RESUMEN
The blood-brain barrier (BBB) plays important roles in both the physiological and pharmacological state of the brain. Transiently enhancing the permeability of the BBB may allow use of more types of medications for neuropsychiatric diseases. Several studies have demonstrated that seizures cause a transient decrease in BBB integrity. We studied the timing of BBB changes following seizures and the role of astrocytes in this process. Rats received 10 applications of electroconvulsive stimulation (ECS). They were then infused with sodium fluorescein, a fluorescent substance that rarely passes the BBB, via the inferior vena cava. After 120min of circulation, the amount of sodium fluorescein in the brain was measured by two methods in vivo fluorescence imaging (total radiant efficiency) and the brain concentration of sodium fluorescein. To assess any changes to the BBB, we measured S100Β in serum, which is a standard marker of BBB breakdown that is expressed by astrocytes. We also examined ultrastructural changes following ECS. Total radiant efficiency and the brain concentration of sodium fluorescein were significantly increased in treated rats compared to controls when sodium fluorescein was injected immediately after ECS but not when the injection was performed more than 15 min after ECS. Astrocytic endfeet showed swelling around brain capillaries following ECS. In conclusion, ECS transiently enhances the permeability of the BBB, which may be accompanied by changes in astrocytic endfeet.
Asunto(s)
Astrocitos/fisiología , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar/fisiología , Electrochoque , Animales , Astrocitos/ultraestructura , Barrera Hematoencefálica/ultraestructura , Femenino , Fluoresceína/farmacocinética , Colorantes Fluorescentes/farmacocinética , Microscopía Electrónica , Actividad Motora , Imagen Óptica , Distribución Aleatoria , Ratas Wistar , Subunidad beta de la Proteína de Unión al Calcio S100/sangre , Convulsiones/metabolismo , Convulsiones/patología , Espectrometría de FluorescenciaRESUMEN
Clinical phenotypes of hereditary diffuse leukoencephalopathy with spheroids (HDLS), a familial progressive neurodegenerative disorder affecting the white matter of the brain, are heterogenous and may include behavioral and personality changes, memory impairment, parkinsonism, seizure, and spasticity. Thus, HDLS is frequently unrecognized and misdiagnosed. Heterozygous mutations located within the kinase domain of the gene encoding the colony-stimulating factor 1 receptor (CSF1R), a cell surface receptor with key roles in development and innate immunity, have been shown in HDLS. These different gene mutations may be related to the various clinical phenotypes. We report here a newly identified family with HDLS harboring a mutation in the CSF1R gene. We examined clinical and neuropathological features in three members of this family. These patients presented with affective incontinence, memory impairment, and executive dysfunction at onset, and revealed nonfluent aphasia, parkinsonism, and seizure as the disease progressed. We identified a novel CSF1R splice site mutation (c.2442+2T>C) in intron 18 for two of the patients. MRI of these patients revealed progressive, frontotemporal-predominant, confluent leukoencephalopathy. We also observed severe myelin loss, axonal degeneration, and abundant axonal spheroids, astrocytes, and microglia in the cerebral white matter, consistent with HDLS neuropathological features. Additionally, we identified atypical neuropathological findings for HDLS, including neuronal loss and gliosis with ballooned neurons and central chromatolysis in the frontal cortex and hippocampus. This report provides further evidence for the clinical and neuropathological heterogeneity of HDLS.
