RESUMEN
In the adult mammalian brain, astrocytes are proposed to be the major Sonic Hedgehog (Shh)-responsive cells. However, the sources of the Shh molecule mediating activation of the pathway are still poorly characterized. The present work investigates the distribution and phenotype of cells expressing Shh mRNA in the adult mouse brain. Using single-molecule fluorescent in situ hybridization (smfISH), we report much broader expression of Shh transcripts in almost all brain regions than originally reported. We identify Shh mRNA in HuC/D+ neuronal populations, including GABAergic (glutamic acid decarboxylase 67, Gad67), cholinergic (choline acetyltransferase, ChAT), dopaminergic (tyrosine hydroxylase, TH), nitrergic (neuronal nitric oxide synthase, nNOS), and in a small population of oligodendroglial cells expressing Sox10 and Olig2 mRNA transcription factors. Further analysis of Shh mRNA in cerebral cortical and hypothalamic neurons suggests that Shh is also expressed by glutamatergic neurons. Interestingly, we did not observe substantial Desert Hedgehog and Indian Hedgehog mRNA signals, nor Shh signals in S100ß+ astrocytes and Iba1+ microglial cells. Collectively, the present work provides the most robust central map of Shh-expressing cells to date and underscores the importance of nitrergic neurons in regulating Shh availability to brain cells. Thus, our study provides a framework for future experiments aimed at better understanding of the functions of Shh signaling in the brain in normal and pathological states, and the characterization of novel regulatory mechanisms of the signaling pathway.
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Proteínas Hedgehog , Neuronas , Ratones , Animales , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Hibridación Fluorescente in Situ , Neuronas/metabolismo , Encéfalo/metabolismo , ARN Mensajero/metabolismo , MamíferosRESUMEN
OBJECTIVE: Astrocytes are glial cells proposed as the main Sonic hedgehog (Shh)-responsive cells in the adult brain. Their roles in mediating Shh functions are still poorly understood. In the hypothalamus, astrocytes support neuronal circuits implicated in the regulation of energy metabolism. In this study, we investigated the impact of genetic activation of Shh signaling on hypothalamic astrocytes and characterized its effects on energy metabolism. METHODS: We analyzed the distribution of gene transcripts of the Shh pathway (Ptc, Gli1, Gli2, and Gli3) in astrocytes using single molecule fluorescence in situ hybridization combined with immunohistofluorescence of Shh peptides by Western blotting in the adult mouse hypothalamus. Based on the metabolic phenotype, we characterized Glast-CreERT2-YFP-Ptc-/- (YFP-Ptc-/-) mice and their controls over time and under a high-fat diet (HFD) to investigate the potential effects of conditional astrocytic deletion of the Shh receptor Patched (Ptc) on metabolic efficiency, insulin sensitivity, and systemic glucose metabolism. Molecular and biochemical assays were used to analyze the alteration of key pathways modulating energy metabolism, insulin sensitivity, glucose uptake, and inflammation. Primary astrocyte cultures were used to evaluate a potential role of Shh signaling in astrocytic glucose uptake. RESULTS: Shh peptides were the highest in the hypothalamic extracts of adult mice and a large population of hypothalamic astrocytes expressed Ptc and Gli1-3 mRNAs. Characterization of Shh signaling after conditional Ptc deletion in the YFP-Ptc-/- mice revealed heterogeneity in hypothalamic astrocyte populations. Interestingly, activation of Shh signaling in Glast+ astrocytes enhanced insulin responsiveness as evidenced by glucose and insulin tolerance tests. This effect was maintained over time and associated with lower blood insulin levels and also observed under a HFD. The YFP-Ptc-/- mice exhibited a lean phenotype with the absence of body weight gain and a marked reduction of white and brown adipose tissues accompanied by increased whole-body fatty acid oxidation. In contrast, food intake, locomotor activity, and body temperature were not altered. At the cellular level, Ptc deletion did not affect glucose uptake in primary astrocyte cultures. In the hypothalamus, activation of the astrocytic Shh pathway was associated with the upregulation of transcripts coding for the insulin receptor and liver kinase B1 (LKB1) after 4 weeks and the glucose transporter GLUT-4 after 32 weeks. CONCLUSIONS: Here, we define hypothalamic Shh action on astrocytes as a novel master regulator of energy metabolism. In the hypothalamus, astrocytic Shh signaling could be critically involved in preventing both aging- and obesity-related metabolic disorders.
