research

Research Description:

Postdoctoral research:

10/2007-present: Post-doctoral fellow UPMC Paris. Primary Investigator: Dr. Christiane Charriaut-Marlangue. Hypoxie et Ischémie du Cerveau en Développement. UMR-CNRS 7102, Universite P. et M. Curie

Mort cellulaire et tolérance Ischémie développementale

http://npa.snv.jussieu.fr/eq/equipe.php?eq=hicd&lang=fr

Doctoral Thesis:

“APOPTOTIC AND ANTIAPOPTOTIC MECHANISMS IN NEURONS AND GLIAL CELLS AFTER DAMAGE TO THE IMMATURE BRAIN”.

Summary

Although it is well established that apoptotic mechanisms play a central role in neuronal death after different types of CNS damage, little is known about the activation of apoptotic pathways in glial cells which have been postulated to be important for the termination of the glial response. It is remarkable that the classically defined main executioner of programmed cell death, caspase-3, has recently been attributed a non-apoptotic role, participating in cellular events including cell proliferation, cell cycle regulation, or cellular differentiation. Accordingly, the general aim of this thesis was to analyze the putative role of the classic apoptotic and anti-apoptotic pathways after acute excitotoxic damage to the immature rat brain. For this purpose, a well-characterized in vivo model of excitotoxicity was used, where damage was induced by the intracortical injection of N-methyl-D-asparate (NMDA) in postnatal day 9 rats. Cell death, apoptotic and anti-apoptotic proteins were analyzed at different survival times after the lesion by enzymatic assays and double immunofluorescence for confocal microscope analysis.

The first set of results demonstrated that although cleaved caspase-3 was found in some damaged neurons showing TUNEL+ nuclei, cleaved caspase-3 was mainly observed in the nucleus of activated astrocytes in the lesioned hemisphere as early as 4 h post-lesion, persisted until the glial scar was formed at 7-14 days, and was marginally associated with TUNEL labeling. Caspase-3 enzymatic activity was significant during the first hours post-lesion and co-localized with the presence of caspase-cleaved fragments of glial fibrillary acidic protein (CCP-GFAP) in astrocytes. However, at longer survival times, when astroglial hypertrophy was observed, astroglial caspase-3 did not generally correlate with GFAP cleavage but instead was associated with de novo expression of vimentin. Moreover, astroglial caspase-3 cleavage was not associated with cell proliferation. These first results provided evidence for a non-traditional role of caspases in astroglial function, suggesting that caspase activation may be important for astroglial cytoskeleton remodeling following injury.

Secondly, in order to evaluate upstream pathways activating caspase-3 in neurons and glial cells, the spatio-temporal activation of the intrinsic and extrinsic apoptosis pathways were analyzed. In damaged neuronal cells of the ipsilateral cortex and hippocampus an important contribution of the intrinsic mitochondrial pathway through caspase-9 activation was shown to account for most of neuronal caspase-3 activation and apoptotic nuclei. However, neuronal caspase-8 showed a diminished correlation with caspase-3 and characteristically showed cortical layer specificity. In glial cells, activation of caspase-9, caspase-8, and also p53, another putative caspase-3 activating molecule, was only significant in cortical layer VI and the corpus callosum, suggesting that astroglial caspase-3 does not generally correlate with classical upstream activating pathways.

In the third part of this thesis, inhibitor of apoptosis proteins (IAPs) and heat shock proteins (HSPs), known inhibitors of cleaved caspase-3 in other cell types, were analyzed. Whereas neuronal cells showed noticeable expression of HSC70/HSP70 and cIAP-2, these poorly correlated with caspase-3 in this cell type. In astrocytes, survivin and the small HSP25/27 showed a strong correlation with caspase-3 in several regions and throughout different survival times, pointing to these proteins as relevant candidates for the blockade of caspase-3 enzymatic activity, which could account for the absence of astroglial cell death.

In conclusion, this thesis has discerned new non-apoptotic roles of caspase-3 in astroglial cells and suggests mechanisms for caspase-3 blockade in these glial cells, improving the understanding of the mechanisms employed by astrocytes to cope with damage in the immature brain.

Neuroscience MASTER:

Evaluación de los efectos del litio en ratas después de una lesión cortical por aspiración: daño cerebral, gliosis y proliferación celular.

D.L. núm. B-44845 -2004.

Autora: Sonia Villapol Salgado

Departament de Biologia Cel•lular, de Fisiologia i d'Immunologia

Presentado el 18 de Octubre del 2004, Facultad de Medicina, UAB.

