RESUMEN
Intracranial hemorrhage in preterm neonates may result in neonatal mortality and functional disabilities, but its pathogenic mechanisms are poorly defined and better therapies are needed. We used a tetracycline-regulated transgenic system to test whether the induction of vascular endothelial growth factor (VEGF) in the germinal matrix leads to intracranial hemorrhage. This genetic strategy initially induced a dense network of loosely adjoined endothelial cells and pericytes near lateral ventricles, similar to the immature vascular rete in human fetal brains. Yet, this rich vascular network transformed into low-vasculature patches correlated with hemorrhage and caspase-3 activation near birth. Gene expression and biochemical analyses suggested that downstream mediators of VEGF in this network include transcriptional factors ETS1 and HIF2α (hypoxia-inducible factor 2α), components of the PDGFß (platelet-derived growth factor ß) and TGFß (transforming growth factor-ß) receptor signaling pathways, matrix metalloproteinase-9 (MMP-9), and cathepsins. Prenatal administration of glucocorticoids markedly reduced mortality and cerebral hemorrhage in mutant animals, as in human neonates. This protective effect was not due to blocking vasculogenesis, but was instead associated with inhibition of neurovascular proteases, notably MMP-9, cathepsin B, and caspase-3. Collectively, these results support a causative role of VEGF in perinatal cerebral hemorrhage and implicate its downstream proteases as potential therapeutic targets.
Asunto(s)
Hemorragia Cerebral/enzimología , Hemorragia Cerebral/patología , Péptido Hidrolasas/biosíntesis , Prosencéfalo/enzimología , Prosencéfalo/patología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Recién Nacidos , Betametasona/farmacología , Betametasona/uso terapéutico , Caspasa 3/metabolismo , Catepsina B/metabolismo , Hemorragia Cerebral/tratamiento farmacológico , Hemorragia Cerebral/genética , Modelos Animales de Enfermedad , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/patología , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Perfilación de la Expresión Génica , Glucocorticoides/farmacología , Glucocorticoides/uso terapéutico , Humanos , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Neovascularización Patológica/tratamiento farmacológico , Fenotipo , Prosencéfalo/irrigación sanguínea , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/uso terapéutico , Tetraciclina/farmacologíaRESUMEN
BACKGROUND: Several studies have indicated that Sonic hedgehog (Shh) regulates the expansion of dopaminergic (DA) progenitors and the subsequent generation of mature DA neurons. This prevailing view has been based primarily on in vitro culture results, and the exact in vivo function of Shh signaling in the patterning and neurogenesis of the ventral midbrain (vMB) remains unclear. METHODS: We characterized the transcriptional codes for the vMB progenitor domains, and correlated them with the expression patterns of Shh signaling effectors, including Shh, Smoothened, Patched, Gli1, Gli2 and Gli3. RESULTS: While Shh and its downstream effectors showed robust expression in the neurogenic niche for DA progenitors at embryonic day (E)8 to E8.5, their expression shifted to the lateral domains from E9.5 to E12.5. Consistent with this dynamic change, conditional mutants with region-specific removal of the Shh receptor Smoothened in the vMB progenitors (Shh-Cre;Smo(fl/fl)) showed a transient reduction in DA progenitors and DA neurons at E10.5, but had more profound defects in neurons derived from the more lateral domains, including those in the red nucleus, oculomotor nucleus, and raphe nuclei. Conversely, constitutive activation of Smoothened signaling in vMB (Shh-Cre;SmoM2) showed transient expansion of the same progenitor population. To further characterize the nature of Shh-Smoothened signaling in vMB, we examined the BAT-GAL reporter and the expression of Wnt1 in vMB, and found that the antagonistic effects of Shh and Wnt signaling critically regulate the development of DA progenitors and DA neurons. CONCLUSION: These results highlight previously unrecognized effects of Shh-Smoothened signaling in the region-specific neurogenesis within the vMB.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Hedgehog/metabolismo , Mesencéfalo/embriología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Linaje de la Célula/genética , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Proteínas Hedgehog/genética , Mesencéfalo/metabolismo , Ratones , Ratones Transgénicos , Neurogénesis/fisiología , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened , Vía de Señalización Wnt/fisiologíaRESUMEN
