RESUMEN
Successful and selective inhibition of the cytosolic protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associating protein 1 (Keap1) can enhance the antioxidant response, with the potential for a therapeutic effect in a range of settings including in neurodegenerative disease (ND). Small molecule inhibitors have been developed, yet many have off-target effects, or are otherwise limited by poor cellular permeability. Peptide-based strategies have also been attempted to enhance specificity, yet face challenges due to susceptibility to degradation and lack of cellular penetration. Herein, these barriers are overcome utilizing a polymer-based proteomimetics. The protein-like polymer (PLP) consists of a synthetic, lipophilic polymer backbone displaying water soluble Keap1-binding peptides on each monomer unit forming a brush polymer architecture. The PLPs are capable of engaging Keap1 and displacing the cellular protective transcription factor Nrf2, which then translocates to the nucleus, activating the antioxidant response element (ARE). PLPs exhibit increased Keap1 binding affinity by several orders of magnitude compared to free peptides, maintain serum stability, are cell-penetrant, and selectively activate the ARE pathway in cells, including in primary cortical neuronal cultures. Keap1/Nrf2-inhibitory PLPs have the potential to impact the treatment of disease states associated with dysregulation of oxidative stress, such as NDs.
Asunto(s)
Proteína 1 Asociada A ECH Tipo Kelch , Factor 2 Relacionado con NF-E2 , Polímeros , Unión Proteica , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/química , Factor 2 Relacionado con NF-E2/metabolismo , Polímeros/química , Humanos , Animales , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Elementos de Respuesta Antioxidante , Neuronas/metabolismo , Neuronas/efectos de los fármacosRESUMEN
Alzheimer's disease (AD) alters astrocytes, but the effect of Aß and Tau pathology is poorly understood. TRAP-seq translatome analysis of astrocytes in APP/PS1 ß-amyloidopathy and MAPTP301S tauopathy mice revealed that only Aß influenced expression of AD risk genes, but both pathologies precociously induced age-dependent changes, and had distinct but overlapping signatures found in human post-mortem AD astrocytes. Both Aß and Tau pathology induced an astrocyte signature involving repression of bioenergetic and translation machinery, and induction of inflammation pathways plus protein degradation/proteostasis genes, the latter enriched in targets of inflammatory mediator Spi1 and stress-activated cytoprotective Nrf2. Astrocyte-specific Nrf2 expression induced a reactive phenotype which recapitulated elements of this proteostasis signature, reduced Aß deposition and phospho-tau accumulation in their respective models, and rescued brain-wide transcriptional deregulation, cellular pathology, neurodegeneration and behavioural/cognitive deficits. Thus, Aß and Tau induce overlapping astrocyte profiles associated with both deleterious and adaptive-protective signals, the latter of which can slow patho-progression.
Asunto(s)
Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Astrocitos/metabolismo , Encéfalo/metabolismo , Neuroprotección/genética , Proteínas tau/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Astrocitos/citología , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Homocigoto , Humanos , Ratones , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Fenotipo , Fosforilación , Proteostasis/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Proteínas tau/metabolismoRESUMEN
The angiogenin (ANG) gene is mutated frequently in individuals with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Delivering human ANG to mice that display ALS-like symptoms extends their lifespan and improves motor function. ANG is a secretory vertebrate RNase that enters neuronal cells and cleaves a subset of tRNAs, leading to the inhibition of translation initiation and the assembly of stress granules. Here, using murine neuronal and astrocytic cell lines, we find that ANG triggers the activation of the Nrf2 (nuclear factor erythroid 2-related factor 2) pathway, which provides a critical cellular defense against oxidative stress. This activation, which occurred in astrocytes but not in neurons, promoted the survival of proximal neurons that had oxidative injury. These findings extend the role of ANG as a neuroprotective agent and underscore its potential utility in ALS management.
