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1.
Front Pharmacol ; 9: 237, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29695962

RESUMO

Brain-derived neurotrophic factor (BDNF) plays important functions in cell survival and differentiation, neuronal outgrowth and plasticity. In Alzheimer's disease (AD), BDNF signaling is known to be impaired, partially because amyloid ß (Aß) induces truncation of BDNF main receptor, TrkB-full length (TrkB-FL). We have previously shown that such truncation is mediated by calpains, results in the formation of an intracellular domain (ICD) fragment and causes BDNF loss of function. Since calpains are Ca2+-dependent proteases, we hypothesized that excessive intracellular Ca2+ build-up could be due to dysfunctional N-methyl-d-aspartate receptors (NMDARs) activation. To experimentally address this hypothesis, we investigated whether TrkB-FL truncation by calpains and consequent BDNF loss of function could be prevented by NMDAR blockade. We herein demonstrate that a NMDAR antagonist, memantine, prevented excessive calpain activation and TrkB-FL truncation induced by Aß25-35. When calpains were inhibited by calpastatin, BDNF was able to increase the dendritic spine density of neurons exposed to Aß25135. Moreover, NMDAR inhibition by memantine also prevented Aß-driven deleterious impact of BDNF loss of function on structural (spine density) and functional outcomes (synaptic potentiation). Collectively, these findings support NMDAR/Ca2+/calpains mechanistic involvement in Aß-triggered BDNF signaling disruption.

2.
Behav Brain Res ; 247: 241-7, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23557695

RESUMO

Behavioral evidence suggests that the organoselenium compound p,p'-methoxyl-diphenyl diselenide [(MeOPhSe)2] ameliorates memory and learning performance in rodents. Here, we investigated the molecular mechanism of (MeOPhSe)2 neuroprotection in cortical neurons exposed to amyloid-ß (Aß) peptide as well as in Aß-infused mice. For this purpose, primary cultures of rat cortical neurons were pre-incubated with 10 µM of (MeOPhSe)2 or vehicle, followed by exposure to 25 µM Aß fragment 25-35 or vehicle. Furthermore, the therapeutic effect of (MeOPhSe)2 (5 mg/kg, oral route, daily for 5 days) on memory deficits was evaluated in mice exposed to Aß fragment 25-35 (3 nmol/3 µl/per site, intracerebroventricular infusion). The results demonstrate that (MeOPhSe)2 prevented Aß-induced cell death in vitro, associated with inhibition of caspase-3 and -9 activities, poly (ADP-ribose) polymerase (PARP) cleavage and c-Jun N-terminal kinase (JNK) activation. Further, (MeOPhSe)2 rescued Aß-induced memory impairment in mice. In conclusion, (MeOPhSe)2 is neuroprotective in vitro and in vivo, suggesting that this organoselenium compound offers a potential treatment option for Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Derivados de Benzeno/farmacologia , Transtornos da Memória/tratamento farmacológico , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Compostos Organosselênicos/farmacologia , Fragmentos de Peptídeos/toxicidade , Animais , Derivados de Benzeno/uso terapêutico , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Citotoxinas/toxicidade , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/metabolismo , Camundongos , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Compostos Organosselênicos/uso terapêutico , Ratos
3.
J Alzheimers Dis ; 33(1): 133-44, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22903129

RESUMO

The purpose of this study was to investigate possible molecular targets involved in the neuroprotective effect of p,p'-methoxyl-diphenyl diselenide [(MeOPhSe)2], using a streptozotocin (STZ)-induced sporadic dementia of Alzheimer's type rat model. Male Wistar rats were injected with STZ (1.0 mg/8 µl; 4 µl/ventricle). After 21 days of STZ injection, regular diet-fed rats were supplemented with 10 ppm of (MeOPhSe)2 during 30 days. At the end of this period, rats performed object recognition and step-down passive avoidance tasks. Apoptosis was assessed by TUNEL staining and active caspase-3. Glial fibrillary acidic protein, ionized calcium binding adaptor molecule 1, and microtubule associated protein 2 were determined by immunofluorescence in rat hippocampus. The results demonstrate that the (MeOPhSe)2 dietary supplementation reversed STZ-induced memory impairment by enhancing memory in sham rats. (MeOPhSe)2 was also effective in reducing STZ-induced apoptosis and preserving dendrites and synapses. Moreover, (MeOPhSe)2 inhibited activation of microglia and astrogliosis induced by STZ in the rat hippocampus. We conclude that the (MeOPhSe)2 neuroprotective action is related to inhibition of apoptosis and suppression of inflammation.


