RESUMEN
Neurodegenerative diseases are characterized by a decline in neuronal function and structure, leading to neuronal death. Understanding the molecular mechanisms of neuronal death is crucial for developing therapeutics. MiRs are small noncoding RNAs that regulate gene expression by degrading target mRNAs or inhibiting translation. MiR dysregulation has been linked to many neurodegenerative diseases, but the underlying mechanisms are not well understood. As mitochondrial dysfunction is one of the common molecular mechanisms leading to neuronal death in many neurodegenerative diseases, here we studied miRs that modulate neuronal death caused by 1-methyl-4-phenylpyridinium (MPP+), an inhibitor of complex I in mitochondria. We identified miR-593-5p, levels of which were increased in SH-SY5Y human neuronal cells, after exposure to MPP+. We found that intracellular Ca2+, but not of reactive oxygen species, mediated this miR-593-5p increase. Furthermore, we found the increase in miR-593-5p was due to enhanced stability, not increased transcription or miR processing. Importantly, we show the increase in miR-593-5p contributed to MPP+-induced cell death. Our data revealed that miR-593-5p inhibits a signaling pathway involving PTEN-induced putative kinase 1 (PINK1) and Parkin, two proteins responsible for the removal of damaged mitochondria from cells, by targeting the coding sequence of PINK1 mRNA. Our findings suggest that miR-593-5p contributes to neuronal death resulting from MPP+ toxicity, in part, by impeding the PINK1/Parkin-mediated pathway that facilitates the clearance of damaged mitochondria. Taken together, our observations highlight the potential significance of inhibiting miR-593-5p as a therapeutic approach for neurodegenerative diseases.
Asunto(s)
MicroARNs , Neuroblastoma , Proteínas Quinasas , Humanos , 1-Metil-4-fenilpiridinio/toxicidad , Apoptosis , Muerte Celular , Línea Celular Tumoral , MicroARNs/genética , MicroARNs/metabolismo , Neuroblastoma/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Parkinson's disease (PD) is a common neurodegenerative disorder characterized by a multifaceted genetic foundation. Genome-Wide Association Studies (GWAS) have played a crucial role in pinpointing genetic variants linked to PD susceptibility. Current study aims to delve into the mechanistic aspects through which the PD-associated Single Nucleotide Polymorphism (SNP) rs329648, identified in prior GWAS, influences the pathogenesis of PD. METHODS AND RESULTS: Employing the CRISPR/Cas9-mediated genome editing mechanism, we demonstrated the association of the disease-associated allele of rs329648 with increased expression of miR-4697-3p in differentiated SH-SY5Y cells. We revealed that miR-4697-3p contributes to the formation of high molecular weight complexes of α-Synuclein (α-Syn), indicative of α-Syn aggregate formation, as evidenced by Western blot analysis. Furthermore, our study unveiled that miR-4697-3p elevates SNCA112 mRNA levels. The resultant protein product, α-Syn 112, a variant of α-Syn with 112 amino acids, is recognized for augmenting α-Syn aggregation. Notably, this regulatory effect minimally impacts the levels of full-length SNCA140 mRNA, as evidenced by qRT-PCR. Additionally, we observed a correlation between the disease-associated allele and miR-4697-3p with increased cell death, substantiated by assessments including cell viability assays, alterations in cell morphology, and TUNEL assays. CONCLUSION: Our research reveals that the disease-associated allele of rs329648 is linked to higher levels of miR-4697-3p. This increase in miR-4697-3p leads to elevated SNCA112 mRNA levels, consequently promoting the formation of α-Syn aggregates. Furthermore, miR-4697-3p appears to play a role in increased cell death, potentially contributing to the pathogenesis of PD.