Asunto(s)
Elementos de Respuesta Antioxidante/efectos de los fármacos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ribonucleasa Pancreática/farmacología , Animales , Astrocitos/citología , Línea Celular , Ratones , Neuronas/citología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Hepatic ischemia/reperfusion injury (IRI) is an unavoidable consequence of liver transplantation that can lead to postoperative hepatic dysfunction. Myeloid cells that include Kupffer cells, monocytes, and neutrophils contribute to the inflammatory response and cellular injury observed during hepatic IRI. We hypothesize that overactivation of the nuclear erythroid 2 p45-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway in myeloid cells leads to decreased cellular damage after hepatic IRI. We constructed transgenic mice with constitutively active nuclear erythroid 2 p45-related factor 2 (caNrf2) that over activates the Nrf2-ARE pathway in myeloid cells (lysozyme M cre recombinase [LysMcre]+/caNrf2+, n = 9), and their littermate controls lacking transgene expression (LysMcre+/caNrf2-, n = 11). The mice underwent either sham or partial hepatic ischemia surgery, with 60 minutes of ischemia followed by 6 hours of reperfusion. After IRI, LysMcre+/caNrf2+ mice demonstrated significantly decreased serum alanine aminotransferase and decreased areas of necrosis. Immunohistochemistry and immunoblot of caspase 3 showed a significantly decreased cleaved to full-length caspase 3 ratio in LysMcre+/caNrf2+ animals. Lymphocyte antigen 6 complex locus G and CD68 staining demonstrated reduced inflammatory cell infiltration. LysMcre+/caNrf2+ animals also had significantly decreased gene expression of proinflammatory cytokines, including interleukin (IL) 1ß, IL6, tumor necrosis factor α, chemokine (C-C motif) ligand 2, and chemokine (C-X-C motif) ligand 10, and significantly decreased levels of 8-isoprostanes. In our model, Nrf2 overactivation in myeloid cells leads to decreased hepatocellular damage, necrosis, apoptosis, inflammation, and oxidative stress. Pharmacologic targeting of the Nrf2-ARE pathway in myeloid cells may be a novel strategy to mitigate hepatic IRI. Liver Transplantation 22 1115-1128 2016 AASLD.
Asunto(s)
Enfermedad Hepática en Estado Terminal/cirugía , Trasplante de Hígado/efectos adversos , Hígado/patología , Factor 2 Relacionado con NF-E2/metabolismo , Daño por Reperfusión/metabolismo , Alanina Transaminasa/sangre , Animales , Elementos de Respuesta Antioxidante , Apoptosis , Caspasa 3/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Macrófagos del Hígado/metabolismo , Hígado/citología , Ratones , Ratones Transgénicos , Monocitos/metabolismo , Factor 2 Relacionado con NF-E2/genética , Necrosis , Neutrófilos/metabolismo , Estrés Oxidativo , Daño por Reperfusión/sangre , Daño por Reperfusión/etiología , Transducción de Señal , Regulación hacia ArribaRESUMEN
The brain is very sensitive to changes in redox status; thus maintaining redox homeostasis in the brain is critical for the prevention of accumulating oxidative damage. Aging is the primary risk factor for developing neurodegenerative diseases. In addition to age, genetic and environmental risk factors have also been associated with disease development. The primary reactive insults associated with the aging process are a result of oxidative stress (OS) and nitrosative stress (NS). Markers of increased oxidative stress, protein and DNA modification, inflammation, and dysfunctional proteostasis have all been implicated in contributing to the progression of neurodegeneration. The ability of the cell to combat OS/NS and maintain a clearance mechanism for misfolded aggregating proteins determines whether or not it will survive. A critical pathway in this regard is the Nrf2 (nuclear factor erythroid 2-related factor 2)- antioxidant response element (ARE) pathway. Nrf2 activation has been shown to mitigate a number of pathologic mechanisms associated with Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and multiple sclerosis. This review will focus on the role of Nrf2 in these diseases and the potential for Nrf2 activation to attenuate disease progression.
Asunto(s)
Factor 2 Relacionado con NF-E2/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Estrés Oxidativo/fisiología , Transducción de Señal/fisiología , Animales , Encéfalo/metabolismo , Humanos , Oxidación-ReducciónRESUMEN
The presence of senile plaques is one of the major pathologic hallmarks of the brain with Alzheimer's disease (AD). The plaques predominantly contain insoluble amyloid ß-peptide, a cleavage product of the larger amyloid precursor protein (APP). Two enzymes, named ß and γ secretase, generate the neurotoxic amyloid-ß peptide from APP. Mature APP is also turned over endogenously by autophagy, more specifically by the endosomal-lysosomal pathway. A defective lysosomal system is known to be pathogenic in AD. Modulation of NF-E2 related factor 2 (Nrf2) has been shown in several neurodegenerative disorders, and Nrf2 has become a potential therapeutic target for various neurodegenerative disorders, including AD, Parkinson's disease, and amyotrophic lateral sclerosis. In the current study, we explored the effect of genetic ablation of Nrf2 on APP/Aß processing and/or aggregation as well as changes in autophagic dysfunction in APP/PS1 mice. There was a significant increase in inflammatory response in APP/PS1 mice lacking Nrf2. This was accompanied by increased intracellular levels of APP, Aß (1-42), and Aß (1-40), without a change total full-length APP. There was a shift of APP and Aß into the insoluble fraction, as well as increased poly-ubiquitin conjugated proteins in mice lacking Nrf2. APP/PS1-mediated autophagic dysfunction is also enhanced in Nrf2-deficient mice. Finally, neurons in the APP/PS1/Nrf2-/- mice had increased accumulation of multivesicular bodies, endosomes, and lysosomes. These outcomes provide a better understanding of the role of Nrf2 in modulating autophagy in an AD mouse model and may help design better Nrf2 targeted therapeutics that could be efficacious in the treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Autofagia/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/fisiología , Enfermedad de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Modelos Animales de Enfermedad , Endosomas/metabolismo , Eliminación de Gen , Lisosomas/metabolismo , Ratones Transgénicos , Terapia Molecular DirigidaRESUMEN
AIMS: The purpose of this study was to determine whether 3'-5'-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2-4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. RESULTS: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1-dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. INNOVATION: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. CONCLUSION: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ayuno/fisiología , Regulación de la Expresión Génica , Factor 2 Relacionado con NF-E2/genética , Sirtuina 1/metabolismo , Región de Flanqueo 5' , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Elementos de Respuesta Antioxidante , Línea Celular , AMP Cíclico/metabolismo , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Masculino , Ratones , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/genética , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Unión Proteica , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Two novel chlorinated pyrones, halomadurones A and B, and two novel brominated analogues, halomadurones C and D, were isolated from a marine Actinomadura sp. cultivated from the ascidian Ecteinascidia turbinata. Additionally, a non-halogenated analogue, 2-methyl-6-((E)-3-methyl-1,3-hexadiene)-γ-pyrone, was synthesized to understand the role of the halogens for activity. Halomadurones C and D demonstrated potent nuclear factor E2-related factor antioxidant response element (Nrf2-ARE) activation, which is an important therapeutic approach for treatment of neurodegenerative diseases.
Asunto(s)
Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Animales , Elementos de Respuesta Antioxidante/genética , Bacterias/genética , Bacterias/metabolismo , Células Cultivadas , Halógenos/metabolismo , Ratones , Enfermedades Neurodegenerativas/tratamiento farmacológico , Pironas/metabolismoRESUMEN
Activation of the peroxisome proliferator activated receptor-gamma (PPAR)- γ is proposed as a neuroprotective strategy to treat neurodegenerative disorders. In this study, we examined if LSN862 (LSN), a novel non-thiazoledinedione partial PPAR- γ agonist, was neuroprotective in a mouse model of Parkinson's disease (PD) and assessed possible mechanisms of action. LSN (3, 10, or 30 mg/kg) or vehicle was orally administered daily to C57BL/6 and antioxidant response element-human placental alkaline phosphatase (ARE-hPAP) reporter mice 3 days prior to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 30 mg/kg, i.p. × 5 days) or PBS administration. LSN elicited a dose-dependent preservation of dopaminergic nigrostriatal innervation that was not associated with inhibition of MPTP metabolism or activation of Nrf2-ARE, although changes in NQO1 and SOD2 mRNA were observed. A significant dose-dependent downregulation in MAC-1 and GFAP positive cells was observed in MPTP + LSN-treated mice as well as significant downregulation of mRNA expression levels of these inflammatory markers. MPTP-induced increases in PPAR- γ and PGC1 α expression were ameliorated by LSN dosing. Our results demonstrate that oral administration of LSN is neuroprotective against MPTP-induced neurodegeneration, and this effect is associated with downregulation of neuroinflammation, decreased oxidative stress, and modulation of PPAR- γ and PGC1 α expression. These results suggest that LSN can be a candidate alternative non-thiazoledinedione partial PPAR- γ agonist for neuroprotective treatment of PD.
RESUMEN
Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate cytoprotective genes which may be useful in the treatment of neurodegenerative disease. In order to better understand the structure activity relationship of phenolic diterpenes from Salvia officinalis L., we isolated carnosic acid, carnosol, epirosmanol, rosmanol, 12-methoxy-carnosic acid, sageone, and carnosaldehyde using polyamide column, centrifugal partition chromatography, and semi-preparative high performance liquid chromatography. Isolated compounds were screened in vitro for their ability to active the Nrf2 and general cellular toxicity using mouse primary cortical cultures. All compounds except 12-methoxy-carnosic acid were able to activate the antioxidant response element. Furthermore both carnosol and carnoasldehyde were able to induce Nrf2-dependent gene expression as well as protect mouse primary cortical neuronal cultures from H(2)O(2) induced cell death.
Asunto(s)
Diterpenos/farmacología , Factor 2 Relacionado con NF-E2/agonistas , Fenoles/farmacología , Extractos Vegetales/farmacología , Salvia officinalis/química , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Diterpenos/química , Diterpenos/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/farmacología , Ratones , Estructura Molecular , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Fenoles/química , Fenoles/aislamiento & purificación , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
The nuclear factor erythroid 2-related factor 2 (Nrf2) governs the expression of antioxidant and phase II detoxifying enzymes. Nrf2 activation can prevent or reduce cellular damage associated with several types of injury in many different tissues and organs. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons and subsequent muscular atrophy. We have previously shown that Nrf2 activation in astrocytes delays neurodegeneration in ALS mouse models. To further investigate the role of Nrf2 in ALS we determined the effect of absence of Nrf2 or its restricted overexpression in neurons or type II skeletal muscle fibers on symptoms onset and survival in mutant hSOD1 expressing mice. We did not observe any detrimental effect associated with the lack of Nrf2 in two different mutant hSOD1 animal models of ALS. However, restricted Nrf2 overexpression in neurons or type II skeletal muscle fibers delayed disease onset but failed to extend survival in hSOD1(G93A) mice. These results highlight the concept that not only the pharmacological target but also the cell type targeted may be relevant when considering a Nrf2-mediated therapeutic approach for ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Fibras Musculares de Contracción Rápida/metabolismo , Factor 2 Relacionado con NF-E2/genética , Neuronas/metabolismo , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Factor 2 Relacionado con NF-E2/metabolismo , Neuronas/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/metabolismo , Análisis de Supervivencia , Factores de TiempoRESUMEN
Parkinson's disease (PD) is the second most common age-related neurodegenerative disorder typified by tremor, rigidity, akinesia and postural instability due in part to the loss of dopamine within the nigrostriatal system. The pathologic features of this disorder include the loss of substantia nigra dopamine neurons and attendant striatal terminals, the presence of large protein-rich neuronal inclusions containing fibrillar α-synuclein and increased numbers of activated microglia. Evidence suggests that both misfolded α-synuclein and oxidative stress play an important role in the pathogenesis of sporadic PD. Here we review evidence that α-synuclein activates glia inducing inflammation and that Nrf2-directed phase-II antioxidant enzymes play an important role in PD. We also provide new evidence that the expression of antioxidant enzymes regulated in part by Nrf2 is increased in a mouse model of α-synuclein overexpression. We show that misfolded α-synuclein directly activates microglia inducing the production and release of the proinflammatory cytokine, TNF-α, and increasing antioxidant enzyme expression. Importantly, we demonstrate that the precise structure of α-synuclein is important for induction of this proinflammatory pathway. This complex α-synuclein-directed glial response highlights the importance of protein misfolding, oxidative stress and inflammation in PD and represents a potential locus for the development of novel therapeutics focused on induction of the Nrf2-directed antioxidant pathway and inhibition of protein misfolding.