Assuntos
Derivados de Benzeno/uso terapêutico , Doenças Neurodegenerativas/prevenção & controle , Neuroglia/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Compostos Organosselênicos/uso terapêutico , Estreptozocina/toxicidade , Animais , Derivados de Benzeno/farmacologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Masculino , Memória/efeitos dos fármacos , Memória/fisiologia , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Fármacos Neuroprotetores/farmacologia , Compostos Organosselênicos/farmacologia , Ratos , Ratos Wistar
4.
Neurobiol Aging ; 34(2): 551-61, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22621777

RESUMO

Synapses are considered the earliest site of Alzheimer's disease (AD) pathology, where synapse density is reduced, and synaptic loss is highly correlated with cognitive impairment. Tauroursodeoxycholic acid (TUDCA) has been shown to be neuroprotective in several models of AD, including neuronal exposure to amyloid ß (Aß) and amyloid precursor protein (APP)/presenilin 1 (PS1) double-transgenic mice. Here, we show that TUDCA modulates synaptic deficits induced by Aß in vitro. Specifically, TUDCA reduced the downregulation of the postsynaptic marker postsynaptic density-95 (PSD-95) and the decrease in spontaneous miniature excitatory postsynaptic currents (mEPSCs) frequency, while increasing the number of dendritic spines. This contributed to the induction of more robust and synaptically efficient neurons, reflected in inhibition of neuronal death. In vivo, TUDCA treatment of APP/PS1 mice abrogated the decrease in PSD-95 reactivity in the hippocampus. Taken together, these results expand the neuroprotective role of TUDCA to a synaptic level, further supporting the use of this molecule as a potential therapeutic strategy for the prevention and treatment of AD.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/farmacologia , Fármacos Neuroprotetores/farmacologia , Sinapses/efeitos dos fármacos , Ácido Tauroquenodesoxicólico/farmacologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Ratos , Ratos Wistar , Sinapses/metabolismo
5.
Phytother Res ; 26(5): 692-6, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22002659

RESUMO

Seven lignans, previously isolated from Pycnanthus angolensis or obtained by derivatization, namely the dibenzylbutane-type lignans threo-4,4'-dihydroxy-3-methoxylignan (1), 4'-hydroxy-3,3',4-trimethoxylignan (2), (-)-dihydroguaiaretic acid (3), 3,3',4,4'-tetramethoxylignan (4), 4,4'-diacetyl-3,3'-dimethoxylignan (5), heliobuphthalmin (6) and the butyrolactone lignan hinokinin (7), were evaluted for their ability as apoptosis inducers in human hepatoma HuH-7 cells. Cell viability assays, morphological evaluation of apoptosis and enzymatic analyses of caspase activity in HuH-7 cells were carried out. Using the lactate dehydrogenase lactate dehydrogenase (LDH) assay, it was demonstrated that the lignans (1-7) tested significantly reduced viability of HuH-7 cells. Morphologic evaluation of HuH-7 cells using Hoechst staining and fluorescence microscopy revealed that lignans 1-7 were strong inducers of apoptosis. In fact, HuH-7 cells developed morphological changes of apoptosis, including chromatin condensation, nuclear fragmentation and formation of apoptotic bodies. However, lignans 2 and 7 were the most promising compounds in this study, inducing 2.4- and 2.5-fold increases in apoptotic cells as compared to controls. Caspase-3-like activity assays confirmed the morphologic data.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Fator de Indução de Apoptose/farmacologia , Apoptose/efeitos dos fármacos , Lignanas/farmacologia , Myristicaceae/química , Extratos Vegetais/farmacologia , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Fator de Indução de Apoptose/química , Fator de Indução de Apoptose/isolamento & purificação , Benzodioxóis , Caspase 3/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Dioxóis/farmacologia , Humanos , L-Lactato Desidrogenase/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Lactonas/química , Lactonas/isolamento & purificação , Lactonas/farmacologia , Lignanas/química , Lignanas/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Plantas Medicinais/química
6.
Artigo em Inglês | MEDLINE | ID: mdl-21600750

RESUMO

We hypothesized that reducing weight properties of conjugated linoleic acid (CLA) are due to adipocyte apoptosis and that CLA differentially modulates the apoptotic responses in hepatic lipotoxicity from rats fed saturated fat diets. Obese Zucker rats were fed atherogenic diets (2%w/w of cholesterol) formulated with high (15%w/w) saturated fat, from vegetable or animal origin, supplemented or not with 1% of a mixture (1:1) of cis-9, trans-11 and trans-10, cis-12 CLA isomers for 14 weeks. CLA induced no changes on retroperitoneal fat depot weight, which was in line with similar levels of apoptosis. Interestingly, CLA had a contrasting effect on cell death in the liver according to the dietary fat. CLA increased hepatocyte apoptosis, associated with upregulation of Fas protein in rats fed palm oil, compared to rats receiving palm oil alone. However, rats fed ovine fat alone displayed the highest levels of hepatic cell death, which were decreased in rats fed ovine fat plus CLA. This reducing effect of CLA was related to positively restoring endoplasmic reticulum (ER) ATF-6α, BiP and CHOP protein levels and increasing phosphorylated c-Jun NH(2)-terminal kinase (JNK) and c-Jun, thus suggesting an adaptive response of cell survival. These findings reinforce the role of CLA as regulator of apoptosis in the liver. Moreover, the dietary fat composition is a key factor in activation of apoptosis.


Assuntos
Apoptose/efeitos dos fármacos , Gorduras na Dieta/administração & dosagem , Ácidos Linoleicos Conjugados/administração & dosagem , Fígado/efeitos dos fármacos , Obesidade/metabolismo , Óleos de Plantas/administração & dosagem , Animais , Caspase 3/metabolismo , Gorduras na Dieta/farmacologia , Gorduras Insaturadas na Dieta/administração & dosagem , Gorduras Insaturadas na Dieta/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Isomerismo , Ácidos Linoleicos Conjugados/química , Ácidos Linoleicos Conjugados/farmacologia , Fígado/citologia , Fígado/metabolismo , Masculino , Obesidade/induzido quimicamente , Óleo de Palmeira , Óleos de Plantas/farmacologia , Ratos , Ratos Zucker , Ovinos
7.
Phytother Res ; 25(12): 1819-24, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21495101

RESUMO

Nine flavonoids isolated from the ethyl acetate extract of Pycnanthus angolensis were assayed for their potential apoptosis induction activities in human hepatoma HuH-7 cells. These flavonoids include eight isoflavones, namely irilone (1), tectorigenine (2), formononetin (3), genistein (4), 2'-hydroxybiochanin A (5), mixture of biochanin A (6) and prunetin (7), and 4',7-dihydroxy-2'-methoxyisoflavan (8), and the flavanone liguiritigentin (9). Their chemical structures were characterized by spectroscopic methods including 2D NMR experiments. Methodology for cell death detection included the LDH assay, Hoechst staining, TUNEL staining and general caspase-3-like activity assay. The compounds tested showed higher apoptosis induction profiles in HuH-7 cells compared with the control. Caspase activity assays confirmed the apoptosis inducing activity of these flavonoids.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Isoflavonas/farmacologia , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Humanos , Isoflavonas/química , Neoplasias Hepáticas/metabolismo , Myristicaceae/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia
8.
CNS Neurol Disord Drug Targets ; 9(6): 679-92, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20942793

RESUMO

Neurodegenerative diseases trigger neuronal cell death by a variety of endogenous suicide pathways. Although cell death may occur through highly heterogeneous processes, specific cell organelles and stress sensors have shown promise as potential therapeutic targets. The plasma membrane senses stress through residing receptors, which can directly or indirectly activate apoptosis. Importantly, several events involved in neuronal death also affect mitochondria homeostasis, leading to calcium uptake, opening of the permeability transition pore, and release of apoptogenic factors. In addition, nuclear DNA damage triggers cell death, where p53 is activated to modulate the expression of selected apoptosis target genes. Signaling proteins implicated in apoptosis pathways are enriched at the Golgi complex, including death receptors and the phosphoinositide 3-kinase. Finally, neurodegenerative diseases progress with accumulation of misfolded proteins, deficiently removed by intracellular proteases or chaperones, and transport abnormalities due to disturbance of cytoskeletal organization in degenerating neurons. The challenge is to decode the complex signaling network of inter-organellar crosstalk leading to cell death and identify therapeutic approaches for delaying or preventing neurodegenerative diseases.


Assuntos
Apoptose/fisiologia , Morte Celular/fisiologia , Doenças Neurodegenerativas/fisiopatologia , Neurônios/fisiologia , Organelas/metabolismo , Estresse Fisiológico/fisiologia , Citoesqueleto/metabolismo , Descoberta de Drogas/métodos , Humanos , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia
9.
J Alzheimers Dis ; 22(2): 557-68, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20847398

RESUMO

Amyloid-ß (Aß) peptide- induced neurotoxicity is typically associated with apoptosis. In previous studies, we have shown that tauroursodeoxycholic acid (TUDCA), an endogenous anti-apoptotic bile acid, modulates Aß-induced apoptosis. Here, we investigated stress signaling events triggered by soluble Aß and further explored alternative pathways of neuroprotection by TUDCA in differentiated rat neuronal-like PC12 cells. Morphologic evaluation of apoptosis confirmed that Aß-induced nuclear fragmentation was prevented by TUDCA. In addition, Aß exposure resulted in activation of the early stress c-Jun N-terminal kinase (JNK) pathway, JNK nuclear translocation, and caspase-2 activation. Knock-down experiments of JNK established caspase-2 as a specific downstream target of JNK in Aß-induced apoptosis. Furthermore, active caspase-2 cleaved golgin-160 and was localized to the Golgi complex. Importantly, TUDCA abrogated Aß-induced JNK/caspase-2 signaling. In conclusion, we show that JNK is the proximal stress sensor for soluble Aß-induced toxicity, which translocates to the nucleus, activates caspase-2, and is strongly modulated by TUDCA in PC12 neuronal cells. Active caspase-2 cleaves golgin-160, suggesting caspase-2-dependent transduction of Aß apoptotic signaling through the Golgi complex. These data provide new information linking apoptotic properties of Aß peptide to distinct subcellular mechanisms of toxicity. Further characterization of this signaling pathway and exact targets of modulation are likely to provide new perspectives for modulation of amyloid-induced apoptosis by TUDCA.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Apoptose/efeitos dos fármacos , Caspase 2/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Colagogos e Coleréticos/farmacologia , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Células PC12/efeitos dos fármacos , Ratos , Ácido Tauroquenodesoxicólico/farmacologia , Fatores de Tempo
10.
Bioorg Med Chem Lett ; 19(15): 4255-8, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19525111

RESUMO

Three known (1-3) and a novel (4) monoterpene indole alkaloids have been isolated from the methanol extract of leaves of Tabernaemontana elegans and their structures were elucidated by a series of spectroscopic experiments, involving NMR, MS, UV, and IR techniques. The isolated monoterpene indole alkaloids along with previously described beta-carbolines (5-7) from the same specimen were studied for their apoptosis induction activity in human hepatoma HuH-7 cells. Methodology for apoptosis induction studies included cell viability assays, nuclear morphology assessments, and general caspase-3-like activity assays. The monoterpene indole alkaloids, tabernaemontanine (1) and vobasine (3) showed the most promising apoptosis induction profile in HuH-7 cells.


Assuntos
Apoptose , Carbolinas/química , Alcaloides Indólicos/síntese química , Extratos Vegetais/metabolismo , Folhas de Planta/metabolismo , Tabernaemontana/metabolismo , Alcaloides , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Alcaloides Indólicos/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Moleculares , Conformação Molecular
11.
J Lipid Res ; 50(9): 1721-34, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19417220

RESUMO

Bile acids are a group of molecular species of acidic steroids with peculiar physical-chemical and biological characteristics. At high concentrations they become toxic to mammalian cells, and their presence is pertinent in the pathogenesis of several liver diseases and colon cancer. Bile acid cytoxicity has been related to membrane damage, but also to nondetergent effects, such as oxidative stress and apoptosis. Strikingly, hydrophilic ursodeoxycholic acid (UDCA), and its taurine-conjugated form (TUDCA), show profound cytoprotective properties. Indeed, these molecules have been described as potent inhibitors of classic pathways of apoptosis, although their precise mode of action remains to be clarified. UDCA, originally used for cholesterol gallstone dissolution, is currently considered the first choice therapy for several forms of cholestatic syndromes. However, the beneficial effects of both UDCA and TUDCA have been tested in other experimental pathological conditions with deregulated levels of apoptosis, including neurological disorders, such as Alzheimer's, Parkinson's, and Huntington's diseases. Here, we review the role of bile acids in modulating the apoptosis process, emphasizing the anti-apoptotic effects of UDCA and TUDCA, as well as their potential use as novel and alternate therapeutic agents for the treatment of apoptosis-related diseases.


