The MDM2-p53 pathway is involved in preconditioning-induced neuronal tolerance to ischemia

Article number: 1610 (2018)[EN]Brain preconditioning (PC) refers to a state of transient tolerance against a lethal insult that can be evoked by a prior mild event. It is thought that PC may induce different pathways responsible for neuroprotection, which may involve the attenuation of cell damage pathways, including the apoptotic cell death. In this context, p53 is a stress sensor that accumulates during brain ischemia leading to neuronal death. The murine double minute 2 gene (MDM2), a p53-specific E3 ubiquitin ligase, is the main cellular antagonist of p53, mediating its degradation by the proteasome. Here, we study the role of MDM2-p53 pathway on PC-induced neuroprotection both in cultured neurons (in vitro) and rat brain (in vivo). Our results show that PC increased neuronal MDM2 protein levels, which prevented ischemia-induced p53 stabilization and neuronal death. Indeed, PC attenuated ischemia-induced activation of the p53/PUMA/caspase-3 signaling pathway. Pharmacological inhibition of MDM2-p53 interaction in neurons abrogated PC-induced neuroprotection against ischemia. Finally, the relevance of the MDM2-p53 pathway was confirmed in rat brain using a PC model in vivo. These findings demonstrate the key role of the MDM2-p53 pathway in PC-induced neuroprotection against a subsequent ischemic insult and poses MDM2 as an essential target in ischemic tolerance

El complejo MDM2-P53 es esencial en la tolerancia isquémica neuronal inducida por el precondicionamiento

[ES] Un estímulo subtóxico en el cerebro (PC) promueve una situación de resistencia transitoria, que lo protege frente a un daño isquémico agudo posterior. Este estado de tolerancia isquémica se debe a la activación de mecanismos endógenos que promueven la supervivencia neuronal (Dirnagl et al., 2009, 2003; Gidday, 2006; Kirino, 2002; Wang et al., 2015). De hecho, se ha observado que aquellos pacientes que sufren, previo al ictus, uno o varios AITs presentan un mejor pronóstico funcional que aquellos pacientes sin AIT (Schaller, 2005; Wang et al., 2017; Wegener et al., 2004). Por ello, en la actualidad, se considera al AIT como el correlato clínico de una situación de PC. Así, el PC constituye una herramienta experimental eficaz en la identificación de las vías de neuroprotección asociadas a la tolerancia neuronal y frente a la isquemia. El empleo de dos modelos de PC distintos (NMDA-PC e IPC) ha permitido identificar, tanto in vitro como in vivo, las vías de señalización p53/PUMA/Caspasa-3 y Akt/MDM2/p53 con un papel clave en el balance entre la supervivencia y la muerte neuronal tras la isquemia. Así, el PC induce la fosforilación temprana de Akt, cuya activación promueve la fosforilación de MDM2 y posterior translocación al núcleo celular. De este modo, se fomenta la formación del complejo MDM2-p53, clave para la desestabilización de la proteína p53. Todo esto previene la activación de la vía proapoptótica p53/PUMA/Caspasa-3 y favorece la supervivencia neuronal tras la isquemia (Vecino et al., 2018). Resultados previos de nuestro grupo han identificado que el polimorfismo humano o SNP Arg72Pro de Tp53 modula la susceptibilidad de las neuronas a la isquemia y condiciona el pronóstico funcional de pacientes de ictus isquémico y hemorrágico (Gomez-Sanchez et al., 2011). Además, este SNP determina la neovascularización y recuperación neurológica tras el ictus hemorrágico (Rodríguez et al., 2017). En este sentido, hemos identificado la función moduladora del polimorfismo Arg72Pro de Tp53 en la supervivencia neuronal asociada al PC y su relevancia clínica en el pronóstico funcional de pacientes que experimentan un AIT previo al ictus (Ramos-Araque et al., 2019)

Regulation of Bcl-xL–ATP synthase interaction by mitochondrial cyclin B1–cyclin-dependent kinase-1 determines neuronal survival

The survival of postmitotic neurons needs continuous degradation of cyclin B1, a mitotic protein accumulated aberrantly in the damaged brain areas of Alzheimer's disease and stroked patients. Degradation of cyclin B1 takes place in the proteasome after ubiquitylation by the anaphase-promoting complex/cyclosome (APC/C)–cadherin 1 (Cdh1), an E3 ubiquitin ligase that is highly active in neurons. However, during excitotoxic damage—a hallmark of neurological disorders—APC/C–Cdh1 is inactivated, causing cyclin B1 stabilization and neuronal death through an unknown mechanism. Here, we show that an excitotoxic stimulus in rat cortical neurons in primary culture promotes cyclin B1 accumulation in the mitochondria, in which it binds to, and activates, cyclin-dependent kinase-1 (Cdk1). The cyclin B1–Cdk1 complex in the mitochondria phosphorylates the anti-apoptotic protein B-cell lymphoma extra-large (Bcl-xL), leading to its dissociation from the β subunit of F1Fo–ATP synthase. The subsequent inhibition of ATP synthase activity causes complex I oxidative damage, mitochondrial inner membrane depolarization, and apoptotic neuronal death. These results unveil a previously unrecognized role for mitochondrial cyclin B1 in the oxidative damage associated with neurological disorders.This work was funded by Instituto de Salud Carlos III Grants PI12/00685 and RD12/0014/0007 (A.A.P.), RD12/0043/0021 (J.P.B.), FI10/00492 (M.V.-P.d.T.), and CP14/00010 (M.D.-E.), Ministerio de Economia y Competitividad Grant SAF2013-41177-R (J.P.B.), SP3-People-MC-ITN programme of the European Commission Grant 608381 (J.P.B.), and the European Regional Development Fund.Peer Reviewe

Preconditioning-activated AKT controls neuronal tolerance to ischemia through the MDM2-p53 pathway

EN][One of the most important mechanisms of preconditioning-mediated neuroprotection is the attenuation of cell apoptosis, inducing brain tolerance after a subsequent injurious ischemia. In this context, the antiapoptotic PI3K/AKT signaling pathway plays a key role by regulating cell differentiation and survival. Active AKT is known to increase the expression of murine double minute-2 (MDM2), an E3-ubiquitin ligase that destabilizes p53 to promote the survival of cancer cells. In neurons, we recently showed that the MDM2-p53 interaction is potentiated by pharmacological preconditioning, based on subtoxic stimulation of NMDA glutamate receptor, which prevents ischemia-induced neuronal apoptosis. However, whether this mechanism contributes to the neuronal tolerance during ischemic preconditioning (IPC) is unknown. Here, we show that IPC induced PI3K-mediated phosphorylation of AKT at Ser473, which in turn phosphorylated MDM2 at Ser166. This phosphorylation triggered the nuclear stabilization of MDM2, leading to p53 destabilization, thus preventing neuronal apoptosis upon an ischemic insult. Inhibition of the PI3K/AKT pathway with wortmannin or by AKT silencing induced the accumulation of cytosolic MDM2, abrogating IPC-induced neuroprotection. Thus, IPC enhances the activation of PI3K/AKT signaling pathway and promotes neuronal tolerance by controlling the MDM2-p53 interaction. Our findings provide a new mechanistic pathway involved in IPC-induced neuroprotection via modulation of AKT signaling, suggesting that AKT is a potential therapeutic target against ischemic injury