RESUMO
BACKGROUND: Huntington's disease (HD) is a fatal genetic disease caused by polyglutamine aggregation encoded by an expanded CAG repeat in the huntingtin gene (HTT). In this study, we cultured neurospheres derived from R6/2 mice, a representative animal model of HD, as an in vitro model. GuideRNAs were designed to induce large deletion or frameshift indel mutation of CAG expansion. These gRNAs and Cas9 were delivered to the R6/2 neurospheres and disease-related phenotypes were observed. METHODS AND RESULTS: Deletion or indel mutation of the CAG repeat was confirmed by PCR, T7E1 assay and sequencing of the edited neurospheres. Edited neurospheres showed decreased polyglutamine aggregation compared with control HD neurospheres. In the edited neurosphere, we confirmed the upregulation of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and brain-derived neurotrophic factor (BDNF), whose reduced expressions are closely involved in the disease progression. In addition, flow cytometry result showed an increase in cell viability with an overall decrease in necrotic and apoptotic populations among edited R6/2 neurospheres. Additional siRNA experiments confirmed that the increased viability was decreased through inhibition of PGC-1α or BDNF. CONCLUSION: Our study confirmed that CAG repeat of R6/2 mouse-derived neurospheres can be edited through CRISPR-Cas9. Editing of CAG repeat sequence decreases polyglutamine aggregation and cellular apoptosis of HD neurospheres, which may be related to the increased expressions of PGC-1α and BDNF. Our data provide the evidence that CRISPR-Cas9 mediated genome editing has therapeutic potential on HD neuronal cells.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Doença de Huntington , Animais , Camundongos , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Edição de Genes , Doença de Huntington/metabolismoRESUMO
BACKGROUND: Tropomyosin-receptor kinase fused gene (TFG) functions as a regulator of intracellular protein packaging and trafficking at the endoplasmic reticulum exit sites. TFG has recently been proposed as a cause of multisystem proteinopathy. OBJECTIVES: Here, we describe a Korean family presenting with Parkinson's disease or amyotrophic lateral sclerosis caused by a novel variant of TFG (c.1148 G > A, p.Arg383His). METHODS: We collected clinical, genetic, dopamine transporter imaging, nerve conduction, and electromyography data from the seven subjects. To verify the pathogenicity of the R383H variant, we studied cell viability and the abnormal aggregation of α-synuclein and TAR DNA-binding protein 43 (TDP-43) in HeLa cells expressing R383H-TFG. RESULTS: The clinical phenotypes of the R383H-TFG mutation varied; of the five family members, one had Parkinson's disease, three had subclinical parkinsonism, and one (the proband) had amyotrophic lateral sclerosis. The individual with multiple system atrophy was the proband's paternal cousin, but the TFG genotype was not confirmed due to unavailability of samples. Our in vitro studies showed that R383H-TFG overexpression impaired cell viability. In cells co-expressing R383H-TFG and α-synuclein, insoluble α-synuclein aggregates increased in concentration and were secreted from the cells and co-localized with R383H-TFG. The levels of cytoplasmic insoluble aggregates of TDP-43 increased in HeLa cells expressing R383H-TFG and co-localized with R383H-TFG. CONCLUSIONS: Clinical and in vitro studies have supported the pathogenic role of the novel TFG mutation in α-synucleinopathy and TDP-43 proteinopathy. These findings expand the phenotypic spectrum of TFG and suggest a pivotal role of endoplasmic reticulum dysfunction during neurodegeneration. © 2021 International Parkinson and Movement Disorder Society.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas , Sinucleinopatias , Esclerose Lateral Amiotrófica/genética , Células HeLa , Humanos , Mutação , Proteínas/genética , República da CoreiaRESUMO
Huntington's disease (HD) is a fatal genetic disease caused by abnormal aggregation of mutant huntingtin protein (mHtt). Reduction of mHtt aggregation decreases cell death of the brain and is a promising therapeutic strategy of HD. MicroRNAs are short non-coding nucleotides which modulate various genes and dysregulated in many diseases including HD. MicroRNA miR-27a was reported to be reduced in the brain of R6/2 HD mouse model and modulate multidrug resistance protein-1 (MDR-1). Using subventricular zone-derived neuronal stem cells (NSCs), we used in vitro HD model to test the effect of miR-27a on MDR-1 and mHtt aggregation. R6/2-derived NSCs can be differentiated under condition of growth factor deprivation, and the progression of differentiation leads to a decrease of MDR-1 level and efflux function of cells. Immunocytochemistry result also confirmed that mHtt aggregation was increased with differentiation. We transfected miR-27a in the R6/2-derived differentiated NSCs, and examined phenotype of HD, mHtt aggregation. As a result, miR-27a transfection resulted in reduction of mHtt aggregation in HD cells. In addition, MDR-1, which can transport mHtt, protein level was increased by miR-27a transfection. Conversely, knock-down of MDR-1 through MDR-1 siRNA increased mHtt aggregation in vitro. Our results indicate that miR-27a could reduce mHtt level of the HD cell by augmenting MDR-1 function.