Objetivo – El litio es la mayor droga usada para tratamientos en desórdenes maníaco-depresivos y enfermedades bipolares. También se ha utilizado en enfermedades neurodegenerativas y como elemento inductor de la neuroprotección tras un daño cerebral. Estudios in vitro e in vivo han demostrado los efectos neuroprotectores y antiapoptóticos del litio. En el estudio actual, hemos estudiado el efecto de un tratamiento crónico con litio tras una lesión traumática por aspiración en el cortex cerebral, así como su influencia en la respuesta de las células nerviosas.

Material y Métodos – El litio fue incorporado en la dieta de un grupo de ratas. La administración fue diaria y durante 14 días previos a la lesión por aspiración cortical, así como de una a tres semanas después de ser lesionadas y hasta su sacrificio. Las ratas fueron examinadas después de las lesiones a dos tiempos de supervivencia de 7 y 21 días. Todos los animales fueron inyectados con 5-bromo-2´-deoxyuridina (BrdU) cada 24 horas a partir de una o tres semanas antes de su sacrificio. Los cerebros fueron procesados para la evaluación inmunohistoquímica de GFAP, lectina de tomate y BrdU. Del mismo modo, se realizaron dobles marcajes para BrdU/GFAP y BrdU/LT para la detección de proliferación astroglial o microglial respectivamente. Se realizaron tinciones de Nissl para calcular la extensión del daño cerebral ocasionado por la lesión.

Resultados – El daño neuronal fue significativamente menor en ratas tratadas con litio transcurridas las 3 semanas postlesión. Se observó como en los animales tratados con litio se incrementaba la astrogliosis en el cortex cerebral lesionado. En cuanto a la microgliosis no se detectaron cambios con el tratamiento. También se demostró que el tratamiento crónico con litio es capaz de incrementar las células BrdU positivas en el borde de lesión a los 7 días postlesión y en la zona de penumbra transcurridos los 21 dpl.

Conclusiones – Tras un tratamiento crónico con litio en ratas adultas con una lesión cortical por aspiración, se han demostrado los efectos neuroprotectores del litio que se traducen en una reducción del daño cerebral. También tiene lugar un aumento en la proliferación celular en la corteza lesionada, así como un incremento de la astrogliosis.

OTHER RESEARCH:

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"10th Meeting of the European Neuroendocrine Association” ENEA, 12-14 de September - 2002 Munich (Germany).

GHRELIN PHSOPHORLATES CREB TO INDUCE PIT-1 TRANSCRIPTION

Villapol S., García A., Cañibano C., Llovo R., Smith RG (2), Diéguez C & Álvarez CV

Departament of Physiology, Faculty of Medicine, University of Santiago de Compostela, 15782 Spain; 2) Huffington center on Aging, Baylor College of Medicine, Houston, Texas 77030.

Somatotroph cell-specific expression of the GH gene is dependent on a pituitary-specific transcription factor (Pit1). This factor is transcribed in a highly restricted manner in the anterior pituitary gland. GH secretagogues are an explanding class of synthetic pectide and nonpeptide molecules that stimulate the pituitary gland to secrete GH through their own specific receptor, the GH-secretagogue receptor. The cloning of the receptor for these nonclassical GH releasing molecules, together with the more recent characterization of an endogenous ligand, named ghrelin, have unambiguously demonstrated the existence of a physiological system that regulates GH secretion. We had previously demonstrated that Ghrelin directly regulates Pit1 transcription in infant rats pituitary primary cultures and in HEK-293-GHSR, a cell line expressing the Ghrelin receptor (GHSR). The effect was mapped to some CRE-elements on the promoter (García et al., Mol Endocrinol. 2001, 15:1484). CREB and AP-1 dimmers has been described as transcription factors that can bind and activate this DNA sequences, CRE, in the promoters of the genes that contains it. To evaluate the role of CREB in the Ghrelin-induced Pit1 expression, we did a time course experiment and studied phospho-CREB protein levels. The effect of Ghrelin was maintained up to thrity minutes. We co-transfected a dominant negative form of CREB, killer-CREB, plus the 231-Pit-1 promoter upstream of a luciferase expression vector. In this conditions, the transduction mechanisms leading to this phosphorylation we used inhibitors of PKC and PKA signaling pathways and studied Ghrelin-dependent CREB phosphorylation. We demonstrated that an irreversible inhibitor of adenylate cyclase, MDL-12330, completely abolished the Ghrelin effect, suggesting the need for cAMP to phosphorylate CREB.

Abstract Reference No.: ABS-8932-00363