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Resumen del trabajo presentado en el 19 Congreso Nacional de la SENC celebrado en Lleida del 3 al 5 de noviembre de 2021, Ischemia-induced oxidative stress compromises genome integrity, which results in DNA damage and neuronal loss after stroke. We described that reactive oxygen species (ROS) generated during ischemia upregulate WRAP53 (WD40 encoding RNA antisense to p53) and trigger its translocation to the nucleus, where it promotes DNA repair [1]. However, the molecular mechanism remains unknown. Transcription factor Sp1 acts as a pleiotropic oxidative stress response protein in neurons. Particularly, ROS-induced Sp1 expression promotes neuroprotection against ischemia [2].i nterestingly, Wrap53 promoter contains putative consensus sequences (GC boxes) for Sp1. We analyze the role of Sp1 as a modulating factor of WRAP53-mediated neuronal survival and its impact on brain repair after stroke., Funded:ISCIII (FI19/00160;PI18/00265;RD16/0019/0018), FEDER, EU Horizon 2020 Research and Innovation Programme (Grant Agreement 686009), Junta de Castilla y León (CSI151P20;Escalera de Excelencia CLU-2017-03 Cofinanciado por P.O.FEDER de Castilla y León 14-20)

Stroke is a major cause of death and long-term disability in the adult. Neuronal apoptosis plays an essential role in the pathophysiology of ischemic brain damage and impaired functional recovery after stroke. The tumor suppressor protein p53 regulates key cellular processes, including cell cycle arrest, DNA repair, senescence, and apoptosis. Under cellular stress conditions, p53 undergoes post-translational modifications, which control protein localization, stability, and proapoptotic activity. After stroke, p53 rapidly accumulates in the ischemic brain, where it activates neuronal apoptosis through both transcriptional-dependent and -independent programs. Over the last years, subcellular localization of p53 has emerged as an important regulator of ischemia-induced neuronal apoptosis. Upon an ischemic insult, p53 rapidly translocates to the mitochondria and interacts with B-cell lymphoma-2 family proteins, which activate the mitochondrial apoptotic program, with higher efficacy than through its activity as a transcription factor. Moreover, the identification of a human single nucleotide polymorphism at codon 72 of the Tp53 gene that controls p53 mitochondrial localization and cell susceptibility to apoptosis supports the important role of the p53 mitochondrial program in neuronal survival and functional recovery after stroke. In this article, we review the relevance of mitochondrial and nuclear localization of p53 on neuronal susceptibility to cerebral ischemia and its impact on functional outcome of stroke patients. Consejería de Educación, Junta de Castilla y León, Grant/Award Numbers: CLU-2017-03, CSI151P20; EU Horizon 2020Research and Innovation Programme,Grant/Award Number: GA 686009;Ministerio de Ciencia e Innovación,Grant/Award Number:SAF2017-90794-REDT to AA; Junta deCastilla y León; Horizon 2020; EuropeanUnion; European Regional DevelopmentFund; Instituto de Salud Carlos III, Grant/Award Numbers: PI18/00265,RD16/0019/0018

Trabajo presentado en la 23rd European Society for Neurochemistry (ESN) Biennial Meeting - 7th Conference on Molecular Mechanisms of Regulation in the Nervous System, celebrada en Milán 8Italia), del 1 al 4 de septiembre de 2019

Trabajo presentado al 23rd European Society for Neurochemistry (ESN) Biennial Meeting and 7th Conference on Molecular Mechanisms of Regulation in the Nervous System, celebrados en Milán (Italia) del 1 al 4 de septiembre de 2019.

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016., The WD40 domain-containing protein Wrap53 (WD40 encoding RNA Antisense to p53) is a scaffold protein implicated in Cajal Bodies maintenance, telomere elongation and DNA repair. WRAP53 loss of function has been related to carcinogenesis and premature aging. Double-strands breaks may result from ischemic stroke, thereby contributing to neuronal death and subsequent brain dysfunction. An adequate DNA damage response is essential to survive after cerebral ischemia and preserve the integrity of the transcribed genome in neurons. Although DNA repair pathways are active after ischemia, the molecular mechanisms underlying neuronal survival remain unknown. To investigate the role of Wrap53 in neuronal survival after ischemia, primary mouse cortical neurons were subjected to an experimental protocol of ischemia in vitro (oxygen and glucose deprivation) for 3 hours and were further incubated in regular medium (reoxygenation). We first observed that ischemia promoted DNA damage, as revealed by the accumulation of γH2AX and 53BP-1 in the neurons. Furthermore, we found a time-dependent increase in WRAP53 gene expression and protein abundance from 4 hours after the ischemic insult. In parallel, ischemia induced the traffic of Wrap53 to the nucleus, which has been associated to cell survival in tumor cells. Moreover, depletion of Wrap53 by siRNA increased neuronal susceptibility to ischemia-induced apoptosis. Our results demonstrate that ischemia-induced Wrap53 nuclear accumulation plays an essential role in neuronal survival., Funded by ISCIII (PI15/00473; RD12/0014/0007), FEDER, and Junta de Castilla y León (ISM).