Asunto(s)
Antioxidantes/farmacología , Activación de Macrófagos/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/inmunología , Enfermedad de Parkinson/inmunología , alfa-Sinucleína/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Western Blotting , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/metabolismo , Microscopía de Fuerza Atómica , Factor 2 Relacionado con NF-E2/fisiología , Estrés Oxidativo/efectos de los fármacos , Placenta/enzimología , Embarazo , Conformación Proteica , ARN/biosíntesis , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Necrosis Tumoral alfa/metabolismo , alfa-Sinucleína/químicaRESUMEN
Alpha synuclein (SYN) is a central player in the pathogenesis of sporadic and familial Parkinson's disease (PD). SYN aggregation and oxidative stress are associated and enhance each other's toxicity. It is unknown whether the redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays a role against the toxicity of SYN. To examine this, mice selectively overexpressing Nrf2 in astrocytes (GFAP-Nrf2) were crossed with mice selectively expressing human mutant SYN (hSYN(A53T)) in neurons. Increased astrocytic Nrf2 delayed the onset and extended the life span of the hSYN(A53T) mice. This correlated with increased motor neuron survival, reduced oxidative stress, and attenuated gliosis in the spinal cord, as well as a dramatic decrease in total hSYN(A53T) and phosphorylated (Ser129) hSYN(A53T) in Triton-insoluble aggregates. Furthermore, Nrf2 in astrocytes delayed chaperone-mediated autophagy and macroautophagy dysfunction observed in the hSYN(A53T) mice. Our data suggest that Nrf2 in astrocytes provides neuroprotection against hSYN(A53T)-mediated toxicity by promoting the degradation of hSYN(A53T) through the autophagy-lysosome pathway in vivo. Thus, activation of the Nrf2 pathway in astrocytes is a potential target to develop therapeutic strategies for treating pathologic synucleinopathies including PD.
Asunto(s)
Astrocitos/metabolismo , Modelos Animales de Enfermedad , Neuronas Motoras/patología , Factor 2 Relacionado con NF-E2/biosíntesis , alfa-Sinucleína/metabolismo , Animales , Elementos de Respuesta Antioxidante/genética , Autofagia/genética , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Sistema Nervioso Central/ultraestructura , Femenino , Gliosis/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/genética , Fármacos Neuroprotectores/metabolismo , Estrés Oxidativo/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Fosforilación , alfa-Sinucleína/genéticaRESUMEN
Tanacetum parthenium produces biologically active sesquiterpene lactones (SL). Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate a series of genes termed the antioxidant response element (ARE). Activation of Nrf2/ARE may be useful for the treatment of neurodegenerative disease. In this study we isolated 11 SL from T. parthenium with centrifugal partition chromatography and semipreparative HPLC. Compounds were screened in vitro for their ability to activate the ARE on primary mouse cortical cultures as well as for their toxicity towards the cultures. All SL containing the α-methylene-γ-lactone moiety were able to activate the ARE and cause cellular toxicity. The structure-activity relationship among the SL isolated indicates that the guaianolides were more active and when lacking the endoperoxide functionality less toxic then the germacranolides.
Asunto(s)
Elementos de Respuesta Antioxidante , Corteza Cerebral/efectos de los fármacos , Lactonas/farmacología , Sesquiterpenos de Guayano/farmacología , Tanacetum parthenium/química , Activación Transcripcional , Animales , Supervivencia Celular , Células Cultivadas , Corteza Cerebral/citología , Cromatografía Líquida de Alta Presión , Evaluación Preclínica de Medicamentos , Lactonas/química , Lactonas/aislamiento & purificación , Ratones , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Componentes Aéreos de las Plantas/química , Extractos Vegetales/química , Extractos Vegetales/farmacología , Cultivo Primario de Células , Sesquiterpenos de Germacrano/química , Sesquiterpenos de Germacrano/aislamiento & purificación , Sesquiterpenos de Germacrano/farmacología , Sesquiterpenos de Guayano/química , Sesquiterpenos de Guayano/aislamiento & purificación , Relación Estructura-Actividad , Pruebas de Toxicidad/métodosRESUMEN
Alexander disease is a fatal neurodegenerative disease caused by dominant mutations in glial fibrillary acidic protein (GFAP). The disease is characterized by protein inclusions called Rosenthal fibers within astrocyte cell bodies and processes, and an antioxidant response mediated by the transcription factor Nrf2. We sought to test whether further elevation of Nrf2 would be beneficial in a mouse model of Alexander disease. Forcing overexpression of Nrf2 in astrocytes of R236H GFAP mutant mice decreased GFAP protein in all brain regions examined (olfactory bulb, hippocampus, cerebral cortex, brainstem, cerebellum, and spinal cord) and decreased Rosenthal fibers in olfactory bulb, hippocampus, corpus callosum, and brainstem. Nrf2 overexpression also restored body weights of R236H mice to near wild-type levels. Nrf2 regulates several genes involved in homeostasis of the antioxidant molecule glutathione, and the neuroprotective effects of Nrf2 in other neurological disorders may reflect restoration of glutathione to normal levels. However, glutathione levels in R236H mice were not decreased. Nrf2 overexpression did not change glutathione levels or ratio of reduced to oxidized glutathione (indicative of oxidative stress) in olfactory bulb, where Nrf2 dramatically reduced GFAP. Depletion of glutathione through knock-out of the GCLM (glutamate-cysteine ligase modifier subunit) also did not affect GFAP levels or body weight of R236H mice. These data suggest that the beneficial effects of Nrf2 are not mediated through glutathione.
