RESUMO
We found that the orally administered thermolysin digest of ß-conglycinin exhibits antidepressant-like effects in tail suspension and forced swim tests in mice. A comprehensive peptide analysis of the digest using liquid chromatography/mass spectrometry was performed, and LSSTQAQQSY emerged as a candidate antidepressant-like peptide. Orally administered synthetic LSSTQAQQSY exhibited antidepressant-like effects at a dose of 0.3 mg/kg; therefore, we named the decapeptide soy-deprestatin. In contrast, intraperitoneally administered soy-deprestatin was ineffective. We then hypothesized that it acted on the gut, and its signal was transferred to the brain. Indeed, orally administered soy-deprestatin exhibited antidepressant-like activity in sham-treated, but not vagotomized, mice. Oral administration of soy-deprestatin also increased the c-Fos expression in the nucleus of the solitary tract, which receives inputs from the vagus nerve. These results suggested that the antidepressant-like effects were mediated by the vagus nerve. Thermolysin digest- and soy-deprestatin-induced antidepressant-like effects were also blocked by antagonists of serotonin 5-HT1A, dopamine D1, or GABAA receptors. We also clarified the order of receptor activation as 5-HT1A, D1, and GABAA, using selective agonists and antagonists. Taken together, soy-deprestatin may exhibit antidepressant-like effects after oral administration via a novel pathway mediated by 5-HT1A, followed by D1 and GABAA systems. This is the first orally active peptide demonstrating antidepressant-like effects via gut-brain communication.-Mori, Y., Asakura, S., Yamamoto, A., Odagiri, S., Yamada, D., Sekiguchi, M., Wada, K., Sato, M., Kurabayashi, A., Suzuki, H., Kanamoto, R., Ohinata, K. Characterization of soy-deprestatin, a novel orally active decapeptide that exerts antidepressant-like effects via gut-brain communication.
Assuntos
Antidepressivos/farmacologia , Encéfalo/metabolismo , Mucosa Intestinal/metabolismo , Oligopeptídeos/farmacologia , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Proteínas de Soja/farmacologia , Administração Oral , Animais , Antidepressivos/química , Masculino , Camundongos , Oligopeptídeos/química , Receptor 5-HT1A de Serotonina/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de GABA-A/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina/química , Proteínas de Soja/químicaRESUMO
Identification of the proteinaceous components of the pathological inclusions is an important step in understanding the associated disease mechanisms. We immunohistochemically examined two previously reported cases with eosinophilic neuronal cytoplasmic inclusions (NCIs)(case 1, Mori et al. Neuropathology 2010; 30: 64853; case 2, Kojima et al. Acta Pathol Jpn 1990; 40: 78591) using 67 antibodies against proteins related to cytoskeletal constituents, ubiquitin-proteasome system, autophagy-lysosome pathway and stress granule formation. Regional distribution pattern of eosinophilic NCIs in case 1 was substantially different from that in case 2. However, NCIs in both cases were immunonegative for ubiquitin and p62 and were immunopositive for stress granule markers as well as autophagy-related proteins, including valosin-containing protein. Considering that eukaryotic stress granules are cleared by autophagy and valosin-containing protein function, our findings suggest that eosinophilic NCIs in the present two cases may represent the process of autophagic clearance of stress granules.