Asunto(s)
MicroARNs , Enfermedad de Parkinson , Polimorfismo de Nucleótido Simple , ARN Mensajero , alfa-Sinucleína , Humanos , Alelos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Línea Celular Tumoral , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Regulación de la Expresión Génica/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , MicroARNs/genética , MicroARNs/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Polimorfismo de Nucleótido Simple/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson's disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5' untranslated region (5' UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5' UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound's selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/genética , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Elementos de Respuesta , alfa-Sinucleína/genética , Regiones no Traducidas 3' , Animales , Línea Celular Tumoral , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Ratones , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/química , ARN Mensajero/química , ARN Mensajero/genética , alfa-Sinucleína/metabolismoRESUMEN
Spinal cord injury (SCI) is a devastating neurological condition for which there are no effective therapies. Following an initial injury, there is a cascade of multiple downstream events termed secondary injury. Thus, therapeutic approaches targeting a single pathway may not offer the best solution for treating SCI. One of the most attractive properties of microRNAs (miR) as potential therapeutics is that they are highly effective in regulating complex biological pathways by targeting multiple genes and pathways. The current study investigated the role of miR-7-5p (miR-7), which was previously shown to have neuroprotective functions, in promoting motor function recovery following SCI. We used an adeno-associated virus 1 (AAV1) vector to deliver the gene encoding miR-7 to the spinal cord of adult mice and found that this virus was mainly transduced into the neurons of the spinal cord. Transduction of AAV1-miR-7 improved hindlimb locomotor function following SCI over an 8-week observation period. This improvement was accompanied by reduced neuronal loss in the lesion. In addition, the beneficial effect of miR-7 was associated with enhanced levels of TH-positive axons in the lesion. Taken together, we suggest that miR-7 improves motor function recovery after SCI by protecting neuronal death and increasing axon levels. These findings suggest that miR-7 could be developed as a potential treatment for SCI in human.
Asunto(s)
MicroARNs/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Actividad Motora , Recuperación de la Función , Traumatismos de la Médula Espinal/patologíaRESUMEN
Hyperphosphorylated α-synuclein in Lewy bodies and Lewy neurites is a characteristic neuropathological feature of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). The catalytic subunit of the specific phosphatase, protein phosphatase 2A (PP2A) that dephosphorylates α-synuclein, is hypomethylated in these brains, thereby impeding the assembly of the active trimeric holoenzyme and reducing phosphatase activity. This phosphatase deficiency contributes to the accumulation of hyperphosphorylated α-synuclein, which tends to fibrillize more than unmodified α-synuclein. Eicosanoyl-5-hydroxytryptamide (EHT), a fatty acid derivative of serotonin found in coffee, inhibits the PP2A methylesterase so as to maintain PP2A in a highly active methylated state and mitigates the phenotype of α-synuclein transgenic (SynTg) mice. Considering epidemiologic and experimental evidence suggesting protective effects of caffeine in PD, we sought, in the present study, to test whether there is synergy between EHT and caffeine in models of α-synucleinopathy. Coadministration of these two compounds orally for 6 mo at doses that were individually ineffective in SynTg mice and in a striatal α-synuclein preformed fibril inoculation model resulted in reduced accumulation of phosphorylated α-synuclein, preserved neuronal integrity and function, diminished neuroinflammation, and improved behavioral performance. These indices were associated with increased levels of methylated PP2A in brain tissue. A similar profile of greater PP2A methylation and cytoprotection was found in SH-SY5Y cells cotreated with EHT and caffeine, but not with each compound alone. These findings suggest that these two components of coffee have synergistic effects in protecting the brain against α-synuclein-mediated toxicity through maintenance of PP2A in an active state.
Asunto(s)
Cafeína/farmacología , Café/química , Enfermedad por Cuerpos de Lewy/metabolismo , Neuroprotección/efectos de los fármacos , Enfermedad de Parkinson/metabolismo , Serotonina/análogos & derivados , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Ácidos Grasos/metabolismo , Enfermedad por Cuerpos de Lewy/tratamiento farmacológico , Enfermedad por Cuerpos de Lewy/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Fosforilación , Proteína Fosfatasa 2/metabolismo , Serotonina/farmacología , alfa-Sinucleína/metabolismoRESUMEN
Mitochondrial dysfunction is one of the major contributors to neurodegenerative disorders including Parkinson disease. The mitochondrial permeability transition pore is a protein complex located on the mitochondrial membrane. Under cellular stress, the pore opens, increasing the release of pro-apoptotic proteins, and ultimately resulting in cell death. MicroRNA-7 (miR-7) is a small non-coding RNA that has been found to exhibit a protective role in the cellular models of Parkinson disease. In the present study, miR-7 was predicted to regulate the function of mitochondria, according to gene ontology analysis of proteins that are down-regulated by miR-7. Indeed, miR-7 overexpression inhibited mitochondrial fragmentation, mitochondrial depolarization, cytochrome c release, reactive oxygen species generation, and release of mitochondrial calcium in response to 1-methyl-4-phenylpyridinium (MPP(+)) in human neuroblastoma SH-SY5Y cells. In addition, several of these findings were confirmed in mouse primary neurons. Among the mitochondrial proteins identified by gene ontology analysis, the expression of voltage-dependent anion channel 1 (VDAC1), a constituent of the mitochondrial permeability transition pore, was down-regulated by miR-7 through targeting 3'-untranslated region of VDAC1 mRNA. Similar to miR-7 overexpression, knockdown of VDAC1 also led to a decrease in intracellular reactive oxygen species generation and subsequent cellular protection against MPP(+). Notably, overexpression of VDAC1 without the 3'-UTR significantly abolished the protective effects of miR-7 against MPP(+)-induced cytotoxicity and mitochondrial dysfunction, suggesting that the protective effect of miR-7 is partly exerted through promoting mitochondrial function by targeting VDAC1 expression. These findings point to a novel mechanism by which miR-7 accomplishes neuroprotection by improving mitochondrial health.
