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The heat shock factor 1 (HSF1)-mediated stress response pathway and autophagy processes play important roles in the maintenance of proteostasis. Autophagy processes are subdivided into three subtypes: macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. Recently, molecular chaperones and co-factors were shown to be involved in the selective degradation of substrates by these three autophagy processes. This evidence suggests that autophagy processes are regulated in a coordinated manner by the HSF1-mediated stress response pathway. Recently, various studies have demonstrated that proteostasis pathways including HSF1 and autophagy are implicated in longevity. Furthermore, they serve as therapeutic targets for aging-related diseases such as cancer and neurodegenerative diseases. In the future, these studies will underpin the development of therapies against various diseases.
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Autofagia , Autofagia Mediada por Chaperones , Macroautofagia , Microautofagia , LongevidadRESUMEN
Parkinson's disease (PD) is characterized by dopaminergic (DA) cell loss and the accumulation of pathological alpha synuclein (asyn), but its precise pathomechanism remains unclear, and no appropriate animal model has yet been established. Recent studies have shown that a heterozygous mutation of glucocerebrosidase (gba) is one of the most important genetic risk factors in PD. To create mouse model for PD, we crossed asyn Bacterial Artificial Chromosome transgenic mice with gba heterozygous knockout mice. These double-mutant (dm) mice express human asyn in a physiological manner through its native promoter and showed an increase in phosphorylated asyn in the regions vulnerable to PD, such as the olfactory bulb and dorsal motor nucleus of the vagus nerve. Only dm mice showed a significant reduction in DA cells in the substantia nigra pars compacta, suggesting these animals were suitable for a prodromal model of PD. Next, we investigated the in vivo mechanism by which GBA insufficiency accelerates PD pathology, focusing on lipid metabolism. Dm mice showed an increased level of glucosylsphingosine without any noticeable accumulation of glucosylceramide, a direct substrate of GBA. In addition, the overexpression of asyn resulted in decreased GBA activity in mice, while dm mice tended to show an even further decreased level of GBA activity. In conclusion, we created a novel prodromal mouse model to study the disease pathogenesis and develop novel therapeutics for PD and also revealed the mechanism by which heterozygous gba deficiency contributes to PD through abnormal lipid metabolism under conditions of an altered asyn expression in vivo.
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Glucosilceramidasa/genética , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , Animales , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Metabolismo de los Lípidos/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Porción Compacta de la Sustancia Negra/metabolismo , Porción Compacta de la Sustancia Negra/patología , Síntomas ProdrómicosRESUMEN
Parkinson's disease is one of the most common movement disorders and is characterized by dopaminergic cell loss and the accumulation of pathological α-synuclein, but its precise pathogenetic mechanisms remain elusive. To develop disease-modifying therapies for Parkinson's disease, an animal model that recapitulates the pathology and symptoms of the disease, especially in the prodromal stage, is indispensable. As subjects with α-synuclein gene (SNCA) multiplication as well as point mutations develop familial Parkinson's disease and a genome-wide association study in Parkinson's disease has identified SNCA as a risk gene for Parkinson's disease, the increased expression of α-synuclein is closely associated with the aetiology of Parkinson's disease. In this study we generated bacterial artificial chromosome transgenic mice harbouring SNCA and its gene expression regulatory regions in order to maintain the native expression pattern of α-synuclein. Furthermore, to enhance the pathological properties of α-synuclein, we inserted into SNCA an A53T mutation, two single-nucleotide polymorphisms identified in a genome-wide association study in Parkinson's disease and a Rep1 polymorphism, all of which are causal of familial Parkinson's disease or increase the risk of sporadic Parkinson's disease. These A53T SNCA bacterial artificial chromosome transgenic mice showed an expression pattern of human α-synuclein very similar to that of endogenous mouse α-synuclein. They expressed truncated, oligomeric and proteinase K-resistant phosphorylated forms of α-synuclein in the regions that are specifically affected in Parkinson's disease and/or dementia with Lewy bodies, including the olfactory bulb, cerebral cortex, striatum and substantia nigra. Surprisingly, these mice exhibited rapid eye movement (REM) sleep without atonia, which is a key feature of REM sleep behaviour disorder, at as early as 5 months of age. Consistent with this observation, the REM sleep-regulating neuronal populations in the lower brainstem, including the sublaterodorsal tegmental nucleus, nuclei in the ventromedial medullary reticular formation and the pedunculopontine nuclei, expressed phosphorylated α-synuclein. In addition, they also showed hyposmia at 9 months of age, which is consistent with the significant accumulation of phosphorylated α-synuclein in the olfactory bulb. The dopaminergic neurons in the substantia nigra pars compacta degenerated, and their number was decreased in an age-dependent manner by up to 17.1% at 18 months of age compared to wild-type, although the mice did not show any related locomotor dysfunction. In conclusion, we created a novel mouse model of prodromal Parkinson's disease that showed RBD-like behaviour and hyposmia without motor symptoms.