Assuntos
Autofagia/fisiologia , Grânulos Citoplasmáticos/metabolismo , Corpos de Inclusão/metabolismo , Ubiquitina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Autopsia , Grânulos Citoplasmáticos/patologia , Grânulos Citoplasmáticos/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica , Corpos de Inclusão/patologia , Corpos de Inclusão/ultraestrutura , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Autophagosomal formation is an initial step for macroautophagy. Similar to the yeast autophagy-related gene 8 (ATG8), mammalian ATG8 is responsible for autophagosomal formation, and categorized into LC3 and GABARAPs/GATE-16. Recent studies have shown that impairment of the autophagy-lysosome system is associated with formation of cytoplasmic inclusions observed in various neurodegenerative disorders including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Although abnormal α-synuclein accumulation is a cardinal neuropathological feature in PD, DLB and multiple system atrophy (MSA), it is unclear whether autophagy is altered in MSA. We here demonstrated that the level of matured GABARAPs was significantly decreased in the cerebellum of MSA relative to controls, and that the higher levels of matured and lipidated LC3 were detected in detergent-insoluble fraction of MSA. Immunohistochemical analysis showed that the vast majority of glial cytoplasmic inclusions, a hallmark of MSA, were positive for LC3, whereas they were unstained or barely stained with anti-GABARAPs or anti-GATE-16 antibodies. Our data suggest that autophagy maturation is impaired through the repressed levels of autophagosomal proteins in MSA.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia/fisiologia , Encéfalo/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Atrofia de Múltiplos Sistemas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose , Família da Proteína 8 Relacionada à Autofagia , Western Blotting , Feminino , Humanos , Imuno-Histoquímica , Corpos de Inclusão/metabolismo , Masculino , Pessoa de Meia-Idade , Neuroglia/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Histone deacetylase 6 (HDAC6) is a multifunctional cytoplasmic protein that plays an especially critical role in the formation of aggresomes, where aggregates of excess protein are deposited. Previous immunohistochemical studies have shown that HDAC6 accumulates in Lewy bodies in Parkinson's disease and dementia with Lewy bodies (DLB) as well as in glial cytoplasmic inclusions in multiple system atrophy (MSA). However, it is uncertain whether the level and activity of HDAC6 are altered in the brains of patients with neurodegenerative dementia. In the present study, we demonstrated that the level of HDAC6 was not altered in the temporal cortex of patients with Alzheimer's disease and DLB in comparison with controls. In contrast, the level of HDAC6 was significantly increased in the temporal cortex of patients with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in the cerebellar white matter of patients with MSA. However, the level of acetylated α-tubulin, one of the substrates of HDAC6, was not altered in FTLD-TDP and MSA relative to controls. These findings suggest that the induced level of HDAC6 in the brain is insufficient for manifestation of its activity in FTLD-TDP and MSA.
Assuntos
Encéfalo/enzimologia , Proteínas de Ligação a DNA/metabolismo , Degeneração Lobar Frontotemporal/enzimologia , Histona Desacetilases/biossíntese , Atrofia de Múltiplos Sistemas/enzimologia , Doença de Alzheimer/enzimologia , Desacetilase 6 de Histona , Humanos , Doença por Corpos de Lewy/enzimologia , Lobo Temporal/enzimologiaRESUMO
Macroautophagy is a dynamic process whereby cytoplasmic components are initially sequestered within autophagosomes. Recent studies have shown that the autophagosome membrane can selectively recognize ubiquitinated proteins and organelles through interaction with adapter proteins such as p62 and NBR1. Both proteins are structurally similar at the amino acid level, and bind with ubiquitin and ubiquitinated proteins. Although p62 is incorporated into a wide spectrum of pathological inclusions in various neurodegenerative diseases, abnormalities of NBR1 have not been reported in these diseases. Our immunohistochemical examination revealed that the vast majority of Lewy bodies (LBs) in Parkinson's disease and dementia with LBs (DLB) as well as of glial cytoplasmic inclusions in multiple system atrophy (MSA) were positive for NBR1. Neuronal and glial inclusions in tauopathies and TAR DNA-binding protein of 43 kDa proteinopathies were rarely immunolabeled, or were unstained. Using cultured cells bearing LB-like inclusions, formation of α-synuclein aggregates was repressed in cells with NBR1 knockdown. Immunoblot analysis showed that the level of NBR1 was significantly increased by 2.5-fold in MSA, but not in DLB. These findings suggest that NBR1 is involved in the formation of cytoplasmic inclusions in α-synucleinopathy.
