RESUMEN
The occurrence of endoplasmic reticulum (ER) stress in the sporadic form of amyotrophic lateral sclerosis (ALS) is unknown, despite it has been recently documented in experimental models of the familial form. Here we show that spinal cord from patients with sporadic ALS showed signs of ER stress, such as increased levels of ER chaperones such as protein-disulfide isomerase, and increased phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). Among the potential causes of such ER stress proteasomal impairment was confirmed in the same samples by demonstrating increased ubiquitin immunoreactivity and increased protein lipoxidative (125%), glycoxidative (55%) and direct oxidative damage (62%) over control values, as evidenced by mass-spectrometry and immunological methods. We found that protein oxidative damage was strongly associated to ALS-specific changes in fatty acid concentrations, specifically of n-3 series (as docosahexaenoic acid), and in the amount of mitochondrial components as respiratory complexes I and III, suggesting a mitochondrial dysfunction leading to increased free radical production. Oxidative stress was also evidenced in frontal cortex, suggesting that this region is affected early in ALS. As those events were partially reproduced by threohydroxyaspartate exposure in organotypic spinal cord cultures, we concluded that changes in fatty acid composition, mitochondrial function and proteasome activity, which may be driven by excitotoxicity, lead to oxidative stress and finally contribute to ER stress in sporadic ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Retículo Endoplásmico/fisiología , Adulto , Anciano , Esclerosis Amiotrófica Lateral/metabolismo , Ácidos Grasos/análisis , Femenino , Lóbulo Frontal/química , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/fisiología , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/química , Técnicas de Cultivo de Órganos , Oxidación-Reducción , Estrés Oxidativo , Complejo de la Endopetidasa Proteasomal/fisiología , Médula Espinal/química , Médula Espinal/fisiopatología , Ubiquitina/metabolismoRESUMEN
Abnormal solubility and aggregation of alpha-synuclein have been observed in the frontal cortex in three cases with Pick's disease (PiD) when compared with age-matched controls. Bands of 45 kDa and higher molecular weight were detected in the SDS-soluble fractions only in PiD. Patterns in PiD differed from that observed in the cerebral cortex in Lewy body diseases which were examined in parallel. Immunoblots to alpha-synuclein nitrated in tyrosines revealed bands of 45 and 60 kDa in Dxc- and SDS-soluble fractions in the frontal cortex (which is vulnerable to PiD) but not in the occipital cortex (which is resistant to this degenerative disease). Moreover, nitrated alpha-synuclein was found in Lewy bodies and neurites in synucleinopathies but diffusely in the cytoplasm of scattered neurons in PiD. These findings demonstrate abnormal and distinct alpha-synuclein solubility and aggregation, and alpha-synuclein nitration without formation of Lewy bodies in the frontal cortex in PiD.
Asunto(s)
Lóbulo Frontal/metabolismo , Enfermedad de Pick/metabolismo , Enfermedad de Pick/patología , alfa-Sinucleína/metabolismo , Anciano , Western Blotting/métodos , Estudios de Casos y Controles , Femenino , Humanos , Inmunohistoquímica/métodos , Masculino , Peso Molecular , Enfermedad de Parkinson/metabolismo , Cambios Post Mortem , Tirosina/metabolismoRESUMEN
Group I metabotropic glutamate receptors (mGluR1) regulate synaptic transmission through the stimulation of phospholipase Cbeta1 (PLCbeta1) and then by the activation of protein kinase C (PKC). Considering these properties, it is conceivable that major cortical functional deficits may be attributed to abnormal mGluR processing and signaling. The present work examines mGluRI expression and signaling in the frontal cortex (area 8) of 3 cases with Pick disease (PiD), a neurodegenerative disease with abnormal phospho-tau accumulation, in comparison with 3 age-matched controls by means of glutamate binding assays, enzymatic activity, gel electrophoresis and Western blotting, solubility and immunoprecipitation assays, and confocal microscopy. Reduced expression levels of PLCbeta1 and reduced PLCbeta1 activity have been found in PiD. The expression levels of the nonrelated phospholipase PLCgamma, a substrate of tyrosine kinase, are also reduced in PiD. This is accompanied by a marked decrease in the expression of cPKCalpha and increased expression of the inner band (76 kDa) of the nPKCdelta doublet at the expense of a decrease of the phosphorylated (active) form (78 kDa). In contrast, L-[3H]glutamate-specific binding to mGluRs is augmented in PiD cases, mainly because of the higher mGluR1 and mGluRs expression levels detected. No modifications in PLCbeta1 solubility have been observed in PiD and no interactions between PLCbeta1 and tau have been demonstrated in diseased and control cases. Moreover, double-labeling immunofluorescence and confocal microscopy have shown no colocalization of phospho-tau (AT8 antibody) and PLCbeta1 in phospho-tau inclusions, including Pick bodies. These results demontrate for the first time abnormal mGluR signaling in the cerebral cortex in PiD and selective vulnerability of phospholipases and PKC to PiD.
Asunto(s)
Lóbulo Frontal/metabolismo , Enfermedad de Pick/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Anciano , Western Blotting , Femenino , Técnica del Anticuerpo Fluorescente , Lóbulo Frontal/patología , Ácido Glutámico/metabolismo , Humanos , Inmunoprecipitación , Isoenzimas/metabolismo , Masculino , Microscopía Confocal , Fosfolipasa C beta , Fosfolipasa C gamma , Enfermedad de Pick/patología , Proteína Quinasa C/metabolismo , Proteína Quinasa C-alfa , Proteína Quinasa C-delta , Fosfolipasas de Tipo C/metabolismoRESUMEN
The aim of the present work was to analyze the status of metabotropic glutamate receptors (mGluRs) in the frontal cortex (area 8) from ten cases with common form DLB (cDLB) and eleven cases with pure AD in comparison with five age-matched controls. mGluRs, determined by radioligand binding assays, were significantly decreased in cerebral cortex in cDLB. This decrease was already present in cases with early AD changes not involving the frontal cortex, but dramatically correlated with AD neuropathological changes, at its greatest in isocortical stages, which was associated with a decrease in the expression levels of mGluR1 detected by Western blotting. Moreover, mGluRs analyzed in pure AD were lower than those obtained in cDLB and also correlated with progression of illness. On the other hand, the expression levels of phospholipase Cbeta1 (PLCbeta1) isoform, which is the effector of group I mGluRs, was decreased in parallel in cDLB cases. Finally, the PLCbeta1 decrease was associated with reduced GTP- and l-glutamate-stimulated PLC activity in both cDLB and AD cases. These results show that group I mGluRs/PLC signaling are down-regulated and desensitized in the frontal cortex in cDLB and AD cases and that these modifications worsen with progression of AD changes in the cerebral neocortex. Therefore, group I mGluR dysfunction may be implicated in the pathogenesis of cognitive impairment and dementia in common form of DLB and pure AD.