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Encéfalo/metabolismo , Modelos Animales de Enfermedad , Ratones , Trastornos del Olfato/genética , Enfermedad de Parkinson/genética , Síntomas Prodrómicos , Trastorno de la Conducta del Sueño REM/genética , alfa-Sinucleína/genética , Animales , Recuento de Células , Cromosomas Artificiales Bacterianos , Electroencefalografía , Electromiografía , Endopeptidasa K/metabolismo , Ratones Transgénicos , Trastornos del Olfato/fisiopatología , Enfermedad de Parkinson/fisiopatología , Polimorfismo de Nucleótido Simple , Trastorno de la Conducta del Sueño REM/fisiopatología , Sueño , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND: Microglia play crucial roles in the maintenance of brain homeostasis. Activated microglia show a biphasic influence, promoting beneficial repair and causing harmful damage via M2 and M1 microglia, respectively. It is well-known that microglia are initially activated to the M2 state and subsequently switch to the M1 state, called M2-to-M1 class switching in acute ischemic models. However, the activation process of microglia in chronic and sporadic hypertension remains poorly understood. We aimed to clarify the process using a chronic hypertension model, the deoxycorticosterone acetate (DOCA)-salt-treated Wistar rats. METHODS: After unilateral nephrectomy, the rats were randomly divided into DOCA-salt, placebo, and control groups. DOCA-salt rats received a weekly subcutaneous injection of DOCA (40 mg/kg) and were continuously provided with 1% NaCl in drinking water. Placebo rats received a weekly subcutaneous injection of vehicle and were provided with tap water. Control rats received no administration of DOCA or NaCl. To investigate the temporal expression profiles of M1- and M2-specific markers for microglia, the animals were subjected to the immunohistochemical and biochemical studies after 2, 3, or 4 weeks DOCA-salt treatment. RESULTS: Hypertension occurred after 2 weeks of DOCA and salt administration, when round-shaped microglia with slightly shortened processes were observed juxtaposed to the vessels, although the histopathological findings were normal. After 3 weeks of DOCA and salt administration, M1-state perivascular and parenchyma microglia significantly increased, when local histopathological findings began to be observed but cerebrovascular destruction did not occur. On the other hand, M2-state microglia were never observed around the vessels at this period. Interestingly, prior to M1 activation, about 55% of perivascular microglia transiently expressed Ki-67, one of the cell proliferation markers. CONCLUSIONS: We concluded that the resting perivascular microglia directly switched to the pro-inflammatory M1 state via a transient proliferative state in DOCA-salt rats. Our results suggest that the activation machinery of microglia in chronic hypertension differs from acute ischemic models. Proliferative microglia are possible initial key players in the development of hypertension-induced cerebral vessel damage. Fine-tuning of microglia proliferation and activation could constitute an innovative therapeutic strategy to prevent its development.