Assuntos
Modelos Animais de Doenças , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Doença de Huntington/terapia , MicroRNAs/genética , Agregados Proteicos , Animais , Proteína Huntingtina/química , Proteína Huntingtina/genética , Doença de Huntington/genética , Camundongos , Camundongos Endogâmicos C57BL , Agregados Proteicos/genéticaRESUMO
Adipose tissue stem cells (ATSCs) are considered as a promising source in the field of cell therapy and regenerative medicine. In addition to direct cell replacement using stem cells, intercellular molecule exchange by stem cell secretory factors showed beneficial effects by reducing tissue damage and augmentation of endogenous repair. Delayed cutaneous wound healing is implicated in many conditions such as diabetes, aging, stress and alcohol consumption. However, the effects of cell-free extract of ATSCs (ATSC-Ex) containing secretome on wound healing process have not been investigated. In this study, ATSC-Ex was topically applied on the cutaneous wound and healing speed was examined. As a result, wound closure was much faster in the cell-free extract treated wound than control wound at 4, 6, 8 days after application of ATSC-Ex. Dermal fibroblast proliferation, migration and extracellular matrix (ECM) production are critical aspects of wound healing, and the effects of ATSC-Ex on human dermal fibroblast (HDF) was examined. ATSC-Ex augmented HDF proliferation in a dose-dependent manner and migration ability was enhanced by extract treatment. Representative ECM proteins, collagen type I and matrix metalloproteinase-1, are significantly up-regulated by treatment of ATSC-Ex. Our results suggest that the ATSC-Ex have improving effect of wound healing and can be the potential therapeutic candidate for cutaneous wound healing.
Assuntos
Tecido Adiposo/citologia , Extratos Celulares/química , Extratos Celulares/farmacologia , Fibroblastos/efeitos dos fármacos , Pele/efeitos dos fármacos , Células-Tronco/química , Cicatrização/efeitos dos fármacos , Tecido Adiposo/química , Administração Tópica , Animais , Extratos Celulares/administração & dosagem , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Pele/metabolismo , Células-Tronco/citologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a degenerative disorder that involves the death of motor neurons in the cortex, brain stem, and spinal cord. Adipose-derived stem cells (ADSCs) are considered as a perspective remedy for therapy of neurodegenerative diseases including ALS. Stem cells secrete various factors which can modulate a hostile environment, called paracrine effect. Exosomes are small extracellular vesicles containing cell derived factors and mediate paracrine effect of cells. Thus, exosomes from ADSCs (ADSC-exo) can be a potential candidate of therapeutic effects of stem cells. To investigate the effect of ADSC-exo on the cellular phenotypes of ALS, we used neuronal stem cells (NSCs), which can be differentiated into neuronal cells, isolated from wild type or G93A ALS mice model. ADSC-exo was treated to neuronal cells from G93A ALS mice model. Immunocytochemistry and dot-blot assay result showed that ADSC-exo alleviated aggregation of superoxide dismutase 1 (SOD1). Reduction of cytosolic SOD1 level by ADSC-exo was also confirmed by western blot. Mitochondria display various abnormalities in ALS and the decrease of phospho-CREB and PGC-1α were observed in the G93A cells. ADSC-exo treatment showed normalization of phospho-CREB/CREB ratio and PGC-1α expression level. Our results suggest that ADSC-exo modulates cellular phenotypes of ALS including SOD-1 aggregation and mitochondrial dysfunction, and can be a therapeutic candidate for ALS.