Asunto(s)
Leucoencefalopatías/genética , Mutación , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Adulto , Familia , Resultado Fatal , Lóbulo Frontal/diagnóstico por imagen , Lóbulo Frontal/patología , Hipocampo/diagnóstico por imagen , Hipocampo/patología , Humanos , Intrones , Leucoencefalopatías/diagnóstico por imagen , Leucoencefalopatías/patología , Masculino , Persona de Mediana EdadRESUMEN
In our previous studies, we reported that the activity of an anti-oxidant enzyme, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) became decreased as the result of glycation in vitro and in vivo. Glycated Cu,Zn-SOD produces hydroxyl radicals in the presence of transition metals due to the formation of a Schiff base adduct and a subsequent Amadori product. This results in the site-specific cleavage of the molecule, followed by random fragmentation. The glycation of other anti-oxidant enzymes such as glutathione peroxidase and thioredoxin reductase results in a loss or decrease in enzyme activity under pathological conditions, resulting in oxidative stress. The inactivation of anti-oxidant enzymes induces oxidative stress in aging, diabetes and neurodegenerative disorders. It is well known that the levels of Amadori products and N(e)-(carboxylmethyl)lysine (CML) and other carbonyl compounds are increased in diabetes, a situation that will be discussed by the other authors in this special issue. We and others, reported that the glycation products accumulate in the brains of patients with Alzheimer's disease (AD) patients as well as in cerebrospinal fluid (CSF), suggesting that glycation plays a pivotal role in the development of AD. We also showed that enzymatic glycosylation is implicated in the pathogenesis of AD and that oxidative stress is also important in this process. Specific types of glycosylation reactions were found to be up- or downregulated in AD patients, and key AD-related molecules including the amyloid-precursor protein (APP), tau, and APP-cleaving enzymes were shown to be functionally modified as the result of glycosylation. These results suggest that glycation as well as glycosylation are involved in oxidative stress that is associated with aging, diabetes and neurodegenerative diseases such as AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Superóxido Dismutasa-1/metabolismo , Proteínas tau/metabolismo , Precursor de Proteína beta-Amiloide/química , Animales , Encéfalo/patología , Glicosilación , Humanos , Superóxido Dismutasa-1/química , Proteínas tau/químicaRESUMEN
Impaired clearance of soluble Aß (amyloid-ß) promotes Aß aggregation in brains with Alzheimer's disease (AD), while apolipoprotein-E (ApoE) in microglia mediates Aß clearance. We studied the protease responsible for ApoE(4) degradation in human peripheral monocyte extracts, which are from the same lineage as microglia. We detected the hydrolytic activity for ApoE(4) in high-salt extracts with 2 M NaCl and found that the activity was inhibited by a serine protease inhibitor and an elastase-specific inhibitor, but not by other protease inhibitors. The extracts exhibited higher activity for the elastase substrate, and we followed the activity with ion-exchange and gel-filtration chromatography. Through silver staining, we partially purified a protein of 28 kDa, which was clarified as elastase by liquid chromatography-tandem mass spectrometry. These observations suggest that elastase is the key protease for ApoE(4) degradation. We also detected ApoE(4) hydrolytic activity in high-salt extracts in mouse microglial (BV-2) cell lysates, and showed that the ApoE(4) fragments by the BV-2 extracts differed from the fragments by the monocyte extracts. Though the ApoE(4) degradation by the extracts was not inhibited with elastase-specific inhibitors, it was inhibited by an elastase-specific monoclonal antibody, suggesting that elastase-like proteases in microglia differ from those of monocytes. Immunohistochemistry revealed that both elastase and ApoE were expressed in the senile plaques of brains with AD. In vitro studies also disclosed the localization of elastase in the microglial cell line, BV-2. Our results suggest that elastase-like proteases in the microglial cells surrounding Aß plaques are responsible for ApoE degradation in the brain.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Apolipoproteína E4/metabolismo , Encéfalo/metabolismo , Elastasa de Leucocito/metabolismo , Microglía/metabolismo , Proteolisis , Anciano , Enfermedad de Alzheimer/patología , Encéfalo/patología , Línea Celular , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Microglía/patología , Monocitos/enzimología , Monocitos/patologíaRESUMEN
Lysosome-associated membrane protein-2 (LAMP-2) is the gene responsible for Danon disease, which is characterized by cardiomyopathy, autophagic vacuolar myopathy, and variable mental retardation. To elucidate the function of LAMP-2 in the central nervous system, we investigated the neuropathological changes in Lamp-2-deficient mice. Immunohistochemical observations revealed that Lamp-1 and cathepsin D-positive lysosomal structures increased in the large neurons of the mouse brain. Ubiquitin-immunoreactive aggregates and concanavalin A-positive materials were detected in these neurons. By means of ultrastructural studies, we found various-shaped accumulations, including lipofuscin, glycolipid-like materials, and membranous structures, in the neurons and glial cells of Lamp-2-deficient brains. In deficient mice, glycogen granules accumulated in hepatocyte lysosomes but were not observed in neurons. These pathological features indicate lysosomal storage disease; however, the findings are unlikely a consequence of deficiency of a single lysosomal enzyme. Although previous study results have shown a large amount of autophagic vacuoles in parenchymal cells of the visceral organs, these findings were rarely detected in the brain tissue except for some axons in the substantia nigra, in which abundant activated microglial cells with increased lipid peroxidation were observed. Thus, LAMP-2 in the central nervous system has a possible role in the degradation of the various macromolecules in lysosomes and an additional function concerning protection from oxidative stress, especially in the substantia nigra.
