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1.
Bioorg Med Chem Lett ; 30(6): 126967, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32005415

RESUMO

Inhibition of the nuclear receptor Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) is a promising strategy for the treatment of autoimmune diseases. In this paper, we describe a series of allosteric, cysteine-dependent, inverse agonists of RORγt. Site-directed mutagenesis and molecular dynamics simulations are supportive of a mechanism of action through specific binding to Cys476 on alpha helix 11 of the ligand binding domain (LBD). Representative compounds in the series selectively inhibit RORγt, potently suppress interleukin-17A (IL-17A) production by human CD4+ T cells, and inhibit T helper 17 (Th17) differentiation from human naïve CD4+ T cells. The advanced compound 13 is orally bioavailable and active at a dose of 3 mg/kg in a murine collagen-induced model of rheumatoid arthritis. Collectively, these data are supportive of the development of compound 13 in autoimmune diseases.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Cisteína/química , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Pirimidinas/química , Sítio Alostérico , Animais , Diferenciação Celular/efeitos dos fármacos , Colágeno/metabolismo , Modelos Animais de Doenças , Humanos , Hidroxicolesteróis/metabolismo , Interleucina-17/metabolismo , Ligantes , Camundongos , Simulação de Dinâmica Molecular , Mutagênese/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Células Th17
2.
Biochim Biophys Acta ; 1860(11 Pt A): 2537-2552, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27474998

RESUMO

BACKGROUND: Semi-synthetic oleanane triterpenoid antioxidant inflammation modulators (tpAIMs) are small molecules that interact with KEAP1 cysteine residue 151 (C151) and activate NRF2. Exploration of the structure-activity relationship between the tpAIMs and KEAP1 is limited by the predominantly hydrocarbon nature of the oleanane triterpenoid pentacyclic ring structure. Therefore, we used novel, chemically-tractable, synthetic antioxidant inflammation modulators (sAIMs) to probe the stereoselectivity of the ligand-protein interaction. METHODS: We measured several parameters of NRF2 activation to assess the potency of sAIM enantiomers with natural (tpAIM-like) 4(S),5(S),10(R) or unnatural 4(R),5(R),10(S) configurations. Additionally, we determined the crystal structure of the KEAP1 BTB domain in complex with two different sAIMs. RESULTS: We found that the potencies of sAIM enantiomers in the natural configuration were similar to those of the tpAIM, RTA 405. Strikingly, sAIM enantiomers in the unnatural configuration were 10- to 40-fold less potent than their natural counterparts. Crystallographic studies of sAIMs in complex with the KEAP1 BTB domain demonstrated that these ligands form a covalent bond with C151 and revealed the presence of additional hydrogen bonds, Van der Waals interactions, and pi-stacking interactions. CONCLUSIONS: Although KEAP1 C151 is required for NRF2 activation by tpAIMs and sAIMs, interactions with other KEAP1 residues are critical for the stereospecific recognition and potency of these ligands. GENERAL SIGNIFICANCE: This work demonstrates that reversible cyanoenone Michael acceptors, such as the tpAIMs and sAIMs, can be specifically tuned to regulate redox sensitive cysteine residues on key signaling molecules, an approach with significant promise for innovative drug development.


Assuntos
Antioxidantes/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ácido Oleanólico/química , Relação Quantitativa Estrutura-Atividade , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antioxidantes/química , Sítios de Ligação , Células HEK293 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Camundongos , Simulação de Acoplamento Molecular , Fator 2 Relacionado a NF-E2/química , Bibliotecas de Moléculas Pequenas/química
3.
PLoS One ; 10(8): e0135257, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26301506