Assuntos
Adipócitos/citologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/terapia , Exossomos/metabolismo , Células-Tronco/citologia , Tecido Adiposo/citologia , Animais , Células Cultivadas , Citoplasma/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Neurônios Motores/metabolismo , Neurônios/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fenótipo , Superóxido Dismutase-1/metabolismoRESUMO
Exosomes are the extracellular vesicles secreted by various cells. Exosomes mediate intercellular communication by delivering a variety of molecules between cells. Cancer cell derived exosomes seem to be related with tumor progression and metastasis. Tumor microenvironment is thought to be acidic and this low pH controls exosome physiology, leading to tumor progression. Despite the importance of microenvironmental pH on exosome, most of exosome studies have been performed without regard to pH. Therefore, the difference of exosome stability and yield of isolation by different pH need to be studied. In this research, we investigated the yield of total exosomal protein and RNA after incubation in acidic, neutral and alkaline conditioned medium. Representative exosome markers were investigated by western blot after incubation of exosomes in different pH. As a result, the concentrations of exosomal protein and nucleic acid were significantly increased after incubation in the acidic medium compared with neutral medium. The higher levels of exosome markers including CD9, CD63 and HSP70 were observed after incubation in an acidic environment. On the other hand, no exosomal protein, exosomal RNA and exosome markers have been detected after incubation in an alkaline condition. In summary, our results indicate that the acidic condition is the favorable environment for existence and isolation of exosomes.
Assuntos
Técnicas de Cultura de Células/métodos , Fracionamento Celular/métodos , Meios de Cultura/química , Exossomos/química , Exossomos/ultraestrutura , Concentração de Íons de Hidrogênio , Animais , Bovinos , Células HEK293 , HumanosRESUMO
Huntington's disease (HD) is the most common hereditary neurodegenerative diseases, in which the loss of striatal neuron caused by the aggregation of mutant huntingtin protein (mHtt) is the main pathological feature. Our previous studies have demonstrated that human adipose stem cells (hASC) and its extracts can slow down the progression of HD in vitro and in vivo. hASC are readily accessible adult stem cells, and the cytosolic extracts contain a number of neurotrophic factors. Here, we further explored the role of the hASC extracts in neuronal death and mitochondrial function in HD. Our results showed that the hASC extracts prevent mHtt-induced cell toxicity and cell apoptosis. Moreover, the hASC extracts recovered mHtt-induced mitochondrial oxidative stress and reduced mitochondrial membrane potential. The hASC extracts blocked the interaction between p53 and mHtt, and decreased the endogenous p53 levels at both transcriptional and post-translational levels, resulting in the instability of p53 and increased neuronal survival. Taken together, these findings implicate protective roles of hASC extracts in mHtt-induced mitochondrial apoptosis, providing insights into the molecular mechanism of the hASC in the therapeutic strategy of HD.