Asunto(s)
Enfermedades por Almacenamiento Lisosomal/patología , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/patología , Mesencéfalo/patología , Neuronas/patología , Animales , Modelos Animales de Enfermedad , Glucógeno/metabolismo , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Lisosomas/metabolismo , Masculino , Mesencéfalo/metabolismo , Ratones , Ratones Noqueados , Neuronas/metabolismoRESUMEN
Lysosomes are the primary catabolic compartment for the degradation of intracellular proteins through autophagy. The presence of abnormal intracellular α-synuclein-positive aggregates in Parkinson's disease (PD) indicates that the degradative capacity of lysosomes is impaired in PD. Specific dysfunction of chaperone-mediated autophagy (CMA) in PD is suggested by reductions in the CMA membrane receptor, lysosomal-associated membrane protein (LAMP) 2A, although whether LAMP2A is the only LAMP2 isoform affected by PD is unknown. Messenger RNA (mRNA) and protein expression of all three LAMP2 isoforms was assessed in brain extracts from regions with and without PD-related increases in α-synuclein in autopsy samples from subjects in the early pathological stage of PD (n = 9), compared to age- and postmortem delay-matched controls (n = 10). In the early stages of PD, mRNA expression of all LAMP2 isoforms was not different from controls, with LAMP2B and LAMP2C protein levels also unchanged in PD. The selective loss of LAMP2A protein directly correlated with the increased levels of α-synuclein and decreased levels of the CMA chaperone heat shock cognate protein 70 in the same PD samples, as well as with the accumulation of cytosolic CMA substrate proteins. Our data show that LAMP2 protein isoforms are differentially affected in the early stages of PD, with LAMP2A selectively reduced in association with increased α-synuclein, and suggests that dysregulation of CMA-mediated protein degradation occurs before substantial α-synuclein aggregation in PD.
Asunto(s)
Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Isoformas de Proteínas/metabolismo , Anciano , Anciano de 80 o más Años , Encéfalo/metabolismo , Colesterol/metabolismo , Femenino , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Metabolismo de los Lípidos , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Estadísticas no Paramétricas , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Danon disease is an X-linked disorder clinically characterized by the triad of hypertrophic cardiomyopathy, myopathy, and intellectual disability. Cardiomyopathy is a severe and life-threatening problem, for which cardiac transplantation is the only therapeutic option. The most striking finding in muscle biopsy samples is small basophilic granules scattered in myofibers, which are in fact small autophagic vacuoles surrounded by membranes with sarcolemmal features characterized by the recruitment of sarcolemmal proteins and acetylcholine esterase and by the presence of basal lamina on its luminal side. The mechanism underlying the formation of these autophagic vacuoles with unique sarcolemmal features (AVSF) still remains a mystery and its origin is unknown. In heart, cardiomyocytes show dramatically increased vacuolation and degenerative features, including myofibrillar disruption and lipofuscin accumulation. In brain, pale granular neurons and neurons with lipofuscin-like granules may be seen. Danon disease is caused by loss-of-function mutations in the LAMP2 gene, which encodes lysosome-associated membrane protein 2 (LAMP-2), a single-spanned transmembrane protein localized in the limiting membranes of lysosomes and late endosomes. Most mutations lead to splicing defects or protein truncation, resulting in a loss of transmembrane and/or cytoplasmic domains, leading to LAMP-2 protein deficiency. LAMP-2 is required for the maturation of autophagosomes by fusion with lysosomes; therefore, LAMP-2 deficiency leads to a failure in macroautophagy. There are three LAMP-2 isoforms, LAMP-2A, -2B, and -2C. Clinical features of Danon disease are thought to be mediated by loss of the LAMP-2B isoform which is the major isoform expressed in muscle. It is also known that LAMP-2 plays a role in chaperone-mediated autophagy and RNA- and DNA-targeting autophagy. However, the precise pathophysiological mechanism through which LAMP-2 deficiency causes Danon disease is still not fully understood and its elucidation would promote the development of new therapies.
