RESUMEN
Parkinson's disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanisms involved in the degeneration of neurons remain unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also reported to be involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unknown, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here, we show, using four independent methods, that DJ-1 associates with RNA targets in cells and the brain, including mitochondrial genes, genes involved in glutathione metabolism, and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations. Further, we show that DJ-1 binds RNA but dissociates after oxidative stress. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds multiple RNA targets in an oxidation-dependent manner.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Oncogénicas/metabolismo , Trastornos Parkinsonianos/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Encéfalo/metabolismo , Línea Celular , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Genes Mitocondriales , Genes Recesivos , Glutatión/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/deficiencia , Proteínas Oncogénicas/genética , Estrés Oxidativo , Fosfohidrolasa PTEN/metabolismo , Trastornos Parkinsonianos/genética , Peroxirredoxinas , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Desglicasa DJ-1 , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Mutations in DJ-1 lead to a monogenic form of early onset recessive parkinsonism. DJ-1 can respond to oxidative stress, which has been proposed to be involved in the pathogenesis of sporadic Parkinson disease (PD). We have recently reported that DJ-1 interacts with mRNA in an oxidation-dependent manner. Here, we confirm interaction of DJ-1 and RNA in human brain using immunoprecipitation followed by quantitative real time PCR. We confirmed previous reports that DJ-1 is more oxidized in cortex from cases of sporadic PD compared to controls. In the same samples, protein and RNA expression was measured for four DJ-1 target genes GPx4, MAPK8IP1, ND2 and ND5. While no alterations in mRNA expression were observed, an increase in protein expression was observed in PD cases for GPx4 and MAPK8IP1. In the same patients, we saw decreased mRNA and protein levels of two mitochondrial targets, ND2 and ND5. These results suggest that these proteins undergo regulation at the post-transcriptional level that may involve translational regulation by DJ-1.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Oncogénicas/genética , Enfermedad de Parkinson/genética , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Encéfalo/metabolismo , Electroforesis en Gel Bidimensional/métodos , Glutatión Peroxidasa/genética , Humanos , Inmunoprecipitación/métodos , NADH Deshidrogenasa/genética , Enfermedad de Parkinson/patología , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Proteína Desglicasa DJ-1 , Proteína Fosfatasa 2/genética , Proteínas Proto-Oncogénicas/genética , Selenoproteína W/genética , Proteína Letal Asociada a bcl/genéticaRESUMEN
BACKGROUND: Parkinson's disease (PD) is a debilitating neurological disorder for which prognostic and diagnostic biomarkers are lacking. Cerebrospinal fluid (CSF) is an accessible body fluid that comes into direct contact with the central nervous system (CNS) and acts as a nuclease-free repository where RNA transcripts shed by brain tissues can reside for extended periods of time. OBJECTIVE: We studied the RNA species present in the CSF of PD patients to identify novel diagnostic biomarkers. METHODS: Small volumes of CSF from 27 PD patients and 30 healthy age- and sex-matched controls were used for RNA extraction followed by next-generation sequencing (RNA-seq) using the Illumina platform. CSF contains a number of fragmented RNA species that were individually sequenced and analyzed. Comparing PD to control subjects, we observed a pool of dysregulated sequencing tags that were further analyzed and validated by quantitative real-time PCR (qRT-PCR). RESULTS: A total of 201 differentially expressed sequencing tags (DETs), including 92 up-regulated and 109 down-regulated DETs were identified. We validated the following DETs by real time PCR in the patient samples: Dnmt1, Ezh2, CCR3, SSTR5,PTPRC, UBC, NDUFV2, BMP7, SCN9, SCN9 antisense (AC010127.3), and long noncoding RNAs AC079630 and UC001lva.4 (close to the LRRK2 gene locus), as potential PD biomarkers. CONCLUSIONS: The CSF is a unique environment that contains many species of RNA. Our work demonstrates that CSF can potentially be used to identify biomarkers for the detection and tracking of disease progression and evaluation of therapeutic outcomes.