Assuntos
Adipócitos/metabolismo , Células-Tronco Adultas/metabolismo , Mitocôndrias/metabolismo , Animais , Apoptose/fisiologia , Extratos Celulares/farmacologia , Linhagem Celular , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Doença de Huntington/terapia , Potencial da Membrana Mitocondrial , Camundongos , Células-Tronco Multipotentes/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo , Proteína Supressora de Tumor p53/metabolismoRESUMO
OBJECTIVE: In comparison with amyotrophic lateral sclerosis (ALS), the contribution of neuroinflammation in spinobulbar muscular atrophy (SBMA) has been less explored. We investigated the role of neuroinflammation in the pathogenesis of ALS and SBMA by analyzing systemic inflammatory markers and osteopontin (Spp1). METHODS: This study involved 105 ALS, 77 SBMA, and 55 healthy controls. We measured their systemic inflammatory markers, serum Spp1, and cytokine levels (interferon-γ, interleukin [IL]-1ß, IL-6, IL-8, IL-10, tumor necrosis factor-α, and IL-17A), investigated correlations between Spp1 levels and clinical features, and evaluated ALS survival rates according to Spp1 levels. RESULTS: In the ALS group, systemic inflammatory markers were significantly higher than in the control and SBMA groups. Spp1 levels were observed to be higher in ALS patients, but the difference was not statistically significant among the study groups. Cytokine profiles were comparable. In ALS, higher Spp1 levels were correlated with lower ALS Functional Rating Scale-Revised (ALSFRS-R) scores (r = -0.25, p = 0.02) and faster disease progression rate (r = 0.37, p < 0.001). After adjusting for other prognostic indicators, high Spp1 levels were independently associated with shorter survival in ALS patients (hazard ratio 13.65, 95% confidence interval 2.57-72.53, p < 0.01). INTERPRETATION: Neuroinflammation does not appear to be a primary contributor to the pathogenesis of SBMA. Serum Spp1 levels may serve as a reliable biomarker for disease progression and prognosis in ALS. These findings expand our understanding of these two distinct motor neuron disorders and offer a potential biomarker for future studies.
Assuntos
Esclerose Lateral Amiotrófica , Progressão da Doença , Osteopontina , Humanos , Esclerose Lateral Amiotrófica/sangue , Esclerose Lateral Amiotrófica/fisiopatologia , Esclerose Lateral Amiotrófica/diagnóstico , Osteopontina/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Biomarcadores/sangue , Adulto , Doenças Neuroinflamatórias/sangue , Citocinas/sangueRESUMO
A recent study suggested that a cell-free extract of human adipose stem cells (hASCs-E) has beneficial effects on neurological diseases by modulating the host environment. Here, we investigated the effects of hASCs-E in several experimental models of stroke in vitro (oxygen and glucose deprivation, OGD) and in vivo (transient or permanent focal cerebral ischemia and intracerebral hemorrhage, ICH). Ischemia was induced in vitro in Neuro2A cells, and the hASCs-E was applied 24h before the OGD or concurrently. Focal cerebral ischemia was induced by unilateral intraluminal thread occlusion of the middle cerebral artery (MCA) in rats for 90min or permanently, or by unilateral MCA microsurgical direct electrocoagulation in mice. The ICH model was induced with an intracerebral injection of collagenase in rats. The hASCs-E was intraperitoneally administered 1h after the stroke insults. Treatment of the hASCs-E led to a substantially high viability in the lactate dehydrogenase and WST-1 assays in the in vitro ischemic model. The cerebral ischemic and ICH model treated with hASCs-E showed decreased ischemic volume and reduced brain water content and hemorrhage volume. The ICH model treated with hASCs-E exhibited better performance on the modified limb placing test. The expression of many genes related to inflammation, immune response, and cell-death was changed substantially in the ischemic rats or neuronal cells treated with the hASCs-E. These results reveal a neuroprotective role of hASCs-E in animal models of stroke, and suggest the feasible application of stem cell-based, noninvasive therapy for treating stroke.