Assuntos
Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/farmacologia , Ácido Ursodesoxicólico/farmacologia , Animais , Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/uso terapêutico , Humanos , Transdução de Sinais/efeitos dos fármacos , Ácido Ursodesoxicólico/metabolismo , Ácido Ursodesoxicólico/uso terapêutico
12.
Mol Med ; 14(5-6): 309-17, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18368144

RESUMO

The rTg4510 mouse is a tauopathy model, characterized by massive neurodegeneration in Alzheimer's disease (AD)-relevant cortical and limbic structures, deficits in spatial reference memory, and progression of neurofibrillary tangles (NFT). In this study, we examined the role of apoptosis in neuronal loss and associated tau pathology. The results showed that DNA fragmentation and caspase-3 activation are common in the hippocampus and frontal cortex of young rTg4510 mice. These changes were associated with cleavage of tau into smaller intermediate fragments, which persist with age. Interestingly, active caspase-3 was often co-localized with cleaved tau. In vitro, fibrillar Abeta(1-42) resulted in nuclear fragmentation, caspase activation, and caspase-3-induced cleavage of tau. Notably, incubation with the antiapoptotic molecule tauroursodeoxycholic acid abrogated apoptosis-mediated cleavage of tau in rat cortical neurons. In conclusion, caspase-3-cleaved intermediate tau species occurred early in rTg54510 brains and preceded cell loss in Abeta-exposed cultured neurons. These results suggest a potential role of apoptosis in neurodegeneration.


Assuntos
Apoptose , Mutação , Proteínas tau/genética , Substituição de Aminoácidos , Animais , Caspase 3/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos Wistar , Proteínas tau/metabolismo
13.
Trends Mol Med ; 14(2): 54-62, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18218342

RESUMO

The potential role of apoptosis in Alzheimer's disease (AD) has been an area of intense research in recent years. Ursodeoxycholic acid (UDCA) and its taurine-conjugate, tauroursodeoxycholic acid (TUDCA) are endogenous bile acids that act as potent inhibitors of apoptosis. Their therapeutic effects have been tested in many experimental pathological conditions, including neurological disorders, such as AD. TUDCA regulates precise transcriptional and post-transcriptional events that impact mitochondrial function in neurons. TUDCA not only stabilizes the mitochondrial membrane and prevents Bax translocation, inhibiting the release of cytochrome c and the activation of caspases, but also interferes with upstream factors, including cell cycle-related proteins. In addition, TUDCA is capable of inducing survival pathways. Here, we review the role of apoptosis in AD and discuss the therapeutic potential of TUDCA in treating this disease.


Assuntos
Doença de Alzheimer/metabolismo , Apoptose , Ácido Tauroquenodesoxicólico/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Humanos , Modelos Biológicos , Ácido Tauroquenodesoxicólico/metabolismo , Ácido Ursodesoxicólico/metabolismo
14.
J Neurochem ; 98(5): 1610-8, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16923170

RESUMO

Early onset familial Alzheimer's disease (FAD) is linked to autosomal dominant mutations in the amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2) genes. These are critical mediators of total amyloid beta-peptide (Abeta) production, inducing cell death through uncertain mechanisms. Tauroursodeoxycholic acid (TUDCA) modulates exogenous Abeta-induced apoptosis by interfering with E2F-1/p53/Bax. Here, we used mouse neuroblastoma cells that express either wild-type APP, APP with the Swedish mutation (APPswe), or double-mutated human APP and PS1 (APPswe/DeltaE9), all exhibiting increased Abeta production and aggregation. Cell viability was decreased in APPswe and APPswe/DeltaE9 but was partially reversed by z-VAD.fmk. Nuclear fragmentation and caspase 2, 6 and 8 activation were also readily detected. TUDCA reduced nuclear fragmentation as well as caspase 2 and 6, but not caspase 8 activities. p53 activity, and Bcl-2 and Bax changes, were also modulated by TUDCA. Overexpression of p53, but not mutant p53, in wild-type and mutant neuroblastoma cells was sufficient to induce apoptosis, which, in turn, was reduced by TUDCA. In addition, inhibition of the phosphatidylinositide 3'-OH kinase pathway reduced TUDCA protection against p53-induced apoptosis. In conclusion, FAD mutations are associated with the activation of classical apoptotic pathways. TUDCA reduces p53-induced apoptosis and modulates expression of Bcl-2 family.


