RESUMEN
Hallmarks of chronic neurodegenerative disease include progressive synaptic loss and neuronal cell death, yet the cellular pathways that underlie these processes remain largely undefined. We provide evidence that dual leucine zipper kinase (DLK) is an essential regulator of the progressive neurodegeneration that occurs in amyotrophic lateral sclerosis and Alzheimer's disease. We demonstrate that DLK/c-Jun N-terminal kinase signaling was increased in mouse models and human patients with these disorders and that genetic deletion of DLK protected against axon degeneration, neuronal loss, and functional decline in vivo. Furthermore, pharmacological inhibition of DLK activity was sufficient to attenuate the neuronal stress response and to provide functional benefit even in the presence of ongoing disease. These findings demonstrate that pathological activation of DLK is a conserved mechanism that regulates neurodegeneration and suggest that DLK inhibition may be a potential approach to treat multiple neurodegenerative diseases.
Asunto(s)
Leucina Zippers , Quinasas Quinasa Quinasa PAM/metabolismo , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/patología , Transducción de Señal , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/patología , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones Transgénicos , Neuroprotección , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Médula Espinal/enzimología , Médula Espinal/patología , Superóxido Dismutasa/metabolismoRESUMEN
Recent advances in genetics and neuropathology support the idea that amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTD) are two ends of a disease spectrum. Although several animal models have been developed to investigate the pathogenesis and disease progression in ALS and FTD, there are significant limitations that hamper our ability to connect these models with the neurodegenerative processes in human diseases. With the technical breakthrough in reprogramming biology, it is now possible to generate patient-specific induced pluripotent stem cells (iPSCs) and disease-relevant neuron subtypes. This review provides a comprehensive summary of studies that use iPSC-derived neurons to model ALS and FTD. We discuss the unique capabilities of iPSC-derived neurons that capture some key features of ALS and FTD, and underscore their potential roles in drug discovery. There are, however, several critical caveats that require improvements before iPSC-derived neurons can become highly effective disease models. This article is part of a Special Issue entitled SI: Exploiting human neurons.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Demencia Frontotemporal/fisiopatología , Células Madre Pluripotentes Inducidas/fisiología , Neuronas/fisiología , Esclerosis Amiotrófica Lateral/genética , Animales , Demencia Frontotemporal/genética , HumanosRESUMEN
Persistent accumulation of misfolded proteins causes endoplasmic reticulum (ER) stress, a prominent feature in many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Here we report the identification of homeodomain interacting protein kinase 2 (HIPK2) as the essential link that promotes ER-stress-induced cell death via the IRE1α-ASK1-JNK pathway. ER stress, induced by tunicamycin or SOD1(G93A), activates HIPK2 by phosphorylating highly conserved serine and threonine residues (S359/T360) within the activation loop of the HIPK2 kinase domain. In SOD1(G93A) mice, loss of HIPK2 delays disease onset, reduces cell death in spinal motor neurons, mitigates glial pathology, and improves survival. Remarkably, HIPK2 activation positively correlates with TDP-43 proteinopathy in NEFH-tTA/tetO-hTDP-43ΔNLS mice, sporadic ALS and C9ORF72 ALS, and blocking HIPK2 kinase activity protects motor neurons from TDP-43 cytotoxicity. These results reveal a previously unrecognized role of HIPK2 activation in ER-stress-mediated neurodegeneration and its potential role as a biomarker and therapeutic target for ALS. VIDEO ABSTRACT.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Neuronas Motoras/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Biomarcadores/análisis , Proteínas Portadoras/genética , Muerte Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Retículo Endoplásmico/genética , Ratones Transgénicos , Neuroglía/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Médula Espinal/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), we found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, we screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf) as a target of FUS-R521C-associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, our results provide insight into how gain-of-function FUS mutations affect critical neuronal functions.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Daño del ADN , Empalme del ARN , Proteína FUS de Unión a ARN/genética , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Células Cultivadas , Cricetinae , Femenino , Histona Desacetilasa 1/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Corteza Motora/metabolismo , Corteza Motora/patología , Neuronas Motoras/metabolismo , Mutación Missense , Unión Proteica , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Receptor trkB/metabolismo , Transducción de Señal , Médula Espinal/metabolismo , Médula Espinal/patología , Sinapsis/metabolismo , TranscriptomaRESUMEN
Several critical events dictate the successful establishment of nascent vasculature in yolk sac and in the developing embryos. These include aggregation of angioblasts to form the primitive vascular plexus, followed by the proliferation, differentiation, migration, and coalescence of endothelial cells. Although transforming growth factor-ß (TGF-ß) is known to regulate various aspects of vascular development, the signaling mechanism of TGF-ß remains unclear. Here we show that homeodomain interacting protein kinases, HIPK1 and HIPK2, are transcriptional corepressors that regulate TGF-ß-dependent angiogenesis during embryonic development. Loss of HIPK1 and HIPK2 leads to marked up-regulations of several potent angiogenic genes, including Mmp10 and Vegf, which result in excessive endothelial proliferation and poor adherens junction formation. This robust phenotype can be recapitulated by siRNA knockdown of Hipk1 and Hipk2 in human umbilical vein endothelial cells, as well as in endothelial cell-specific TGF-ß type II receptor (TßRII) conditional mutants. The effects of HIPK proteins are mediated through its interaction with MEF2C, and this interaction can be further enhanced by TGF-ß in a TAK1-dependent manner. Remarkably, TGF-ß-TAK1 signaling activates HIPK2 by phosphorylating a highly conserved tyrosine residue Y-361 within the kinase domain. Point mutation in this tyrosine completely eliminates the effect of HIPK2 as a transcriptional corepressor in luciferase assays. Our results reveal a previously unrecognized role of HIPK proteins in connecting TGF-ß signaling pathway with the transcriptional programs critical for angiogenesis in early embryonic development.
