Función del complejo Cdk1-ciclina B1 en la excitotoxicidad

Tesis Doctoral realizada por el licenciado Miguel Veas-Pérez de Tudela Rodríguez en el Departamento de Bioquímica y Biología Molecular y en el Instituto de Biología Funcional y Genómica de la Universidad de Salamanca para optar al grado de Doctor.Una característica fundamental de las neuronas, células post-mitóticas, es que su supervivencia depende de la contínua degradación de la ciclina mitótica B1, proteína de ciclo celular que se acumula en áreas cerebrales dañadas de pacientes que han sufrido la enfermedad de Alzheimer o un infarto cerebral. La degradación de ciclina B1 tiene lugar en el proteasoma tras la ubiquitinación por parte del complejo promotor de la anafase ciclosoma (APC/C)-Cdh1, una E3 ubiquitina ligasa muy activa en neuronas. Sin embargo, durante un daño excitotóxico, el complejo APC/C-Cdh1 se inactiva, causando la estabilización y la acumulación de ciclina B1 y la muerte neuronal por un mecanismo aún desconocido. Nosotros describimos, por primera vez, que la estimulación de los receptores de glutamato induce la acumulación de la ciclina B1 en la mitocondria, que activa la Cdk1 formándose el complejo Cdk1-ciclinaB1. Éste complejo fosforila a Bcl-xL, que se disocia de la subunidad beta de la F1Fo-ATP sintasa, provocando su inhibición; lo que induce hiperpolarización mitocondrial y, en consecuencia, el estrés oxidativo responsable de la disfunción del complejo I y el fallo energético que culminan en la muerte neuronal excitotóxica. La cascada de señalización aquí descrita, ha permitido identificar nuevas dianas moleculares para, en un futuro, poder establecer nuevas vías de actuación terapéuticas en desórdenes neurológicos que cursan con excitotoxicidad, tales como el ictus o la enfermedad de Alzheimer.Peer Reviewe

Cdk5-mediated inhibition of APC/C-Cdh1 switches on the cyclin D1-Cdk4-pRb pathway causing aberrant S-phase entry of postmitotic neurons

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that regulates cell cycle progression in proliferating cells. To enter the S-phase, APC/C must be inactivated by phosphorylation of its cofactor, Cdh1. In post-mitotic cells such as neurons APC/C-Cdh1 complex is highly active and responsible for the continuous degradation of mitotic cyclins. However, the specific molecular pathway that determines neuronal cell cycle blockade in post-mitotic neurons is unknown. Here, we show that activation of glutamatergic receptors in rat cortical primary neurons endogenously triggers cyclin-dependent kinase-5 (Cdk5)-mediated phosphorylation of Cdh1 leading to its cytoplasmic accumulation and disassembly from the APC3 core protein, causing APC/C inactivation. Conversely, pharmacological or genetic inhibition of Cdk5 promotes Cdh1 ubiquitination and proteasomal degradation. Furthermore, we show that Cdk5-mediated phosphorylation and inactivation of Cdh1 leads to p27 depletion, which switches on the cyclin D1-cyclin-dependent kinase-4 (Cdk4)-retinoblastoma protein (pRb) pathway to allow the S-phase entry of neurons. However, neurons do not proceed through the cell cycle and die by apoptosis. These results indicate that APC/C-Cdh1 actively suppresses an aberrant cell cycle entry and death of neurons, highlighting its critical function in neuroprotection.This work was funded by The 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.), The E.U. SP3-People-MC-ITN programme (608381; J.P.B.), and the European Regional Development Fund.Peer Reviewe

Cell cycle proteins and oxidative stress in neurodegeneration

Resumen del trabajo presentado a la 5th Conference on Advances in Molecular Mechanisms Underlying Neurological Disorders (Joint conference of the European Society for Neurochemistry and the Biochemical Society) en la University of Bath (UK) del 23 al 26 de junio de 2013.Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ubiquitin ligase that destabilizes cell cycle proteins. In post-mitotic neurons, APC/C is activated by Cdh1 and regulates axonal growth, synaptic plasticity, metabolism and survival. The APC/C-Cdh1 substrate, cyclin B1, has been found to accumulate in degenerating brain areas in Alzheimer’s disease and stroke. In primary cultured neurons, stimulation of glutamate receptors promoted the activation of cyclin-dependent kinase-5 (Cdk5), a Cdk important for synaptic plasticity and neurotoxicity, leading to Cdh1 phosphorylation and inactivation, which triggers cyclin B1 stabilization. Cyclin B1 interacted directly with mitochondrial Bcl-xL and promoted mitochondrial dysfunction and oxidative stress, leading to activation of the intrinsic apoptotic pathway. All these effects were counteracted by Cdk activity inhibition, cyclin B1 silencing (siRNA) or Cdh1 expression in the neurons. Our results reveal Cdh1 as a novel Cdk5 substrate that mediates cyclin B1 neuronal accumulation in excitotoxicity and neurological disease.This work was funded by the ISCIII (PS09/0366 and RD06/0026/1008).Peer reviewe

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