Asunto(s)
Autofagia/genética , Enfermedad por Depósito de Glucógeno de Tipo IIb/genética , Enfermedad por Depósito de Glucógeno de Tipo IIb/patología , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Humanos , FenotipoRESUMEN
The seeding of amyloid-ß 40 (Aß40) oligomers from monomers is the initial step of Aß aggregation, and many reports have suggested that cholesterol enhances this step. We studied the potential of secosteroid vitamin D derivatives for Aß40 aggregation in vitro. The quartz-crystal microbalance technique demonstrated that vitamin D3 does not show any effect on Aß40 aggregation while vitamin D2 promoted it and docking simulation but that vitamin D2 has high potential in this regard. Thus, stacking of the Phe19 benzene ring in Aß40 and the C22-C23 double bond in vitamin D2 may alter the energy of these molecules. Electron microscopy revealed the potential of vitamin D2 to increase Aß40 aggregation. Thioflavin-T assays indicated that Vitamin D2 induced increased fluorescence at 490 nm, as typically observed for amyloid fibrils but also for protofibrils; in both cases this reflects of the increase of ß-sheet contents. Aß40 aggregation was further confirmed in ELISA, SDS-PAGE and dot blot analysis which revealed changes in protease K resistance. These results suggest a possible mechanism, of how vitamin D2 could increase Aß40 aggregation and the docking simulation explains, why the same is not observed with vitamin D3.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Colecalciferol/metabolismo , Ergocalciferoles/metabolismo , Fragmentos de Péptidos/metabolismo , Placa Amiloide/complicaciones , Péptidos beta-Amiloides/ultraestructura , Benzotiazoles , Colecalciferol/química , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Ergocalciferoles/química , Humanos , Técnicas In Vitro , Microscopía Electrónica , Modelos Químicos , Simulación del Acoplamiento Molecular , Fragmentos de Péptidos/ultraestructura , Placa Amiloide/química , Placa Amiloide/ultraestructura , Mapeo de Interacción de Proteínas , Tecnicas de Microbalanza del Cristal de Cuarzo , Tiazoles/metabolismo , Factores de TiempoRESUMEN
Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered "RNautophagy," an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term "DNautophagy," is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa.
Asunto(s)
ADN/metabolismo , Lisosomas/metabolismo , Animales , Citosol/metabolismo , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
Regulated degradation of cellular components by lysosomes is essential to maintain biological homeostasis. In mammals, three forms of autophagy, macroautophagy, microautophagy and chaperone-mediated autophagy (CMA), have been identified. Here, we showed a novel type of autophagy, in which RNA is taken up directly into lysosomes for degradation. This pathway, which we term "RNautophagy," is ATP-dependent, and unlike CMA, is independent of HSPA8/Hsc70. LAMP2C, a lysosomal membrane protein, serves as a receptor for this pathway. The cytosolic tail of LAMP2C specifically binds to almost all total RNA derived from mouse brain. The cytosolic sequence of LAMP2C and its affinity for RNA are evolutionarily conserved from nematodes to humans. Our findings shed light on the mechanisms underlying RNA homeostasis in higher eukaryotes.