Resumen del póster presentado al XXXIX Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Salamanca del 5 al 8 de septiembre de 2016., The highly variable prediction of functional outcome after stroke could be the effect of different genetic backgrounds to apoptosis. MDM2 protein is the main negative regulator of p53, which plays an important role on neuronal apoptosis after cerebral ischemia. Recently, we found that the Arg72Pro single nucleotide polymorphism (SNP) of p53 regulates the pro-apoptotic activity of the protein and conditions neuronal vulnerability to ischemia-induced apoptosis and functional outcome of stroke patients. In MDM2 a SNP in the promoter gene (309T>G) modulates levels of Mdm2 expression. However, the role of Mdm2 309T>G SNP in stroke prognosis remains unknown. Here we study the association of the Mdm2 309T>G SNP and the functional prognosis after stroke in blood samples from 408 patients with ischemic stroke and 206 with intracerebral hemorrhage. Functional outcome at 3 and 12 months was evaluated by the modified Rankin scale. Mononuclear cells from healthy individuals were collected to measure levels of MDM2 mRNA and protein. We found that mononuclear cells harboring the Mdm2 TT genotype have lower levels of MDM2 mRNA and protein than those with the Mdm2 GG and Mdm2 TG genotypes, which may affect p53 stabilization and then cell vulnerability to ischemia-induced apoptosis. Furthermore, patients harboring the Mdm2 TT genotype showed poor functional outcome at 3 and 12 months following ischemic (p=0.003) and hemorrhagic (pG SNP modulates the MMD2p53 signaling pathway and then cell susceptibility to apoptosis, which conditions the functional outcome of patients after stroke.

Resumen del póster presentado al XXXVI Congreso de la Sociedad Española de Bioquímica y Biología Molecular celebrado en Madrid del 3 al 6 de septiembre de 2013., Durante la isquemia cerebral se interrumpe el aporte de glucosa y de oxígeno a la región cerebral afectada, lo que desencadena una compleja cascada de señalización celular, denominada "cascada isquémica", que culmina en la muerte neuronal. En los últimos años se ha descrito que la proteína supresora de tumores p53 está implicada en la muerte neuronal isquémica. Se ha identificado un dominio rico en prolina en p53 que es esencial para su actividad apoptótica. Esta región contiene un sitio polimórfico, específico de humanos, situado en el exón 4, codón 72 del gen Tp53 (Arg72Pro) que da lugar a las variantes polimórficas Arg72-p53 y Pro72-p53. Recientemente, nuestro grupo ha demostrado que el pronóstico funcional de pacientes de ictus está condicionado por el polimorfismo Arg72Pro de Tp53. Así, los pacientes que portan el genotipo Arg/Arg presentan peor pronóstico funcional a los tres meses del ictus que los que portan el alelo Pro. En el presente trabajo nos propusimos investigar el mecanismo molecular responsable de la diferente susceptibilidad genética de los pacientes al ictus. Cultivos primarios de neuronas corticales de ratón knockout de p53 se transfectaron con vectores BAC (bacterial artificial chromosome) que expresaban las variantes polimórficas humanas Arg72-p53 o Pro72-p53. Las neuronas se sometieron a isquemia experimental (privación de oxígeno y glucosa) durante 2 horas y posteriormente se incubaron en presencia de oxígeno y glucosa (reoxigenación) durante 1, 4, 8, 14 o 24 horas. La variante polimórfica Arg72-p53 incrementó la susceptibilidad de las neuronas al daño mitocondrial causado por la isquemia, respecto a Pro72-p53, a pesar de que la isquemia indujo la expresión de ambas proteínas de manera similar. Es consecuencia, Arg72-p53 aumentó la susceptibilidad de las neuronas a la apoptosis causada por la isquemia y posterior reoxigenación. Dicho efecto se produjo como consecuencia de la interacción directa de Arg72-p53 con Bcl-xL, lo que provocó la salida de citocromo c de la mitocondria y la activación de las caspasas 9 y 3. En conclusión, la variante polimórfica Arg72-p53 aumenta la susceptibilidad de las neuronas a la isquemia, lo que condiciona el pronóstico funcional de pacientes de ictus.