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Astrocitos/metabolismo , Glucosa/metabolismo , Proteínas Hedgehog/metabolismo , Receptores Patched/metabolismo , Envejecimiento , Animales , Astrocitos/patología , Metabolismo Energético/genética , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Hipotálamo/metabolismo , Hipotálamo/patología , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Neuronas/metabolismo , Obesidad , Receptores Patched/deficiencia , Receptores Patched/genética , Transducción de Señal , Activación TranscripcionalRESUMEN
The regeneration of myelin is known to restore axonal conduction velocity after a demyelinating event. Remyelination failure in the central nervous system contributes to the severity and progression of demyelinating diseases such as multiple sclerosis. Remyelination is controlled by many signaling pathways, such as the Sonic hedgehog (Shh) pathway, as shown by the canonical activation of its key effector Smoothened (Smo), which increases the proliferation of oligodendrocyte precursor cells via the upregulation of the transcription factor Gli1. On the other hand, the inhibition of Gli1 was also found to promote the recruitment of a subset of adult neural stem cells and their subsequent differentiation into oligodendrocytes. Since Smo is also able to transduce Shh signals via various non-canonical pathways such as the blockade of Gli1, we addressed the potential of non-canonical Smo signaling to contribute to oligodendroglial cell maturation in myelinating cells using the non-canonical Smo agonist GSA-10, which downregulates Gli1. Using the Oli-neuM cell line, we show that GSA-10 promotes Gli2 upregulation, MBP and MAL/OPALIN expression via Smo/AMP-activated Protein Kinase (AMPK) signaling, and efficiently increases the number of axonal contact/ensheathment for each oligodendroglial cell. Moreover, GSA-10 promotes the recruitment and differentiation of oligodendroglial progenitors into the demyelinated corpus callosum in vivo. Altogether, our data indicate that non-canonical signaling involving Smo/AMPK modulation and Gli1 downregulation promotes oligodendroglia maturation until axon engagement. Thus, GSA-10, by activation of this signaling pathway, represents a novel potential remyelinating agent.
RESUMEN
In the mature rodent brain, Sonic Hedgehog (Shh) signaling regulates stem and progenitor cell maintenance, neuronal and glial circuitry and brain repair. However, the sources and distribution of Shh mediating these effects are still poorly characterized. Here, we report in the adult mouse brain, a broad expression pattern of Shh recognized by the specific monoclonal C9C5 antibody in a subset (11-12%) of CC1+ mature oligodendrocytes that do not express carbonic anhydrase II. These cells express also Olig2 and Sox10, two oligodendrocyte lineage-specific markers, but not PDGFRα, a marker of oligodendrocyte progenitors. In agreement with oligodendroglial cells being a source of Shh in the adult mouse brain, we identify Shh transcripts by single molecule fluorescent in situ hybridization in a subset of cells expressing Olig2 and Sox10 mRNAs. These findings also reveal that Shh expression is more extensive than originally reported. The Shh-C9C5-associated signal labels the oligodendroglial cell body and decorates by intense puncta the processes. C9C5+ cells are distributed in a grid-like manner. They constitute small units that could deliver locally Shh to its receptor Patched expressed in GFAP+ and S100ß+ astrocytes, and in HuC/D+ neurons as shown in PtcLacZ/+ reporter mice. Postnatally, C9C5 immunoreactivity overlaps the myelination peak that occurs between P10 and P20 and is down regulated during ageing. Thus, our data suggest that C9C5+CC1+ oligodendroglial cells are a source of Shh in the mouse postnatal brain.
