by the elevated plus-maze and cliff-avoidance tests, thereby suggesting that a deficiency of full-length DISC1 may result in lower anxiety and/or higher impulsivity. Based on these results, we suggest that full-length Disc1-deficient mice and DISC1-specific antibodies are powerful tools for dissecting the pathophysiological functions of DISC1.
Asunto(s)
Conducta Animal , Exones/genética , Marcación de Gen , Proteínas del Tejido Nervioso/genética , Envejecimiento/efectos de los fármacos , Envejecimiento/patología , Aminas/metabolismo , Animales , Anticuerpos/inmunología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Conducta Animal/efectos de los fármacos , Clozapina/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/ultraestructura , Inmunohistoquímica , Aprendizaje por Laberinto/efectos de los fármacos , Ratones , Ratones Endogámicos , Proteínas del Tejido Nervioso/inmunología , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Transmisión Sináptica/efectos de los fármacos , Factores de TiempoRESUMEN
We conducted field surveys at 25 sites in three Japanese catchments to provide conservative estimates of the safe concentration of zinc (Zn) for the protection of riverine macroinvertebrate diversity. The relationships between the Zn concentration and six macroinvertebrate metrics for taxon richness were determined by using regression analysis; this included a piecewise regression model, where two lines are joined at an unknown point. For each metric the piecewise regression model with a zero slope below a threshold concentration was selected as the best model to explain the influence of Zn. Under the assumption that macroinvertebrate diversity reductions of <10% are acceptable, the safe concentrations of Zn were estimated to be 84, 115, 84, 80, 85, and 70 µg/L for total taxon richness, Ephemeroptera, Plecoptera, and Trichoptera (EPT) richness, mayfly richness, caddisfly richness, chironomid richness, and estimated total taxon richness at the riffle scale, respectively. These concentrations are more than twice the water quality standard for Zn in Japan (30 µg/L), suggesting that the standard is likely overprotective for macroinvertebrate diversity. Field studies are useful for evaluating the level of protectiveness of safe concentrations (water quality standards) based on individual-level effects from laboratory toxicity tests, and this evaluation process will have a crucial role in implementing more purpose-driven ecological risk managements that aim to protect natural populations and communities.
Asunto(s)
Biodiversidad , Invertebrados/efectos de los fármacos , Contaminantes Químicos del Agua/farmacología , Zinc/farmacología , Animales , Monitoreo del Ambiente/métodos , Japón , Análisis de Componente Principal , Análisis de Regresión , Ríos/química , Calidad del AguaRESUMEN
In postnatally developing and adult brains, interneurons of the olfactory bulb (OB) are continuously generated at the subventricular zone of the forebrain. The newborn neuroblasts migrate tangentially to the OB through a well defined pathway, the rostral migratory stream (RMS), where the neuroblasts undergo collective migration termed "chain migration." The cell-intrinsic regulatory mechanism of neuroblast chain migration, however, has not been uncovered. Here we show that mice lacking the actin-binding Akt substrate Girdin (a protein that interacts with Disrupted-In-Schizophrenia 1 to regulate neurogenesis in the dentate gyrus) have profound defects in neuroblast chain migration along the RMS. Analysis of two gene knock-in mice harboring Girdin mutants identified unique amino acid residues in Girdin's C-terminal domain that are responsible for the regulation of neuroblast chain migration but revealed no apparent requirement of Girdin phosphorylation by Akt. Electron microscopic analyses demonstrated the involvement of Girdin in neuroblast cell-cell interactions. These findings suggest that Girdin is an important intrinsic factor that specifically governs neuroblast chain migration along the RMS.
Asunto(s)
Encéfalo/fisiología , Movimiento Celular/fisiología , Proteínas de Microfilamentos/fisiología , Células-Madre Neurales/fisiología , Bulbo Olfatorio/fisiología , Proteínas de Transporte Vesicular/fisiología , Animales , Encéfalo/anatomía & histología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Encéfalo/ultraestructura , Movimiento Celular/genética , Células Cultivadas , Técnicas de Sustitución del Gen/métodos , Uniones Intercelulares/genética , Uniones Intercelulares/ultraestructura , Interneuronas/metabolismo , Interneuronas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/ultraestructura , Bulbo Olfatorio/anatomía & histología , Bulbo Olfatorio/crecimiento & desarrollo , Bulbo Olfatorio/metabolismo , Bulbo Olfatorio/ultraestructura , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
RATIONALE: It is well established that the migration and proliferation of vascular smooth muscle cells (VSMCs) have major roles in the vascular remodeling process. Our previous study showed that the Akt substrate Girdin, which is expressed in VSMCs and endothelial cells, is essential for postnatal angiogenesis. However, the function of Girdin and its Akt-mediated phosphorylation in VSMCs and their in vivo roles in vascular remodeling remain to be elucidated. OBJECTIVE: We investigated the function of Girdin and its Akt-mediated phosphorylation using cultured VSMCs and animal models of vascular remodeling. METHODS AND RESULTS: The depletion of Girdin by RNA interference disrupted the rearrangement of the actin cytoskeleton in VSMCs, resulting in impaired cell migration. The depletion of Girdin also inhibited VSMC proliferation. Girdin expression was highly upregulated and its serine at position 1416 was phosphorylated in the neointima of carotid arteries after balloon injury in a rat model. The introduction of an adenovirus harboring short hairpin RNA against Girdin attenuated the proliferation of VSMCs and neointima formation without affecting reendothelialization. Furthermore, we found that neointima formation after femoral wire injury was significantly attenuated in Girdin S1416A knock-in mice, in which the Akt phosphorylation site of Girdin was mutated, thus indicating a major role for Girdin phosphorylation in vascular remodeling. CONCLUSIONS: These findings indicate that Girdin and its Akt-mediated phosphorylation have major roles in the migration and proliferation of VSMCs and vascular remodeling, making the Akt/Girdin signaling pathway a potential target for the development of new therapeutics for vascular diseases.