RESUMEN
Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen-glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.
Asunto(s)
Infarto Encefálico/metabolismo , Dinaminas/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Infarto Encefálico/genética , Infarto Encefálico/patología , Muerte Celular , Línea Celular , Modelos Animales de Enfermedad , Dinaminas/genética , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Ratones , Mitocondrias/genética , Mitocondrias/patología , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Transporte de ProteínasRESUMEN
Detection of neuronal cell death is a standard requirement in cell culture models of neurodegenerative diseases. Although plenty of viability assays are available for in vitro applications, most of these are endpoint measurements providing only little information on the kinetics of cell death. Here, we validated the xCELLigence system based on impedance measurement for real-time detection of cell death in a neuronal cell line of immortalized hippocampal neurons (HT-22 cells), neuronal progenitor cells (NPC) and differentiated primary cortical neurons. We found a good correlation between impedance measurements and endpoint viability assays in HT-22 cells and NPC, for detecting proliferation, cell death kinetics and also neuroprotective effects of pharmacological inhibitors of apoptosis. In primary neurons we could not detect dendritic outgrowth during differentiation of the cells. Cell death in primary neurons was detectable by the xCELLigence system, however, the changes in the cell index on the basis of impedance measurements depended to a great extent on the severity of the insult. Cell death induced by ionomycin, e.g. shows as a fast paced process involving a strong cellular disintegration, which allows for impedance-based detection. Cell death accompanied by less pronounced morphological changes like glutamate induced cell death, however, is not well accessible by this approach. In conclusion, our data show that impedance measurement is a convenient and reliable method for the detection of proliferation and kinetics of cell death in neuronal cell lines, whereas this method is less suitable for the assessment of neuronal differentiation and viability of primary neurons.
Asunto(s)
Muerte Celular/fisiología , Impedancia Eléctrica , Neuronas/patología , Muerte Celular/efectos de los fármacos , Línea Celular , Ácido Glutámico/toxicidad , Humanos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacologíaRESUMEN
Glutamate toxicity involves increases in intracellular calcium levels and enhanced formation of reactive oxygen species (ROS) causing neuronal dysfunction and death in acute and chronic neurodegenerative disorders. The molecular mechanisms mediating glutamate-induced ROS formation are, however, still poorly defined. Using a model system that lacks glutamate-operated calcium channels, we demonstrate that glutamate-induced acceleration of ROS levels occurs in two steps and is initiated by lipoxygenases (LOXs) and then significantly accelerated through Bid-dependent mitochondrial damage. The Bid-mediated secondary boost of ROS formation downstream of LOX activity further involves mitochondrial fragmentation and release of mitochondrial apoptosis-inducing factor (AIF) to the nucleus. These data imply that the activation of Bid is an essential step in amplifying glutamate-induced formation of lipid peroxides to irreversible mitochondrial damage associated with further enhanced free radical formation and AIF-dependent execution of cell death.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Ácido Glutámico/toxicidad , Mitocondrias/metabolismo , Neuronas/metabolismo , Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/antagonistas & inhibidores , Línea Celular Transformada , Hipocampo/citología , Humanos , Inhibidores de la Lipooxigenasa/farmacología , Lipooxigenasas/química , Lipooxigenasas/metabolismo , Neuronas/citología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Muerte Celular/fisiología , Mitocondrias/metabolismo , Neuronas/fisiología , Animales , Factor Inductor de la Apoptosis/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/antagonistas & inhibidores , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Caspasas/metabolismo , Activación Enzimática , Silenciador del Gen , Ácido Glutámico/toxicidad , Humanos , Ratones , Microscopía Fluorescente , Microscopía por Video , Neuronas/citología , Neuronas/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
We identify a chaperone complex composed of (1) the synaptic vesicle cysteine string protein (CSP), thought to function in neurotransmitter release, (2) the ubiquitous heat-shock protein cognate Hsc70, and (3) the SGT protein containing three tandem tetratricopeptide repeats. These three proteins interact with each other to form a stable trimeric complex that is located on the synaptic vesicle surface, and is disrupted in CSP knockout mice. The CSP/SGT/Hsc70 complex functions as an ATP-dependent chaperone that reactivates a denatured substrate. SGT overexpression in cultured neurons inhibits neurotransmitter release, suggesting that the CSP/SGT/Hsc70 complex is important for maintenance of a normal synapse. Taken together, our results identify a novel trimeric complex that functions as a synapse-specific chaperone machine.
Asunto(s)
Exocitosis/fisiología , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas de la Membrana/fisiología , Chaperonas Moleculares/fisiología , Proteínas del Tejido Nervioso/fisiología , Neurotransmisores/metabolismo , Proteínas/fisiología , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Adenosina Trifosfato/fisiología , Animales , Química Encefálica , Proteínas Portadoras , Células Cultivadas , Proteínas del Choque Térmico HSC70 , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/química , Hipocampo/citología , Sustancias Macromoleculares , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Modelos Biológicos , Chaperonas Moleculares/química , Proteínas del Tejido Nervioso/química , Unión Proteica , Pliegue de Proteína , Proteínas/química , Ratas , Ratas Wistar , Vesículas Sinápticas/química , Técnicas del Sistema de Dos HíbridosRESUMEN
PDZ domains play a pivotal role in the synaptic localization of ion channels, receptors, signaling enzymes, and cell adhesion molecules. These domains mediate protein-protein interactions via the recognition of a conserved sequence motif at the extreme C terminus of their target proteins. By means of a yeast two-hybrid screen using the C terminus of the G protein-coupled alpha-latrotoxin receptor CL1 as bait, three PDZ domain proteins of the Shank family were identified. These proteins belong to a single protein family characterized by a common domain organization. The PDZ domain is highly conserved among the family members, significantly different from other known PDZ domains, and specifically binds to the C terminus of CL1. Shank1 and CL1 are expressed primarily in brain, and both proteins co-enrich in the postsynaptic density. Furthermore, Shank1 induces a clustering of CL1 in transfected cells, strongly supporting an interaction of both proteins in vivo.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Clonación Molecular , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores de Péptidos/química , Receptores de Péptidos/genética , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The cysteine string protein (csp) is a synaptic vesicle protein found to be essential for normal neurotransmitter release. The precise function of csp in the synaptic vesicle cycle is still enigmatic. By interacting with the heat-shock cognate hsc70, a cochaperone-chaperone complex with an unknown function is formed. We report here that the formation of this complex is mediated by two distinct domains in hsc70. The ATPase domain and the substrate-binding domain must cooperate to create a binding site for csp. The C-terminal domain of hsc70 seems to function as a regulator for the formation of the cochaperone-chaperone complex. We also show that the interaction of csp with heat-shock proteins is confined to hsc70 and hsp70. Other heat-shock proteins, like hsp60 and hsp90, do not interact with csp.