Assuntos
Encéfalo/metabolismo , Corpos de Inclusão/metabolismo , Corpos de Lewy/metabolismo , Doenças Neurodegenerativas/metabolismo , Neuroglia/metabolismo , Proteínas/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Autofagia , Encéfalo/patologia , Feminino , Humanos , Imuno-Histoquímica , Corpos de Inclusão/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Corpos de Lewy/patologia , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/patologia , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologiaRESUMO
Recent studies have shown that eosinophilic intranuclear inclusions (INI) in the brain of patients with intranuclear inclusion body disease (INIBD) are immunopositive for ubiquitin and ubiquitin-related proteins (URP). However, the extent and frequency of URP-immunoreactive inclusions in INIBD are uncertain. We immunohistochemically examined the brain, spinal cord and dorsal root ganglia from five patients with INIBD, using a virtual slide system with sequential staining of the same sections with hematoxylin and eosin and by immunolabeling with antibodies against ubiquitin and URP (NEDD8, NUB1, SUMO-1 and SUMO-2). Intranuclear inclusions were widely distributed in neurons and glial cells in all the cases. Sequential staining revealed that 100% of INI in neurons and glial cells were positive for ubiquitin. Moreover, the majority or a significant proportion of INI were positive for NEDD8, NUB1, SUMO-1 and SUMO-2. However, the proportions of NEDD8-, NUB1- and SUMO-1-positive inclusions were significantly higher in neurons than in glial cells (P < 0.05). These findings suggest that proteins related to ubiquitination and proteasomal degradation are involved in the formation of INI in INIBD.
Assuntos
Encefalopatias/metabolismo , Corpos de Inclusão Intranuclear/metabolismo , Doenças Neurodegenerativas/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Ubiquitina/metabolismo , Idoso , Encéfalo/metabolismo , Encéfalo/patologia , Encefalopatias/patologia , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Humanos , Corpos de Inclusão Intranuclear/patologia , Pessoa de Meia-Idade , Doenças Neurodegenerativas/patologia , Neuroglia/patologia , Neurônios/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , UbiquitinaçãoRESUMO
Marinesco bodies (MBs) are spherical eosinophilic intranuclear inclusions in pigmented neurons in the substantia nigra and locus ceruleus. Previous immunohistochemical studies have shown that MBs are positive for ubiquitin, p62 and SUMO-1, suggesting the involvement of ubiquitination and related proteins in the formation or disaggregation of MBs. However, the involvement is not thoroughly understood. Therefore, we immunohistochemically examined the midbrain from five control subjects ranged from 53 to 84 years old. MBs were positive for various proteins implicated in the ubiquitin-proteasome system (ubiquitin, p62, EDD1, NEDD8, NUB1, SUMO-1 and SUMO-2), aggresome formation (HDAC6) and autophagy (ubiquitin, p62, LC3, GABARAP and GATE-16). These findings suggest that proteins related to ubiquitination, proteasomal degradation and autophagy are involved in the formation or disaggregation of MBs.