Asunto(s)
MicroARNs/genética , Interferencia de ARN , Canal Aniónico 1 Dependiente del Voltaje/metabolismo , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Expresión Génica , Ontología de Genes , Humanos , Potencial de la Membrana Mitocondrial , Ratones Endogámicos C57BL , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Membranas Mitocondriales/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Tamaño Mitocondrial , Especies Reactivas de Oxígeno/metabolismo , Canal Aniónico 1 Dependiente del Voltaje/genéticaRESUMEN
Parkinson disease is associated with decreased activity of the mitochondrial electron transport chain. This defect can be recapitulated in vitro by challenging dopaminergic cells with 1-methyl-4-phenylpyridinium (MPP(+)), a neurotoxin that inhibits complex I of electron transport chain. Consequently, oxidative phosphorylation is blocked, and cells become dependent on glycolysis for ATP production. Therefore, increasing the rate of glycolysis might help cells to produce more ATP to meet their energy demands. In the present study, we show that microRNA-7, a non-coding RNA that protects dopaminergic neuronal cells against MPP(+)-induced cell death, promotes glycolysis in dopaminergic SH-SY5Y and differentiated human neural progenitor ReNcell VM cells, as evidenced by increased ATP production, glucose consumption, and lactic acid production. Through a series of experiments, we demonstrate that targeted repression of RelA by microRNA-7, as well as subsequent increase in the neuronal glucose transporter 3 (Glut3), underlies this glycolysis-promoting effect. Consistently, silencing Glut3 expression diminishes the protective effect of microRNA-7 against MPP(+). Further, microRNA-7 fails to prevent MPP(+)-induced cell death when SH-SY5Y cells are cultured in a low glucose medium, as well as when differentiated ReNcell VM cells or primary mouse neurons are treated with the hexokinase inhibitor, 2-deoxy-d-glucose, indicating that a functional glycolytic pathway is required for this protective effect. In conclusion, microRNA-7, by down-regulating RelA, augments Glut3 expression, promotes glycolysis, and subsequently prevents MPP(+)-induced cell death. This protective effect of microRNA-7 could be exploited to correct the defects in oxidative phosphorylation in Parkinson disease.
Asunto(s)
1-Metil-4-fenilpiridinio/química , Muerte Celular , MicroARNs/metabolismo , Factor de Transcripción ReIA/metabolismo , Adenosina Trifosfato/química , Animales , Diferenciación Celular , Línea Celular Tumoral , Supervivencia Celular , Desoxiglucosa/química , Glucosa/química , Transportador de Glucosa de Tipo 3/metabolismo , Glucólisis , Células HEK293 , Humanos , Ácido Láctico/química , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Neuronas/metabolismo , Fosforilación Oxidativa , Oxígeno/química , Enfermedad de Parkinson/metabolismo , FosforilaciónRESUMEN
Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Mitochondrial complex I impairment in PD is modeled in vitro by the susceptibility of dopaminergic neurons to the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+). In the present study, we demonstrate that microRNA-7 (miR-7), which is expressed in tyrosine hydroxylase-positive nigral neurons in mice and humans, protects cells from MPP+-induced toxicity in dopaminergic SH-SY5Y cells, differentiated human neural progenitor ReNcell VM cells, and primary mouse neurons. RelA, a component of nuclear factor-κB (NF-κB), was identified to be downregulated by miR-7 using quantitative proteomic analysis. Through a series of validation experiments, it was confirmed that RelA mRNA is a target of miR-7 and is required for cell death following MPP+ exposure. Further, RelA mediates MPP+-induced suppression of NF-κB activity, which is essential for MPP+-induced cell death. Accordingly, the protective effect of miR-7 is exerted through relieving NF-κB suppression by reducing RelA expression. These findings provide a novel mechanism by which NF-κB suppression, rather than activation, underlies the cell death mechanism following MPP+ toxicity, have implications for the pathogenesis of PD, and suggest miR-7 as a therapeutic target for this disease.
Asunto(s)
1-Metil-4-fenilpiridinio/antagonistas & inhibidores , MicroARNs/fisiología , Fármacos Neuroprotectores/metabolismo , Enfermedad de Parkinson Secundaria/prevención & control , Factor de Transcripción ReIA/biosíntesis , 1-Metil-4-fenilpiridinio/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/fisiología , Regulación hacia Abajo , Humanos , Ratones , MicroARNs/biosíntesis , MicroARNs/genética , FN-kappa B/biosíntesis , Neuronas/efectos de los fármacos , Enfermedad de Parkinson Secundaria/inducido químicamente , Sustancia Negra/metabolismo , Factor de Transcripción ReIA/genética , Transfección , alfa-Sinucleína/genéticaRESUMEN
α-Synuclein is a key pathogenic protein that aggregates in hallmark lesions in Parkinson's disease and other α-synucleinopathies. Prior in vitro studies demonstrated that it is a substrate for cross-linking by transglutaminase 2 (TG2) into higher-order species. Here we investigated whether this increased aggregation occurs in vivo and whether TG2 exacerbates α-synuclein toxicity in Mus musculus and Saccharomyces cerevisiae. Compared with α-synuclein transgenic (Syn(Tg)) mice, animals double transgenic for human α-synuclein and TG2 (TG2(Tg)/Syn(Tg)) manifested greater high-molecular-weight insoluble species of α-synuclein in brain lysates and developed α-synuclein aggregates in the synaptic vesicle fraction. In addition, larger proteinase K-resistant aggregates developed, along with increased thioflavin-S-positive amyloid fibrils. This correlated with an exaggerated neuroinflammatory response, as seen with more astrocytes and microglia. Further neuronal damage was suggested by greater morphological disruption of nerve fibers and a trend toward decreased c-Fos immunoreactive neurons. Finally, the performance of TG2(Tg)/Syn(Tg) animals on motor behavioral tasks was worse relative to Syn(Tg) mice. Greater toxicity of α-synuclein was also demonstrated in yeast cells coexpressing TG2. Our findings demonstrate that TG2 promotes the aggregation of α-synuclein in vivo and that this is associated with aggravated toxicity of α-synuclein and its downstream neuropathologic consequences.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Unión al GTP/metabolismo , Agregación Patológica de Proteínas , Saccharomyces cerevisiae/metabolismo , Transglutaminasas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/fisiología , Humanos , Locomoción , Ratones , Ratones Endogámicos C57BL , Fibras Nerviosas/metabolismo , Fibras Nerviosas/patología , Agregado de Proteínas , Proteína Glutamina Gamma Glutamiltransferasa 2 , Vesículas Sinápticas/metabolismoRESUMEN
DJ-1, which is linked to recessively inherited Parkinson's disease when mutated, is a multi-functional protein with anti-oxidant and transcription regulatory activities. However, the mechanism(s) through which DJ-1 and the genes it regulates provide neuroprotection is not fully understood. Here, we show that wild-type DJ-1 induces the expression of thioredoxin 1 (Trx1), a protein disulfide oxidoreductase, whereas pathogenic mutant isoforms L166P and M26I cannot. Conversely, DJ-1 knockdown in SH-SY5Y cells and DJ-1 knockout in mice result in significant decrease in Trx1 protein and mRNA expression levels. The importance of Trx1 in the cytoprotective function of DJ-1 is confirmed using a pharmacological inhibitor of Trx reductase, 1-chloro-2,4-dinitrobenzene, and Trx1 siRNA. Both approaches result in partial loss of DJ-1-mediated protection. Additionally, knockdown of Trx1 significantly abrogates DJ-1-dependent, hydrogen peroxide-induced activation of the pro-survival factor AKT. Promoter analysis of the human Trx1 gene identified an antioxidant response element (ARE) that is required for DJ-1-dependent induction of Trx1 expression. The transcription factor Nuclear factor erythroid-2 related factor 2 (Nrf2), which is a critical inducer of ARE-mediated expression, is regulated by DJ-1. Overexpression of DJ-1 results in increased Nrf2 protein levels, promotes its translocation into the nucleus and enhances its recruitment onto the ARE site in the Trx1 promoter. Further, Nrf2 knockdown abolishes DJ-1-mediated Trx1 induction and cytoprotection against hydrogen peroxide, indicating the critical role of Nrf2 in carrying out the protective functions of DJ-1 against oxidative stress. These findings provide a new mechanism to support the antioxidant function of DJ-1 by increasing Trx1 expression via Nrf2-mediated transcriptional induction.