Signaling mechanisms involving Wnt/beta-catenin and sonic hedgehog (Shh) are known to regulate the development of ventral midbrain (vMB) dopamine neurons. However, the interactions between these two mechanisms and how such interactions can be targeted to promote a maximal production of dopamine neurons are not fully understood. Here we show that conditional mouse mutants with region-specific activation of beta-catenin signaling in vMB using the Shh-Cre mice show a marked expansion of Sox2-, Ngn2-, and Otx2-positive progenitors but perturbs their cell cycle exit and reduces the generation of dopamine neurons. Furthermore, activation of beta-catenin in vMB also results in a progressive loss of Shh expression and Shh target genes. Such antagonistic effects between the activation of Wnt/beta-catenin and Shh can be recapitulated in vMB progenitors and in mouse embryonic stem cell cultures. Notwithstanding these antagonistic interactions, cell-type-specific activation of beta-catenin in the midline progenitors using the tyrosine hydroxylase-internal ribosomal entry site-Cre (Th-IRES-Cre) mice leads to increased dopaminergic neurogenesis. Together, these results indicate the presence of a delicate balance between Wnt/beta-catenin and Shh signaling mechanisms in the progression from progenitors to dopamine neurons. Persistent activation of beta-catenin in early progenitors perturbs their cell cycle progression and antagonizes Shh expression, whereas activation of beta-catenin in midline progenitors promotes the generation of dopamine neurons.
Asunto(s)
Dopamina/metabolismo , Proteínas Hedgehog/metabolismo , Mesencéfalo/citología , Neurogénesis/fisiología , Neuronas/fisiología , Transducción de Señal/fisiología , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Bromodesoxiuridina/metabolismo , Recuento de Células/métodos , Diferenciación Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Hedgehog/genética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Mesencéfalo/embriología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Células Madre/fisiología , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Proteína Wnt1/genética , beta Catenina/genéticaRESUMEN
Stem cell-based replacement therapy has emerged as a potential strategy to alleviate specific features of movement disorder in Parkinson's disease. However, the current strategy to produce dopamine (DA) neurons from embryonic stem cells has many limitations, including the difficulty of generating DA neurons with high yields. Further insights into the mechanisms that control the neurogenesis of DA neurons will reduce or mitigate such limitations. It is well established that the ventral midbrain (vMB) contains the neurogenic niche that produces DA neurons. However, it is unclear how the microenvironment within this niche controls DA neurogenesis. Here, we show that beta-catenin controls DA neurogenesis by maintaining the integrity of the neurogenic niche and the progression from progenitors to DA neurons. Using conditional gene targeting approaches, we show that regional deletion of beta-catenin in the vMB by using Shh-Cre disrupts adherent junctions of progenitors and the integrity of radial glia in the vMB, which leads to a severe reduction in DA neurogenesis and perturbs the migration and segregation of DA neurons. By contrast, Th-IRES-Cre removes beta-catenin in a subset of neural progenitor cells without perturbing the cellular and structural integrity of the vMB. Interestingly, loss of beta-catenin in Th-IRES-Cre;beta-Ctn(fl/fl) mutants negatively regulates neurogenesis by interfering with the progression of committed progenitors to DA neurons. Taken together, these results provide new insights into the indispensable functions of beta-catenin at multiple stages during DA neurogenesis. They also suggest that beta-catenin-mediated signaling pathways can be targeted to promote and expand DA neurons in cell-based therapeutic strategies.
Asunto(s)
Uniones Adherentes/fisiología , Dopamina/metabolismo , Mesencéfalo/fisiología , Neuronas/fisiología , beta Catenina/fisiología , Animales , Diferenciación Celular , Movimiento Celular/fisiología , Mesencéfalo/embriología , Ratones , Ratones Noqueados , Mutación , Neurogénesis , Neuroglía/citología , Neuroglía/fisiología , Nicho de Células Madre , Células Madre/citología , Células Madre/fisiología , beta Catenina/genéticaRESUMEN
Toosendanin, a triterpenoid derivative isolated from the barks of Melia toosendan Sieb et Zucc, has been used as an anthelmintic vermifuge against ascaris for more than fifty years in China. In the present study, we investigated the growth inhibition and apoptosis-induced effect of toosendanin on human cancer cells. The result showed that toosendanin significantly suppressed the proliferation of tested human cancer cell lines. The IC(50) values were less than 1.7 x 10(-7) M and U937 was the most sensitive cell line with a IC(50) of 5.4 x 10(-9) M. Flow cytometric analysis revealed that treatment of U937 cells with toosendanin resulted in a dose- and time-dependent accumulation of cells in the S phase with a concomitant decrease in cells processing to G(0)/G(1) phase. The growth inhibition of U937 cells after exposure to toosendanin was subsequently associated with the induction of apoptosis, as evidence by the typical condensed and fragmented nuclei, DNA fragmentation, and exposure of phosphatidylserine on the outer leaflet of plasma membrane. All these results indicated that toosendanin could serve as a potential candidate for anticancer drug.