Assuntos
Autofagia/fisiologia , Corpos de Inclusão/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Substância Negra/patologia , Ubiquitina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismoAssuntos
Encéfalo/patologia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Corpos de Inclusão Intranuclear/patologia , Doenças Neurodegenerativas/patologia , Peptídeos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Relacionadas à Autofagia , Autopsia , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Doenças Neurodegenerativas/metabolismoRESUMO
Information from the peripheral organs is thought to be transmitted to the brain by humoral factors and neurons such as afferent vagal or spinal nerves. The common hepatic branch of the vagus (CHBV) is one of the main vagus nerve branches, and consists of heterogeneous neuronal fibers that innervate multiple peripheral organs such as the bile duct, portal vein, paraganglia, and gastroduodenal tract. Although, previous studies suggested that the CHBV has a pivotal role in transmitting information on the status of the liver to the brain, the details of its central projections remain unknown. The purpose of the present study was to investigate the brain regions activated by the CHBV. For this purpose, we injected L-arginine or anorexia-associated peptide cholecystokinin-8 (CCK), which are known to increase CHBV electrical activity, into the portal vein of transgenic Arc-dVenus mice expressing the fluorescent protein Venus under control of the activity-regulated cytoskeleton-associated protein (Arc) promotor. The brain slices were prepared from these mice and the number of Venus positive cells in the slices was counted. After that, c-Fos expression in these slices was analyzed by immunohistochemistry using the avidin-biotin-peroxidase complex method. Intraportal administration of L-arginine increased the number of Venus positive or c-Fos positive cells in the insular cortex. This action of L-arginine was not observed in CHBV-vagotomized Arc-dVenus mice. In contrast, intraportal administration of CCK did not increase the number of c-Fos positive or Venus positive cells in the insular cortex. Intraportal CCK induced c-Fos expression in the dorsomedial hypothalamus, while intraportal L-arginine did not. This action of CCK was abolished by CHBV vagotomy. Intraportal L-arginine reduced, while intraportal CCK increased, the number of c-Fos positive cells in the nucleus tractus solitarii in a CHBV-dependent manner. The present results suggest that the CHBV can activate different brain regions depending on the nature of the peripheral stimulus.
RESUMO
Here we found that the chymotryptic digest of soy ß-conglycinin, a major storage protein, exhibited anxiolytic-like effects in mice. We then searched for anxiolytic-like peptides in the digest. Based on a comprehensive peptide analysis of the chymotryptic digest by high performance liquid chromatograph connected to an LTQ Orbitrap mass spectrometer and the structure-activity relationship of known peptides, we explored anxiolytic-like peptides present in the digest. FLSSTEAQQSY, which corresponds to 323-333 of the ß-conglycinin α subunit [ßCGα(323-333)] emerged as a candidate. Oral administration of synthetic ßCGα(323-333) exhibited anxiolytic-like effects in the elevated plus-maze and open-field test in male mice. Orally administered ßCGα(323-333) exhibited anxiolytic-like effects in sham-operated control mice but not in vagotomized mice. In addition, oral administration of ßCGα(323-333) increased the expression of c-Fos, a marker of neuronal activity, in the nucleus of the solitary tract, which receives inputs from the vagus nerve. These results suggest that the anxiolytic-like effects were mediated by the vagus nerve. The anxiolytic-like effects of ßCGα(323-333) were also blocked by antagonists of the serotonin 5-HT1A, dopamine D1 and GABAA receptors. However ßCGα(323-333) had no affinity for these receptors, suggesting it stimulates the release of endogenous neurotransmitters to activate the receptors. Taken together, a soy-derived undecapeptide, ßCGα(323-333), may exhibit anxiolytic-like effects after oral administration via the vagus nerve and 5-HT1A, D1 and GABAA systems.