Asunto(s)
Biomarcadores/líquido cefalorraquídeo , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Enfermedad de Parkinson/líquido cefalorraquídeo , ARN/líquido cefalorraquídeo , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma/genéticaRESUMEN
Mutations in the DJ-1 gene are associated with recessive, early onset Parkinson's disease (PD). We reported previously that one of the point mutations, L166P, destabilizes the protein and thus produces an effective knockout of the gene. Here, we have expanded this analysis to include a series of mutations and polymorphisms identified throughout the gene. The M26I point mutation was also unstable, although the effect was not as dramatic as with L166P. Protein levels were rescued in part, but not completely, by proteasome inhibition. Other variants, such as R98Q, were generally stable. We noted that M26I and L166P are both in helical regions near the dimer interface. However, M26I retains the ability to dimerize. We also examined the subcellular localization of DJ-1 and found that most mutations were similar to the wild-type (wt) protein in that a few cells showed mitochondrial staining. However, in all cases, the proportion of cells with mitochondrial DJ-1 staining was increased in oxidative conditions, suggesting that oxidation promotes the mitochondrial localization of DJ-1.
Asunto(s)
Acetilcisteína/análogos & derivados , Proteínas Oncogénicas/genética , Mutación Puntual , Polimorfismo Genético , Acetilcisteína/farmacología , Cloruro de Amonio/farmacología , Análisis de Varianza , Animales , Western Blotting/métodos , Línea Celular Tumoral , Cicloheximida/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Dimerización , Técnica del Anticuerpo Fluorescente/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación/métodos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Modelos Moleculares , Neuroblastoma , Proteínas Oncogénicas/química , Proteína Desglicasa DJ-1 , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Transfección/métodosRESUMEN
The formation of cysteine-sulfinic acid has recently become appreciated as a modification that links protein function to cellular oxidative status. Human DJ-1, a protein associated with inherited parkinsonism, readily forms cysteine-sulfinic acid at a conserved cysteine residue (Cys106 in human DJ-1). Mutation of Cys106 causes the protein to lose its normal protective function in cell culture and model organisms. However, it is unknown whether the loss of DJ-1 protective function in these mutants is due to the absence of Cys106 oxidation or the absence of the cysteine residue itself. To address this question, we designed a series of substitutions at a proximal glutamic acid residue (Glu18) in human DJ-1 that alter the oxidative propensity of Cys106 through changes in hydrogen bonding. We show that two mutations, E18N and E18Q, allow Cys106 to be oxidized to Cys106-sulfinic acid under mild conditions. In contrast, the E18D mutation stabilizes a cysteine-sulfenic acid that is readily reduced to the thiol in solution and in vivo. We show that E18N and E18Q can both partially substitute for wild-type DJ-1 using mitochondrial fission and cell viability assays. In contrast, the oxidatively impaired E18D mutant behaves as an inactive C106A mutant and fails to protect cells. We therefore conclude that formation of Cys106-sulfinic acid is a key modification that regulates the protective function of DJ-1.
Asunto(s)
Cisteína/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Oncogénicas/metabolismo , Ácidos Sulfínicos/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular Tumoral , Cisteína/genética , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Proteínas Mitocondriales/genética , Mutación , Proteínas Oncogénicas/genética , Oxidación-Reducción , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Proteína Desglicasa DJ-1RESUMEN
PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.
