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1.
Cell Mol Life Sci ; 79(8): 437, 2022 Jul 21.
Artículo en Inglés | MEDLINE | ID: mdl-35864382

RESUMEN

The neurodegenerative condition FENIB (familiar encephalopathy with neuroserpin inclusion bodies) is caused by heterozygous expression of polymerogenic mutant neuroserpin (NS), with polymer deposition within the endoplasmic reticulum (ER) of neurons. We generated transgenic neural progenitor cells (NPCs) from mouse fetal cerebral cortex stably expressing either the control protein GFP or human wild type, polymerogenic G392E or truncated (delta) NS. This cellular model makes it possible to study the toxicity of polymerogenic NS in the appropriated cell type by in vitro differentiation to neurons. Our previous work showed that expression of G392E NS in differentiated NPCs induced an adaptive response through the upregulation of several genes involved in the defence against oxidative stress, and that pharmacological reduction of the antioxidant defences by drug treatments rendered G392E NS neurons more susceptible to apoptosis than control neurons. In this study, we assessed mitochondrial distribution and found a higher percentage of perinuclear localisation in G392E NS neurons, particularly in those containing polymers, a phenotype that was enhanced by glutathione chelation and rescued by antioxidant molecules. Mitochondrial membrane potential and contact sites between mitochondria and the ER were reduced in neurons expressing the G392E mutation. These alterations were associated with a pattern of ER stress that involved the ER overload response but not the unfolded protein response. Our results suggest that intracellular accumulation of NS polymers affects the interaction between the ER and mitochondria, causing mitochondrial alterations that contribute to the neuronal degeneration seen in FENIB patients.


Asunto(s)
Antioxidantes , Neuronas , Animales , Antioxidantes/metabolismo , Antioxidantes/farmacología , Estrés del Retículo Endoplásmico , Epilepsias Mioclónicas , Trastornos Heredodegenerativos del Sistema Nervioso , Humanos , Ratones , FN-kappa B/metabolismo , Neuronas/metabolismo , Neuropéptidos , Polímeros , Serpinas , Neuroserpina
2.
Exp Cell Res ; 313(3): 588-601, 2007 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-17156776

RESUMEN

Isolation and expansion of neural stem cells (NSCs) of human origin are crucial for successful development of cell therapy approaches in neurodegenerative diseases. Different epigenetic and genetic immortalization strategies have been established for long-term maintenance and expansion of these cells in vitro. Here we report the generation of a new, clonal NSC (hc-NSC) line, derived from human fetal cortical tissue, based on v-myc immortalization. Using immunocytochemistry, we show that these cells retain the characteristics of NSCs after more than 50 passages. Under proliferation conditions, when supplemented with epidermal and basic fibroblast growth factors, the hc-NSCs expressed neural stem/progenitor cell markers like nestin, vimentin and Sox2. When growth factors were withdrawn, proliferation and expression of v-myc and telomerase were dramatically reduced, and the hc-NSCs differentiated into glia and neurons (mostly glutamatergic and GABAergic, as well as tyrosine hydroxylase-positive, presumably dopaminergic neurons). RT-PCR analysis showed that the hc-NSCs retained expression of Pax6, Emx2 and Neurogenin2, which are genes associated with regionalization and cell commitment in cortical precursors during brain development. Our data indicate that this hc-NSC line could be useful for exploring the potential of human NSCs to replace dead or damaged cortical cells in animal models of acute and chronic neurodegenerative diseases. Taking advantage of its clonality and homogeneity, this cell line will also be a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in human NSC biology.


Asunto(s)
Transformación Celular Viral , Corteza Cerebral/citología , Células Madre Fetales/fisiología , Neuronas/fisiología , Proteína Oncogénica p55(v-myc)/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Células Clonales/fisiología , Regulación hacia Abajo , Células Madre Fetales/enzimología , Regulación del Desarrollo de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Proteína Oncogénica p55(v-myc)/genética , Fenotipo , Telomerasa , Tubulina (Proteína)/metabolismo
3.
J Neurosci Res ; 74(5): 760-8, 2003 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-14635227

RESUMEN

Hepatocyte growth factor/scatter factor (HGF/SF) is a growth factor with pleiotropic effects on different cell types. It acts as a mitogen and motility factor for many epithelial cells. HGF/SF and its receptor Met are present in the developing and adult mammalian brain and control neuritogenesis of sympathetic and sensory neurons. We report that the striatal progenitor ST14A cells express the Met receptor, which is activated after binding with HGF/SF. The interaction between Met and HGF/SF triggers a signaling cascade that leads to increased levels of c-Jun, c-Fos, and Egr-1 proteins, in agreement with data reported on the signaling events evoked by HGF in other cellular types. We also studied the effects of the exposure of ST14A cells to HGF/SF. By time-lapse photography, we observed that a 24-hr treatment with 50 ng/ml HGF/SF induced modification in cell morphology, with a decrease in cell-cell interactions and increase of cell motility. In contrast, no effect on cell proliferation was observed. To investigate which intracellular pathway is primarily involved we used PD98059 and LY294002, two specific inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAP-kinase/ERK-kinase) and phosphoinositide 3-OH kinase (PI3-K), respectively. Cell motility in HGF/SF treated cultures was inhibited by LY294002 but not by PD98059, suggesting that PI3-K plays a key role in mediating the HGF/SF-induced dissociation of ST14A cells. Previous evidence of HGF stimulation of motility in nervous system has been obtained on postmitotic neurons, which have already acquired their specificity. Data reported here of a motogenic response of ST14A cell line, which displays properties of neuronal progenitors, seem of interest because they suggest that HGF could play a role in very early steps of neurogenesis.


Asunto(s)
Movimiento Celular/efectos de los fármacos , Factor de Crecimiento de Hepatocito/farmacología , Neuronas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-met/fisiología , Células Madre/efectos de los fármacos , Animales , Western Blotting , Comunicación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Cuerpo Estriado/citología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/embriología , Embrión de Mamíferos , Genes Inmediatos-Precoces/efectos de los fármacos , Inmunohistoquímica , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neuronas/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Pruebas de Precipitina , Ratas , Células Madre/fisiología
4.
Mutagenesis ; 17(3): 241-9, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-11971996

RESUMEN

Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression.


Asunto(s)
Aflatoxina B1 , Carcinógenos , Ciclo Celular/efectos de los fármacos , Western Blotting , Bromodesoxiuridina/farmacología , Línea Celular , AMP Cíclico/metabolismo , Citometría de Flujo , Humanos , Análisis Multivariante , Mutación , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Factores de Tiempo , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis
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