Assuntos
Ansiolíticos/administração & dosagem , Antígenos de Plantas/administração & dosagem , Ansiedade/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Globulinas/administração & dosagem , Glycine max , Proteínas de Armazenamento de Sementes/administração & dosagem , Proteínas de Soja/administração & dosagem , Nervo Vago/efeitos dos fármacos , Administração Oral , Animais , Ansiedade/metabolismo , Ansiedade/psicologia , Encéfalo/metabolismo , Relação Dose-Resposta a Droga , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Nervo Vago/metabolismoRESUMO
In Lewy body disease (LBD) such as dementia with LBs and Parkinson's disease, several lines of evidence show that disrupted proteolysis occurs. p62/SQSTM1 (p62) is highly involved with intracellular proteolysis and is a component of ubiquitin-positive inclusions in various neurodegenerative disorders. However, it is not clear whether p62 deficiency affects inclusion formation and abnormal protein accumulation. To answer this question, we used a mouse model of LBD that lacks p62, and found that LB-like inclusions were observed in transgenic mice that overexpressed α-synuclein (Tg mice) with or without the p62 protein. p62 deficiency enhanced α-synuclein pathology with regard to the number of inclusions and staining intensity compared with Tg mice that expressed p62. To further investigate the molecular mechanisms associated with the loss of p62 in Tg mice, we assessed the mRNA and protein levels of several molecules, and found that the neighbor of the brca1 gene (NBr1), which is functionally and structurally similar to p62, is increased in Tg mice without p62 compared with control Tg mice. These findings suggest that p62 and NBR1 affect the pathogenesis of neurodegenerative diseases through the cooperative modulation of α-synuclein aggregation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Encéfalo/patologia , Proteínas de Choque Térmico/deficiência , Corpos de Inclusão/patologia , Doença por Corpos de Lewy/patologia , alfa-Sinucleína/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Encéfalo/metabolismo , Proteínas de Choque Térmico/genética , Humanos , Corpos de Inclusão/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Doença por Corpos de Lewy/metabolismo , Camundongos , Camundongos Transgênicos , Atividade Motora , Proteínas/metabolismo , Proteólise , Proteína Sequestossoma-1 , Estresse Fisiológico , alfa-Sinucleína/genéticaRESUMO
The histopathological hallmark of Parkinson's disease (PD) is the presence of fibrillar aggregates referred to as Lewy bodies (LBs), in which α-synuclein is a major constituent. Pale bodies, the precursors of LBs, may serve the material for that LBs continue to expand. LBs consist of a heterogeneous mixture of more than 90 molecules, including PD-linked gene products (α-synuclein, DJ-1, LRRK2, parkin, and PINK-1), mitochondria-related proteins, and molecules implicated in the ubiquitin-proteasome system, autophagy, and aggresome formation. LB formation has been considered to be a marker for neuronal degeneration because neuronal loss is found in the predilection sites for LBs. However, recent studies have indicated that nonfibrillar α-synuclein is cytotoxic and that fibrillar aggregates of α-synuclein (LBs and pale bodies) may represent a cytoprotective mechanism in PD.
Assuntos
Corpos de Lewy/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Animais , Humanos , Corpos de Lewy/fisiologia , Doença por Corpos de Lewy/metabolismo , Doença por Corpos de Lewy/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/prevenção & controle , Doença de Parkinson/prevenção & controle , alfa-Sinucleína/efeitos adversos , alfa-Sinucleína/toxicidadeRESUMO
Oxidative stress has been proposed as a potential mechanism for neurodegenerative diseases, such as Alzheimer disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS). In response to oxidative stress, the levels of numerous cytoprotective products are increased via alteration of the Kelch-like ECH-associated protein 1 (Keap1) and NF-E2-related factor 2 (Nrf2) system. One of the Nrf2 targets, p62, has been known to be incorporated into a wide spectrum of cytoplasmic inclusions in neurodegenerative diseases and interact with Keap1. However, it remains unclear whether Keap1 is associated with the pathogenesis of neurodegenerative diseases. In this study, we investigated the relationship between p62 and Keap1 in the brains of patients with AD, PD, dementia with Lewy bodies (DLB), and ALS. Biochemical analyses showed that p62 and Keap1 interacted with each other in AD and DLB brains and were extracted into similar detergent-soluble and -insoluble fractions. Pathologic examination demonstrated that anti-Keap1 antibodies immunostained Lewy bodies in PD and DLB, neurofibrillary tangles in AD, and skeinlike inclusions in ALS. Further analysis showed that the levels of common Nrf2 target genes were increased in AD compared with those in controls. However, there were no statistical significances in the levels of Nrf2 target genes in DLB relative to controls. Our pathologic and biochemical results suggest a molecular basis for stress response to be involved in the formation of cytoplasmic inclusions observed in several neurodegenerative diseases.