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Anticuerpos Monoclonales/inmunología , Encéfalo/metabolismo , Proteínas Hedgehog/inmunología , Proteínas Hedgehog/metabolismo , Neuronas/metabolismo , Oligodendroglía/metabolismo , Animales , Encéfalo/inmunología , Células Cultivadas , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/inmunología , Oligodendroglía/inmunología , Receptores Patched/inmunología , Receptores Patched/metabolismoRESUMEN
During cerebellar development, granule neuron progenitors (GNPs) proliferate by transducing Sonic Hedgehog (SHH) signaling via the primary cilium. Precise regulation of ciliogenesis, thus, ensures proper GNP pool expansion. Here, we report that Atoh1, a transcription factor required for GNPs formation, controls the presence of primary cilia, maintaining GNPs responsiveness to SHH. Loss of primary cilia abolishes the ability of Atoh1 to keep GNPs in a proliferative state. Mechanistically, Atoh1 promotes ciliogenesis by transcriptionally regulating Cep131, which facilitates centriolar satellite (CS) clustering to the basal body. Importantly, ectopic expression of Cep131 counteracts the effects of Atoh1 loss in GNPs by restoring proper localization of CS and ciliogenesis. This Atoh1-CS-primary cilium-SHH pro-proliferative pathway is also conserved in SHH-type medulloblastoma, a pediatric brain tumor arising from the GNPs. Together, our data reveal how Atoh1 modulates the primary cilium to regulate GNPs development.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Neuronas/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Neoplasias Cerebelosas/metabolismo , Meduloblastoma/metabolismo , Ratones Transgénicos , NeurogénesisRESUMEN
Neural stem cells (NSCs) enter quiescence in early embryonic stages to create a reservoir of dormant NSCs able to enter proliferation and produce neuronal precursors in the adult mammalian brain. Various approaches of fluorescent-activated cell sorting (FACS) have emerged to allow the distinction between quiescent NSCs (qNSCs), their activated counterpart (aNSCs), and the resulting progeny. In this article, we review two FACS techniques that can be used alternatively. We also show that their association with transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice allows an unprecedented overlook on the cell cycle dynamics of adult NSCs.
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Encéfalo/citología , Ciclo Celular , Separación Celular/métodos , Citometría de Flujo/métodos , Microscopía Fluorescente/métodos , Células-Madre Neurales/citología , Animales , Encéfalo/fisiología , Proliferación Celular , Células Cultivadas , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Células-Madre Neurales/fisiologíaRESUMEN
Identifying the mechanisms controlling quiescence and activation of neural stem cells (NSCs) is crucial for understanding brain repair. Here, we demonstrate that Hedgehog (Hh) signaling actively regulates different pools of quiescent and proliferative NSCs in the adult ventricular-subventricular zone (V-SVZ), one of the main brain neurogenic niches. Specific deletion of the Hh receptor Patched in NSCs during adulthood upregulated Hh signaling in quiescent NSCs, progressively leading to a large accumulation of these cells in the V-SVZ. The pool of non-neurogenic astrocytes was not modified, whereas the activated NSC pool increased after a short period, before progressively becoming exhausted. We also showed that Sonic Hedgehog regulates proliferation of activated NSCs in vivo and shortens both their G1 and S-G2/M phases in culture. These data demonstrate that Hh orchestrates the balance between quiescent and activated NSCs, with important implications for understanding adult neurogenesis under normal homeostatic conditions or during injury.
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Proteínas Hedgehog/metabolismo , Ventrículos Laterales/citología , Ventrículos Laterales/metabolismo , Células-Madre Neurales/metabolismo , Fase de Descanso del Ciclo Celular , Transducción de Señal , Animales , Ciclo Celular , Eliminación de Gen , Ratones , Ratones Noqueados , Ratones Transgénicos , Neurogénesis , Neuronas , Receptores Patched/genética , Nicho de Células MadreRESUMEN
Smoothened (Smo) is the signal transducer of the Hedgehog (Hh) pathway and its stimulation is considered a potential powerful tool in regenerative medicine to treat severe tissue injuries. Starting from GSA-10, a recently reported Hh activator acting on Smo, we have designed and synthesized a new class of quinolone-based compounds. Modification and decoration of three different portions of the original scaffold led to compounds able to induce differentiation of multipotent mesenchymal cells into osteoblasts. The submicromolar activity of several of these new quinolones (0.4-0.9 µM) is comparable to or better than that of SAG and purmorphamine, two reference Smo agonists. Structure-activity relationships allow identification of several molecular determinants important for the activity of these compounds.