Assuntos
Adipócitos/química , Encéfalo/efeitos dos fármacos , Sistema Livre de Células , Fármacos Neuroprotetores/farmacologia , Células-Tronco/química , Acidente Vascular Cerebral/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acidente Vascular Cerebral/patologia , Transcriptoma/efeitos dos fármacosAssuntos
Olho/patologia , Regulação da Expressão Gênica/efeitos da radiação , Hipocampo , Proteínas do Tecido Nervoso/biossíntese , Neurogênese/efeitos da radiação , Pele/patologia , Sinapses , Raios Ultravioleta/efeitos adversos , Animais , Olho/metabolismo , Olho/fisiopatologia , Hipocampo/metabolismo , Hipocampo/patologia , Hipocampo/fisiopatologia , Camundongos , Camundongos Pelados , Pele/metabolismo , Pele/fisiopatologia , Sinapses/metabolismo , Sinapses/patologiaRESUMO
OBJECTIVE: Biomarkers are needed to predict prognosis and disease activity in patients with Guillain-Barré syndrome (GBS). The complement system is a key player in the pathogenesis of GBS. This study aimed to assess the potential utility of serum complement proteins as novel biomarkers in GBS. METHODS: We reviewed the medical records of 76 GBS patients with C3 and C4 measurements during hospitalization between 2010 and 2021. Clinical outcomes were correlated with baseline serum C3, C4, and seven additional predictors: four existing biomarkers (GM1, albumin, immunoglobulin G, neutrophil-lymphocyte ratio) and three clinical factors from the modified Erasmus GBS outcome score model. Five complement activation products (C3a, C4a, C5a, soluble C5b-9, factor Bb) were measured in 35 patients and were compared with C3 and C4 levels. Longitudinal changes in C3 and C4 levels were compared with the disease course in 12 patients. RESULTS: Higher C3, but not C4, was associated with poorer outcomes: lower Medical Research Council sum scores (MRCSS), higher GBS disability score (GBSDS), longer hospitalization, and more frequent treatment-related fluctuations. Age, MRCSS at admission, and baseline serum C3 were significant independent indicators of 1- and 3-month GBSDS. We found that C3 was positively correlated with C3a (r = 0.32) and C5a (r = 0.37), which indicates an activated complement cascade with high C3. Longitudinal change of C3 coincided with clinical severity of the disease course. INTERPRETATION: This study highlights the use of serum C3 as a novel mechanistic biomarker in GBS. Larger prospective studies are needed to validate our findings.
Assuntos
Síndrome de Guillain-Barré , Humanos , Síndrome de Guillain-Barré/diagnóstico , Complemento C3/análise , Prognóstico , Imunoglobulina G , Biomarcadores , Progressão da DoençaRESUMO
Dietary habits have a great impact on one's health, especially in cognitive decline. Tomato and lemon contain diverse bioactive compounds and possess various effects, including the enhancement of cognitive function. We observed the protective effect of tomato, lemon extract and the mixture of them on H2O2-induced cytotoxicity of PC12 cells. To measure the in vivo effect in a murine model, each extract was orally administered to forty 1-year-old mice for 6 weeks, and a novel object recognition (NOR) test was performed to observe cognitive function, and hippocampal neurogenesis was observed through a doublecortin (DCX) stain. PC12 cell death by oxidative stress was reduced by pretreating with each extract, and a synergistic reduction was observed in the mixture. Newly generated DCX-positive neurons were synergistically increased in the hippocampus by the mixture. NOR test showed a tendency to significantly improve age-related cognitive dysfunction by consuming the mixture of tomato and lemon. In conclusion, tomato and lemon extracts can reduce cellular oxidative stress and increase NOR, likely due to enhanced neurogenesis, while the mixture of the two showed synergistic anti-oxidative effects and hippocampal neurogenesis.
RESUMO
Immune-mediated neuropathies are a heterogenous group of inflammatory peripheral nerve disorders. They can be classified according to the domain where the autoimmune process begins: the internode, paranode, or node. However, conventional diagnostic tools, electrodiagnosis (EDX), and autoantibody testing do not fully address this issue. In this institutional cohort study, we investigated the value of dermal myelinated fiber analysis for target domain-based classification. Twenty-seven consecutive patients with immune-mediated neuropathies underwent skin biopsies. The sections were stained with antibodies representative of myelinated fiber domains and were scanned using a confocal microscope. Clinical and pathological features of each patient were reviewed comprehensively. Quantitative morphometric parameters were subjected to clustering analysis, which stratified patients into 3 groups. Cluster 1 ("internodopathy") was characterized by prominent internodal disruption, intact nodes and paranodes, demyelinating EDX pattern, and absence of nodal-paranodal antibodies. Cluster 2 ("paranodopathy") was characterized by paranodal disruption and corresponding antibodies. Morphological changes were restricted to the nodes in cluster 3; we designated this cluster as "nodopathy." This report highlights the utility of skin biopsy as a diagnostic aid to gain pathogenic insight and classify patients with immune-mediated neuropathies.