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Oncogénicas/metabolismo , Tiorredoxinas/biosíntesis , Animales , Antioxidantes , Línea Celular Tumoral , Citoprotección , Dinitroclorobenceno/farmacología , Regulación de la Expresión Génica , Células HeLa , Humanos , Peróxido de Hidrógeno/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Factor 2 Relacionado con NF-E2/genética , Proteínas Oncogénicas/genética , Estrés Oxidativo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Proteína Desglicasa DJ-1 , Proteínas Proto-Oncogénicas c-akt/biosíntesis , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Elementos de Respuesta , Transducción de Señal , Tiorredoxinas/antagonistas & inhibidores , Tiorredoxinas/genéticaRESUMEN
α-Synuclein (α-Syn) is a key protein that accumulates as hyperphosphorylated aggregates in pathologic hallmark features of Parkinson's disease (PD) and other neurodegenerative disorders. Phosphorylation of this protein at serine 129 is believed to promote its aggregation and neurotoxicity, suggesting that this post-translational modification could be a therapeutic target. Here, we demonstrate that phosphoprotein phosphatase 2A (PP2A) dephosphorylates α-Syn at serine 129 and that this activity is greatly enhanced by carboxyl methylation of the catalytic C subunit of PP2A. α-Syn-transgenic mice raised on a diet supplemented with eicosanoyl-5-hydroxytryptamide, an agent that enhances PP2A methylation, dramatically reduced both α-Syn phosphorylation at Serine 129 and α-Syn aggregation in the brain. These biochemical changes were associated with enhanced neuronal activity, increased dendritic arborizations, and reduced astroglial and microglial activation, as well as improved motor performance. These findings support the notion that serine 129 phosphorylation of α-Syn is of pathogenetic significance and that promoting PP2A activity is a viable disease-modifying therapeutic strategy for α-synucleinopathies such as PD.
Asunto(s)
Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Serotonina/análogos & derivados , alfa-Sinucleína/metabolismo , Animales , Astrocitos/metabolismo , Astrocitos/patología , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Células Cultivadas , Dendritas/genética , Dendritas/metabolismo , Dendritas/patología , Modelos Animales de Enfermedad , Inmunohistoquímica , Metilación , Ratones , Ratones Transgénicos , Microglía/metabolismo , Microglía/patología , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Neuronas/patología , Fosforilación/fisiología , Serotonina/metabolismo , alfa-Sinucleína/genéticaRESUMEN
The mutation or overexpression of alpha-synuclein protein plays a pivotal role in the pathogenesis of Parkinson's disease. In our preliminary experiments, we found that alpha-synuclein induced the expression of matrix metalloproteinases (MMPs) (MMP-1, -3, -8, and -9) in rat primary cultured microglia. Thus, the current study was undertaken to determine the roles of MMPs in alpha-synuclein-induced microglial activation. The inhibition of MMP-3, -8, or -9 significantly reduced NO and reactive oxygen species levels and suppressed the expression of TNF-alpha and IL-1beta. Notably, MMP-8 inhibitor suppressed TNF-alpha production more efficaciously than MMP-3 or MMP-9 inhibitors. Inhibition of MMP-3 or -9 also suppressed the activities of MAPK, NF-kappaB, and AP-1. Previously, protease-activated receptor-1 (PAR-1) has been associated with the actions of MMPs, and thus, we further investigated the role of PAR-1 in alpha-synuclein-induced inflammatory reactions. A PAR-1-specific inhibitor and a PAR-1 antagonist significantly suppressed cytokine levels, and NO and reactive oxygen species production in alpha-synuclein-treated microglia. Subsequent PAR-1 cleavage assay revealed that MMP-3, -8, and -9, but not alpha-synuclein, cleaved the synthetic peptide containing conventional PAR-1 cleavage sites. These results suggest that MMPs secreted by alpha-synuclein-stimulated microglia activate PAR-1 and amplify microglial inflammatory signals in an autocrine or paracrine manner. Furthermore, our findings suggest that modulation of the activities of MMPs and/or PAR-1 may provide a new therapeutic strategy for Parkinson's disease.