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Encéfalo/patología , Proliferación Celular/fisiología , Hipertensión/complicaciones , Hipertensión/patología , Microglía/clasificación , Microglía/patología , Animales , Antígenos CD/metabolismo , Presión Sanguínea/efectos de los fármacos , Encéfalo/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Carboximetilcelulosa de Sodio/farmacología , Proliferación Celular/efectos de los fármacos , Acetato de Desoxicorticosterona/toxicidad , Modelos Animales de Enfermedad , Lateralidad Funcional , Hipertensión/diagnóstico por imagen , Hipertensión/etiología , Antígeno Ki-67/metabolismo , Imagen por Resonancia Magnética , Masculino , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Mineralocorticoides/toxicidad , Nefrectomía/efectos adversos , Ratas , Ratas Wistar , Cloruro de Sodio/toxicidad , Factores de TiempoRESUMEN
The formation of intracellular aggregates containing α-synuclein (α-Syn) is one of the key steps in the progression of Parkinson's disease and dementia with Lewy bodies. Recently, it was reported that pathological α-Syn fibrils can undergo cell-to-cell transmission and form Lewy body-like aggregates. However, little is known about how they form α-Syn aggregates from fibril seeds. Here, we developed an assay to study the process of aggregate formation using fluorescent protein-tagged α-Syn-expressing cells and examined the aggregate forming activity of exogenous α-Syn fibrils. α-Syn fibril-induced formation of intracellular aggregates was suppressed by a cathepsin B specific inhibitor, but not by a cathepsin D inhibitor. α-Syn fibrils pretreated with cathepsin B in vitro enhanced seeding activity in cells. Knockdown of cathepsin B also reduced fibril-induced aggregate formation. Moreover, using LAMP-1 immunocytochemistry and live-cell imaging, we observed that these aggregates initially occurred in the lysosome. They then rapidly grew larger and moved outside the boundary of the lysosome within one day. These results suggest that the lysosomal protease cathepsin B is involved in triggering intracellular aggregate formation by α-Syn fibrils.
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Catepsina B/metabolismo , Lisosomas/metabolismo , Agregación Patológica de Proteínas/metabolismo , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Catepsina B/antagonistas & inhibidores , Catepsina D/metabolismo , HumanosRESUMEN
The detection of spontaneous magnetic signals can be used for the non-invasive electrophysiological evaluation of induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). We report that deep learning with a dataset that combines magnetic signals estimated using numerical simulation and actual noise data is effective in the detection of weak biomagnetic signals. To verify the feasibility of this method, we measured artificially generated magnetic signals that mimic cellular magnetic fields using a superconducting quantum interference device and attempted peak detection using a long short-term memory network. We correctly detected 80.0% of the peaks and the method achieved superior detection performance compared with conventional methods. Next, we attempted peak detection for magnetic signals measured from mouse iPS-CMs. The number of detected peaks was consistent with the spontaneous beats counted using microscopic observation and the average peak waveform achieved good similarity with the prediction. We also observed the synchronization of peak positions between simultaneously measured field potentials and magnetic signals. Furthermore, the magnetic measurements of cell samples treated with isoproterenol showed potential for the detection of chronotropic effects. These results suggest that the proposed method is effective and has potential application in the safety assessment of regenerative medicine and drug screening.