Assuntos
Doenças do Sistema Nervoso Periférico , Nós Neurofibrosos , Humanos , Nós Neurofibrosos/patologia , Estudos de Coortes , Doenças do Sistema Nervoso Periférico/diagnóstico , Doenças do Sistema Nervoso Periférico/patologia , Axônios/patologia , Pele/patologia , BiópsiaRESUMO
Ginseng is a popular herbal medicine and known to have protective and therapeutic effects in various diseases. Ginsenosides are active gradients representing the diverse pharmacological efficacy of ginseng. Huntington's disease (HD) is incurable genetic disorder associated with mutant huntingtin (mHtt) aggregation in the central nervous system. This study was conducted to investigate the effects of ginsenoside Rg3 and Rf on mHtt aggregation, cell viability, mitochondrial function, and apoptotic molecules on HD model. To investigate the effect of ginsenosides on HD, neural stem cells were isolated from the R6/2 mouse brain and used as a cellular model of HD. Nuclear aggregation of mHtt was measured by immunocytochemistry, and expressions of mitochondrial biogenesis and apoptotic molecules were investigated by western blot. As a result, the number of mHtt aggregates positive cells has decreased by ginsenoside Rg3 and Rf treatment in cellular model of HD. Mitochondrial biogenesis-related molecules such as PGC-1α and phosphorylated CREB were increased or showed increased tendency by ginsenoside Rg3 and Rf. Apoptotic molecules, p53, Bax, and cleaved caspase-3, were down-regulated by treatment of ginsenoside Rg3 and Rf. In addition, Lysotracker staining result showed that cellular lysosomal content was reduced by ginsenoside Rg3 and Rf. Given that ginsenoside Rg3 and Rf have the potential to reduce mHtt aggregation and cellular apoptosis, these ginsenosides can be possible therapeutic candidates for treating HD phenotypes.
Assuntos
Ginsenosídeos/farmacologia , Proteína Huntingtina/genética , Doença de Huntington/tratamento farmacológico , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Doença de Huntington/genética , Doença de Huntington/patologia , Proteínas Mutantes/genética , Células-Tronco Neurais/efeitos dos fármacosRESUMO
Massive vaccination against COVID-19 has become a global priority. Simultaneously, concerns regarding the safety of vaccines are growing. We describe two patients who developed sensory Guillain-Barre syndrome (GBS) shortly after the first dose of the ChAdOx1 vaccine. We also summarize 12 published cases of GBS after ChAdOx1 vaccination, highlighting their unique clinical and paraclinical features. We propose a possible association between the risk of GBS and the ChAdOx1 vaccine and recommend surveillance for GBS following vaccination. Population-based studies are needed to determine causality and whether specific subpopulations are susceptible.
Assuntos
Vacinas contra COVID-19/efeitos adversos , COVID-19/prevenção & controle , Síndrome de Guillain-Barré/induzido quimicamente , Síndrome de Guillain-Barré/diagnóstico por imagem , Adulto , ChAdOx1 nCoV-19 , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Huntington's disease (HD) is one of the most devastating genetic neurodegenerative disorders with no effective medical therapy. ß-Lapachone (ßL) is a natural compound obtained from the bark of the Lapacho tree and has been reported to have beneficial effects on various diseases. Sirt1 is a deacetylase of the sirtuin family and deacetylates proteins including the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) which is associated with mitochondrial respiration and biogenesis. To examine the effectiveness of ßL on HD, ßL was orally applied to R6/2 HD mice and behavioral phenotypes associated with HD, such as impairment of rota-rod performance and increase of clasping behavior, as well as changes of Sirt1 expression, CREB phosphorylation and PGC-1α deacetylation were examined. Western blot results showed that Sirt1 and p-CREB levels were significantly increased in the brains of ßL-treated R6/2 mice. An increase in deacetylation of PGC-1α, which is thought to increase its activity, was observed by oral administration of ßL. In an in vitro HD model, ßL treatment resulted in an attenuation of MitoSOX red fluorescence intensity, indicating an amelioration of mitochondrial reactive oxygen species by ßL. Furthermore, improvements in the rota-rod performance and clasping score were observed in R6/2 HD mice after oral administration of ßL compared to that of vehicle control-treated mice. Taken together, our data show that ßL is a potential therapeutic candidate for the treatment of HD-associated phenotypes, and increases in Sirt1 level, CREB phosphorylation and PGC-103B1 deacetylation can be the possible underlying mechanism of the effects of ßL.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Doença de Huntington/tratamento farmacológico , Naftoquinonas/farmacologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fenótipo , Sirtuína 1/metabolismo , Acetilação/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Naftoquinonas/uso terapêutico , Fosforilação/efeitos dos fármacos , Superóxidos/metabolismoRESUMO
Adipose-derived stem cells (ADSC) have a therapeutic potential for the treatment of neurodegenerative disorders such as Alzheimer's disease (AD). Exosomes are extracellular vesicles secreted from various types of cells, and stem cell-derived exosomes are known to have beneficial effects in many diseases. Many studies have suggested that amyloid beta (Aß) peptides have a pivotal role in AD progression, by mitochondrial dysfunction of neuronal cells. We examined the therapeutic potential of exosomes derived from ADSCs (ADSC-Exo) in preventing the disease phenotypes induced by the Aß cascade in an AD in vitro model. Neuronal stem cells (NSCs) from the brains of TG2576 AD mice were used to examine the effects of ADSC-Exo on AD phenotypes. NSCs from AD mice can be grown as a neurosphere and differentiated. Differentiated NSCs of TG2576 mice showed increase of Aß42 and Aß40 levels, and Aß42/40 ratio. Apoptotic molecules such as p53, Bax and caspase-3 were increased and Bcl2, an anti-apoptotic molecule, was decreased in AD cells compared with wild-type littermate cells. Lower viable cell population and higher necrotic cells were examined in AD neuronal cells. ELISA result showed that ADSC-Exo treatment resulted in reduced Aß42 levels, Aß40 levels, and the Aß42/40 ratio of AD cells. Increased apoptotic molecules, p53, Bax, pro-caspase-3 and cleaved-caspase-3, and decreased Bcl-2 protein level were normalized by ADSC-Exo treatment. Flow cytometry analysis revealed that increased cell apoptosis of AD neuronal cells was reduced by ADSC-Exo. In addition, neurite growth, which is impaired by Aß in the brains of patients with AD, was augmented by ADSC-Exo treatment. Taken together, these findings implicate the disease-modulating effects of ADSC-Exo in the transgenic mice-derived AD in vitro model, and ADSC-Exo can be a therapeutic source to ameliorate the progression of Aß-induced neuronal death and AD.
Assuntos
Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Exossomos/metabolismo , Células-Tronco Mesenquimais/citologia , Neurônios/patologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Apoptose , Caspase 3/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Neuritos/efeitos da radiação , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Proteína X Associada a bcl-2/metabolismoRESUMO
Exposure of the skin to ultraviolet (UV) radiation causes extracellular matrix (ECM) collapse in the dermis, owing to an increase in matrix metalloproteinase (MMP) production in both the epidermis and dermis, and a decrease in type I collagen expression in the dermis. Recently, black rice (Oryza sativa L.) was reported to have a wide range of pharmacological effects in various settings. However, the effects of black rice extract (BRE) on UVirradiated skin cells have not yet been characterized. BRE treatment did not affect cell morphology and viability of HaCaT and human dermal fibroblasts (HDF). We demonstrated that BRE downregulated basal and UVinduced MMP1 expression in HaCaT cells. Furthermore, BRE significantly increased type I procollagen expression, and decreased MMP1 and MMP3 expression in UVirradiated HDF. The underlying mechanisms of these results involve a decrease in p38 and cJun Nterminal kinase activity, and suppression of UVinduced activation of activator protein1 (AP1). BRE reduced UVinduced reactive oxygen species production in HaCaT cells in a dosedependent manner. Indeed, mass spectrometry revealed that BRE contained antioxidative flavonoid components such as cyanidin3OßDglycoside and taxifolin7Oglucoside. These findings suggest that BRE attenuates UVinduced ECM damage by modulating mitogenactivated protein kinase and AP1 signaling, and could be used as an active ingredient for preventing photoaging of the skin.
Assuntos
Metaloproteinases da Matriz/metabolismo , Oryza , Extratos Vegetais/farmacologia , Pró-Colágeno/metabolismo , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Antioxidantes/química , Antioxidantes/farmacologia , Linhagem Celular , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Metaloproteinase 1 da Matriz/análise , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/análise , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinases da Matriz/análise , Oryza/química , Extratos Vegetais/química , Pró-Colágeno/análise , Espécies Reativas de Oxigênio/metabolismo , Pele/metabolismo , Envelhecimento da Pele/efeitos dos fármacos , Envelhecimento da Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
Human adipose stem cells (hASC) have therapeutic potential for the treatment of neurodegenerative disorders. Mitochondrial dysfunction is frequently observed in most neurodegenerative disorders, including Alzheimer's disease. We explored the therapeutic potential of hASC cytosolic extracts to attenuate neuronal death induced by mitochondrial dysfunction in an Alzheimer's disease (AD) in vitro models. Amyloid beta (Aß) was used to induce cytotoxity in an immortal hippocampal cell line (HT22) and neuronal stem cells from the brain of TG2576 transgenic mice were also used to test the protective role of hASC cytosolic extracts. Cell viability and flow cytometry results demonstrated that the hASC extract prevents the toxicity and apoptosis in AD in vitro models. Moreover, JC-1 and MitoSoxRed staining followed by fluorescence microscopy and flow cytometry results showed that the hASC extract ameliorated the effect of Aß-induced mitochondrial oxidative stress and reduced the mitochondrial membrane potential. Western blot result showed that hASC extract modulated mitochondria-associated proteins, such as Bax and Bcl2, and down-regulated cleaved caspase-3. In addition, hASC extract decreased Aß generation and reversed up-regulated p53 and foxo3a protein level in AD in vitro model cell derived from TG2576 mice. Taken together, these findings implicate a protective role of the hASC extract in the Aß-induced mitochondrial apoptosis via regulation of P53/foxo3a pathway, providing insight into the molecular mechanisms of hASC extract and a therapeutic strategy to ameliorate neuronal death induced by Aß.
Assuntos
Adipócitos/química , Peptídeos beta-Amiloides/metabolismo , Apoptose , Proteína Forkhead Box O3/metabolismo , Mitocôndrias/patologia , Células-Tronco/química , Proteína Supressora de Tumor p53/metabolismo , Doença de Alzheimer/metabolismo , Animais , Caspase 3/metabolismo , Sobrevivência Celular , Citosol/química , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação da Expressão Gênica , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Neurônios/metabolismo , Estresse OxidativoRESUMO
The skin senses external environment, including ultraviolet light (UV). Hippocampus is a brain region that is responsible for memory and emotion. However, changes in hippocampus by UV irradiation to the skin have not been studied. In this study, after 2 weeks of UV irradiation to the mouse skin, we examined molecular changes related to cognitive functions in the hippocampus and activation of the hypothalamic-pituitary-adrenal (HPA) axis. UV exposure to the skin decreased doublecortin-positive immature neurons and synaptic proteins, including N-methyl-D-aspartate receptor 2 A and postsynaptic density protein-95, in the hippocampus. Moreover, we observed that UV irradiation to the skin down-regulated brain-derived neurotrophic factor expression and ERK signaling in the hippocampus, which are known to modulate neurogenesis and synaptic plasticity. The cutaneous and central HPA axes were activated by UV, which resulted in significant increases in serum levels of corticosterone. Subsequently, UV irradiation to the skin activated the glucocorticoid-signaling pathway in the hippocampal dentate gyrus. Interestingly, after 6 weeks of UV irradiation, mice showed depression-like behavior in the tail suspension test. Taken together, our data suggest that repeated UV exposure through the skin may negatively affect hippocampal neurogenesis and synaptic plasticity along with HPA axis activation.