Asunto(s)
Autofagia , Lisosomas/metabolismo , ARN/metabolismo , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Secuencia Conservada , Células HeLa , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Ratones , Ratones Noqueados , Homología de Secuencia de AminoácidoAsunto(s)
Envejecimiento/genética , Enfermedad por Depósito de Glucógeno de Tipo IIb/genética , Enfermedad por Depósito de Glucógeno de Tipo IIb/patología , Proteínas de Membrana de los Lisosomas/deficiencia , Lisosomas/patología , Adolescente , Adulto , Amilasas/metabolismo , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas , Lisosomas/metabolismo , Lisosomas/ultraestructura , Masculino , Microscopía Electrónica de Transmisión , Proteínas de Microtúbulos/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Ubiquitina/metabolismo , Adulto Joven , Proteínas tau/metabolismoRESUMEN
Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide that acts as a neuromodulator in the CNS. Recently, secretion of several functional molecules has been identified in VIP-stimulated astrocytes in vitro. However, the relationship between VIP and its specific receptors in neurological disorders remains unknown. To investigate the role of the VIP system under pathological conditions, we performed a cold injury on the right cerebrum of adult C57BL/6 mice and observed expression patterns for VIP and its receptor, VPAC2. Immunohistochemical studies revealed VPAC2 expression in reactive astrocytes around the core lesion by post-injury day 7, which then returned to contralateral levels at post-injury day 14. By contrast, VIP immunoreactivity was detected in activated microglial cells, suggesting that microglia-astrocyte interactions in the VIP/VPAC2 system are important for the tissue repair process. In primary cultured astrocytes stimulated with N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate sodium salt (dbcAMP) to mimic reactive astrocytosis, VPAC2 mRNA expression was highly up-regulated compared to that of the other VIP receptors, PAC1 and VPAC1. VPAC2 activation by the selective VPAC2 agonist, Ro25-1553, induced reactive morphological and biochemical changes from a polygonal shape to a stellate shape in cultured astrocytes. Further, Ro25-1553 increased cell surface expression of the glutamate transporters GLAST and GLT-1, which can limit excitotoxic neuronal cell death. In summary, the transient expression of VPAC2 in reactive astrocytes and the up-regulation of functional glutamate transporters suggest that the VIP/VPAC2 system induces reactive astrocytosis and plays a key role in neuroprotection against excitotoxicity in neurological disorders.
Asunto(s)
Transportador 1 de Aminoácidos Excitadores/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Gliosis/metabolismo , Receptores de Tipo II del Péptido Intestinal Vasoactivo/metabolismo , Péptido Intestinal Vasoactivo/metabolismo , Animales , Astrocitos/metabolismo , Western Blotting , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiologíaRESUMEN
Lipid peroxidation has many deleterious effects on cells, and in the nervous system is considered to be involved in the pathogenesis of neurodegenerative diseases. To suppress lipid peroxidation, cells have various defense systems such as glutathione and thioredoxin, and defects in these defense systems will result in disturbance of normal cellular functions. Here we report that deficiency of ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1) leads to vulnerability to lipid peroxidation both in vivo and in vitro, through analyses of the UCH-L1-deficient mutant mouse gracile axonal dystrophy (gad). In the gracile fasciculus of gad mice, punctate deposits were observed to be immunoreactive for 4-hydroxy-2-nonenal, a by-product of lipid peroxidation. The motor deficits of gad mice were worsened by a diet deficient in vitamin E. When neurons from dorsal root ganglions (DRG) were cultured in the vitamin E-free medium, cell death was increased in the neurons of gad mice. These data suggest that UCH-L1 has a function in protecting DRG neurons from lipid peroxidation. Further, we describe newly identified properties: that UCH-L1 is localized on the inside of the plasma membrane of DRG neurons, and that UCH-L1 binds to phosphatidic acid according to the redox status and presence of mono-ubiquitin protein. These findings will provide clues for elucidating the physiological function of UCH-L1.
Asunto(s)
Peroxidación de Lípido , Ubiquitina Tiolesterasa/metabolismo , Animales , Membrana Celular/enzimología , Células Cultivadas , Ganglios Espinales/enzimología , Inmunohistoquímica , Ratones , Ratones Mutantes , Ácidos Fosfatidicos/metabolismo , Ubiquitina Tiolesterasa/químicaRESUMEN
2-[Bis(pentafluorophenyl)boryl]azobenzenes bearing hydrogen, methoxy, dimethylamino, trifluoromethyl, fluoro, n-butyl, and tert-butyldimethylsiloxy groups at the 4'-position or methoxy and bromo groups at the 4-position have been synthesized. The 4-bromo group of the 2-boryl-4-bromoazobenzene derivative was converted to phenyl and diphenylamino groups by palladium-catalyzed reactions. The absorption and fluorescence properties have been investigated using UV/Vis and fluorescence spectroscopy. The 2-borylazobenzenes emitted an intense green, yellow, and orange fluorescence, in marked contrast to the usual azobenzene fluorescence. The 4'-siloxy derivative showed the highest fluorescence quantum yield (0.90) among those reported for azobenzenes to date. The correlation between the substituent and the fluorescence properties was elucidated by studying the effect of the substituent on the relaxation process and from DFT and TD-DFT calculations. An electron-donating group at the 4'-position was found to be important for an intense emission. Application of fluorescent azobenzenes as a fluorescent vital stain for the visualization of living tissues was also investigated by microinjection into Xenopus embryos, suggesting these compounds are nontoxic towards embryos.
Asunto(s)
Compuestos Azo/síntesis química , Boranos/síntesis química , Fluorescencia , Colorantes Fluorescentes/síntesis química , Animales , Compuestos Azo/química , Compuestos Azo/toxicidad , Boranos/química , Boranos/toxicidad , Catálisis , Cristalografía por Rayos X , Embrión no Mamífero/efectos de los fármacos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/toxicidad , Microinyecciones , Modelos Químicos , Estructura Molecular , Paladio/química , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Xenopus/embriologíaRESUMEN
Parkinson disease (PD) is the most common neurodegenerative movement disorder. An increase in the amount of alpha-synuclein protein could constitute a cause of PD. Alpha-synuclein is degraded at least partly by chaperone-mediated autophagy (CMA). The I93M mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is associated with familial PD. However, the relationship between alpha-synuclein and UCH-L1 in the pathogenesis of PD has remained largely unclear. In this study, we found that UCH-L1 physically interacts with LAMP-2A, the lysosomal receptor for CMA, and Hsc70 and Hsp90, which can function as components of the CMA pathway. These interactions were abnormally enhanced by the I93M mutation and were independent of the monoubiquitin binding of UCH-L1. In a cell-free system, UCH-L1 directly interacted with the cytosolic region of LAMP-2A. Expression of I93M UCH-L1 in cells induced the CMA inhibition-associated increase in the amount of alpha-synuclein. Our findings may provide novel insights into the molecular links between alpha-synuclein and UCH-L1 and suggest that aberrant interaction of mutant UCH-L1 with CMA machinery, at least partly, underlies the pathogenesis of PD associated with I93M UCH-L1.