Asunto(s)
Calcineurina/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Proteínas Quinasas/deficiencia , Línea Celular , Supervivencia Celular/efectos de los fármacos , Dinaminas , Activación Enzimática/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Modelos Biológicos , Fenotipo , Fosforilación/efectos de los fármacos , Proteínas Quinasas/metabolismo , Rotenona/farmacologíaRESUMEN
Several mutations have been found in the leucine-rich repeat kinase 2 gene (LRRK2), encoding the protein dardarin, which are associated with autosomal dominant Parkinson disease. We have previously shown that mutant LRRK2/dardarin is toxic to neurons and neuron-like cell lines in culture and that some mutations are also associated with an inclusion-body phenotype. There is a homologous kinase, LRRK1, which has a similar domain structure but is not known to carry mutations causing Parkinson disease. In the current study, we introduced mutations at equivalent residues in both LRRK2 and LRRK1 to determine their effects in cells. We show that mutations in dardarin are more prone to form inclusion bodies in transfected cells and are more toxic than equivalent mutations in LRRK1. This work suggests that dardarin/LRRK2 is inherently more damaging than LRRK1.
Asunto(s)
Enfermedad de Parkinson/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Western Blotting , Encéfalo/enzimología , Células COS , Células Cultivadas , Chlorocebus aethiops , Clonación Molecular , Citosol/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Inmunoprecipitación , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Microscopía Confocal , Mutación/fisiología , Fosfoproteínas/metabolismo , Fosforilación , Plásmidos/genética , Proteínas Serina-Treonina Quinasas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Mutations in the LRRK2 gene, coding for dardarin, cause dominantly inherited Parkinson's disease (PD). Dardarin is a large protein, and mutations are found throughout the gene including the kinase domain. However, it is not clear if kinase activity is important for the damaging effects of pathogenic mutations. In this study, we noted two cellular phenotypes associated with mutant dardarin. First, pathogenic mutations increase the tendency of dardarin to form inclusion bodies. Secondly, neurons and neuronal cell lines undergo cell death after expression of mutant protein. Manipulating activity by replacing the kinase domain with a 'kinase-dead' version blocks inclusion body formation and strongly delays cell death. This predicts that kinase inhibitors will be useful therapeutic agents in patients with LRRK2 mutations and, perhaps, in sporadic PD. We also show that dardarin protein is expressed within human midbrain neurons and that C-terminal epitopes are also found in some Lewy bodies.
Asunto(s)
Enfermedad de Parkinson/genética , Proteínas Serina-Treonina Quinasas/genética , Sustitución de Aminoácidos , Encéfalo/enzimología , Encéfalo/patología , ADN Complementario/genética , Humanos , Inmunohistoquímica , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/patología , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Mutación , Enfermedad de Parkinson/enzimología , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Several mutations in PTEN-induced putative kinase 1 (PINK1) gene have been reported to be associated with recessive parkinsonism. The encoded protein is predicted to be a Ser/Thr protein kinase targeted to mitochondria. In this study, we have investigated the effects of mutations on PINK1 kinase activity in vitro and on expression levels and localization in mammalian cells. We chose to examine two point mutations: G309D, which was originally reported to be stable and properly localized in cells and L347P, which is of interest because it is present at an appreciable carrier frequency in the Philippines. We were able to confirm kinase activity and produce artificial "kinase-dead" mutants that are stable but lack activity. The L347P mutation grossly destabilizes PINK1 and drastically reduces kinase activity, whereas G309D has much more modest effects on these parameters in vitro. This finding is in line with predictions based on homology modeling. We also examined the localization of PINK1 in transfected mammalian cells by using constructs that were tagged with myc or GFP at either end of the protein. These results show that PINK1 is processed at the N terminus in a manner consistent with mitochondrial import, but the mature protein also exists in the cytosol. The physiological relevance of this observation is not yet clear, but it implies that a portion of PINK1 may be exported after processing in the mitochondria.