Assuntos
Corpos de Inclusão/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Neurodegenerativas/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Idoso , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Encéfalo/patologia , Química Encefálica/fisiologia , Feminino , Humanos , Corpos de Inclusão/química , Corpos de Inclusão/patologia , Proteína 1 Associada a ECH Semelhante a Kelch , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/patologia , Neuroglia/química , Neuroglia/patologia , Neurônios/química , Neurônios/patologia , Ligação Proteica/fisiologiaRESUMO
Incorporation of ubiquitin and ubiquitin-related proteins including p62 into neuronal intranuclear inclusions (NIIs) has been reported in a variety of neurodegenerative diseases. However, involvement of autophagy-specific proteins (NBR1 and LC3) in NIIs has not been mentioned. We immunohistochemically examined the brain of patients with Machado-Joseph disease (MJD; n=5), dentatorubral-pallidoluysian atrophy (DRPLA; n=5) and intranuclear inclusion body disease (INIBD; n=5), using antibodies against ubiquitin, p62, NBR1 and LC3. The proportion of p62-, NBR1- and LC3-positive inclusions relative to the number of ubiquitin-positive inclusions was calculated in each case. NIIs were positive for p62 in MJD (19.3%), DRPLA (49.7%) and INIBD (99.8%). As for autophagy-specific proteins, NIIs were positive for NBR1 in MJD (4.2%), DRPLA (5.5%) and INIBD (13.2%) and negative for LC3 in MJD, DRPLA and INIBD, except for one case of INIBD. These findings suggest that autophagy-lysosome pathway is not involved in the formation/degradation of NIIs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Corpos de Inclusão Intranuclear/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Proteínas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/metabolismo , Estudos de Casos e Controles , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Pessoa de Meia-Idade , Doenças Neurodegenerativas/patologia , Proteína Sequestossoma-1RESUMO
Microscopic globular bodies (MGBs) are brilliantly and homogenously eosinophilic spherical inclusions, 1-10 µm in diameter. They are mainly distributed in the cerebral neocortex and hippocampus in normal individuals ranged in age from first to tenth decade. Ultrastructurally, MGBs are composed of electron-dense granular material and are located in dendrites. However, immunohistochemical profile of MGBs is uncertain. Therefore, we immunohistochemically examined the hippocampus from five control subjects ranged from 25 to 76 years. The marginal portion of MGBs was positive for lysosomal proteases (cathepsins B, D and L), and markers of dendrite (MAP2) and dendritic spine (drebrin). In some cases, MGBs were entirely immunostained with anti-cathepsin D. Among the cathepsins, MGBs were most frequently immunolabeled with anticathepsin D. They were negative for ubiquitin, ubiquitin-proteasome system (p62, NUB1 and EDD1), autophagosome (LC3), cytoskeletal proteins (neurofilament, actin, tubulin and cytokeratin), tau, α-synuclein and TDP-43. These findings suggest that MGBs are sequestered by lysosome- protease system, but not by ubiquitin-proteasome system or autophagosome.
Assuntos
Encéfalo/citologia , Encéfalo/metabolismo , Corpos de Inclusão/metabolismo , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The granular retrosplenial cortex (GRS) in the rat has a distinct microcolumn-type structure. The apical tufts of dendritic bundles at layer I, which are formed by layer II neurons, co-localize with patches of thalamic terminations from anteroventral (AV) thalamic nucleus. To further understand this microcolumn-type structure in the GRS, one of remaining questions is whether this structure extends into other layers, such as layers III/IV. Other than layer I, previous tracer injection study showed that AV thalamic nucleus also projects to layer III/IV in the GRS. In this study, we examined the morphology of branches in the GRS from the AV thalamus in single axon branch resolution in order to determine whether AV axon branches in layer III/IV are branches of axons with extensive branch in layer I, and, if so, whether the extent of these arborizations in layer III/IV vertically matches with that in layer I. For this purpose, we used a small volume injection of biotinylated dextran-amine into the AV thalamus and reconstructing labeled single axon branches in the GRS. We found that the AV axons consisted of heterogeneous branching types. Type 1 had extensive arborization occurring only in layer Ia. Type 2 had additional branches in III/IV. Types 1 and 2 had extensive ramifications in layer Ia, with lateral extensions within the previously reported extensions of tufts from single dendritic bundles (i.e., 30-200 µm; mean 78 µm). In type 2 branches, axon arborizations in layer III/IV were just below to layer Ia ramifications, but much wider (148-533 µm: mean, 341 µm) than that in layer Ia axon branches and dendritic bundles, suggesting that layer-specific information transmission spacing existed even from the same single axons from the AV to the GRS. Thus, microcolumn-type structure in the upper layer of the GRS was not strictly continuous from layer I to layer IV. How each layer and its components interact each other in different spatial scale should be solved future.