Asunto(s)
Metaloproteinasas de la Matriz/biosíntesis , Microglía/metabolismo , Receptor PAR-1/metabolismo , alfa-Sinucleína/fisiología , Secuencia de Aminoácidos , Animales , Comunicación Autocrina/inmunología , Línea Celular Tumoral , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Humanos , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Lipopolisacáridos/fisiología , Inhibidores de la Metaloproteinasa de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Microglía/enzimología , Microglía/patología , Datos de Secuencia Molecular , Comunicación Paracrina/inmunología , Procesamiento Proteico-Postraduccional/inmunología , Ratas , Ratas Sprague-Dawley , Receptor PAR-1/agonistas , Receptor PAR-1/fisiologíaRESUMEN
alpha-Synuclein is a key protein in Parkinson's disease (PD) because it accumulates as fibrillar aggregates in pathologic hallmark features in affected brain regions, most notably in nigral dopaminergic neurons. Intraneuronal levels of this protein appear critical in mediating its toxicity, because multiplication of its gene locus leads to autosomal dominant PD, and transgenic animal models overexpressing human alpha-synuclein manifest impaired function or decreased survival of dopaminergic neurons. Here, we show that microRNA-7 (miR-7), which is expressed mainly in neurons, represses alpha-synuclein protein levels through the 3'-untranslated region (UTR) of alpha-synuclein mRNA. Importantly, miR-7-induced down-regulation of alpha-synuclein protects cells against oxidative stress. Further, in the MPTP-induced neurotoxin model of PD in cultured cells and in mice, miR-7 expression decreases, possibly contributing to increased alpha-synuclein expression. These findings provide a mechanism by which alpha-synuclein levels are regulated in neurons, have implications for the pathogenesis of PD, and suggest miR-7 as a therapeutic target for PD and other alpha-synucleinopathies.
Asunto(s)
MicroARNs/fisiología , alfa-Sinucleína/antagonistas & inhibidores , Regiones no Traducidas 3'/fisiología , Animales , Encéfalo/metabolismo , Línea Celular , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , Enfermedad de Parkinson/etiología , Inhibidores de Proteasoma , alfa-Sinucleína/genética , alfa-Sinucleína/toxicidadRESUMEN
α-Synuclein is a key protein in the pathogenesis of Parkinson's disease as it accumulates in fibrillar form in affected brain regions. Misfolded α-synuclein seeds recruit monomeric α-synuclein to form aggregates, which can spread to anatomically connected brain regions, a phenomenon that correlates with clinical disease progression. Thus, downregulating α-synuclein levels could reduce seeding and inhibit aggregate formation and propagation. We previously reported that microRNA-7 (miR-7) protects neuronal cells by downregulating α-synuclein expression through its effect on the 3'-untranslated region of SNCA mRNA; however, whether miR-7 blocks α-synuclein seeding and propagation in vivo remains unknown. Here, we induced miR-7 overexpression in the mouse striatum unilaterally by infusing adeno-associated virus 1 (AAV-miR-7) followed by inoculation with recombinant α-synuclein preformed fibrils (PFF) a month later. Compared with control mice injected with non-targeting AAV-miR-NT followed by PFF, AAV-miR-7 pre-injected mice exhibited lower levels of monomeric and high-molecular-weight α-synuclein species in the striatum, and reduced amount of phosphorylated α-synuclein in the striatum and in nigral dopamine neurons. Accordingly, AAV-miR-7-injected mice had less pronounced degeneration of the nigrostriatal pathway and better behavioral performance. The neuroinflammatory reaction to α-synuclein PFF inoculation was also significantly attenuated. These data suggest that miR-7 inhibits the formation and propagation of pathological α-synuclein and protects against neurodegeneration induced by PFF. Collectively, these findings support the potential of miR-7 as a disease modifying biologic agent for Parkinson's disease and related α-synucleinopathies.
Asunto(s)
MicroARNs , Enfermedades Neurodegenerativas/genética , Sinucleinopatías , Animales , Encéfalo/metabolismo , Neuronas Dopaminérgicas/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
α-Synuclein (α-Syn) is a key pathogenic protein in α-synucleinopathies including Parkinson disease (PD) and Dementia with Lewy Bodies. The aggregation of α-Syn is believed to be deleterious and a critical step leading to neuronal dysfunction and death. One of the factors that may contribute to the initial steps of this aggregation is crosslinking through transglutaminase 2 (TG2). We previously demonstrated that overexpression of TG2 exacerbates α-Syn toxicity in mice and yeast by increasing the higher-order species of α-Syn. Herein, we investigated whether deletion of the TG2 encoding gene could mitigate the toxicity of α-Syn in vivo. Compared with α-Syn transgenic (SynTg) mice, TG2 null /α-Syn transgenic mice (TG2KO/SynTg) exhibited a reduced amount of phosphorylated α-Syn aggregates and fewer proteinase K-resistant α-Syn aggregates in sections of brain tissue. Neuritic processes that are depleted in SynTg mice compared to wild-type mice were preserved in double TG2KO/SynTg mice. Additionally, the neuroinflammatory reaction to α-Syn was attenuated in TG2KO/SynTg animals. These neuropathological markers of diminished α-Syn toxicity in the absence of TG2 were associated with better motor performance on the rotarod and balance beam. These results suggest that deleting TG2 reduces the toxicity of α-Syn in vivo and improves the behavioral performance of SynTg mice. Accordingly, these findings collectively support pharmacological inhibition of TG2 as a potential disease modifying therapeutic strategy for α-synucleinopathies.