Assuntos
Doença de Alzheimer/genética , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Mutação , Ácido Tauroquenodesoxicólico/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Precursor de Proteína beta-Amiloide/genética , Animais , Apoptose/fisiologia , Western Blotting , Caspases/metabolismo , Linhagem Celular Tumoral , Inibidores de Cisteína Proteinase/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Marcação In Situ das Extremidades Cortadas/métodos , Proteínas de Membrana/genética , Camundongos , Neuroblastoma , Presenilina-1 , Presenilina-2 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transfecção/métodos , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2/metabolismo
15.
Mol Endocrinol ; 20(10): 2292-303, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16728529

RESUMO

Tauroursodeoxycholic acid (TUDCA) prevents amyloid beta-peptide (Abeta)-induced neuronal apoptosis, by modulating both classical mitochondrial pathways and specific upstream targets. In addition, activation of nuclear steroid receptors (NSRs), such as the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) differentially regulates apoptosis in the brain. In this study we investigated whether TUDCA, a cholesterol-derived endogenous molecule, requires NSRs for inhibiting Abeta-induced apoptosis in primary neurons. Our results confirmed that TUDCA significantly reduced Abeta-induced apoptosis; in addition, the fluorescently labeled bile acid molecule was detected diffusely in both cytoplasm and nucleus of rat cortical neurons. Interestingly, experiments using small interfering RNAs (siRNAs) revealed that, in contrast to GR siRNA, MR siRNA abolished the antiapoptotic effect of TUDCA. Abeta incubation reduced MR nuclear translocation while increasing nuclear GR levels. Notably, pretreatment with TUDCA markedly altered Abeta-induced changes in NSRs, including MR dissociation from its cytosolic chaperone, heat shock protein 90, and subsequent translocation to the nucleus. Furthermore, when a carboxy terminus-deleted form of MR was used, nuclear trafficking of both MR and the bile acid was abrogated, suggesting that they translocate to the nucleus as a steroid-receptor complex. Transfection experiments with wild-type or mutant MR confirmed that this interaction was required for TUDCA protection against Abeta-induced apoptosis. Finally, in cotransfection experiments with NSR response element reporter and overexpression constructs, pretreatment with TUDCA significantly modulated Abeta-induced changes in MR and GR transactivation. In conclusion, these results provide novel insights into the specific cellular mechanism of TUDCA antiapoptotic function against Abeta-induced apoptosis and suggest targets for potential therapeutic intervention.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Apoptose/efeitos dos fármacos , Neurônios/citologia , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Ativação Transcricional/efeitos dos fármacos , Animais , Apoptose/fisiologia , Núcleo Celular/metabolismo , Células Cultivadas , Immunoblotting , Imunoprecipitação , Microscopia de Fluorescência , RNA Interferente Pequeno/metabolismo , Ratos , Ativação Transcricional/fisiologia
16.
Eur J Gastroenterol Hepatol ; 18(1): 21-9, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16357615

RESUMO

OBJECTIVES: Apoptosis may play a role in the pathogenesis of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH). In this study, we investigated the modulation of apoptosis-related liver proteins in steatohepatitis. METHODS: Hepatocyte apoptosis was evaluated by the TUNEL assay in liver tissue of 12 patients with NASH, 12 with ASH and in histologically normal controls. In addition, caspase-3 processing was evaluated by immunoblot analysis. Expression of death receptors, Bcl-2 family members, and NF-kappaB inhibitor (IkappaB) were determined by western blot. Liver biopsies were also graded for inflammation and fibrosis. RESULTS: Apoptotic hepatocytes were markedly increased in NASH (P<0.05) and ASH (P<0.001) as compared to controls. Active caspase-3 was also elevated in steatohepatitis (P<0.01), coinciding with upregulation of pro-apoptotic Bax (P<0.001). Further, production of tumour necrosis factor-receptor 1 was increased up to 4-fold (P<0.05). Degradation of IkappaB increased >70% in steatohepatitis (P<0.001). Notably, Bcl-2 was also strongly expressed (>100-fold; P<0.001). These data were significantly correlated with relative degrees of portal and lobular inflammation. CONCLUSION: The results show that liver injury in NASH and ASH is associated with apoptosis and NF-kappaB activation. Anti-apoptotic Bcl-2 is strongly expressed, probably reflecting an adaptive response to obesity or alcohol-related stress.


Assuntos
Apoptose , Fígado Gorduroso/patologia , Hepatócitos/patologia , Fígado/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adulto , Caspase 3 , Caspases/metabolismo , Fígado Gorduroso/metabolismo , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso Alcoólico/patologia , Feminino , Humanos , Proteínas I-kappa B/metabolismo , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Masculino , Pessoa de Meia-Idade , NF-kappa B/antagonistas & inibidores , Estudos Prospectivos , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo
17.
Hepatology ; 42(4): 925-34, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16175607

RESUMO

Ursodeoxycholic acid (UDCA) inhibits classical mitochondrial pathways of apoptosis by either directly stabilizing mitochondrial membranes or modulating specific upstream targets. Furthermore, UDCA regulates apoptosis-related genes from transforming growth factor beta1 (TGF-beta1)-induced hepatocyte apoptosis by a nuclear steroid receptor (NSR)-dependent mechanism. In this study, we further investigated the potential role of the glucocorticoid receptor (GR) in the anti-apoptotic function of UDCA. Our results with short interference RNA (siRNA) technology confirmed that UDCA significantly reduces TGF-beta1-induced apoptosis of primary rat hepatocytes through a GR-dependent effect. Immunoprecipitation assays and confocal microscopy showed that UDCA enhanced free GR levels with subsequent GR nuclear translocation. Interestingly, when a carboxy-terminus deleted form of GR was used, UDCA no longer increased free GR and/or GR translocation, nor did it protect against TGF-beta1-induced apoptosis. In co-transfection experiments with GR response element reporter and overexpression constructs, UDCA did not enhance the transactivation of GR with TGF-beta1. Finally, using a fluorescently labeled UDCA molecule, the bile acid appeared diffuse in the cytosol but was aggregated in the nucleus of hepatocytes. Both siRNA assays and transfection experiments with either wild-type or mutant forms of GR showed that nuclear trafficking occurs through a GR-dependent mechanism. In conclusion, these results further clarify the anti-apoptotic mechanism(s) of UDCA and suggest that GR is crucial for the nuclear translocation of this bile acid for reducing apoptosis.


Assuntos
Apoptose/fisiologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Receptores de Glucocorticoides/metabolismo , Ácido Ursodesoxicólico/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Masculino , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1
18.
J Hepatol ; 42(6): 897-906, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15885361

RESUMO

BACKGROUND/AIMS: Ursodeoxycholic acid (UDCA) and its taurine-conjugated derivative, TUDCA, modulate cell death and cell cycle regulators, such as E2F-1 and p53. However, precise pathways underlying UDCA's effects are not fully understood. The aim of this study was to identify specific cellular targets of UDCA. METHODS: The expression profile of primary rat hepatocytes incubated with UDCA was determined using Affymetrix GeneChip Rat 230A arrays. Hybridization data were processed to identify genes with significant expression changes. RT-PCR and immunoblot analyses of a selected target confirmed microarray data. RESULTS: The results showed that >440 genes were modulated with UDCA by >1.5-fold; approximately 25% were significantly different from controls. Genes affected by UDCA included new regulatory molecules, such as Apaf-1. RT-PCR and immunoblotting confirmed a decrease in Apaf-1. Other altered genes were directly involved in cell cycle (cyclin D1, cadherin 1, HMG-box containing protein 1) and apoptosis (prothymosin-alpha) events. The E2F-1/p53/Apaf-1 pathway appears to be targeted by UDCA. Finally, transcripts for proteins with kinase activity and transcription factors were specifically modulated by TUDCA. CONCLUSIONS: This study expands our knowledge of the biological effects of UDCA in hepatocytes. Most of the identified genes represent novel potential targets of UDCA, which may ultimately explain its therapeutic properties.


Assuntos
Colagogos e Coleréticos/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Ácido Ursodesoxicólico/farmacologia , Animais , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Masculino , Ratos , Ratos Sprague-Dawley , Ácido Tauroquenodesoxicólico/farmacologia
19.
Am J Gastroenterol ; 99(9): 1708-17, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15330907

RESUMO

OBJECTIVES: The increasing incidence of nonalcoholic (NASH) and alcoholic steatohepatitis (ASH), associated with lack of effective treatment, has prompted intensive studies on disease pathogenesis. Apoptosis is recognized as common in liver injury and may also contribute to tissue inflammation, fibrogenesis, and development of cirrhosis. In this study, we identified mechanisms of apoptosis induction in human steatohepatitis, and evaluated potential associations between apoptosis, liver pathology, and clinical presentation in NASH and ASH. METHODS: Hepatocyte apoptosis was evaluated by the TUNEL assay in 20 patients with NASH (all ambulatory), 40 with ASH (20 ambulatory, 20 hospitalized), and 20 controls. Liver biopsies were also graded for inflammation and fibrosis. Immunohistochemistry was performed for death receptors (Fas and TNF-R1), activated caspase-3, NF-kappaB p65, antiapoptotic Bcl-2 protein, and uncoupling protein 2 (UCP-2). RESULTS: TUNEL-positive hepatocytes were markedly increased in NASH (p < 0.05) and ASH (p < 0.01). Similar results were obtained for activated caspase-3, confirming the occurrence of apoptosis. The Fas receptor was upregulated in ASH, especially in hospitalized patients (p < 0.01), whereas TNF-R1 was expressed both in NASH and ASH (p < 0.01). In addition, patients with ASH showed a remarkable expression of active NF-kappaB, as compared to NASH and controls (p < 0.01). Degrees of inflammation and fibrosis correlated with NF-kappaB p65 expression, which in turn coincided with apoptosis albeit Bcl-2 and UCP-2 expression. CONCLUSIONS: Liver injury in NASH and ASH is associated with increased hepatocyte apoptosis mediated by death receptors. Further, apoptosis correlates with active NF-kappaB expression, and disease severity. This potential mechanistic link might provide multiple interesting targets for therapeutic intervention.


Assuntos
Apoptose/fisiologia , Hepatócitos/patologia , NF-kappa B/metabolismo , Receptor fas/metabolismo , Adulto , Assistência Ambulatorial , Biomarcadores/análise , Biópsia por Agulha , Estudos de Casos e Controles , Células Cultivadas , Fígado Gorduroso/patologia , Fígado Gorduroso/terapia , Fígado Gorduroso Alcoólico/patologia , Fígado Gorduroso Alcoólico/terapia , Feminino , Hospitalização , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Probabilidade , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/análise , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Regulação para Cima
20.
J Neurochem ; 90(3): 567-75, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15255934

RESUMO

Amyloid beta-peptide (Abeta)-induced cell death may involve activation of the E2F-1 transcription factor and other cell cycle-related proteins. In previous studies, we have shown that tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, modulates Abeta-induced apoptosis by interfering with crucial events of the mitochondrial pathway. In this study, we examined the role of E2F and p53 activation in the induction of apoptosis by Abeta, and investigated novel molecular targets for TUDCA. The results showed that despite Bcl-2 up-regulation, PC12 neuronal cells underwent significant apoptosis after incubation with the active fragment Abeta (25-35), as assessed by DNA fragmentation, nuclear morphology and caspase-3-like activation. In addition, transcription through the E2F-1 promoter was significantly induced and associated with loss of the retinoblastoma protein. In contrast, levels of E2F-1, p53 and Bax proteins were markedly increased. Overexpression of E2F-1 in PC12 cells was sufficient to induce p53 and Bax proteins, as well as nuclear fragmentation. Notably, TUDCA modulated Abeta-induced apoptosis, E2F-1 induction, p53 stabilization and Bax expression. Further, TUDCA protected PC12 cells against p53- and Bax-dependent apoptosis induced by E2F-1 and p53 overexpression, respectively. In conclusion, the results demonstrate that Abeta-induced apoptosis of PC12 cells proceeds through an E2F-1/p53/Bax pathway, which, in turn, can be specifically inhibited by TUDCA, thus underscoring its potential therapeutic use.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Fragmentos de Peptídeos/toxicidade , Proteínas Proto-Oncogênicas/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Expressão Gênica/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Proteínas Nucleares/metabolismo , Células PC12 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteína X Associada a bcl-2
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