Asunto(s)
Enfermedad de Alexander/metabolismo , Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Factor 2 Relacionado con NF-E2/metabolismo , Factores de Edad , Enfermedad de Alexander/genética , Enfermedad de Alexander/patología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Arginina/genética , Astrocitos/metabolismo , Astrocitos/patología , Peso Corporal/genética , Encéfalo/patología , Cromatografía Líquida de Alta Presión/métodos , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/genética , Glutamato-Cisteína Ligasa/deficiencia , Glutatión/metabolismo , Histidina/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Factor 2 Relacionado con NF-E2/genética , Fibras Nerviosas/metabolismo , Fibras Nerviosas/patología , ARN Mensajero/metabolismoRESUMEN
Nuclear factor erythroid 2-related factor 2 (Nrf2) is the master transcription factor of the antioxidant response element pathway, coordinating the induction of detoxifying and antioxidant enzymes. Nrf2 is normally sequestered in the cytoplasm by Kelch-like ECH-associating protein 1 (Keap1). To identify novel small molecules that will disturb Nrf2-Keap1 binding and promote activation of the Nrf2- antioxidant response element pathway, we generated a quantum model based on the structures of known Nrf2- antioxidant response element activators. We used the quantum model to perform in silico screening on over 18 million commercially available chemicals to identify the structures predicted to activate the Nrf2- antioxidant response element pathway based on the quantum model. The top hits were tested in vitro, and half of the predicted hits activated the Nrf2-antioxidant response element pathway significantly in primary cell culture. In addition, we identified a new family of Nrf2-antioxidant response element-activating structures that all have comparable activity to tBHQ and protect against oxidative stress and dopaminergic toxins in vitro. The improved ability to identify potent activators of Nrf2 through the combination of in silico and in vitro screening described here improves the speed and cost associated with screening Nrf2-antioxidant response element -activating compounds for drug development.
Asunto(s)
Antioxidantes/química , Factor 2 Relacionado con NF-E2/agonistas , Animales , Antioxidantes/síntesis química , Antioxidantes/farmacología , Astrocitos/citología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Evaluación Preclínica de Medicamentos , Ratones , Modelos Químicos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Neuronas/citología , Teoría Cuántica , Elementos de Respuesta , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that binds to the antioxidant response element, a cis-acting regulatory element that increases expression of detoxifying enzymes and antioxidant proteins. Kelch-like ECH associating protein 1 (Keap1) protein is a negative regulator of Nrf2. Previous work has shown that genetic overexpression of Nrf2 is protective in vitro and in vivo. To modulate the Nrf2-ARE system without overexpressing Nrf2, we used short interfering RNA (siRNA) directed against Keap1. Keap1 siRNA administration in primary astrocytes increased the levels of Nrf2-ARE driven genes and protected against oxidative stress. Moreover, Keap1 siRNA resulted in a persistent upregulation of the Nrf2-ARE pathway and protection against oxidative stress in primary astrocytes. Keap1 siRNA injected into the striatum was also modestly protective against MPTP-induced dopaminergic terminal damage. These data indicate that activation of endogenous intracellular levels of Nrf2 is sufficient to protect in models of oxidative stress and Parkinson's disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antioxidantes/metabolismo , Astrocitos/efectos de los fármacos , Proteínas del Citoesqueleto/metabolismo , Terapia Genética/métodos , Intoxicación por MPTP/prevención & control , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Elementos de Respuesta , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Sitios de Unión , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Células Cultivadas , Citoprotección , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Relación Dosis-Respuesta a Droga , Proteína 1 Asociada A ECH Tipo Kelch , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Intoxicación por MPTP/genética , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/patología , Ratones , Ratones Transgénicos , Oxidantes/toxicidad , Proteínas Asociadas a Pancreatitis , Factores de Tiempo , Transfección , terc-Butilhidroperóxido/toxicidadRESUMEN
Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. To investigate the role of antioxidant defenses in ALS we used knockout mice for the glutamate-cysteine ligase modifier subunit (GCLM-/-), which have a 70-80% reduction in total glutathione. Although GCLM(-/-) mice are viable and fertile, the life span of GCLM(-/-)/hSOD1(G93A) mice decreased in 55% when compared to GCLM(+/+)/hSOD1(G93A) mice. Decreased life span in GCLM(-/-)/hSOD1(G93A) mice was associated to increased oxidative stress, aggravated mitochondrial pathology and increased association of hSOD1 with the mitochondria. Interestingly, when the GCLM(-/-) animals were mated with a different ALS-model which overexpress the experimental mutation hSOD1(H46R/H48Q), no effect was observed in survival of GCLM(-/-)/hSOD1(H46R/H48Q) mice; and little or no mitochondrial pathology was observed. Since a specific disease modifier, such as glutathione deficiency, may affect only certain hSOD1 mutants, these findings contribute to our understanding of the potential difference in the molecular pathways by which different hSOD1 mutants generate disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Glutatión/deficiencia , Mitocondrias/patología , Superóxido Dismutasa/genética , Alanina/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Glutatión/antagonistas & inhibidores , Glicina/genética , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Neuronas Motoras/enzimología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Ultraviolet (UV) B irradiation can severely damage the skin and even induce tumorigenesis. It exerts its effects by direct DNA modification and by formation of reactive oxygen species (ROS). We developed a strategy to genetically activate target gene expression of the transcription factor NF-E2-related factor 2 (Nrf2) in keratinocytes in vivo based on expression of a constitutively active Nrf2 mutant. Activation of Nrf2 target genes strongly reduced UVB cytotoxicity through enhancement of ROS detoxification. Remarkably, the protective effect was extended to neighboring cells. Using different combinations of genetically modified mice, we demonstrate that Nrf2 activates the production, recycling, and release of glutathione and cysteine by suprabasal keratinocytes, resulting in protection of basal cells in a paracrine, glutathione/cysteine-dependent manner. Most importantly, we found that endogenous Nrf2 controls selective protection of suprabasal keratinocytes from UVB-induced apoptosis through activation of cytoprotective genes. This finding explains the preferential UVB-induced apoptosis of basal cells, which is important for elimination of mutated stem cells as well as for preservation of skin integrity. Taken together, our results identify Nrf2 as a key regulator in the UV response of the skin.
Asunto(s)
Citoprotección/fisiología , Glutatión/metabolismo , Queratinocitos/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Rayos Ultravioleta , Animales , Apoptosis/efectos de la radiación , Femenino , Regulación de la Expresión Génica , Humanos , Queratinocitos/efectos de la radiación , Ratones , Ratones Transgénicos , Mutación/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The molecular mechanisms leading to neurodegeneration in Parkinson's disease remain elusive. Deletion and mutations of DJ-1 (PARK7) have been reported to cause autosomal recessive familial Parkinson's disease. Wildtype DJ-1 scavenges H(2)O(2) by cysteine oxidation in response to oxidative stress, and thus confers neuroprotection. Activation of the transcription factor NF-E2-related factor-2 (Nrf2) has also been shown to be important for protection against oxidative stress in many models of neurodegenerative diseases. Previous data indicate that DJ-1 affects the transcriptional functions and stability of Nrf2. However, this observation has not been confirmed. In the current study, the role of DJ-1 in the regulation of Nrf2 is examined in primary cultured neurons, astrocytes and in vivo. The prototypical Nrf2 activator tBHQ protected primary cortical neurons derived from DJ-1-knockout (KO) as well as DJ-1 wildtype mice by activation of Nrf2-ARE pathway. Nrf2 nuclear translocation, robust increases in canonical Nrf2-driven genes and proteins, and dramatic activation of the ARE reporter gene, hPAP, were observed after tBHQ treatment. These results were further confirmed by siRNA-mediated DJ-1 knockdown in primary cortical astrocytes from ARE-hPAP mice and tBHQ administration into the striatum of mouse brain. In addition, overexpression of Nrf2 with adenovirus preferentially in astrocytes from DJ-1-KO mice enhanced survival of neurons under oxidative insults. These findings indicate that activation of the Nrf2-ARE pathway is independent of DJ-1, and Nrf2 activation is a potential therapeutic target to prevent neurodegeneration in sporadic and DJ-1 familial Parkinson's disease.