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Aprendizaje Profundo , Células Madre Pluripotentes Inducidas , Animales , Ratones , Miocitos Cardíacos , Isoproterenol/farmacología , Evaluación Preclínica de Medicamentos , Diferenciación CelularRESUMEN
Neuropeptide Y (NPY) is a neural peptide distributed widely in the brain and has various functions in each region. We previously reported that NPY neurons in the nucleus accumbens (NAc) are involved in the regulation of anxiety behavior. Anterograde and retrograde tracing studies suggest that neurons in the NAc project to several areas, such as the lateral hypothalamus (LH) and ventral pallidum (VP), and receive afferent projections from the cortex, thalamus, and amygdala. However, the neural connections between accumbal NPY neurons and other brain areas in mice remain unclear. In this study, we sought to clarify these anatomical connections of NPY neurons in the NAc by investigating their neural outputs and inputs. To selectively map NPY neuronal efferents from the NAc, we injected Cre-dependent adeno-associated viruses (AAVs) into the NAc of NPY-Cre mice. This revealed that NAc NPY neurons exclusively projected to the LH. We confirmed this by injecting cholera toxin b subunit (CTb), a retrograde tracer, into the LH and found that approximately 7-10% of NPY neurons in the NAc were double-labeled for mCherry and CTb. Moreover, retrograde tracing using recombinant rabies virus (rRABV) also identified NAc NPY projections to the LH. Finally, we investigated monosynaptic input to the NPY neurons in the NAc using rRABV. We found that NPY neurons in the NAc received direct synaptic connections from the midline thalamic nuclei and posterior basomedial amygdala. These findings provide new insight into the neural networks of accumbal NPY neurons and should assist in elucidating their functional roles.
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α-Synuclein (αSyn), the major constituent of Lewy bodies and Lewy neurites, is generally expressed in presynapses and is involved in synaptic function. However, we previously demonstrated that some neurons, including those in the olfactory bulb, show high αSyn expression levels in the cell body under normal conditions. αSyn is also known to be important for adult neurogenesis. Thus, in present study, we examined the role of αSyn in juxtaglomerular neurons (JGNs) with high αSyn expression in the mouse olfactory bulb. Most αSyn-enriched JGNs expressed sex-determining region Y-box 2 (Sox2), which functions to maintain neural immature identity. Interestingly, in αSyn homozygous (-/-) knockout (KO) mice, Sox2-positive JGNs were significantly increased compared with heterozygous (+/-) KO mice. Following global brain ischemia using wild-type mice, there was also a significant decrease in Sox2-positive JGNs, and in the co-expression ratio of Sox2 in αSyn-enriched JGNs. By contrast, the co-expression ratio of neuronal nuclei (NeuN, mature neuronal marker) was significantly increased in αSyn-enriched JGNs. However, this ischemia-induced decrease of Sox2-positive JGNs was not observed in αSyn homozygous KO mice. Overall, these data suggest that αSyn functions to promote the maturation of immature JGNs in the mouse olfactory bulb.
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Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Bulbo Olfatorio/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Cuerpos de Lewy/metabolismo , Ratones Noqueados , Neurogénesis/fisiología , Sinapsis/metabolismo , alfa-Sinucleína/genéticaRESUMEN
Ubiquitin signals play various roles in proteolytic and non-proteolytic functions. Ubiquitin signals are recognized as targets of the ubiquitin-proteasome system and the autophagy-lysosome pathway. In autophagy, ubiquitin signals are required for selective incorporation of cargoes, such as proteins, organelles, and microbial invaders, into autophagosomes. Autophagy receptors possessing an LC3-binding domain and a ubiquitin binding domain are involved in this process. Autophagy activity can decline as a result of genetic variation, aging, or lifestyle, resulting in the onset of various neurodegenerative diseases. This review summarizes the selective autophagy of neurodegenerative disease-associated protein aggregates via autophagy receptors and discusses its therapeutic application for neurodegenerative diseases.