Asunto(s)
Autofagia , Proteínas de Membrana de los Lisosomas/metabolismo , Mutación , Enfermedad de Parkinson/metabolismo , Ubiquitina Tiolesterasa/metabolismo , alfa-Sinucleína/metabolismo , Animales , Autofagia/genética , Células COS , Chlorocebus aethiops , Proteínas del Choque Térmico HSC70/genética , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas/genética , Ratones , Células 3T3 NIH , Enfermedad de Parkinson/genética , Estructura Terciaria de Proteína/genética , Ubiquitina Tiolesterasa/genética , alfa-Sinucleína/genéticaRESUMEN
G-protein coupled receptors (GPCRs) form the largest superfamily of membrane proteins. About 50% of medicines are thought to target GPCRs. We recently developed a novel strategy to screen GPCRs that are highly or selectively expressed in particular cells of the brain. Since recent literature suggests causative roles of glial cell dysfunction in many neuropsychiatric disorders, we first characterized GPCRs expressed in cultured astrocytes and neural progenitor cells (NPCs) using the method. Among approximately 300 GPCRs expressed in the adult mouse brain, we found that type 2 neurotensin receptor (Ntsr2) was abundantly expressed in cultured astrocytes. In situ hybridization of Ntsr2 and co-immunostaining of GFAP confirmed that the molecule was expressed in astrocytes of the adult mouse brain. Mice lacking Ntsr2 showed altered emotional behaviors. Application of a Ntsr2 agonist modified the behavior of wild type mice. In NPCs, PACAP receptor (PAC1) was identified as one of the highly expressed GPCRs. Previously the PACAP/PAC1 system was reported to induce differentiation of NPCs. We observed that PACAP and PAC1 were co-localized in NPCs of the mouse embryonic cortex and that activation of the PACAP/PAC1 system potentiated growth factor-induced proliferation of the glial progenitors. Furthermore, VPAC2, a structurally related GPCR to PAC1, was detected in reactive astrocytes in vivo. These observations suggest potential roles of Ntsr2, PAC1 and VPAC2 in the development, expression and maintenance of the brain function. Further study on glial GPCRs should provide important information for the role of astrocytes in the processing of neural information.
Asunto(s)
Encéfalo/fisiología , Neuroglía/fisiología , Receptores Acoplados a Proteínas G/fisiología , Animales , Astrocitos/fisiología , Encéfalo/crecimiento & desarrollo , Ratones , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Receptores de Neurotensina/fisiología , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/fisiología , Receptores de Tipo II del Péptido Intestinal Vasoactivo/fisiologíaRESUMEN
The Pituitary adenylate cyclase-activating peptide (PACAP) ligand/type 1 receptor (PAC1) system regulates neurogenesis and gliogenesis. It has been well established that the PACAP/PAC1 system induces differentiation of neural progenitor cells (NPCs) through the Gs-mediated cAMP-dependent signaling pathway. However, it is unknown whether this ligand/receptor system has a function in proliferation of NPCs. In this study, we identified that PACAP and PAC1 were highly expressed and co-localized in NPCs of mouse cortex at embryonic day 14.5 (E14.5) and found that the PACAP/PAC1 system potentiated growth factor-induced proliferation of mouse cortical NPCs at E14.5 via Gq-, but not Gs-, mediated PLC/IP3-dependent signaling pathway in an autocrine manner. Moreover, PAC1 activation induced elongation of cellular processes and a stellate morphology in astrocytes that had the bromodeoxyuridine (BrdU)-incorporating ability of NPCs. Consistent with this notion, we determined that the most BrdU positive NPCs differentiated to astrocytes through PAC1 signaling. These results suggest that the PACAP/PAC1 system may play a dual role in neural/glial progenitor cells not only differentiation but also proliferation in the cortical astrocyte lineage via Ca2+-dependent signaling pathways through PAC1.
Asunto(s)
Astrocitos/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Células Madre/metabolismo , Animales , Astrocitos/citología , Comunicación Autocrina/fisiología , Bromodesoxiuridina , Señalización del Calcio/fisiología , Linaje de la Célula/fisiología , Forma de la Célula/efectos de los fármacos , Forma de la Célula/fisiología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/efectos de los fármacos , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Madre/citología , Fosfolipasas de Tipo C/efectos de los fármacos , Fosfolipasas de Tipo C/metabolismoRESUMEN
The I93M mutation in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was reported in one German family with autosomal dominant Parkinson's disease (PD). The causative role of the mutation has, however, been questioned. We generated transgenic (Tg) mice carrying human UCHL1 under control of the PDGF-B promoter; two independent lines were generated with the I93M mutation (a high- and low-expressing line) and one line with wild-type human UCH-L1. We found a significant reduction in the dopaminergic neurons in the substantia nigra and the dopamine content in the striatum in the high-expressing I93M Tg mice as compared with non-Tg mice at 20 weeks of age. Although these changes were absent in the low-expressing I93M Tg mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment profoundly reduced dopaminergic neurons in this line as compared with wild-type Tg or non-Tg mice. Abnormal neuropathologies were also observed, such as silver staining-positive argyrophilic grains in the perikarya of degenerating dopaminergic neurons, in I93M Tg mice. The midbrains of I93M Tg mice contained increased amounts of insoluble UCH-L1 as compared with those of non-Tg mice, perhaps resulting in a toxic gain of function. Collectively, our data represent in vivo evidence that expression of UCHL1(I93M) leads to the degeneration of dopaminergic neurons.