Asunto(s)
Proteínas Portadoras/fisiología , Quinasas Quinasa Quinasa PAM/metabolismo , Neovascularización Fisiológica/genética , Proteínas Serina-Treonina Quinasas/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Uniones Adherentes/enzimología , Uniones Adherentes/ultraestructura , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proliferación Celular , Secuencia Conservada , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Proteínas de Dominio MADS/metabolismo , Factores de Transcripción MEF2 , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 10 de la Matriz/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Factores Reguladores Miogénicos/metabolismo , Fosforilación , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/química , Proteolisis , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Inhibition of basal JNK activity by JNK inhibitor SP600125 or JNK1siRNA repressed presenilin-1 (PS1) expression in SK-N-SH cells by augmenting the level of p53, a repressor of the PS1 gene (1). We now showed that repression of PS1 transcription by JNK inhibitor SP600125 inhibited gamma-secretase mediated processing of amyloid precursor protein (APP) resulting in the accumulation of C99 fragment and the reduction of secreted Abeta40 level without altering the expression of nicastrin (NCT). Co-treatment of cells with SP600125 and p53 inhibitor, pifithrin-alpha, partially nullified the suppressive effects of SP610025 on PS1 expression and secreted Abeta40 level. Suppression of JNK1 by JNK1siRNA also decreased Abeta40 level. Furthermore, overexpression of the repressors p53, ZNF237 and CHD3 of the PS1 gene also suppressed the processing of APP through repression of PS1 transcription by deacetylation of histone at the PS1 promoter. Transcriptional activator Ets2 increased PS1 protein and secreted Abeta40 levels without affecting the expression of NCT by activating PS1 transcription via hyper-acetylation of histone at the PS1 promoter. Therefore, regulation of PS1 transcription modulates gamma-secretase activity.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Presenilina-1/metabolismo , Péptidos beta-Amiloides/metabolismo , Análisis de Varianza , Antracenos/farmacología , Benzotiazoles/farmacología , Western Blotting , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica/fisiología , Vectores Genéticos/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tolueno/análogos & derivados , Tolueno/farmacologíaRESUMEN
Presenilin-1 (PS1) is a multifunctional protein involved in many cellular functions including the processing of type 1 transmembrane proteins and regulation of calcium signaling. Although PS1 is important in many aspects of cellular functions, little is known about the PS1 gene regulation in the context of intracellular signal pathways. We tested the role of c-jun-NH2-terminal kinase (JNK) on PS1 gene expression using a JNK specific inhibitor, SP600125. SP600125 efficiently suppressed basal JNK activity in SK-N-SH cell line as shown by inhibition of phosphor-JNK and phosphor-c-jun, and also decreased PS1 expression. Previously we reported that Ets1/2 bind to the PS1 promoter to activate PS1 transcription and p53 represses PS1 transcription without direct binding to the PS1 promoter [Pastorcic, M., Das, H.K., 2000. Regulation of transcription of the human presenilin-1 gene by ets transcription factors and the p53 protooncogene. J Biol Chem. 275, 34938-45.]. Involvement of protein-protein interaction between p53 and other transcription factors was speculated to be a mechanism by which p53 represses PS1 expression. Therefore, we tested whether the interaction between p53 and Ets1/2 is involved in JNK-mediated inhibition of PS1 expression. In this report we showed that p53 level was upregulated by SP600125 in SK-N-SH cell line. In addition, protein-protein interaction between p53 and Ets1/2 was enhanced with a concomitant dissociation of Ets1/2 from the PS1 promoter resulting in the suppression of PS1 transcription. We also showed that suppression of JNK1 by JNK1 siRNA increased p53 protein level and decreased PS1 expression. This observation was supported by the fact that overexpression of p53 in SK-N-SH cell line promoted dissociation of Ets1/2 from the PS1 promoter and suppressed PS1 expression. Furthermore, p53 inhibitor pifithrin-alpha partially nullified the suppressive effects of SP600125 on PS1 expression. We also showed that transfection of p53 was required for SP600125-mediated suppression of PS1 expression in p53-deficient PC3 cell line suggesting that inhibition of basal JNK activity suppresses PS1 expression through a p53-dependent mechanism.
Asunto(s)
Expresión Génica/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Presenilina-1/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Regulación hacia Arriba/fisiología , Análisis de Varianza , Antracenos/farmacología , Línea Celular Tumoral , Inmunoprecipitación de Cromatina/métodos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Neuroblastoma , Presenilina-1/genética , ARN Interferente Pequeño/farmacología , Transcripción Genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
This study was conducted to determine the toxic effects of bisphenol A (BPA) and ethynyl estradiol (EE), well-known endocrine disruptors, on Chironomus riparius under controlled laboratory conditions. Mortality, enzyme activities, and growth/development parameters were studied as acute, biochemical, and physiological toxicities, respectively. The results of the present study showed activation of catalase and glutathione-S-transferase after BPA and EE exposure, as well as increased emergence failure after EE exposure. This study on the effects of BPA and EE on C. riparius can be an important addition to the knowledge that has been obtained regarding the toxicology of BPA and EE in aquatic organism, on which limited data are available. The data obtained from this study, however, are not sufficient to establish any correlation or casual relationship between these two compounds and the response of C. riparius. Thus, further research is required to come up with direct experimental demonstrations of the wider relationship between the biochemical effects of BPA and EE on C. riparius and their consequences at higher levels of biological organization.
Asunto(s)
Chironomidae/fisiología , Disruptores Endocrinos/toxicidad , Estrógenos no Esteroides/toxicidad , Etinilestradiol/toxicidad , Larva/efectos de los fármacos , Fenoles/toxicidad , Acetilcolinesterasa/metabolismo , Animales , Compuestos de Bencidrilo , Relación Dosis-Respuesta a Droga , Glutatión Transferasa/metabolismo , Larva/enzimología , Larva/crecimiento & desarrollo , Peroxidasas/metabolismo , Razón de Masculinidad , Pruebas de ToxicidadRESUMEN
The multilevel biomarker approach, wherein different biological responses ranging from molecular to physiological are evaluated, is essential to determine the general health status of an organism in pollutant biomonitoring programs. Furthermore, it permits extrapolation of the relationship between responses at different levels of biological organization. The aim of this study was to develop, under laboratory conditions, a multilevel biomarker approach for evaluating the toxicological response of nonylphenol (NP) in Chironoms riparius. To investigate the effect of NP on C. riparius, an acute toxicity test was performed measuring 24-h median lethal concentration. Responses on molecular, biochemical, and physiological levels were subsequently investigated on sublethal exposure. To assess molecular-level effects, we investigated DNA damage and the expression of heat shock protein 70 (HSP70) gene, whereas biochemical-level responses were determined by investigation of various enzymes activities. Growth and development were investigated as physiological-level responses. The biomarkers found to be most sensitive to NP treatment were HSP70 gene expression and DNA strand break. After having been studied with lower concentration levels with longer exposure period, these biomarkers could be considered early warning signs of exposure to low concentrations of chemical exposure. Statistically significant correlations were observed between DNA damage and the development descriptor; however, a mechanistic study appears to be necessary to establish causal relationships. This approach could be applied in environmental biomonitoring programs, and the data obtained from this study should constitute an important contribution to knowledge of the toxicology of NP in C. riparius, about which little data is available.
Asunto(s)
Chironomidae/efectos de los fármacos , Fenoles/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Catalasa/metabolismo , Chironomidae/crecimiento & desarrollo , Chironomidae/metabolismo , Daño del ADN , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Glutatión Transferasa/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Larva/metabolismo , Dosificación Letal Mediana , MasculinoRESUMEN
To identify a sensitive biomarker of freshwater monitoring, we evaluated pollutant-induced expression of heat shock proteins (HSPs) and hemoglobins (Hbs) genes in the larvae of the aquatic midge Chironomus tentans (Diptera, Chironomidae). As pollutants, we examined nonylphenol, bisphenol-A, 17alpha-ethynyl estradiol, bis(2-ethylhexyl) phthalate, endosulfan, paraquat dichloride, chloropyriphos, fenitrothion, cadmium chloride, lead nitrate, potassium dichromate, benzo[a]pyrene and carbon tetrachloride. We also investigated larval growth as a physiological descriptor by measuring changes in the body fresh weight and dry weight after chemical exposure. The response of the HSPs gene expression by chemical exposure was rapid and sensitive to low chemical concentrations but it was not stressor specific. Interestingly, an increase in the expression of HSPs genes was observed not only in a stress inducible form (HSP70), but also in a constitutively (HSC70) expressed form. The expression of Hb genes showed chemical-specific responses: that is, alkyl phenolic compounds increased the expression of hemoglobin genes, whereas pesticides decreased the expression. As expected, molecular-level markers were more sensitive than physiological endpoints, suggesting that gene expression could be developed as an early warning biomarker in this animal. The overall results suggest that the expression of HSP and Hb genes in Chironomus could give useful information for diagnosing general health conditions in fresh water ecosystem. The expression of Hb genes, in particular, seems to be a promising biomarker, especially in view of the potential of Chironomus larvae as a biomonitoring species and of the physiological particularities of their respiratory pigments.