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1.
Mol Cell Proteomics ; 22(5): 100543, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37030595

RESUMEN

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIß (CaMKIIß). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIß, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.


Asunto(s)
Muerte Celular , Neuronas , Sinapsis , Animales , Masculino , Ratones , Ratas , Calpaína/metabolismo , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , Neuronas/fisiología , Neuroprotección , Proteoma/análisis , Ratas Wistar , Accidente Cerebrovascular/patología , Sinapsis/patología , Sinapsis/fisiología
2.
Cell Death Dis ; 10(3): 213, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30824683

RESUMEN

Excitotoxicity, caused by overstimulation or dysregulation of ionotropic glutamate receptors (iGluRs), is a pathological process directing neuronal death in many neurological disorders. The aberrantly stimulated iGluRs direct massive influx of calcium ions into the affected neurons, leading to changes in expression and phosphorylation of specific proteins to modulate their functions and direct their participation in the signalling pathways that induce excitotoxic neuronal death. To define these pathways, we used quantitative proteomic approaches to identify these neuronal proteins (referred to as the changed proteins) and determine how their expression and/or phosphorylation dynamically changed in association with excitotoxic cell death. Our data, available in ProteomeXchange with identifier PXD008353, identified over 100 changed proteins exhibiting significant alterations in abundance and/or phosphorylation levels at different time points (5-240 min) in neurons after glutamate overstimulation. Bioinformatic analyses predicted that many of them are components of signalling networks directing defective neuronal morphology and functions. Among them, the well-known neuronal survival regulators including mitogen-activated protein kinases Erk1/2, glycogen synthase kinase 3 (GSK3) and microtubule-associated protein (Tau), were selected for validation by biochemical approaches, which confirmed the findings of the proteomic analysis. Bioinformatic analysis predicted Protein Kinase B (Akt), c-Jun kinase (JNK), cyclin-dependent protein kinase 5 (Cdk5), MAP kinase kinase (MEK), Casein kinase 2 (CK2), Rho-activated protein kinase (Rock) and Serum/glucocorticoid-regulated kinase 1 (SGK1) as the potential upstream kinases phosphorylating some of the changed proteins. Further biochemical investigation confirmed the predictions of sustained changes of the activation states of neuronal Akt and CK2 in excitotoxicity. Thus, future investigation to define the signalling pathways directing the dynamic alterations in abundance and phosphorylation of the identified changed neuronal proteins will help elucidate the molecular mechanism of neuronal death in excitotoxicity.


Asunto(s)
Ácido Glutámico/toxicidad , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Quinasa de la Caseína II/química , Quinasa de la Caseína II/genética , Quinasa de la Caseína II/metabolismo , Muerte Celular , Supervivencia Celular , Células Cultivadas , Cromatografía Liquida , Biología Computacional , Ácido Glutámico/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/química , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas del Tejido Nervioso/química , Neuronas/citología , Neuronas/patología , Fosforilación , Proteómica , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo , Transducción de Señal/genética , Programas Informáticos , Espectrometría de Masas en Tándem , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismo
3.
Pharmacol Ther ; 160: 159-79, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26899498

RESUMEN

Excitotoxicity, a pathological process caused by over-stimulation of ionotropic glutamate receptors, is a major cause of neuronal loss in acute and chronic neurological conditions such as ischaemic stroke, Alzheimer's and Huntington's diseases. Effective neuroprotective drugs to reduce excitotoxic neuronal loss in patients suffering from these neurological conditions are urgently needed. One avenue to achieve this goal is to clearly define the intracellular events mediating the neurotoxic signals originating from the over-stimulated glutamate receptors in neurons. In this review, we first focus on the key cellular events directing neuronal death but not involved in normal physiological processes in the neurotoxic signalling pathways. These events, referred to as pathologically activated events, are potential targets for the development of neuroprotectant therapeutics. Inhibitors blocking some of the known pathologically activated cellular events have been proven to be effective in reducing stroke-induced brain damage in animal models. Notable examples are inhibitors suppressing the ion channel activity of neurotoxic glutamate receptors and those disrupting interactions of specific cellular proteins occurring only in neurons undergoing excitotoxic cell death. Among them, Tat-NR2B9c and memantine are clinically effective in reducing brain damage caused by some acute and chronic neurological conditions. Our second focus is evaluation of the suitability of the other inhibitors for use as neuroprotective therapeutics. We also discuss the experimental approaches suitable for bridging our knowledge gap in our current understanding of the excitotoxic signalling mechanism in neurons and discovery of new pathologically activated cellular events as potential targets for neuroprotection.


Asunto(s)
Muerte Celular/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Animales , Humanos , Memantina/farmacología , Memantina/uso terapéutico , Péptidos/farmacología , Péptidos/uso terapéutico , Transducción de Señal/efectos de los fármacos
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