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Envejecimiento/genética , Enfermedad de Alzheimer/genética , Esclerosis Amiotrófica Lateral/genética , Autofagia/genética , Enfermedad de Huntington/genética , Enfermedad de Parkinson/genética , Ubiquitina/genética , Envejecimiento/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulación de la Expresión Génica , Humanos , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Lisosomas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
α-Synuclein, the major component of Lewy bodies (LBs) and Lewy neurites (LNs), is expressed in presynapses under physiologically normal conditions and is involved in synaptic function. Abnormal intracellular aggregates of misfolded α-synuclein such as LBs and LNs are pathological hallmarks of synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). According to previous studies using pathological models overexpressing α-synuclein, high expression of this protein in neurons is a critical risk factor for neurodegeneration. Therefore, it is important to know the endogenous expression levels of α-synuclein in each neuronal cell type. We previously reported differential expression profiles of α-synuclein in vitro and in vivo. In the wild-type mouse brain, particularly in vulnerable regions affected during the progression of idiopathic PD, α-synuclein is highly expressed in neuronal cell bodies of some early PD-affected regions, such as the olfactory bulb, the dorsal motor nucleus of the vagus, and the substantia nigra pars compacta. Synaptic expression of α-synuclein is mostly accompanied by expression of vesicular glutamate transporter-1, an excitatory synapse marker protein. In contrast, α-synuclein expression in inhibitory synapses differs among brain regions. Recently accumulated evidence indicates the close relationship between differential expression profiles of α-synuclein and selective vulnerability of certain neuronal populations. Further studies on the regulation of α-synuclein expression will help to understand the mechanism of LB pathology and provide an innovative therapeutic strategy to prevent PD and DLB onset.
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Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Cuerpos de Lewy/metabolismo , Neuronas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/citología , Humanos , Enfermedad por Cuerpos de Lewy/patología , Ratones , Enfermedad de Parkinson/patología , Pliegue de ProteínaRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is primarily used as an animal model of autoimmune demyelinating disease, multiple sclerosis. In this study, we found the proliferative rate of oligodendrocyte progenitor cells (OPCs) in the medulla elevated twofold above control levels during EAE and new generation of mature oligodendrocytes was increased as well. Although astrocytes showed hypertrophic reactive phenotype, a new generation of them was rare. Astrocyte- and tanycyte-like neural stem cells (NSCs), multipotent NSCs, did not augment their low proliferative rate. Thus, the present study demonstrates that resident OPCs derived from NSCs contribute to remyelination in the medulla oblongata in EAE mice.
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Encefalomielitis Autoinmune Experimental/patología , Bulbo Raquídeo/patología , Células-Madre Neurales/citología , Oligodendroglía/citología , Remielinización/fisiología , Animales , Diferenciación Celular , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Soluble oligomers of amyloid-ß (Aß) peptides (AßOs) contribute to neurotoxicity in Alzheimer's disease (AD). However, it currently remains unknown whether an increase in AßOs is the common phenotype in cellular and animal models. Furthermore, it has not yet been established whether experimental studies conducted using models overexpressing mutant genes of the amyloid precursor protein (APP) are suitable for investigating the underlying molecular mechanism of AD. We herein employed the Flp-In™ T-REx™-293 (T-REx 293) cellular system transfected with a single copy of wild-type, Swedish-, Dutch-, or London-type APP, and quantified the levels of Aß monomers (Aß1-40 and Aß1-42) and AßOs using an enzyme-linked immunosorbent assay (ELISA). The levels of extracellular AßOs were significantly higher in Dutch- and London-type APP-transfected cells than in wild-type APP-transfected cells. Increased levels were also observed in Swedish-type APP-transfected cells. On the other hand, intracellular levels of AßOs were unaltered among wild-type and mutant APP-transfected cells. Intracellular levels of Aß monomers were undetectable, and no common abnormality was observed in their extracellular levels or ratios (Aß1-42/Aß1-40) among the cells examined. We herein demonstrated that increased levels of extracellular AßOs are the common phenotype in cellular models harboring different types of APP mutations. Our results suggest that extracellular AßOs play a key role in the pathogenesis of AD.