Asunto(s)
Dopamina/metabolismo , Mutación , Neuronas/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
UCH-L3 belongs to the ubiquitin C-terminal hydrolase family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system. A murine Uchl3 deletion mutant displays retinal degeneration, muscular degeneration, and mild growth retardation. To elucidate the function of UCH-L3, we investigated histopathological changes and expression of apoptosis- and oxidative stress-related proteins during retinal degeneration. In the normal retina, UCH-L3 was enriched in the photoreceptor inner segment that contains abundant mitochondria. Although the retina of Uchl3-deficient mice showed no significant morphological abnormalities during retinal development, prominent retinal degeneration became manifested after 3 weeks of age associated with photoreceptor cell apoptosis. Ultrastructurally, a decreased area of mitochondrial cristae and vacuolar changes were observed in the degenerated inner segment. Increased immunoreactivities for manganese superoxide dismutase, cytochrome c oxidase I, and apoptosis-inducing factor in the inner segment indicated mitochondrial oxidative stress. Expression of cytochrome c, caspase-1, and cleaved caspase-3 did not differ between wild-type and mutant mice; however, immunoreactivity for endonuclease G was found in the photoreceptor nuclei in the mutant retina. Hence, loss of UCH-L3 leads to mitochondrial oxidative stress-related photoreceptor cell apoptosis in a caspase-independent manner. Thus, Uchl3-deficient mice represent a model for adult-onset retinal degeneration associated with mitochondrial impairment.
Asunto(s)
Apoptosis/fisiología , Estrés Oxidativo/fisiología , Células Fotorreceptoras/patología , Retina/metabolismo , Degeneración Retiniana/patología , Ubiquitina Tiolesterasa/metabolismo , Animales , Western Blotting , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Microscopía Electrónica de Transmisión , Mitocondrias/patología , Mitocondrias/ultraestructura , Células Fotorreceptoras/citología , Retina/patología , Retina/ultraestructura , Degeneración Retiniana/enzimologíaRESUMEN
Glutamate transporters are thought to have an important role in central nervous system (CNS) development. We investigated the expression of the sodium-dependent high-affinity glutamate transporters EAAT1, EAAT2, and EAAT3 in 11 human autopsied cases without neurological disorders and in four cases with type II lissencephaly including Walker Warburg's syndrome (WWS) and Fukuyama-type congenital muscular dystrophy (FCMD), both of which are classified as migration disorders of the human brain. Expression of glutamate transporter subtypes was differentially regulated during normal human corticogenesis. Although EAAT1 and EAAT2 were mainly localized to the cortical astrocytes in the postnatal brain, EAAT1 was enriched in the proliferative zones and radial glia from 13 gestational weeks (GW) to 20 GW. EAAT2 was abundant in the intermediate zone until 23 GW, and transiently expressed in the radial fibers of the transitional form of radial glia into mature astrocytes as well as partly in the corticofugal axonal bundles. EAAT3 immunoreactivity was robust in the apical dendrites of the pyramidal neurons in the marginal zone and cortical plate during corticogenesis, and decreased postnatally. In the individuals with type II lissencephaly, glutamate transporters were expressed in the extrusion of neuroglial tissue. Bundles of EAAT2-immunoreactive radial fibers were prominent in the specimens at 20 GW. Thus, glutamate transporters are differentially regulated during normal and impaired corticogenesis. Altered glutamate transporter expression in type II lissencephaly suggests that glutamate metabolism is involved in the formation of the normal cortex and contributes to the disorganized cortex seen in migration disorders.