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Diseño de Fármacos , Osteogénesis/efectos de los fármacos , Quinolonas/química , Quinolonas/farmacología , Animales , Técnicas de Química Sintética , Evaluación Preclínica de Medicamentos , Proteínas Hedgehog/metabolismo , Ratones , Modelos Moleculares , Células 3T3 NIH , Quinolonas/síntesis química , Relación Estructura-ActividadRESUMEN
The most relevant therapeutic approaches to treat CML rely on the administration of tyrosine kinase inhibitors (TKIs) like Imatinib, which are able to counteract the activity of Bcr-Abl protein increasing patient's life expectancy and survival. Unfortunately, there are some issues TKIs are not able to address; first of all TKIs are not so effective in increasing survival of patients in blast crisis, second they are not able to eradicate leukemic stem cells (LSC) which represent the major cause of disease relapse, and third patients often develop resistance to TKIs due to mutations in the drug binding site. For all these reasons it's of primary interest to find alternative strategies to treat CML. Literature shows that Hedgehog signaling pathway is involved in LSC maintenance, and pharmacological inhibition of Smoothened (SMO), one of the key molecules of the pathway, has been demonstrated to reduce Bcr-Abl positive bone marrow cells and LSC. Consequently, targeting SMO could be a promising way to develop a new treatment strategy for CML overcoming the limitations of current therapies. In our work we have tested some compounds able to inhibit SMO, and among them MRT92 appears to be a very potent SMO antagonist. We found that almost all our compounds were able to reduce Gli1 protein levels in K-562 and in KU-812 CML cell lines. Furthermore, they were also able to increase Gli1 and SMO RNA levels, and to reduce cell proliferation and induce apoptosis/autophagy in both the tested cell lines. Finally, we demonstrated that our compounds were able to modulate the expression of some miRNAs related to Hedgehog pathway such as miR-324-5p and miR-326. Being Hedgehog pathway deeply implicated in the mechanisms of CML we may conclude that it could be a good therapeutic target for CML and our compounds seem to be promising antagonists of such pathway.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Autofagia/efectos de los fármacos , Crisis Blástica/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Mesilato de Imatinib/farmacología , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , MicroARNs/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor SmoothenedRESUMEN
Hedgehog (Hh) is a critical regulator of adipogenesis. Extracellular vesicles are natural Hh carriers, as illustrated by activated/apoptotic lymphocytes specifically shedding microparticles (MP) bearing the morphogen (MP(Hh+)). We show that MP(Hh+) inhibit adipocyte differentiation and orientate mesenchymal stem cells towards a pro-osteogenic program. Despite a Smoothened (Smo)-dependency, MP(Hh+) anti-adipogenic effects do not activate a canonical Hh signalling pathway in contrast to those elicited either by the Smo agonist SAG or recombinant Sonic Hedgehog. The Smo agonist GSA-10 recapitulates many of the hallmarks of MP(Hh+) anti-adipogenic effects. The adipogenesis blockade induced by MP(Hh+) and GSA-10 was abolished by the Smo antagonist LDE225. We further elucidate a Smo/Lkb1/Ampk axis as the non-canonical Hh pathway used by MP(Hh+) and GSA-10 to inhibit adipocyte differentiation. Our results highlight for the first time the ability of Hh-enriched MP to signal via a non-canonical pathway opening new perspectives to modulate fat development.
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Adipocitos/citología , Diferenciación Celular/fisiología , Proteínas Hedgehog/fisiología , Células 3T3-L1 , Animales , Células Cultivadas , Proteínas Hedgehog/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Factores de Transcripción/metabolismoRESUMEN
We report the first comprehensive structure-activity study of calindol (4, (R)-N-[(1H-indol-2-yl)methyl]-1-(1-naphthyl)ethanamine), a positive allosteric modulator, or calcimimetic, of the calcium sensing receptor (CaSR). While replacement of the naphthyl moiety of calindol by other aromatic groups (phenyl, biphenyl) was largely detrimental to calcimimetic activity, incorporation of substituents on the 4, 5 or 7 position of the indole portion of calindol was found to provide either equipotent derivatives compared to calindol (e.g., 4-phenyl, 4-hydroxy, 5-hydroxycalindol 44, 52, 53) or, in the case of 7-nitrocalindol (51), a 6-fold more active calcimimetic displaying an EC50 of 20nM. Unlike calindol, the more active CaSR calcimimetics were shown not to act as antagonists of the closely related GPRC6A receptor, suggesting a more selective profile for these new analogues.
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Diseño de Fármacos , Indoles/farmacología , Naftalenos/farmacología , Receptores Sensibles al Calcio/agonistas , Relación Dosis-Respuesta a Droga , Humanos , Indoles/síntesis química , Indoles/química , Estructura Molecular , Naftalenos/síntesis química , Naftalenos/química , Relación Estructura-ActividadRESUMEN
Basal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated ß-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings suggest that defects in dermo/epidermal interactions could contribute to BCC susceptibility in NBCCS patients.
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Síndrome del Nevo Basocelular/patología , Carcinoma Basocelular/patología , Fibroblastos/citología , Receptores de Superficie Celular/genética , Neoplasias Cutáneas/patología , Microambiente Tumoral , Síndrome del Nevo Basocelular/genética , Síndrome del Nevo Basocelular/metabolismo , Carcinoma Basocelular/etiología , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados/farmacología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Mutación , Técnicas de Cultivo de Órganos , Receptores Patched , Receptor Patched-1 , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.
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Citometría de Flujo/métodos , Ventrículos Laterales/citología , Células-Madre Neurales/citología , Animales , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Ratones , Ratones Transgénicos , Neurogénesis/fisiología , Neuronas/citología , Bulbo OlfatorioRESUMEN
The Smoothened (Smo) receptor, a member of class F G protein-coupled receptors, is the main transducer of the Hedgehog (Hh) signaling pathway implicated in a wide range of developmental and adult processes. Smo is the target of anticancer drugs that bind to a long and narrow cavity in the 7-transmembrane (7TM) domain. X-ray structures of human Smo (hSmo) bound to several ligands have revealed 2 types of 7TM-directed antagonists: those binding mostly to extracellular loops (site 1, e.g., LY2940680) and those penetrating deeply in the 7TM cavity (site 2, e.g., SANT-1). Here we report the development of the acylguanidine MRT-92, which displays subnanomolar antagonist activity against Smo in various Hh cell-based assays. MRT-92 inhibits rodent cerebellar granule cell proliferation induced by Hh pathway activation through pharmacologic (half maximal inhibitory concentration [IC50] = 0.4 nM) or genetic manipulation. Using [(3)H]MRT-92 (Kd = 0.3 nM for hSmo), we created a comprehensive framework for the interaction of small molecule modulators with hSmo and for understanding chemoresistance linked to hSmo mutations. Guided by molecular docking and site-directed mutagenesis data, our work convincingly confirms that MRT-92 simultaneously recognized and occupied both sites 1 and 2. Our data demonstrate the existence of a third type of Smo antagonists, those entirely filling the Smo binding cavity from the upper extracellular part to the lower cytoplasmic-proximal subpocket. Our studies should help design novel potent Smo antagonists and more effective therapeutic strategies for treating Hh-linked cancers and associated chemoresistance.
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Antineoplásicos/farmacología , Membrana Celular/metabolismo , Neoplasias Cerebelosas/metabolismo , Guanidinas/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Meduloblastoma/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Adulto , Animales , Sitios de Unión , Western Blotting , Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Proteínas Hedgehog/metabolismo , Humanos , Técnicas para Inmunoenzimas , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Ratones , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Unión Proteica , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor SmoothenedRESUMEN
In the adult brain, self-renewal is essential for the persistence of neural stem cells (NSCs) throughout life, but its regulation is still poorly understood. One NSC can give birth to two NSCs or one NSC and one transient progenitor. A correct balance is necessary for the maintenance of germinal areas, and understanding the molecular mechanisms underlying NSC division mode is clearly important. Here, we report a function of the Sonic Hedgehog (SHH) receptor Patched in the direct control of long-term NSC self-renewal in the subependymal zone. We show that genetic conditional activation of SHH signaling in adult NSCs leads to their expansion and the depletion of their direct progeny. These phenotypes are associated in vitro with an increase in NSC symmetric division in a process involving NOTCH signaling. Together, our results demonstrate a tight control of adult neurogenesis and NSC renewal driven by Patched.
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Proteínas Hedgehog/metabolismo , Células-Madre Neurales/citología , Sistema de Transporte de Aminoácidos X-AG/genética , Animales , Encéfalo/metabolismo , Proliferación Celular/efectos de los fármacos , Proteínas Hedgehog/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/metabolismo , Neurogénesis , Receptores Patched , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Notch/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Transducción de Señal , Tamoxifeno/farmacología , Regulación hacia Arriba , Proteína con Dedos de Zinc GLI1Asunto(s)
División Celular/fisiología , Proteínas Hedgehog/fisiología , Células-Madre Neurales/fisiología , Adulto , Células Madre Adultas/fisiología , Animales , División Celular Asimétrica/fisiología , Proliferación Celular , Humanos , Receptores Patched , Receptores de Superficie Celular/fisiologíaRESUMEN
The Smoothened (Smo) receptor is a key transducer of the Hedgehog (Hh) signaling pathway, which plays a critical role in tissue maintenance and repair. Recent studies have highlighted the therapeutic value of Smo antagonists for treating Hh-linked cancers. Research on Smo agonists indicates that these molecules are important not only for delineating canonical versus noncanonical Hh signaling but also for understanding the role of Smo in physiological and pathological conditions. This review provides an update on the potential therapeutic importance of Smo modulators, and unravels the increasing complexity of its pharmacology with regard to clinical implications.