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Pituitary gland is a well-known endocrine tissue. The hypothalamo-neurohypophysial system, containing arginine vasopressin and oxytocin, shows a reversible morphological reorganization of both neurons and glial cells during chronic physiological stimulations. Since many signal transducing and cell adhesion molecules (CAMs) are recovered in membrane microdomain (MD) fractions, MDs are considered as signaling platforms of cells. In order to know the molecular background for these endocrine systems, we characterized MD-components derived from rat pituitary and found specific enrichment of several proteins in the fraction. One of them was identified as myelin protein zero (P0) with mass analysis and this result was further confirmed by a result that a specific antibody to this protein reacted to the authentic P0 protein in the myelin fraction of rat sciatic nerve. P0 is one of type-I transmembrane CAMs and a major structural component of mammalian peripheral nerve myelin. In mammals, expression of P0 has been considered to be restricted to peripheral nervous system. This result however indicates that P0 expresses more widely and its enrichment in the MD-fraction from rat pituitary suggests the participation in cell-cell communications.
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Microdominios de Membrana/metabolismo , Proteína P0 de la Mielina/biosíntesis , Hipófisis/metabolismo , Transducción de Señal/fisiología , Animales , Anticuerpos Monoclonales/química , Arginina Vasopresina/metabolismo , Inmunohistoquímica , Microdominios de Membrana/química , Microdominios de Membrana/inmunología , Proteína P0 de la Mielina/química , Proteína P0 de la Mielina/inmunología , Proteína P0 de la Mielina/aislamiento & purificación , Oxitocina/metabolismo , Hipófisis/química , Hipófisis/citología , Hipófisis/inmunología , Ratas , Ratas Wistar , Nervio Ciático/química , Nervio Ciático/citología , Nervio Ciático/inmunología , Nervio Ciático/metabolismoRESUMEN
Na+, K+-ATPase is one of major membrane proteins that has two subunits, alpha and beta. The alpha subunit has the ATPase activity and the ouabain binding site. Among four isoforms of the alpha subunit, expression of alpha1, alpha2, and alpha3, but not alpha4, is observed in matured rat brain. Ouabain is one of cardiac glycosides, and endogenous ouabain-like compounds have been recognized as a new class of steroid hormone. The alpha subunit is considered as their endogenous receptor. Recent studies envisaged the importance of membrane microdomains (MDs) as signaling platforms, which are recovered as a detergent-resistant membrane microdomain fraction (DRM). Although this ATPase has been considered as a non-DRM protein, some amount of the alpha subunit was found to be a component of the DRM prepared from the synaptic plasma membrane fraction (SPM) of rat brain. Ouabain treatment increased the amount of alpha3 isoform, but not alpha1, in the DRM derived from synaptosome fraction and SPM. These results suggest that the localization of the alpha subunit of Na+, K+-ATPase is regulated with isoform-specific mechanisms and the physiological importance of DRM in the signal transduction of the endogenous ouabain-like steroid hormone in neurons.
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Encéfalo/ultraestructura , Inhibidores Enzimáticos/farmacología , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Membranas Sinápticas/efectos de los fármacos , Animales , Animales Recién Nacidos , Relación Dosis-Respuesta a Droga , Isoenzimas/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as "lipid rafts" obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP(2) is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP(2) in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP(2) in SPM-derived DRM, we measured the amount of PIP(2) in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cdelta1 as a PIP(2) binding probe. About 10% of the PIP(2) in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP(2) in DRM prepared from SPM.