RESUMO

Synthetic triterpenoids are antioxidant inflammation modulators (AIMs) that exhibit broad anticancer activity. AIMs bind to KEAP1 and inhibit its ability to promote NRF2 degradation. As a result, NRF2 increases transcription of genes that restore redox balance and reduce inflammation. AIMs inhibit tumor growth and metastasis by increasing NRF2 activity in the tumor microenvironment and by modulating the activity of oncogenic signaling pathways, including NF-κB, in tumor cells. Accumulating evidence suggests that KEAP1 loss or mutation--which results in high levels of sustained NRF2 activity--may promote cancer growth and increase chemoresistance. Loss of KEAP1 also increases the levels of other oncogenic proteins, including IKKß and BCL2. The apparent survival advantage provided to some tumor cells by loss of functional KEAP1 raises the question of whether pharmacological inhibition of KEAP1 could promote tumor growth. To address this issue, we characterized the basal levels of KEAP1 and NRF2 in a panel of human tumor cell lines and profiled the activity of an AIM, RTA 405. We found that in tumor cell lines with low or mutant KEAP1, and in Keap1-/- murine embryonic fibroblasts, multiple KEAP1 targets including NRF2, IKKß, and BCL2 were elevated. Keap1-/- murine embryonic fibroblasts also had higher rates of proliferation and colony formation than their wild-type counterparts. In cells with functional KEAP1, RTA 405 increased NRF2 levels, but not IKKß or BCL2 levels, and did not increase cell proliferation or survival. Moreover, RTA 405 inhibited growth at similar concentrations in cells with different basal NRF2 activity levels and in cells with wild-type or mutant KRAS. Finally, pre-treatment with RTA 405 did not protect tumor cells from doxorubicin- or cisplatin-mediated growth inhibition. Collectively, these data demonstrate that pharmacological activation of NRF2 by AIMs is distinct from genetic activation and does not provide a growth or survival advantage to tumor cells.


Assuntos
Quinase I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fator 2 Relacionado a NF-E2/genética , Neoplasias/genética , Ácido Oleanólico/análogos & derivados , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Resistencia a Medicamentos Antineoplásicos/genética , Fibroblastos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteína 1 Associada a ECH Semelhante a Kelch , Células MCF-7 , Camundongos , Fator 2 Relacionado a NF-E2/biossíntese , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ácido Oleanólico/administração & dosagem , Ácido Oleanólico/síntese química , Estresse Oxidativo/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Transdução de Sinais/efeitos dos fármacos
4.
PLoS One ; 10(4): e0122942, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25897966

RESUMO

Semi-synthetic triterpenoids are antioxidant inflammation modulator (AIM) compounds that inhibit tumor cell growth and metastasis. Compounds in the AIM class bind to Keap1 and attenuate Nrf2 degradation. In the nucleus, Nrf2 increases antioxidant gene expression and reduces pro-inflammatory gene expression. By increasing Nrf2 activity, AIMs reduce reactive oxygen species and inflammation in the tumor microenvironment, which reverses tumor-mediated immune evasion and inhibits tumor growth and metastasis. AIMs also directly inhibit tumor cell growth by modulating oncogenic signaling pathways, such as IKKß/NF-κB. Here, we characterized the in vitro antioxidant, anti-inflammatory, and anticancer activities of RTA 408, a novel AIM that is currently being evaluated in patients with advanced malignancies. At low concentrations (≤ 25 nM), RTA 408 activated Nrf2 and suppressed nitric oxide and pro-inflammatory cytokine levels in interferon-γ-stimulated RAW 264.7 macrophage cells. At higher concentrations, RTA 408 inhibited tumor cell growth (GI50 = 260 ± 74 nM) and increased caspase activity in tumor cell lines, but not in normal primary human cells. Consistent with the direct effect of AIMs on IKKß, RTA 408 inhibited NF-κB signaling and decreased cyclin D1 levels at the same concentrations that inhibited cell growth and induced apoptosis. RTA 408 also increased CDKN1A (p21) levels and JNK phosphorylation. The in vitro activity profile of RTA 408 is similar to that of bardoxolone methyl, which was well-tolerated by patients at doses that demonstrated target engagement. Taken together, these data support clinical evaluation of RTA 408 for cancer treatment.


Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos/farmacologia , Triterpenos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Ativação Enzimática , Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Concentração Inibidora 50 , MAP Quinase Quinase 4/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo
5.
PLoS One ; 9(5): e96544, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24824433

RESUMO

CNS inflammation is a hallmark of neurodegenerative disease, and recent studies suggest that the inflammatory response may contribute to neuronal demise. In particular, increased tumor necrosis factor (TNF) signaling is implicated in the pathology of both Parkinson's disease (PD) and Alzheimer's disease (AD). We have previously shown that localized gene delivery of dominant negative TNF to the degenerating brain region can limit pathology in animal models of PD and AD. TNF is upregulated in Huntington's disease (HD), like in PD and AD, but it is unknown whether TNF signaling contributes to neuronal degeneration in HD. We used in vivo gene delivery to test whether selective reduction of soluble TNF signaling could attenuate medium spiny neuron (MSN) degeneration in the YAC128 transgenic (TG) mouse model of Huntington's disease (HD). AAV vectors encoding cDNA for dominant-negative tumor necrosis factor (DN-TNF) or GFP (control) were injected into the striatum of young adult wild type WT and YAC128 TG mice and achieved 30-50% target coverage. Expression of dominant negative TNF protein was confirmed immunohistologically and biochemically and was maintained as mice aged to one year, but declined significantly over time. However, the extent of striatal DN-TNF gene transfer achieved in our studies was not sufficient to achieve robust effects on neuroinflammation, rescue degenerating MSNs or improve motor function in treated mice. Our findings suggest that alternative drug delivery strategies should be explored to determine whether greater target coverage by DN-TNF protein might afford some level of neuroprotection against HD-like pathology and/or that soluble TNF signaling may not be the primary driver of striatal neuroinflammation and MSN loss in YAC128 TG mice.


Assuntos
Corpo Estriado/metabolismo , Terapia Genética/métodos , Doença de Huntington/terapia , Neurônios/metabolismo , Fator de Necrose Tumoral alfa/genética , Animais , Corpo Estriado/patologia , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Doença de Huntington/genética , Doença de Huntington/patologia , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Microglia/patologia , Destreza Motora/fisiologia , Degeneração Neural/genética , Degeneração Neural/patologia , Degeneração Neural/terapia , Neurônios/patologia , Teste de Desempenho do Rota-Rod , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismo
6.
J Neuroinflammation ; 10: 50, 2013 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-23622116

RESUMO

BACKGROUND: Complex interactions involving genetic susceptibility and environmental factors are thought to underlie the pathogenesis of Parkinson's disease (PD). Although the role of inflammatory processes in modulating risk for development of PD has yet to be fully understood, prospective studies suggest that chronic use of NSAIDs reduce the incidence of PD. Loss-of-function mutations in the DJ-1 gene cause a rare form of familial PD with an autosomal recessive pattern of inheritance; however, DJ-1-/- mice do not display nigrostriatal pathway degeneration, suggesting that additional factors such as inflammation may be needed to induce neurodegeneration on the background of DJ-1 gene mutations. Neuroinflammation causes oxidative stress and, based on evidence that DJ-1 plays a protective role against oxidative stress, we investigated whether DJ-1-/- mice display increased vulnerability to inflammation-induced nigral degeneration. METHODS: We exposed adult wild-type and DJ-1-/- mice to repeated intranasal administration of soluble TNF (inTNF) or repeated intraperitoneal injections of low-dose lipopolysaccharide (LPS) or saline vehicle. We measured locomotor performance using a variety of behavior tasks, striatal dopamine (DA) content by HPLC, DA neuron (TH+ cells) and total neuron (NeuN+ cells) number in the substantia nigra pars compacta and ventral tegmental area by unbiased stereology, number of Iba1-positive microglia, and mRNA levels of inflammatory and oxidative stress genes by quantitative PCR in the midbrain, cortex and isolated peritoneal macrophages of DJ-1-/- and wild-type mice. RESULTS: We found that chronic LPS injections induced similar neuroinflammatory responses in the midbrains of DJ-1-/- mice and wild-type mice and neither group developed locomotor deficits or nigral degeneration. inTNF administration did not appear to induce neuroinflammatory responses in LPS-treated wild-type or DJ-1-/- mice. The lack of vulnerability to inflammation-induced nigral degeneration was not due to enhanced anti-oxidant gene responses in the midbrains of DJ-1-/- mice which, in fact, displayed a blunted response relative to that of wild-type mice. Peripheral macrophages from wild-type and DJ-1-/- mice displayed similar basal and LPS-induced inflammatory and oxidative stress markers in vitro. CONCLUSIONS: Our studies indicate that DJ-1-/- mice do not display increased vulnerability to inflammation-related nigral degeneration in contrast to what has been reported for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine. We conclude that either DJ-1 does not have a critical role in protecting DA neurons against inflammation-induced oxidative stress and/or there is compensatory gene expression in the midbrain of DJ-1-/- mice that renders them resistant to the cytotoxic effects triggered by chronic peripheral inflammation.