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Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Proteínas Hedgehog/metabolismo , Humanos , Ligandos , Terapia Molecular Dirigida , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Receptor SmoothenedRESUMEN
OBJECTIVE: The purpose of this study is to further document alteration of signal transduction pathways, more particularly of hedgehog (Hh) signaling, causing impaired ischemic muscle repair in old mice. APPROACH AND RESULTS: We used 12-week-old (young mice) and 20- to 24-month-old C57BL/6 mice (old mice) to investigate the activity of Hh signaling in the setting of hindlimb ischemia-induced angiogenesis and skeletal muscle repair. In this model, delayed ischemic muscle repair observed in old mice was associated with an impaired upregulation of Gli1. Sonic Hh expression was not different in old mice compared with young mice, whereas desert Hh (Dhh) expression was downregulated in the skeletal muscle of old mice both in healthy and ischemic conditions. The rescue of Dhh expression by gene therapy in old mice promoted ischemia-induced angiogenesis and increased nerve density; nevertheless, it failed to promote myogenesis or to increase Gli1 mRNA expression. After further investigation, we found that, in addition to Dhh, smoothened expression was significantly downregulated in old mice. We used smoothened haploinsufficient mice to demonstrate that smoothened knockdown by 50% is sufficient to impair activation of Hh signaling and ischemia-induced muscle repair. CONCLUSIONS: The present study demonstrates that Hh signaling is impaired in aged mice because of Dhh and smoothened downregulation. Moreover, it shows that hegdehog-dependent regulation of angiogenesis and myogenesis involves distinct mechanisms.
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Envejecimiento/metabolismo , Proteínas Hedgehog/metabolismo , Isquemia/metabolismo , Desarrollo de Músculos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/metabolismo , Neovascularización Fisiológica , Factores de Edad , Envejecimiento/genética , Animales , Células COS , Chlorocebus aethiops , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Terapia Genética , Proteínas Hedgehog/genética , Miembro Posterior , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Isquemia/genética , Isquemia/patología , Isquemia/fisiopatología , Isquemia/terapia , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/inervación , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regeneración , Transducción de Señal , Receptor Smoothened , Transfección , Proteína con Dedos de Zinc GLI1RESUMEN
The Smoothened (Smo) receptor is a major component involved in signal transduction of the Hedgehog (Hh) morphogens both during embryogenesis and in the adult. Smo antagonists represent a promi-sing alternative for the treatment of cancers linked to abnormal Hh signalling. The crystal structure of the human Smo receptor bound to an antitumour agent demonstrates that this receptor belongs to the superfamily of G-protein coupled receptors. The antagonist binds to a pocket localized at the extracellular side formed by the seven transmembrane domains and the complex arrangement of the unusually long extracellular loops. The structure of the Smo receptor will promote the development of small molecules interacting with a key therapeutic target with interests in regenerative medicine and cancer.
Asunto(s)
Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/química , Adulto , Antineoplásicos/uso terapéutico , Ensayos Clínicos como Asunto , Cristalografía , Humanos , Modelos Moleculares , Terapia Molecular Dirigida , Neoplasias/genética , Neoplasias/terapia , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/genética , Receptor SmoothenedRESUMEN
The transgenic plp-GFP mouse line expressing the green fluorescent protein (GFP) driven by the mouse myelin proteolipid protein (plp) gene promoter has been previously used to study the contribution of the plp lineage to oligodendrocyte development in the embryonic brain. Here, we show that the GFP fluorescence reflects the developmental expression of proteolipid protein during the postnatal development until adulthood in brain slices and in primary cultures of plp-GFP(+) cells derived from postnatal animals. In the adult brain, plp-GFP-expressing cells are mature oligodendrocytes but not oligodendroglial progenitors. In the model of focal demyelination induced by lysolecithin (LPC) in the corpus callosum of adult plp-GFP animals, we observed an up-regulation of the morphogen Sonic Hedgehog (Shh) in the LPC-induced lesion but not in the control animals. Moreover, we show that the adenovirus-mediated transfer of Shh in the lesion results in the attenuation of the demyelination extent as evidenced by GFP fluorescence analysis in Shh-treated and control animals. Altogether these data show how plp-GFP fluorescence can be monitored to follow the oligodendrocyte lineage during demyelination and identify Shh morphogen as an important factor during repair.