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Química Encefálica , Lípidos/análisis , Microdominios de Membrana/química , Membranas Sinápticas/química , Animales , Cromatografía Líquida de Alta Presión , Detergentes , Electroforesis en Gel de Poliacrilamida , Fosfatidilinositol 4,5-Difosfato/análisis , Ratas , Ratas WistarRESUMEN
Proteostasis is important for protecting cells from harmful proteins and is mainly controlled by the HSF1 (heat shock transcription factor 1) stress response pathway. This pathway facilitates protein refolding by molecular chaperones; however, it is unclear whether it functions in autophagy or inclusion formation. The autophagy receptor SQSTM1/p62 is involved in selective autophagic clearance and inclusion formation by harmful proteins, and its phosphorylation at S349, S403, and S407 is required for binding to substrates. Here, we demonstrate that casein kinase 1 phosphorylates the SQSTM1 S349 residue when harmful proteins accumulate. Investigation of upstream factors showed that both SQSTM1 S349 and SQSTM1 S403 residues were phosphorylated in an HSF1 dependent manner. Inhibition of SQSTM1 phosphorylation suppressed inclusion formation by ubiquitinated proteins and prevented colocalization of SQSTM1 with aggregation-prone proteins. Moreover, HSF1 inhibition impaired aggregate-induced autophagosome formation and elimination of protein aggregates. Our findings indicate that HSF1 triggers SQSTM1-mediated proteostasis.
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Autofagia , Quinasa de la Caseína I/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Proteína Sequestosoma-1/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Núcleo Celular/metabolismo , Citoprotección , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Ubiquitinadas/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
α-Synuclein, the major constituent of Lewy bodies (LBs), is normally expressed in presynapses and is involved in synaptic function. Abnormal intracellular aggregation of α-synuclein is observed as LBs and Lewy neurites in neurodegenerative disorders, such as Parkinson's disease (PD) or dementia with Lewy bodies. Accumulated evidence suggests that abundant intracellular expression of α-synuclein is one of the risk factors for pathological aggregation. Recently, we reported differential expression patterns of α-synuclein between excitatory and inhibitory hippocampal neurons. Here we further investigated the precise expression profile in the adult mouse brain with special reference to vulnerable regions along the progression of idiopathic PD. The results show that α-synuclein was highly expressed in the neuronal cell bodies of some early PD-affected brain regions, such as the olfactory bulb, dorsal motor nucleus of the vagus, and substantia nigra pars compacta. Synaptic expression of α-synuclein was mostly accompanied by expression of vesicular glutamate transporter-1, an excitatory presynaptic marker. In contrast, expression of α-synuclein in the GABAergic inhibitory synapses was different among brain regions. α-Synuclein was clearly expressed in inhibitory synapses in the external plexiform layer of the olfactory bulb, globus pallidus, and substantia nigra pars reticulata, but not in the cerebral cortex, subthalamic nucleus, or thalamus. These results suggest that some neurons in early PD-affected human brain regions express high levels of perikaryal α-synuclein, as happens in the mouse brain. Additionally, synaptic profiles expressing α-synuclein are different in various brain regions.
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Química Encefálica/fisiología , Encéfalo/metabolismo , Red Nerviosa/metabolismo , alfa-Sinucleína/biosíntesis , Animales , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Cuerpos de Lewy/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Bulbo Olfatorio/metabolismo , Enfermedad de Parkinson/metabolismo , Porción Compacta de la Sustancia Negra/metabolismo , alfa-Sinucleína/análisisRESUMEN
The serotonin 2C receptor (5-HT2CR) is a G-protein-coupled receptor implicated in emotion, feeding, reward, and cognition. 5-HT2CRs are pharmacological targets for mental disorders and metabolic and reward system abnormalities, as alterations in 5-HT2CR expression, RNA editing, and SNPs are involved in these disturbances. To date, 5-HT2CR activity has mainly been measured by quantifying inositol phosphate production and intracellular Ca(2+) release, but these assays are not suitable for in vivo analysis. Here, we developed a 5-HT2CR-Tango assay system, a novel analysis tool of 5-HT2CR activity based on the G-protein-coupled receptor (GPCR)-arrestin interaction. With desensitization of activated 5-HT2CR by arrestin, this system converts the 5-HT2CR-arrestin interaction into EGFP reporter gene signal via the LexA transcriptional activation system. For validation of our system, we measured activity of two 5-HT2CR RNA-editing isoforms (INI and VGV) in HEK293 cells transfected with EGFP reporter gene. The INI isoform displayed both higher basal- and 5-HT-stimulated activities than the VGV isoform. Moreover, an inhibitory effect of 5-HT2CR antagonist SB242084 was also detected by 5-HT2CR-Tango system. This novel tool is useful for in vitro high-throughput targeted 5-HT2CR drug screening and can be applied to future in vivo brain function studies associated with 5-HT2CRs in transgenic animal models.