Assuntos
Inflamação/patologia , Atividade Motora/fisiologia , Degeneração Neural/patologia , Proteínas Oncogênicas/fisiologia , Substância Negra/patologia , Administração Intranasal , Animais , Comportamento Animal/efeitos dos fármacos , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Imuno-Histoquímica , Inflamação/induzido quimicamente , Injeções Intraperitoneais , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Proteínas Oncogênicas/genética , Estresse Oxidativo/fisiologia , Peroxirredoxinas , Equilíbrio Postural/efeitos dos fármacos , Proteína Desglicase DJ-1 , Desempenho Psicomotor/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismo
7.
Kidney Int ; 83(5): 845-54, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23235569

RESUMO

Bardoxolone methyl, a synthetic triterpenoid, improves the estimated glomerular filtration rate (GFR) in patients with chronic kidney disease and type 2 diabetes. Since the contractile activity of mesangial cells may influence glomerular filtration, we evaluated the effect of the synthetic triterpenoid RTA 405, with structural similarity to bardoxolone methyl, on GFR in rats and on mesangial cell contractility in freshly isolated glomeruli. In rats, RTA 405 increased basal GFR, assessed by inulin clearance, and attenuated the angiotensin II-induced decline in GFR. RTA 405 increased the filtration fraction, but did not affect arterial blood pressure or renal plasma flow. Glomeruli from RTA 405-treated rats were resistant to angiotensin II-induced volume reduction ex vivo. In cultured mesangial cells, angiotensin II-stimulated contraction was attenuated by RTA 405, in a dose- and time-dependent fashion. Further, Nrf2-targeted gene transcription (regulates antioxidant, anti-inflammatory, and cytoprotective responses) in mesangial cells was associated with decreased basal and reduced angiotensin II-stimulated hydrogen peroxide and calcium ion levels. These mechanisms contribute to the GFR increase that occurs following treatment with RTA 405 in rats and may underlie the effect of bardoxolone methyl on the estimated GFR in patients.


Assuntos
Angiotensina II/farmacologia , Forma Celular/efeitos dos fármacos , Taxa de Filtração Glomerular/efeitos dos fármacos , Células Mesangiais/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , Animais , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Hemodinâmica/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Inulina/metabolismo , Masculino , Células Mesangiais/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ácido Oleanólico/administração & dosagem , Ácido Oleanólico/farmacologia , Ratos , Ratos Sprague-Dawley , Circulação Renal/efeitos dos fármacos , Fatores de Tempo
8.
J Med Chem ; 55(10): 4837-46, 2012 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-22533790

RESUMO

Novel monocyclic cyanoenones examined to date display unique features regarding chemical reactivity as Michael acceptors and biological potency. Remarkably, in some biological assays, the simple structure is more potent than pentacyclic triterpenoids (e.g., CDDO and bardoxolone methyl) and tricycles (e.g., TBE-31). Among monocyclic cyanoenones, 1 is a highly reactive Michael acceptor with thiol nucleophiles. Furthermore, an important feature of 1 is that its Michael addition is reversible. For the inhibition of NO production, 1 shows the highest potency. Notably, its potency is about three times higher than CDDO, whose methyl ester (bardoxolone methyl) is presently in phase III clinical trials. For the induction of NQO1, 1 also demonstrated the highest potency. These results suggest that the reactivity of these Michael acceptors is closely related to their biological potency. Interestingly, in LPS-stimulated macrophages, 1 causes apoptosis and inhibits secretion of TNF-α and IL-1ß with potencies that are higher than those of bardoxolone methyl and TBE-31.