Asunto(s)
Apoptosis/efectos de los fármacos , Línea Celular Tumoral/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral/citología , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , HumanosRESUMEN
Our previous study showed that toosendanin, a triterpenoid derivative isolated from a Chinese traditional medicine, could induce apoptosis in PC12 cells. In this study we confirmed the apoptosis-inducing effect of toosendanin in PC12 cells with new evidences in morphology and biochemistry: the shrinkage of cytosol, the condensation and fragmentation of nuclei and the formation of DNA ladder. It was also demonstrated that toosendanin decreased the PC12 cell viability in a time- and concentration-dependent manner. To elucidate the pathway linked with the toosendanin-induced apoptosis, the cytochrome c in the cytosol and the cleavage of poly(ADP-ribose) polymerase (PARP) were examined. The obtained results showed that toosendanin caused the release of cytochrome c from mitochondria into the cytosol and then led to the activation of caspase, indicating that the cytochrome c release and caspase activation were involved in the toosendanin-induced apoptosis process. These results suggested the possibility that toosendanin could serve as a candidate for anti-cancer drug.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Citocromos c/metabolismo , Medicamentos Herbarios Chinos/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células PC12 , RatasRESUMEN
Toosendanin (TSN), a triterpenoid derivative extracted from Chinese traditional medicine, has been demonstrated to be an effective cure for experimental botulism. This study is designed to explore its antibotulismic mechanism by Western blotting. The results showed that TSN incubation did not change the electrophoresis pattern and the amounts of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin and synaptobrevin/vesicle-associated membrane protein in rat cerebral synaptosomes, but made the synaptosomes completely resistant to botulinum neurotoxin A (BoNT/A)-mediated cleavage of SNAP-25. After binding of BoNT/A to synaptosomes, TSN still partially antagonized the toxin-mediated cleavage of SNAP-25. However, TSN-incubated synaptosomal membrane fraction did not resist the cleavage of SNAP-25 by the light chain of BoNT/A. It is suggested that the antibotulismic effect of TSN results from blocking the toxin's approach to its enzymatic substrate.
Asunto(s)
Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Toxinas Botulínicas Tipo A/metabolismo , Corteza Cerebral/metabolismo , Medicamentos Herbarios Chinos/farmacología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sinaptosomas/metabolismo , Proteínas de Transporte Vesicular , Animales , Western Blotting , Toxinas Botulínicas Tipo A/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Corteza Cerebral/efectos de los fármacos , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Oxidación-Reducción , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratas , Ratas Sprague-Dawley , Proteínas SNARE , Proteína 25 Asociada a Sinaptosomas , Sinaptosomas/efectos de los fármacosRESUMEN
In the present study, the effects of toosendanin on cell differentiation and apoptosis were investigated in PC12 cells. The results showed that after 24-48 h of culture in a medium containing toosendanin (approximately 1-10x10(-7) M), cell differentiation and outgrowth of neuronal processes were promoted. Combined treatment with toosendanin and a calcium channel blocker, nifedipine or omega-conotoxin GVIA, resulted in a significant inhibition of the toosendanin-induced effects. Pretreatment of PC12 cells with BAPTA-AM also inhibited the toosendanin-induced effects; however, these effects were not inhibited by pertussis toxin and H-7 in the medium. Toosendanin also induced cell apoptosis. Based on the DNA content determined by flow cytometric analysis, the number of apoptotic cells significantly increased when the incubation time in the toosendanin-containing medium was lasted up to 72 h. Toosendanin at a higher concentration (> or =1 x 10(-6) M) caused cell death while it had no effect on cell division at concentrations lower than 1 x 10(-7) M.