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Arrestina/genética , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento/métodos , Receptor de Serotonina 5-HT2C/genética , Receptores Acoplados a Proteínas G/genética , Antagonistas del Receptor de Serotonina 5-HT2/farmacología , Animales , Arrestina/metabolismo , Células COS , Chlorocebus aethiops , Descubrimiento de Drogas/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Receptor de Serotonina 5-HT2C/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
Neuron-enriched acidic protein having a molecular mass of 22 kDa, NAP-22, is a Ca(2+)-dependent calmodulin-binding protein and is phosphorylated with protein kinase C (PKC). This protein is localized to the biological membrane via myristoylation and found in the membrane fraction of the brain and in the synaptic vesicle fraction. Recent studies showed that NAP-22 is localized in the membrane raft domain in a cholesterol-dependent manner and suggest a role for NAP-22 in maturation and/or maintenance of nerve terminals by controlling cholesterol-dependent membrane dynamics. The present study revealed the immunohistochemical distribution of NAP-22 in the peripheral nerves in rat muscles. In all examined muscles, nerve terminals in the motor endplates showed NAP-22 immunoreactivity associated with the membranes of synaptic vesicles and nerve terminals. In the muscle spindles, annulospiral endings, which made spirals around the intrafusal muscles, showed intense NAP-22 immunoreactivity. Autonomic nerve fibers around the intramuscular blood vessels also showed the immunoreactivity for NAP-22. NAP-22 immunoreactivity in these peripheral nerves was observed from birth to adulthood (100 days after birth). Though growth-associated protein-43 (GAP-43) immunoreactivity in these nerves was observed from birth, this immunoreactivity decreased from 20 days after birth. These findings suggest that NAP-22 is distributed and regulates functions in the motor, sensory and autonomic nerve terminals in the peripheral nervous system.
Asunto(s)
Vías Autónomas/química , Proteínas de Unión a Calmodulina/análisis , Proteínas del Citoesqueleto , Neuronas Motoras/química , Husos Musculares/química , Proteínas del Tejido Nervioso/análisis , Neuronas Aferentes/química , Animales , Vías Autónomas/crecimiento & desarrollo , Femenino , Masculino , Neuronas Motoras/fisiología , Husos Musculares/crecimiento & desarrollo , Neuronas Aferentes/fisiología , Terminales Presinápticos/química , Terminales Presinápticos/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
The hypothalamo-neurohypophysial system synthesizes and releases arginine vasopressin (AVP) and oxytocin (OXT) with physiological stimulation. In the present study, we investigated localization of a chondroitin sulfate proteoglycan (CSPG), phosphacan/RPTPbeta, in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of adult rats at both the light and electron microscopic levels. Immunohistochemical analyses demonstrated stronger phosphacan/RPTPbeta immunoreactivity within the SON and PVN compared with adjacent hypothalamic areas. Double labeling experiments showed phosphacan/RPTPbeta immunoreactivity constituting punctate networks to surround the somata and dendrites of AVP- and OXT-secreting magnocellular neurons. Electron microscopic examination further revealed strong phosphacan/RPTPbeta immunoreactivity at extracellular membrane surface of some axons, somata, and dendrites of the SON, but not of synaptic junctions. Interestingly, phosphacan/RPTPbeta immunoreactivity was also observed at extracellular surface membrane between astrocytic processes and neurons rather than between magnocellular neurons. The present results indicate the high expression of the CSPG, phosphacan/RPTPbeta at the extracellular space in the hypothalamic AVP- and OXT-secreting magnocellular neurons.