RESUMO
Iron in the brain is utilized for cellular respiration, neurotransmitter synthesis/degradation, and myelin formation. Iron, especially its ferrous form, also has the potential for catalyzing the Fenton reaction to generate highly cytotoxic hydroxyl radicals. The amount of iron in the brain must therefore be strictly controlled. In this study, we focused on the cellular and subcellular localizations of nonheme ferric (Fe(III)) and ferrous (Fe(II)) iron in the adult female rat brain using light and electron microscopic histochemistry. Although Fe(II) deposition was much less dominant than Fe(III), the brain contained iron in both forms. Among the cellular elements of the brain, oligodendrocytes were numerically the most prominent and heavily iron-storing cells. Pericapillary astrocytes and sporadic microglial cells also showed dense iron accumulation. Large neurons involved in the motor system were relatively strongly iron-positive. Subcellularly, Fe(III) and Fe(II) were mainly localized in lysosomes, and occasionally in the cytosol and mitochondria. Furthermore, capillary endothelial cells had Fe(III)-positive reactions in lysosomes and the cytosol, with Fe(II)-positive reactions on the luminal membrane. With advancing age, both Fe(III) and Fe(II) became more extensively distributed and accumulated more numerously in oligodendrocytes and astrocytes. These findings suggest that age-related increases in Fe(II) accumulation may raise the risk of tissue damage in the normal brain.
Assuntos
Encéfalo/citologia , Encéfalo/ultraestrutura , Compostos Férricos/química , Compostos Ferrosos/química , Ferroproteínas não Heme/química , Oligodendroglia/química , Fatores Etários , Animais , Astrócitos/química , Encéfalo/metabolismo , Feminino , Microglia/química , Microglia/citologia , Perfusão/métodos , Ratos , Ratos WistarRESUMO
The perfusion-Perls and -Turnbull methods supplemented by diaminobenzidine intensification demonstrated the generation and localization of chelatable Fe (II) which can catalyze the generation of cytotoxic hydroxyl radicals (OH.) during the Fenton reaction in rat kidneys exposed to 40 min ischemia or 40 min-ischemia followed by 60 min-reperfusion. The kidneys exposed to 40 min-ischemia showed Fe (II)-deposits largely localized in the deeper half of the cortex, where the deposits densely filled the tubular cell nuclei, with a small amount of them in the cytoplasm of the proximal convoluted tubules (PCT). Intraluminally protruded or exfoliated tubular cell nuclei were also filled with the deposits. The kidneys subjected to 40 min-ischemia/ 60 min-reperfusion showed a more extensive distribution of Fe (II)-deposits, including most depths of the cortex. Furthermore, there were numerous exfoliated, Fe (II)-positive nuclei surrounded by a small amount of cytoplasm in the lumen of the PCT. These cells appeared to undergo apoptotic cell death since the lumen of strongly dilated, down-stream, proximal straight tubules were obstructed with numerous apoptotic cells in the kidneys exposed to 40 min-ischemia and 24 h-reperfusion. Pretreatment with a divalent metal chelator, 2, 2'-dipyridyl, effectively inhibited Fe (II)-staining, decreased the number of exfoliated cells in the kidneys with 40 min-ischemia/ 60 m-reperfusion, and decreased the number of apoptotic cells in the kidneys with 40 min-ischemia/24 h-reperfusion. The generation of highly reactive OH. during the Fe2+-catalyzed Fenton reaction was suggested to play a crucial role in ischemia/reperfusion-induced kidney injury.
