RESUMEN
α-Synuclein (α-syn) phosphorylation at serine 129 (pS129α-syn) is substantially increased in Lewy body disease, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). However, the pathogenic relevance of pS129α-syn remains controversial, so we sought to identify when pS129 modification occurs during α-syn aggregation and its role in initiation, progression and cellular toxicity of disease. Using diverse aggregation assays, including real-time quaking-induced conversion (RT-QuIC) on brain homogenates from PD and DLB cases, we demonstrated that pS129α-syn inhibits α-syn fibril formation and seeded aggregation. We also identified lower seeding propensity of pS129α-syn in cultured cells and correspondingly attenuated cellular toxicity. To build upon these findings, we developed a monoclonal antibody (4B1) specifically recognizing nonphosphorylated S129α-syn (WTα-syn) and noted that S129 residue is more efficiently phosphorylated when the protein is aggregated. Using this antibody, we characterized the time-course of α-syn phosphorylation in organotypic mouse hippocampal cultures and mice injected with α-syn preformed fibrils, and we observed aggregation of nonphosphorylated α-syn followed by later pS129α-syn. Furthermore, in postmortem brain tissue from PD and DLB patients, we observed an inverse relationship between relative abundance of nonphosphorylated α-syn and disease duration. These findings suggest that pS129α-syn occurs subsequent to initial protein aggregation and apparently inhibits further aggregation. This could possibly imply a potential protective role for pS129α-syn, which has major implications for understanding the pathobiology of Lewy body disease and the continued use of reduced pS129α-syn as a measure of efficacy in clinical trials.
Asunto(s)
Amiloide , Enfermedad por Cuerpos de Lewy , Enfermedad de Parkinson , Agregación Patológica de Proteínas , alfa-Sinucleína , Amiloide/metabolismo , Humanos , Enfermedad por Cuerpos de Lewy/genética , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fosforilación , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Serina/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Parkin, the gene responsible for hereditary Parkinson's disease (PD) called "Autosomal Recessive Juvenile Parkinsonism (AR-JP)" was discovered a quarter of a century ago. Owing to its huge gene structure and unique protein functions, parkin has become a subject of interest to those involved in PD research and researchers and clinicians in various fields and is being vigorously studied worldwide in relation to its nature and disease. The gene structure was registered under the gene name "parkin" in the GenBank in 1997. In 1998, deletion and point mutations in the parkin gene were reported, thereby demonstrating parkin is the causative gene for hereditary PD. Although 25 years have passed since the gene's discovery and many researchers have worked tirelessly to elucidate the function of the Parkin protein and the mechanism of its role against neuronal cell death and pathogenesis remain unknown, which raises a major question concerning the current leading hypothesis. In this review, we present the results of related research on the parkin gene in chronological order and discuss unresolved problems concerning its function and pathology as well as new trends in the research conducted to solve them. The relationship between parkin and tumorigenesis has also been addressed from the perspective of Parkin's redox molecule.