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Proteínas de Unión al GTP/genética , Ratones , Ratones Transgénicos , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Mutations in DJ-1 (PARK7) cause recessively inherited Parkinson's disease. DJ-1 is a multifunctional protein with antioxidant and transcription modulatory activity. Its localization in cytoplasm, mitochondria, and nucleus is recognized, but the relevance of this subcellular compartmentalization to its cytoprotective activity is not fully understood. Here we report that under basal conditions DJ-1 is present mostly in the cytoplasm and to a lesser extent in mitochondria and nucleus of dopaminergic neuroblastoma SK-N-BE(2)C cells. Upon oxidant challenge, more DJ-1 translocates to mitochondria within 3 hr and subsequently to the nucleus by 12 hr. The predominant DJ-1 species in both mitochondria and nucleus is a dimer believed to be the functional form. Mutating cysteine 106, 53, or 46 had no impact on the translocation of DJ-1 to mitochondria. To study the relative neuroprotective activity of DJ-1 in mitochondria and nucleus, DJ-1 cDNA constructs fused to the appropriate localization signal were transfected into cells. Compared with 30% protection against oxidant-induced cell death in wild-type DJ-1-transfected cells, mitochondrial targeting of DJ-1 provided a significantly stronger (55%) cytoprotection based on lactate dehydrogenase release. Nuclear targeting of DJ-1 preserved cells equally as well as the wild-type protein. These observations suggest that the time frame for the translocation of DJ-1 from the cytoplasm to mitochondria and to the nucleus following oxidative stress is quite different and that dimerized DJ-1 in mitochondria is functional as an antioxidant not related to cysteine modification. These findings further highlight the multifaceted functions of DJ-1 as a cytoprotector in different cellular compartments.
Asunto(s)
Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitocondrias/metabolismo , Fármacos Neuroprotectores/metabolismo , Proteínas Oncogénicas/metabolismo , Análisis de Varianza , Apoptosis/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cisteína/genética , Humanos , Peróxido de Hidrógeno/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mutación , Neuroblastoma/patología , Proteínas Oncogénicas/genética , Oxidantes/farmacología , Proteína Desglicasa DJ-1 , Transfección/métodosRESUMEN
Parkinson's disease (PD) is a progressive neurodegenerative disorder that is diagnosed largely on clinical grounds due to characteristic motor manifestations that result from the loss of nigrostriatal dopaminergic neurons. While traditional pharmacological approaches to enhance dopamine levels, such as with L-dopa, can be very effective initially, the chronic use of this dopamine precursor is commonly plagued with motor response complications. Additionally, with advancing disease, non-motor manifestations emerge, including psychosis and dementia that compound patient disability. The pathology includes hallmark intraneuronal inclusions known as Lewy bodies and Lewy neurites that contain fibrillar α-synuclein aggregates. Evidence has also accumulated that these aggregates can propagate across synaptically connected brain regions, a phenomenon that can explain the progressive nature of the disease and the emergence of additional symptoms over time. The level of α-synuclein is believed to play a critical role in its fibrillization and aggregation. Accordingly, nucleic acid-based therapeutics for PD include strategies to deliver dopamine biosynthetic enzymes to boost dopamine production or modulate the basal ganglia circuitry in order to improve motor symptoms. Delivery of trophic factors that might enhance the survival of dopamine neurons is another strategy that has been attempted. These gene therapy approaches utilize viral vectors and are delivered stereotaxically in the brain. Alternative disease-modifying strategies focus on downregulating the expression of the α-synuclein gene using various techniques, including modified antisense oligonucleotides, short hairpin RNA, short interfering RNA, and microRNA. The latter approaches also have implications for dementia with Lewy bodies. Other PD genes can also be targeted using nucleic acids. In this review, we detail these various strategies that are still experimental, and discuss the challenges and opportunities of nucleic acid-based therapeutics for PD.
Asunto(s)
Terapia Genética/métodos , Oligonucleótidos Antisentido/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , alfa-Sinucleína/genética , Animales , Humanos , MicroARNs , Enfermedad de Parkinson/genética , ARN Interferente PequeñoRESUMEN
Alpha-Synuclein (α-Syn) is an important protein in the pathogenesis of Parkinson disease (PD) as it accumulates as fibrillar inclusions in affected brain regions including dopaminergic neurons in the substantia nigra. Elevated levels of α-Syn seem to be crucial in mediating its toxicity. Thus, detailed information regarding the regulatory mechanism of α-Syn expression in several layers such as transcription, post-transcription and post-translation is needed in order to devise therapeutic interventions for PD. Previously, we reported that expression of α-Syn is repressed by microRNA-7 (miR-7) through its effect on the 3'-untranslated region (UTR) of α-Syn mRNA. Here, we show that miR-7 also accelerates the clearance of α-Syn and its aggregates by promoting autophagy in differentiated ReNcell VM cells. Further, miR-7 facilitates the degradation of pre-formed fibrils of α-Syn transported from outside the cells. This additional mechanism for reducing α-Syn levels show miR-7 to be an important molecular target for PD and other alpha-synucleinopathies.
Asunto(s)
Autofagia , MicroARNs/metabolismo , Agregación Patológica de Proteínas , Proteolisis , alfa-Sinucleína/metabolismo , Células Cultivadas , Neuronas Dopaminérgicas/metabolismo , Células HEK293 , Humanos , Células-Madre Neurales/metabolismoRESUMEN
DJ-1 is a highly conserved protein that protects neurons against oxidative stress and whose loss of function mutations are linked to recessively inherited Parkinson's disease (PD). While a number of signaling pathways have been shown to be regulated by DJ-1, its role in controlling cell survival through non-coding RNAs remains poorly understood. Here, using a microarray screen, we found that knocking down DJ-1 in human neuroblastoma cells results in down-regulation of microRNA-221 (miR-221). This is one of the most abundant miRNAs in the human brain and promotes neurite outgrowth and neuronal differentiation. Yet the molecular mechanism linking miR-221 to genetic forms of PD has not been studied. Consistent with the microarray data, miR-221 expression is also decreased in DJ-1-/- mouse brains. Re-introduction of wild-type DJ-1, but not its PD-linked pathogenic M26I mutant, restores miR-221 expression. Notably, over-expression of miR-221 is protective against 1-methyl-4-phenylpyridinium (MPP+)-induced cell death, while inhibition of endogenous miR-221 sensitizes cells to this toxin. Additionally, miR-221 down-regulates the expression of several pro-apoptotic proteins at basal conditions and prevents oxidative stress-induced up-regulation of bcl-2-like protein 11 (BIM). Accordingly, miR-221 protects differentiated DJ-1 knock-down ReNcell VM human dopaminergic neuronal cells from MPP+-induced neurite retraction and cell death. DJ-1 is a known activator of the mitogen-activated protein kinase (MAPK)/extracellular-regulated kinase (ERK) pathway and may modulate miR-221 levels in part through this pathway. We found that inhibiting ERK1/2 decreases miR-221 levels, whereas over-expressing ERK1 in DJ-1 knock-down cells increases miR-221 levels. These findings point to a new cytoprotective mechanism by which DJ-1 may increase miR-221 expression through the MAPK/ERK pathway, subsequently leading to repression of apoptotic molecules. The inability of a pathogenic DJ-1 mutant to modulate miR-221 further supports the relevance of this mechanism in neuronal health and its failure in DJ-1-linked PD.
Asunto(s)
MicroARNs/genética , Estrés Oxidativo , Enfermedad de Parkinson/genética , Proteína Desglicasa DJ-1/genética , Animales , Muerte Celular , Supervivencia Celular , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas , Masculino , Ratones Noqueados , MicroARNs/metabolismo , Mutación , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Proteína Desglicasa DJ-1/metabolismoRESUMEN
Nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor that regulates the expression of a number of antioxidant and detoxifying genes that provide cellular protection against various stressors including reactive oxygen species (ROS). Nrf2 activity is tightly regulated by a cytoplasmic inhibitory protein called Kelch-like ECH-associated protein 1 (Keap1). The mechanism that controls Keap1 expression, however, remains poorly understood. In the present study, we demonstrate that microRNA-7 (miR-7), which is highly expressed in the brain, represses Keap1 expression by targeting the 3'-untranslated region (UTR) of its mRNA in human neuroblastoma cells, SH-SY5Y. Subsequently, this event results in an increased Nrf2 activity, as evidenced by an increase in the expression of its transcriptional targets, heme oxygenase 1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM), and an enhanced nuclear localization of Nrf2. In addition, miR-7 decreases the intracellular hydroperoxides level and increases the level of reduced form of glutathione, indicative of oxidative stress relief. We also demonstrate that targeted repression of Keap1 and activation of Nrf2 pathway, in part, underlies the protective effects of miR-7 against 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in SH-SY5Y and differentiated human neural progenitor cells, ReNcell VM. These findings point to a new mechanism by which miR-7 exerts cytoprotective effects by regulating the Nrf2 pathway.