Assuntos
Alcinos/síntese química , Anti-Inflamatórios não Esteroides/síntese química , Anticarcinógenos/síntese química , Nitrilas/síntese química , Alcinos/química , Alcinos/farmacologia , Amidas/química , Amidas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Anticarcinógenos/química , Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Citoproteção , Quinase I-kappa B/antagonistas & inibidores , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , NAD(P)H Desidrogenase (Quinona)/biossíntese , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/biossíntese , Nitrilas/química , Nitrilas/farmacologia , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Fenantrenos/química , Fenantrenos/farmacologia , Tiofenos/química , Tiofenos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
9.
Glia ; 60(2): 189-202, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21989628

RESUMO

The exact biological role of the cytokine tumor necrosis factor (TNF) in the central nervous system (CNS) is not well understood; but overproduction of TNF by activated microglia has been implicated in neuronal death, suggesting that TNF inhibition in the CNS may be a viable neuroprotective strategy. We investigated the role of TNF signaling in regulation of microglia effector functions using molecular, cellular, and functional analyses of postnatal and adult microglia populations in the CNS. No differences were found by flow cytometric analyses in the basal activation state between TNF-null and wild-type mice. Although TNF-null microglia displayed an atypical morphology with cytoplasmic vacuoles in response to stimulation with lipopolysaccharide (LPS), the phagocytic response of TNF-null microglia to Escherichia coli particles in vitro was normal and there were no signs of enhanced caspase 3 activation or apoptosis. Functionally, conditioned media from LPS-stimulated TNF-null microglia was found to have significantly reduced levels of IL-10, IL-6, IL-1ß, IL-12, and CXCL1 relative to wild-type microglia and exerted no cytotoxic effects on neurally differentiated dopaminergic (DA) MN9D cells. In contrast, incubation of wild-type microglia with TNF inhibitors selectively depleted the levels of soluble TNF and its cytotoxicity on MN9D cells. To distinguish whether reduced cytotoxicity by LPS-activated TNF-null microglia could be attributed to deficient autocrine TNF signaling, we employed primary microglia deficient in one or both TNF receptors (TNFR1 and TNFR2) in co-culture with MN9D cells and found that neither receptor is required to elicit LPS-evoked TNF production and cytotoxicity on DA cells.


Assuntos
Microglia/patologia , Degeneração Neural/patologia , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/toxicidade , Animais , Animais Recém-Nascidos , Linhagem Celular Tumoral , Técnicas de Cocultura , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Cultura Primária de Células , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/fisiologia
10.
Mol Ther ; 19(1): 46-52, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20959812

RESUMO

Parkinson's disease (PD) is a progressive neurodegenerative disorder typified by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Recent evidence indicates that neuroinflammation may play a critical role in the pathogenesis of PD, particularly tumor necrosis factor (TNF). We have previously shown that soluble TNF (solTNF) is required to mediate robust degeneration induced by 6-hydroxydopamine (6-OHDA) or lipopolysaccharide. What remains unknown is whether TNF inhibition can attenuate the delayed and progressive phase of neurodegeneration. To test this, rats were injected in the SNpc with lentivirus encoding dominant-negative TNF (lenti-DN-TNF) 2 weeks after receiving a 6-OHDA lesion. Remarkably, when examined 5 weeks after the initial 6-OHDA lesion, no further loss of nigral DA neurons was observed. Lenti-DN-TNF also attenuated microglial activation. Together, these data suggest that TNF is likely a critical mediator of nigral DA neuron death during the delayed and progressive phase of neurodegeneration, and that microglia may be the principal cell type involved. These promising findings provide compelling reasons to perform DN-TNF gene transfer studies in nonhuman primates with the long-term goal of using it in the clinic to prevent the delayed and progressive degeneration of DA neurons that gives rise to motor symptoms in PD.


Assuntos
Dopamina/metabolismo , Terapia Genética/métodos , Neurônios/patologia , Doença de Parkinson/patologia , Doença de Parkinson/terapia , Substância Negra/patologia , Fator de Necrose Tumoral alfa/genética , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Morte Celular/genética , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Microglia/metabolismo , Microglia/patologia , Neurônios/metabolismo , Oxidopamina , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismo , Fator de Necrose Tumoral alfa/biossíntese
11.
J Neurosci ; 28(43): 10825-34, 2008 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-18945890

RESUMO

The loss of nigral dopaminergic (DA) neurons in idiopathic Parkinson's disease (PD) is believed to result from interactions between genetic susceptibility and environmental factors. Evidence that inflammatory processes modulate PD risk comes from prospective studies that suggest that higher plasma concentrations of a number of proinflammatory cytokines correlate with an increased risk of developing PD and chronic nonsteroidal anti-inflammatory drug regimens reduce the incidence of PD. Although loss-of-function mutations in the parkin gene cause early-onset familial PD, Parkin-deficient (parkin-/-) mice do not display nigrostriatal pathway degeneration, suggesting that a genetic factor is not sufficient, and an environmental trigger may be needed to cause nigral DA neuron loss. To test the hypothesis that parkin-/- mice require an inflammatory stimulus to develop nigral DA neuron loss, low-dose lipopolysaccaride (LPS) was administered intraperitoneally for prolonged periods. Quantitative real-time PCR and immunofluorescence labeling of inflammatory markers indicated that this systemic LPS treatment regimen triggered persistent neuroinflammation in wild-type and parkin-/- mice. Although inflammatory and oxidative stress responses to the inflammation regimen did not differ significantly between the two genotypes, only parkin-/- mice displayed subtle fine-motor deficits and selective loss of DA neurons in substantia nigra. Therefore, our studies suggest that loss of Parkin function increases the vulnerability of nigral DA neurons to inflammation-related degeneration. This new model of nigral DA neuron loss may enable identification of early biomarkers of degeneration and aid in preclinical screening efforts to identify compounds that can halt or delay the progressive degeneration of the nigrostriatal pathway.


Assuntos
Inflamação/complicações , Degeneração Neural/etiologia , Substância Negra/patologia , Ubiquitina-Proteína Ligases/deficiência , Animais , Comportamento Animal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Dopamina/metabolismo , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Transtornos Neurológicos da Marcha/etiologia , Transtornos Neurológicos da Marcha/genética , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Inflamação/induzido quimicamente , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Análise Multivariada , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Degeneração Neural/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Polissacarídeos , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Teste de Desempenho do Rota-Rod , Substância Negra/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Tirosina 3-Mono-Oxigenase
12.
PLoS One ; 3(1): e1453, 2008 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-18197263

RESUMO

We investigated whether adipose-derived adult stromal (ADAS) are of neural crest origin and the extent to which Notch 1 regulates their growth and differentiation. Mouse ADAS cells cultured in media formulated for neural stem cells (NSC) displayed limited capacity for self-renewal, clonogenicity, and neurosphere formation compared to NSC from the subventricular zone in the hippocampus. Although ADAS cells expressed Nestin, GFAP, NSE and Tuj1 in vitro, exposure to NSC differentiation supplements did not induce mature neuronal marker expression. In contrast, in mesenchymal stem cell (MSC) media, ADAS cells retained their ability to proliferate and differentiate beyond 20 passages and expressed high levels of Nestin. In neuritizing cocktails, ADAS cells extended processes, downregulated Nestin expression, and displayed depolarization-induced Ca(2+) transients but no spontaneous or evoked neural network activity on Multi-Electrode Arrays. Deletion of Notch 1 in ADAS cell cultures grown in NSC proliferation medium did not significantly alter their proliferative potential in vitro or the differentiation-induced downregulation of Nestin. Co-culture of ADAS cells with fibroblasts that stably expressed the Notch ligand Jagged 1 or overexpression of the Notch intracellular domain (NICD) did not alter ADAS cell growth, morphology, or cellular marker expression. ADAS cells did not display robust expression of neural crest transcription factors or genes (Sox, CRABP2, and TH); and lineage tracing analyses using Wnt1-Cre;Rosa26R-lacZ or -EYFP reporter mice confirmed that fewer than 2% of the ADAS cell population derived from a Wnt1-positive population during development. In summary, although media formulations optimized for MSCs or NSCs enable expansion of mouse ADAS cells in vitro, we find no evidence that these cells are of neural crest origin, that they can undergo robust terminal differentiation into functionally mature neurons, and that Notch 1 is likely to be a key regulator of their cellular and molecular characteristics.


Assuntos
Tecido Adiposo/citologia , Crista Neural/citologia , Neuroglia/citologia , Células Estromais/citologia , Animais , Sequência de Bases , Diferenciação Celular , Linhagem da Célula , Técnicas de Cocultura , Meios de Cultura , Primers do DNA , Feminino , Masculino , Camundongos , Reação em Cadeia da Polimerase
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