Asunto(s)
Enfermedad de Parkinson , Trastornos Parkinsonianos , Humanos , Enfermedad de Parkinson/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas/genética , Trastornos Parkinsonianos/genéticaRESUMEN
Mutations in ATP13A2 (PARK9), an autophagy-related protein, cause Kufor-Rakeb syndrome, an autosomal recessive, juvenile-onset form of parkinsonism. α-Synuclein (α-syn) is a presynaptic neuronal protein that forms toxic aggregates in Parkinson's disease (PD). We studied α-syn aggregation and autophagic flux in ATP13A2-knockdown Drosophila expressing either wild-type (WT) or mutant α-syn. Dopaminergic (DA) neuron loss was studied by confocal microscopy. Sleep and circadian activity were evaluated in young and old flies using a Drosophila activity monitor. Thirty-day-old ATP13A2-RNAi A53T-α-syn flies had increased Triton-insoluble α-syn levels, compared to control A53T-α-syn flies without ATP13A2-RNAi. Whole-brain staining revealed significantly fewer dopaminergic (DA) neurons in the PPL2 cluster of 30-day-old ATP13A2-RNAi flies expressing WT-, A30P-, and A53T-α-syn than in that of controls. In ATP13A2-RNAi A53T-α-syn flies, autophagic flux was decreased, as indicated by increased accumulation of Ref(2)P, the Drosophila p62 homologue. ATP13A2 silencing decreased total locomotor activity in young, and enhanced sleep features, similar to PD (decreasing bout length), in old flies expressing A53T-α-syn. ATP13A2 silencing also altered the circadian locomotor activity of A30P- and A53T-α-syn flies. Thus, ATP13A2 may play a role in the autophagic degradation of A53T-α-syn.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Animales , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Drosophila/genética , Drosophila/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Neuronas Dopaminérgicas/metabolismo , Mutación , Silenciador del GenRESUMEN
The loss of the E3 ligase Parkin, in a familial form of Parkinson's disease, is thought to cause the failure of both the polyubiquitination of abnormal mitochondria and the consequent induction of mitophagy, resulting in abnormal mitochondrial accumulation. However, this has not been confirmed in patient autopsy cases or animal models. More recently, the function of Parkin as a redox molecule that directly scavenges hydrogen peroxide has attracted much attention. To determine the role of Parkin as a redox molecule in the mitochondria, we overexpressed various combinations of Parkin, along with its substrates FAF1, PINK1, and ubiquitin in cell culture systems. Here, we observed that the E3 Parkin monomer was surprisingly not recruited to abnormal mitochondria but self-aggregated with or without self-ubiquitination into the inner and outer membranes, becoming insoluble. Parkin overexpression alone generated aggregates without self-ubiquitination, but it activated autophagy. These results suggest that for damaged mitochondria, the polyubiquitination of Parkin substrates on the mitochondria is not indispensable for mitophagy.
Asunto(s)
Proteínas Quinasas , Ubiquitina-Proteína Ligasas , Animales , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Mitocondrias/genética , Mitocondrias/metabolismo , MitofagiaRESUMEN
Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopamine neurons and the deposition of misfolded proteins known as Lewy bodies (LBs), which contain α-synuclein (α-syn). The causes and molecular mechanisms of PD are not clearly understood to date. However, misfolded proteins, oxidative stress, and impaired autophagy are believed to play important roles in the pathogenesis of PD. Importantly, α-syn is considered a key player in the development of PD. The present study aimed to assess the role of Ellagic acid (EA), a polyphenol found in many fruits, on α-syn aggregation and toxicity. Using thioflavin and seeding polymerization assays, in addition to electron microscopy, we found that EA could dramatically reduce α-syn aggregation. Moreover, EA significantly mitigated the aggregated α-syn-induced toxicity in SH-SY5Y cells and thus enhanced their viability. Mechanistically, these cytoprotective effects of EA are mediated by the suppression of apoptotic proteins BAX and p53 and a concomitant increase in the anti-apoptotic protein, BCL-2. Interestingly, EA was able to activate autophagy in SH-SY5Y cells, as evidenced by normalized/enhanced expression of LC3-II, p62, and pAKT. Together, our findings suggest that EA may attenuate α-syn toxicity by preventing aggregation and improving viability by restoring autophagy and suppressing apoptosis.
Asunto(s)
Ácido Elágico/farmacología , Agregación Patológica de Proteínas/prevención & control , alfa-Sinucleína/metabolismo , Apoptosis/fisiología , Autofagia/fisiología , Línea Celular Tumoral , Neuronas Dopaminérgicas/metabolismo , Ácido Elágico/metabolismo , Humanos , Cuerpos de Lewy/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Enfermedad de Parkinson/metabolismo , Agregado de Proteínas/fisiología , Agregación Patológica de Proteínas/metabolismo , alfa-Sinucleína/fisiologíaRESUMEN
Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by tremor, rigidity, bradykinesia, and postural instability, for which there is no effective treatment available till date. Here, we report the development of nonviral vectors specific for neuronal cells that can deliver short interfering RNA (siRNA) against the α-synuclein gene (SNCA), and prevent PD-like symptoms both in vitro and in vivo. These vectors not only help siRNA duplexes cross the blood-brain barrier in mice, but also stabilize these siRNAs leading to a sustainable 60-90% knockdown of α-synuclein protein. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine rapidly develop PD-like symptoms which were significantly alleviated when SNCA was knocked down using our vectors. Together, our data not only confirm the central role of α-synuclein in the onset of PD, but also provide a proof of principle that these nonviral vectors can be used as novel tools to design effective strategies to combat central nervous system diseases.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Enfermedad de Parkinson/terapia , ARN Interferente Pequeño/administración & dosificación , alfa-Sinucleína/antagonistas & inhibidores , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/efectos adversos , Animales , Línea Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Vectores Genéticos/administración & dosificación , Humanos , Ratones , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/genética , Biblioteca de Péptidos , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND: Parkinson's disease (PD) diagnosis is mainly based on clinical criteria, with a high risk of misdiagnosis. The identification of reliable biomarkers for disease diagnosis and progression has a key role for developing disease-modifying therapies. In this article, we investigated the longitudinal changes of CSF α-synuclein species in early PD patients and explored the potential use of these species as surrogate biomarkers for PD progression. METHODS: We used our newly developed enzyme-linked immunosorbent assay systems for measuring different forms of α-synuclein, such as oligomeric-α-synuclein, phosphorylated-α-synuclein at serine 129, or total-α-synuclein in CSF from the longitudinal Deprenyl and Tocopherol Antioxidative Therapy for Parkinsonism study cohort (n = 121). CSF Alzheimer's disease biomarkers (total-tau, phosphorylated-tau, Aß40 , and Aß42 ) were also measured for this cohort. RESULTS: Interestingly, total-α-synuclein and oligomeric-α-synuclein levels significantly increased during the 2-year Deprenyl and Tocopherol Antioxidative Therapy for Parkinsonism study follow-up period, whereas phosphorylated-α-synuclein at serine 129 levels showed a longitudinal decrease. We have also noted an association between a change of the oligomeric-α-synuclein/total-α-synuclein ratio and a worsening of motor signs, in particular in the postural-instability and gait-difficulty dominant PD group. A strong positive correlation between the changes in CSF total-α-synuclein and oligomeric-α-synuclein during the 2-year Deprenyl and Tocopherol Antioxidative Therapy for Parkinsonism study was also noted (r = 0.84, P < .001). CONCLUSION: Our data show that CSF α-synuclein species have a dynamic pattern along the course of the disease, supporting their possible role as progression biomarkers for PD and their link with PD clinical phenotypes. © 2016 International Parkinson and Movement Disorder Society.
Asunto(s)
Progresión de la Enfermedad , Enfermedad de Parkinson/líquido cefalorraquídeo , alfa-Sinucleína/líquido cefalorraquídeo , Anciano , Antioxidantes/uso terapéutico , Biomarcadores/líquido cefalorraquídeo , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/tratamiento farmacológicoRESUMEN
Compelling evidence indicates that α-synuclein (α-syn) aggregation plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. Identification of compounds that inhibit or reverse the aggregation process may thus represent a viable therapeutic strategy against PD and related disorders. Ginseng is a well-known medicinal plant that has been used in East Asia for more than two thousand years to treat several conditions. It is now understood that the pharmacological properties of ginseng can be attributed to its biologically active components, the ginsenosides, which in turn have been shown to have neuroprotective properties. We therefore sought to determine for the first time, the potential of the most frequently used and studied ginsenosides, namely Rg1, Rg3 and Rb1, as anti-amyloidogenic agents. The effect of Rg1, Rg3 and Rb1 on α-syn aggregation and toxicity was determined by an array of biophysical, biochemical and cell-culture-based techniques. Among the screened ginsenosides, only Rb1 was shown to be a potent inhibitor of α-syn fibrillation and toxicity. Additionally, Rb1 exhibited a strong ability to disaggregate preformed fibrils and to inhibit the seeded polymerization of α-syn. Interestingly, Rb1 was found to stabilize soluble non-toxic oligomers with no ß-sheet content, that were susceptible to proteinase K digestion, and the binding of Rb1 to those oligomers may represent a potential mechanism of action. Thus, Rb1 could represent the starting point for designing new molecules that could be utilized as drugs for the treatment of PD and related disorders.
Asunto(s)
Amiloide/efectos de los fármacos , Ginsenósidos/farmacología , Fármacos Neuroprotectores/farmacología , alfa-Sinucleína/efectos de los fármacos , alfa-Sinucleína/toxicidad , Amiloide/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Endopeptidasa K/metabolismo , Escherichia coli , Humanos , Estructura Molecular , Polimerizacion/efectos de los fármacos , Estructura Secundaria de Proteína , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , alfa-Sinucleína/metabolismoRESUMEN
α-Synuclein (α-syn), a small protein that has the intrinsic propensity to aggregate, is implicated in several neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), which are collectively known as synucleinopathies. Genetic, pathological, biochemical, and animal modeling studies provided compelling evidence that α-syn aggregation plays a key role in the pathogenesis of PD and related synucleinopathies. It is therefore of utmost importance to develop reliable tools that can detect the aggregated forms of α-syn. We describe here the generation and characterization of six novel conformation-specific monoclonal antibodies that recognize specifically α-syn aggregates but not the soluble, monomeric form of the protein. The antibodies described herein did not recognize monomers or fibrils generated from other amyloidogenic proteins including ß-syn, γ-syn, ß-amyloid, tau protein, islet amyloid polypeptide and ABri. Interestingly, the antibodies did not react to overlapping linear peptides spanning the entire sequence of α-syn, confirming further that they only detect α-syn aggregates. In immunohistochemical studies, the new conformation-specific monoclonal antibodies showed underappreciated small micro-aggregates and very thin neurites in PD and DLB cases that were not observed with generic pan antibodies that recognize linear epitope. Furthermore, employing one of our conformation-specific antibodies in a sandwich based ELISA, we observed an increase in levels of α-syn oligomers in brain lysates from DLB compared to Alzheimer's disease and control samples. Therefore, the conformation-specific antibodies portrayed herein represent useful tools for research, biomarkers development, diagnosis and even immunotherapy for PD and related pathologies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Encéfalo/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/inmunología , Proteínas Adaptadoras Transductoras de Señales , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Encéfalo/patología , Escherichia coli , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Glicoproteínas de Membrana/metabolismo , Ratones , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/metabolismo , Sinucleína beta/inmunología , Sinucleína beta/metabolismo , gamma-Sinucleína/inmunología , gamma-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
In the present study, we investigated the role of the main intracellular energy sensor, AMP-activated protein kinase (AMPK), in the in vitro neurotoxicity of α-synuclein (ASYN), one of the key culprits in the pathogenesis of Parkinson's disease. The loss of viability in retinoic acid-differentiated SH-SY5Y human neuroblastoma cells inducibly overexpressing wild-type ASYN was associated with the reduced activation of AMPK and its activator LKB1, as well as AMPK target Raptor. ASYN-overexpressing rat primary neurons also displayed lower activity of LKB1/AMPK/Raptor pathway. Restoration of AMPK activity by metformin or AICAR reduced the in vitro neurotoxicity of ASYN overexpression, acting independently of the prosurvival kinase Akt or the induction of autophagic response. The conditioned medium from ASYN-overexpressing cells, containing secreted ASYN, as well as dopamine-modified or nitrated recombinant ASYN oligomers, all inhibited AMPK activation in differentiated SH-SY5Y cells and reduced their viability, but not in the presence of metformin or AICAR. The RNA interference-mediated knockdown of AMPK increased the sensitivity of SH-SY5Y cells to the harmful effects of secreted ASYN. AMPK-dependent protection from extracellular ASYN was also observed in rat neuron-like pheochromocytoma cell line PC12. These data demonstrate the protective role of AMPK against the toxicity of both intracellular and extracellular ASYN, suggesting that modulation of AMPK activity may be a promising therapeutic strategy in Parkinson's disease.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Neuronas/efectos de los fármacos , alfa-Sinucleína/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Medios de Cultivo Condicionados/farmacología , Fragmentación del ADN , Embrión de Mamíferos , Humanos , Hipoglucemiantes/farmacología , Metformina/farmacología , Neuroblastoma/patología , Neuroblastoma/ultraestructura , Neuronas/ultraestructura , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/farmacología , Ratas , Ribonucleótidos/farmacología , Tretinoina/farmacología , alfa-Sinucleína/genéticaRESUMEN
Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.
Asunto(s)
Encéfalo/metabolismo , Eritrocitos/metabolismo , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Sistema Nervioso Central/metabolismo , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Estructura Secundaria de Proteína , Desplegamiento Proteico , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMEN
Parkinson's disease (PD) is the second most common neurological disorder, pathologically characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) as well as the formation of Lewy bodies composed mainly of α-synuclein (α-syn) aggregates. It has been documented that abnormal aggregation of α-syn is one of the major causes of developing PD. In the current study, administration of ellagic acid (EA), a polyphenolic compound (10 mg/kg bodyweight), significantly decreased α-syn spreading and preserved dopaminergic neurons in a male C57BL/6 mouse model of PD. Moreover, EA altered the autophagic flux, suggesting the involvement of a restorative mechanism meditated by EA treatment. Our data support that EA could play a major role in the clearing of toxic α-syn from spreading, in addition to the canonical antioxidative role, and thus preventing dopaminergic neuronal death.
Asunto(s)
Enfermedad de Parkinson , Masculino , Ratones , Animales , Ratones Endogámicos C57BL , Enfermedad de Parkinson/tratamiento farmacológico , alfa-Sinucleína , Ácido Elágico/farmacología , Modelos AnimalesRESUMEN
α-Synuclein (α-syn) is a presynaptic protein present at most nerve terminals, but its function remains largely unknown. The familial forms of Parkinson's disease associated with multiplications of the α-syn gene locus indicate that overabundance of this protein might have a detrimental effect on dopaminergic transmission. To investigate this hypothesis, we use adeno-associated viral (AAV) vectors to overexpress human α-syn in the rat substantia nigra. Moderate overexpression of either wild-type (WT) or A30P α-syn differs in the motor phenotypes induced, with only the WT form generating hemiparkinsonian impairments. Wild-type α-syn causes a reduction of dopamine release in the striatum that exceeds the loss of dopaminergic neurons, axonal fibers, and the reduction in total dopamine. At the ultrastructural level, the reduced dopamine release corresponds to a decreased density of dopaminergic vesicles and synaptic contacts in striatal terminals. Interestingly, the membrane-binding-deficient A30P mutant does neither notably reduce dopamine release nor it cause ultrastructural changes in dopaminergic axons, showing that α-syn's membrane-binding properties are critically involved in the presynaptic defects. To further determine if the affinity of the protein for membranes determines the extent of motor defects, we compare three forms of α-syn in conditions leading to pronounced degeneration. While membrane-binding α-syns (wild-type and A53T) induce severe motor impairments, an N-terminal deleted form with attenuated affinity for membranes is inefficient in inducing motor defects. Overall, these results demonstrate that α-syn overabundance is detrimental to dopamine neurotransmission at early stages of the degeneration of nigrostriatal dopaminergic axons.
Asunto(s)
Cuerpo Estriado/metabolismo , Dopamina/deficiencia , Proteínas de Filamentos Intermediarios/metabolismo , Actividad Motora/fisiología , Sustancia Negra/metabolismo , Vesículas Sinápticas/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Anfetamina/farmacología , Análisis de Varianza , Animales , Apomorfina/farmacología , Cromatografía Líquida de Alta Presión/métodos , Cuerpo Estriado/efectos de los fármacos , Agonistas de Dopamina/farmacología , Inhibidores de Captación de Dopamina/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/ultraestructura , Electroquímica , Ensayo de Inmunoadsorción Enzimática/métodos , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Femenino , Lateralidad Funcional , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ácido Homovanílico/metabolismo , Humanos , Técnicas In Vitro , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/ultraestructura , Microscopía Electrónica de Transmisión , Actividad Motora/efectos de los fármacos , Mutación/genética , Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , Sustancia Negra/ultraestructura , Vesículas Sinápticas/ultraestructura , Factores de Tiempo , Transducción Genética , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
Abnormal protein aggregation in the brain is linked to the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Recent studies revealed that the oligomeric form of aggregates is most likely the toxic species, and thus could be a good therapeutic target. To screen for potent inhibitors that can inhibit both oligomerisation and fibrillation of α-synuclein (α-syn), we systematically compared the antioligomeric and antifibrillar activities of eight compounds that were extracted from Chinese herbal medicines through three platforms that can monitor the formation of α-syn fibrils and oligomers in cell-free or cellular systems. Our results revealed that baicalein, a flavonoid extracted from the Chinese herbal medicine Scutellaria baicalensis Georgi ("huang qin" in Chinese), is a potent inhibitor of α-syn oligomerisation both in cell-free and cellular systems, and is also an effective inhibitor of α-syn fibrillation in cell-free systems. We further tested the protective effect of baicalein against α-syn-oligomer-induced toxicity in neuronal cells. Our data showed that baicalein inhibited the formation of α-syn oligomers in SH-SY5Y and Hela cells, and protected SH-SY5Y cells from α-syn-oligomer-induced toxicity. We also explored the effect of baicalein on amyloid-ß peptide (Aß) aggregation and toxicity. We found that baicalein can also inhibit Aß fibrillation and oligomerisation, disaggregate pre-formed Aß amyloid fibrils and prevent Aß fibril-induced toxicity in PC12 cells. Our study indicates that baicalein is a good inhibitor of amyloid protein aggregation and toxicity. Given the role of these processes in neurodegenerative diseases such as AD and PD, our results suggest that baicalein has potential as a therapeutic agent for the treatment of these devastating disorders.
Asunto(s)
Medicamentos Herbarios Chinos/química , Inhibidores Enzimáticos , Flavanonas/química , Flavanonas/farmacología , alfa-Sinucleína/antagonistas & inhibidores , Amiloide/biosíntesis , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medicamentos Herbarios Chinos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Estructura Molecular , alfa-Sinucleína/químicaRESUMEN
Although alpha-synuclein is the main constituent of Lewy bodies, cerebrospinal fluid determination on its own does not seem fundamental for the diagnosis of synucleinopathies. We evaluated whether the combination of classical biomarkers, Aß(1-42) , total tau, phosphorylated tau, and α-synuclein can improve discrimination of Parkinson's disease, dementia with Lewy bodies, Alzheimer's disease, and frontotemporal dementia. Aß(1-42) , total tau, phosphorylated tau, and α-synuclein were measured in a series of patients with Parkinson's disease (n = 38), dementia with Lewy bodies (n = 32), Alzheimer's disease (n = 48), frontotemporal dementia (n = 31), and age-matched control patients with other neurological diseases (n = 32). Mean α-synuclein levels in cerebrospinal fluid were significantly lower in the pathological groups than in cognitively healthy subjects. An inverse correlation of α-synuclein with total tau (r = -0.196, P < .01) was observed. In the group of patients with Parkinson's disease, Aß(1-42) , total tau, and phosphorylated tau values were similar to controls, whereas total tau/α-synuclein and phosphorylated tau/α-synuclein ratios showed the lowest values. Cerebrospinal fluid α-synuclein alone did not provide relevant information for Parkinson's disease diagnosis, showing low specificity (area under the curve, 0.662; sensitivity, 94%; specificity, 25%). Instead, a better performance was obtained with the total tau/α-syn ratio (area under the curve, 0.765; sensitivity, 89%; specificity, 61%). Combined determination of α-synuclein and classical biomarkers in cerebrospinal fluid shows differential patterns in neurodegenerative disorders. In particular, total tau/α-synuclein and phosphorylated tau/α-synuclein ratios can contribute to the discrimination of Parkinson's disease. © 2011 Movement Disorder Society.
Asunto(s)
Demencia/líquido cefalorraquídeo , Enfermedades Neurodegenerativas/líquido cefalorraquídeo , Enfermedad de Parkinson/líquido cefalorraquídeo , alfa-Sinucleína/líquido cefalorraquídeo , Proteínas tau/líquido cefalorraquídeo , Anciano , Péptidos beta-Amiloides/líquido cefalorraquídeo , Análisis de Varianza , Demencia/complicaciones , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Neurodegenerativas/complicaciones , Fragmentos de Péptidos/líquido cefalorraquídeo , Fosforilación , Estudios Prospectivos , Curva ROCRESUMEN
BACKGROUND: Parkinson's disease (PD), the second most common progressive neurodegenerative disorder, is characterized by the abnormal accumulation of intraneuronal inclusions enriched in aggregated α-synuclein (α-syn), known as Lewy bodies (LBs) and Lewy neurites (LNs), and significant loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) of the brain. Recent evidence suggests that the intrastriatal inoculation of α-syn preformed fibrils (PFF) in mice brain triggers endogenous α-syn in interconnected brain regions. 1-methyl, 4-phenyl, 1,2,3,6 tetrahydropyridine (MPTP), a mitochondrial neurotoxin, has been used previously to generate a PD mouse model. However, the common methods of MPTP exposure do not induce LB or α-syn aggregation in mice. In the present study, we evaluated the effect of different doses of MPTP (10 mg/kg.b.wt and/or 25 mg/kg.b.wt) on the spread, accumulation, and toxicity of endogenous α-syn in mice administered an intrastriatal injection of human α-syn PFF. METHODS: We inoculated human WT α-syn PFF in mouse striatum. At 6 weeks post PFF injection, we challenged the animal with two different doses of MPTP (10 mg/kg.b.wt and 25 mg/kg.b.wt) once daily for five consecutive days. At 2 weeks from the start of the MPTP regimen, we collected the mice brain and performed immunohistochemical analysis, and Rotarod test to assess motor coordination and muscle strength before and after MPTP injection. RESULTS: A single injection of human WT α-syn PFF in the mice striatum induced the propagation of α-syn, occurring as phosphorylated α-synuclein (pS129), towards the SNpc, within a very short time. Injection of a low dose of MPTP (10 mg/kg.b.wt) at 6 weeks post α-syn PFF inoculation further enhanced the spread, whereas a high dose of MPTP (25 mg/kg.b.wt.) reduced the spread. Majority of the accumulated α-syn were proteinase K resistant, as recognized using a conformation-specific α-syn antibody. Injection of α-syn PFF alone caused 12 % reduction in the number of tyrosine hydroxylase positive neurons while α-syn PFF + a low dose of MPTP caused 33 % reduction (loss), compared to the control mice injected with saline. This combination also reduced the motor coordination. Interestingly, a low dose of MPTP alone did not cause any significant reduction in the number of tyrosine hydroxylase positive neurons compared to saline treatment. Animals that received α-syn PFF and a high dose of MPTP showed massive activation of glial cells and decreased spread of α-syn, majority of which were detected in the nucleus. CONCLUSION: Our results suggest that a combination of human WT α-syn PFF and a low dose of MPTP increases the pathological conversion and propagation of endogenous α-syn, and neurodegeneration, within a very short time. Our model can be used to study the mechanisms of α-syn propagation and screen for potential drugs against PD.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , Neurotoxinas/administración & dosificación , Neurotoxinas/toxicidad , Trastornos Parkinsonianos/metabolismo , alfa-Sinucleína/biosíntesis , alfa-Sinucleína/toxicidad , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Relación Dosis-Respuesta a Droga , Inyecciones Intraventriculares , Masculino , Ratones , Ratones Endogámicos C57BL , Trastornos Parkinsonianos/inducido químicamente , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , alfa-Sinucleína/análisisRESUMEN
Parkinson's disease (PD) is one of the most common neurodegenerative diseases and is characterized by progressive dopaminergic neurodegeneration in the substantia nigra pars compacta area. In the present study, treatment of EA for 1 week at a dose of 10 mg/kg body weight prior to MPTP (25 mg/kg body weight) was carried out. MPTP administration caused oxidative stress, as evidenced by decreased activities of superoxide dismutase and catalase, and the depletion of reduced glutathione with a concomitant rise in the lipid peroxidation product, malondialdehyde. It also significantly increased the pro-inflammatory cytokines and elevated the inflammatory mediators like cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the striatum. Immunohistochemical analysis revealed a loss of dopamine neurons in the SNc area and a decrease in dopamine transporter in the striatum following MPTP administration. However, treatment with EA prior to MPTP injection significantly rescued the dopaminergic neurons and dopamine transporter. EA treatment further restored antioxidant enzymes, prevented the depletion of glutathione and inhibited lipid peroxidation, in addition to the attenuation of pro-inflammatory cytokines. EA also reduced the levels of COX-2 and iNOS. The findings of the present study demonstrate that EA protects against MPTP-induced PD and the observed neuroprotective effects can be attributed to its potent antioxidant and anti-inflammatory properties.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Ácido Elágico/farmacología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/patología , Animales , Cuerpo Estriado/patología , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Glutatión/metabolismo , Mediadores de Inflamación/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico Sintasa de Tipo II/metabolismo , Enfermedad de Parkinson/metabolismoRESUMEN
BACKGROUND: Recent studies indicated that seeded fibril formation and toxicity of α-synuclein (α-syn) play a main role in the pathogenesis of certain diseases including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. Therefore, examination of compounds that abolish the process of seeding is considered a key step towards therapy of several synucleinopathies. METHODS: Using biophysical, biochemical and cell-culture-based assays, assessment of eleven compounds, extracted from Chinese medicinal herbs, was performed in this study for their effect on α-syn fibril formation and toxicity caused by the seeding process. RESULTS: Salvianolic acid B and dihydromyricetin were the two compounds that strongly inhibited the fibril growth and neurotoxicity of α-syn. In an in-vitro cell model, these compounds decreased the insoluble phosphorylated α-syn and aggregation. Also, in primary neuronal cells, these compounds showed a reduction in α-syn aggregates. Both compounds inhibited the seeded fibril growth with dihydromyricetin having the ability to disaggregate preformed α-syn fibrils. In order to investigate the inhibitory mechanisms of these two compounds towards fibril formation, we demonstrated that salvianolic acid B binds predominantly to monomers, while dihydromyricetin binds to oligomeric species and to a lower extent to monomers. Remarkably, these two compounds stabilized the soluble non-toxic oligomers lacking ß-sheet content after subjecting them to proteinase K digestion. CONCLUSIONS: Eleven compounds were tested but only two showed inhibition of α-syn aggregation, seeded fibril formation and toxicity in vitro. These findings highlight an essential beginning for development of new molecules in the field of synucleinopathies treatment.