Assuntos
2,2'-Dipiridil/farmacologia , Quelantes/farmacologia , Compostos Férricos/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Feminino , Histocitoquímica , Concentração de Íons de Hidrogênio , Perfusão , Ratos , Ratos Wistar , Fatores de TempoRESUMO
We reviewed the methods of nonheme-iron histochemistry with special focus on the underlying chemical principles. The term nonheme-iron includes heterogeneous species of iron complexes where iron is more loosely bound to low-molecular weight organic bases and proteins than that of heme (iron-protoporphyrin complex). Nonheme-iron is liberated in dilute acid solutions and available for conventional histochemistry by the Perls and Turnbull and other methods using iron chelators, which depend on the production of insoluble iron compounds. Treatment with strong oxidative agents is required for the liberation of heme-iron, which therefore is not stained by conventional histochemistry. The Perls method most commonly used in laboratory investigations largely stains ferric iron, but stains some ferrous iron as well, while the Turnbull method is specific for the latter. Although the Turnbull method performed on sections fails in staining ferrous iron or stains only such parts of the tissue where iron is heavily accumulated, an in vivo perfusion-Turnbull method demonstrated the ubiquitous distribution of ferrous iron, particularly in lysosomes. The Perls or Turnbull reaction is enhanced by DAB/silver/gold methods for electron microscopy. The iron sulfide method and the staining of redox-active iron with H(2)O(2) and DAB are also applicable for electron microscopy. Although the above histochemical methods have advantages for visualizing iron by conventional light and electron microscopy, the quantitative estimation of iron is not easy. Recent methods depending on the quenching of fluorescent divalent metal indicators by Fe(2+) and dequenching by divalent metal chelators have enabled the quantitative estimation of chelatable Fe(2+) in isolated viable cells.
Assuntos
Histocitoquímica/métodos , Ferro , Microscopia Eletrônica/métodos , Microscopia/métodos , Coloração e Rotulagem/métodos , Animais , Compostos Férricos/análise , Compostos Férricos/metabolismo , Compostos Ferrosos/análise , Compostos Ferrosos/metabolismo , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Ferro/análise , Ferro/história , Ferro/metabolismo , Ferroproteínas não Heme/análise , Ferroproteínas não Heme/metabolismo , Ferroproteínas não Heme/ultraestruturaRESUMO
Redox-active non-heme iron catalyzes hydroxyl radical [Formula: see text] generation through Haber-Weiss reaction. Oxidative tissue damage by OH* has been suggested in the development of stress-induced gastric lesion. Using highly sensitive non-heme iron histochemistry, the perfusion-Perls and -Turnbull methods plus DAB intensification, we studied the distribution of non-heme ferric and ferrous iron (NHF[III] and NHF[II]) in the normal stomach and its changes in the acute gastric lesions induced by restraint water immersion (RWI) stress in the rat. Both NHF[III] and NHF[II] staining increased in the oncotic parietal cells located at the erosive lesion which developed on the gastric mucosal folds after 3 h RWI. It was considered that increase in non-heme iron in these cells catalyzed OH* generation under the presence of O(2)(*-) released from abundant injured mitochondria. This was supported by the increase in H(2)O(2) staining in the erosive region and the obvious reduction of the gastric lesion following administration of deferoxamine before RWI. NHF[II] was stained in the arterial endothelium in the tela submucosa of the normal gastric wall and increase in the entire gastric mucosa after 3 h RWI suggests that the changes in the vascular non-heme iron metabolism were also involved in the response of the stomach to stressful conditions.