Asunto(s)
Trastornos Parkinsonianos/enzimología , Trastornos Parkinsonianos/genética , Mutación Puntual , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Estabilidad de Enzimas , Humanos , Persona de Mediana Edad , Mitocondrias/enzimología , Modelos Moleculares , Proteínas Quinasas/química , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Tyrosinase is a key enzyme in the synthesis of melanin in skin and hair and has also been proposed to contribute to the formation of neuromelanin (NM). The presence of NM, which is biochemically similar to melanin in peripheral tissues, identifies groups of neurons susceptible in Parkinson's disease (PD). Whether tyrosinase is beneficial or detrimental to neurons is unclear; whilst the enzyme activity of tyrosinase generates dopamine-quinones and other oxidizing compounds, NM may form a sink for such radical species. In the present study, we demonstrated that tyrosinase is expressed at low levels in the human brain. We found that mRNA, protein and enzyme activity are all present but at barely detectable levels. In cell culture systems, expression of tyrosinase increases neuronal susceptibility to oxidizing conditions, including dopamine itself. We related these in vitro observations to the human disease by assessing whether there was any genetic association between the gene encoding tyrosinase and idiopathic PD. We found neither genotypic or haplotypic association with three polymorphic markers of the gene. This argues against a strong genetic association between tyrosinase and PD, although the observed contribution to cellular toxicity suggests that a biochemical association is likely.
Asunto(s)
Dopamina/toxicidad , Monofenol Monooxigenasa/fisiología , Neuronas/efectos de los fármacos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Northern Blotting/métodos , Western Blotting/métodos , Encéfalo/citología , Encéfalo/metabolismo , Recuento de Células/métodos , Células Cultivadas , Clonación Molecular/métodos , Interacciones Farmacológicas , Femenino , Técnica del Anticuerpo Fluorescente/métodos , Frecuencia de los Genes/fisiología , Genotipo , Haplotipos , Humanos , Inmunoprecipitación/métodos , Masculino , Ratones , Persona de Mediana Edad , Monofenol Monooxigenasa/farmacología , Neuroblastoma , Neuronas/citología , Neuronas/metabolismo , Polimorfismo Genético , Cambios Post Mortem , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sales de Tetrazolio , Tiazoles , Factores de Tiempo , Transducción Genética/métodos , Transfección/métodosRESUMEN
Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) alpha-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant alpha-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.
Asunto(s)
Dopamina/biosíntesis , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/metabolismo , Neuronas/metabolismo , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Descarboxilasas de Aminoácido-L-Aromático/genética , Descarboxilasas de Aminoácido-L-Aromático/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Neuroblastoma/química , Neuronas/química , Neuronas/citología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Elementos de Respuesta/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sinucleínas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Células Tumorales Cultivadas , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , alfa-SinucleínaRESUMEN
Loss-of-function DJ-1 mutations can cause early-onset Parkinson's disease. The function of DJ-1 is unknown, but an acidic isoform accumulates after oxidative stress, leading to the suggestion that DJ-1 is protective under these conditions. We addressed whether this represents a posttranslational modification at cysteine residues by systematically mutating cysteine residues in human DJ-1. WT or C53A DJ-1 was readily oxidized in cultured cells, generating a pI 5.8 isoform, but an artificial C106A mutant was not. We observed a cysteine-sulfinic acid at C106 in crystalline DJ-1 but no modification of C53 or C46. Oxidation of DJ-1 was promoted by the crystallization procedure. In addition, oxidation-induced mitochondrial relocalization of DJ-1 and protection against cell death were abrogated in C106A but not C53A or C46A. We suggest that DJ-1 protects against neuronal death, and that this is signaled by acidification of the key cysteine residue, C106.
Asunto(s)
Cisteína/análogos & derivados , Cisteína/metabolismo , Mitocondrias/metabolismo , Fármacos Neuroprotectores/metabolismo , Proteínas Oncogénicas/metabolismo , 1-Metil-4-fenilpiridinio/toxicidad , Sustitución de Aminoácidos , Línea Celular Tumoral , Cisteína/química , Cisteína/genética , Humanos , Membranas Intracelulares/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Modelos Moleculares , Neurotransmisores , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Oxidación-Reducción , Estrés Oxidativo , Proteína Desglicasa DJ-1 , Isoformas de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , TransfecciónRESUMEN
Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell.