RESUMEN
Metabolites in the kynurenine pathway, generated by tryptophan degradation, are thought to play an important role in neurodegenerative disorders, including Alzheimer's and Huntington's diseases. In these disorders, glutamate receptor-mediated excitotoxicity and free radical formation have been correlated with decreased levels of the neuroprotective metabolite kynurenic acid. Here, we describe the synthesis and characterization of JM6, a small-molecule prodrug inhibitor of kynurenine 3-monooxygenase (KMO). Chronic oral administration of JM6 inhibits KMO in the blood, increasing kynurenic acid levels and reducing extracellular glutamate in the brain. In a transgenic mouse model of Alzheimer's disease, JM6 prevents spatial memory deficits, anxiety-related behavior, and synaptic loss. JM6 also extends life span, prevents synaptic loss, and decreases microglial activation in a mouse model of Huntington's disease. These findings support a critical link between tryptophan metabolism in the blood and neurodegeneration, and they provide a foundation for treatment of neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Huntington/tratamiento farmacológico , Ácido Quinurénico/análisis , Quinurenina 3-Monooxigenasa/antagonistas & inhibidores , Sulfonamidas/uso terapéutico , Tiazoles/uso terapéutico , Administración Oral , Enfermedad de Alzheimer/fisiopatología , Animales , Química Encefálica , Modelos Animales de Enfermedad , Femenino , Humanos , Ácido Quinurénico/sangre , Masculino , Ratones , Ratones Transgénicos , Sulfonamidas/administración & dosificación , Tiazoles/administración & dosificaciónRESUMEN
α-Synuclein (α-syn) has been implicated in neurological disorders with parkinsonism, including Parkinson's disease and Dementia with Lewy body. Recent studies have shown α-syn oligomers released from neurons can propagate from cell-to-cell in a prion-like fashion exacerbating neurodegeneration. In this study, we examined the role of the endosomal sorting complex required for transport (ESCRT) pathway on the propagation of α-syn. α-syn, which is transported via the ESCRT pathway through multivesicular bodies for degradation, can also target the degradation of the ESCRT protein-charged multivesicular body protein (CHMP2B), thus generating a roadblock of endocytosed α-syn. Disruption of the ESCRT transport system also resulted in increased exocytosis of α-syn thus potentially increasing cell-to-cell propagation of synuclein. Conversely, delivery of a lentiviral vector overexpressing CHMP2B rescued the neurodegeneration in α-syn transgenic mice. Better understanding of the mechanisms of intracellular trafficking of α-syn might be important for understanding the pathogenesis and developing new treatments for synucleinopathies.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Enfermedad por Cuerpos de Lewy/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Línea Celular , Modelos Animales de Enfermedad , Humanos , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Enfermedad por Cuerpos de Lewy/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patologíaRESUMEN
Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are common neurodegenerative disorders of the aging population, characterized by progressive and abnormal accumulation of α-synuclein (α-syn). Recent studies have shown that C-terminus (CT) truncation and propagation of α-syn play a role in the pathogenesis of PD/DLB. Therefore, we explored the effect of passive immunization against the CT of α-syn in the mThy1-α-syn transgenic (tg) mouse model, which resembles the striato-nigral and motor deficits of PD. Mice were immunized with the new monoclonal antibodies 1H7, 5C1, or 5D12, all directed against the CT of α-syn. CT α-syn antibodies attenuated synaptic and axonal pathology, reduced the accumulation of CT-truncated α-syn (CT-α-syn) in axons, rescued the loss of tyrosine hydroxylase fibers in striatum, and improved motor and memory deficits. Among them, 1H7 and 5C1 were most effective at decreasing levels of CT-α-syn and higher-molecular-weight aggregates. Furthermore, in vitro studies showed that preincubation of recombinant α-syn with 1H7 and 5C1 prevented CT cleavage of α-syn. In a cell-based system, CT antibodies reduced cell-to-cell propagation of full-length α-syn, but not of the CT-α-syn that lacked the 118-126 aa recognition site needed for antibody binding. Furthermore, the results obtained after lentiviral expression of α-syn suggest that antibodies might be blocking the extracellular truncation of α-syn by calpain-1. Together, these results demonstrate that antibodies against the CT of α-syn reduce levels of CT-truncated fragments of the protein and its propagation, thus ameliorating PD-like pathology and improving behavioral and motor functions in a mouse model of this disease.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Encéfalo/inmunología , Trastornos del Movimiento/inmunología , Trastornos del Movimiento/terapia , Trastornos Parkinsonianos/inmunología , Trastornos Parkinsonianos/terapia , alfa-Sinucleína/inmunología , Animales , Encéfalo/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Inmunoterapia/métodos , Ratones , Ratones Transgénicos , Distribución Tisular , Resultado del TratamientoRESUMEN
In Parkinson's disease and dementia with Lewy bodies, α-synuclein aggregates to form oligomers and fibrils; however, the precise nature of the toxic α-synuclein species remains unclear. A number of synthetic α-synuclein mutations were recently created (E57K and E35K) that produce species of α-synuclein that preferentially form oligomers and increase α-synuclein-mediated toxicity. We have shown that acute lentiviral expression of α-synuclein E57K leads to the degeneration of dopaminergic neurons; however, the effects of chronic expression of oligomer-prone α-synuclein in synapses throughout the brain have not been investigated. Such a study could provide insight into the possible mechanism(s) through which accumulation of α-synuclein oligomers in the synapse leads to neurodegeneration. For this purpose, we compared the patterns of neurodegeneration and synaptic damage between a newly generated mThy-1 α-synuclein E57K transgenic mouse model that is prone to forming oligomers and the mThy-1 α-synuclein wild-type mouse model (Line 61), which accumulates various forms of α-synuclein. Three lines of α-synuclein E57K (Lines 9, 16 and 54) were generated and compared with the wild-type. The α-synuclein E57K Lines 9 and 16 were higher expressings of α-synuclein, similar to α-synuclein wild-type Line 61, and Line 54 was a low expressing of α-synuclein compared to Line 61. By immunoblot analysis, the higher-expressing α-synuclein E57K transgenic mice showed abundant oligomeric, but not fibrillar, α-synuclein whereas lower-expressing mice accumulated monomeric α-synuclein. Monomers, oligomers, and fibrils were present in α-synuclein wild-type Line 61. Immunohistochemical and ultrastructural analyses demonstrated that α-synuclein accumulated in the synapses but not in the neuronal cells bodies, which was different from the α-synuclein wild-type Line 61, which accumulates α-synuclein in the soma. Compared to non-transgenic and lower-expressing mice, the higher-expressing α-synuclein E57K mice displayed synaptic and dendritic loss, reduced levels of synapsin 1 and synaptic vesicles, and behavioural deficits. Similar alterations, but to a lesser extent, were seen in the α-synuclein wild-type mice. Moreover, although the oligomer-prone α-synuclein mice displayed neurodegeneration in the frontal cortex and hippocampus, the α-synuclein wild-type only displayed neuronal loss in the hippocampus. These results support the hypothesis that accumulating oligomeric α-synuclein may mediate early synaptic pathology in Parkinson's disease and dementia with Lewy bodies by disrupting synaptic vesicles. This oligomer-prone model might be useful for evaluating therapies directed at oligomer reduction.
Asunto(s)
Encéfalo/patología , Degeneración Nerviosa/patología , Neuronas/metabolismo , Sinapsis/patología , alfa-Sinucleína/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Ácido Glutámico/genética , Humanos , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Lisina/genética , Trastornos de la Memoria/etiología , Trastornos de la Memoria/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructura , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , alfa-Sinucleína/genéticaRESUMEN
Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of α-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinson's disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/α-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy.
Asunto(s)
Encéfalo/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Degeneración Nerviosa/metabolismo , Anticuerpos de Cadena Única/metabolismo , alfa-Sinucleína/metabolismo , Secuencias de Aminoácidos , Animales , Apolipoproteínas B/química , Apolipoproteínas B/metabolismo , Autofagia , Conducta Animal , Encéfalo/patología , Línea Celular , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Orden Génico , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Lentivirus/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/metabolismo , Neuronas/patología , Unión Proteica , Transporte de Proteínas , Proteolisis , Ratas , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Transducción Genética , alfa-Sinucleína/genética , alfa-Sinucleína/inmunologíaRESUMEN
The development of disease-modifying therapy for Parkinson disease has been a main drug development challenge, including the need to deliver the therapeutic agents to the brain. Here, we examined the ability of mannitol to interfere with the aggregation process of α-synuclein in vitro and in vivo in addition to its blood-brain barrier-disrupting properties. Using in vitro studies, we demonstrated the effect of mannitol on α-synuclein aggregation. Although low concentration of mannitol inhibited the formation of fibrils, high concentration significantly decreased the formation of tetramers and high molecular weight oligomers and shifted the secondary structure of α-synuclein from α-helical to a different structure, suggesting alternative potential pathways for aggregation. When administered to a Parkinson Drosophila model, mannitol dramatically corrected its behavioral defects and reduced the amount of α-synuclein aggregates in the brains of treated flies. In the mThy1-human α-synuclein transgenic mouse model, a decrease in α-synuclein accumulation was detected in several brain regions following treatment, suggesting that mannitol promotes α-synuclein clearance in the cell bodies. It appears that mannitol has a general neuroprotective effect in the transgenic treated mice, which includes the dopaminergic system. We therefore suggest mannitol as a basis for a dual mechanism therapeutic agent for the treatment of Parkinson disease.
Asunto(s)
Antiparkinsonianos/química , Barrera Hematoencefálica/efectos de los fármacos , Manitol/química , Enfermedad de Parkinson/tratamiento farmacológico , alfa-Sinucleína/metabolismo , Amiloide/antagonistas & inhibidores , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestructura , Animales , Antiparkinsonianos/farmacología , Benzotiazoles , Drosophila , Femenino , Colorantes Fluorescentes/química , Proteínas HSP70 de Choque Térmico/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Locomoción , Masculino , Manitol/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Neocórtex/metabolismo , Neocórtex/patología , Multimerización de Proteína/efectos de los fármacos , Estructura Secundaria de Proteína , Tiazoles/química , alfa-Sinucleína/antagonistas & inhibidores , alfa-Sinucleína/química , alfa-Sinucleína/ultraestructuraRESUMEN
The aggregation of α-synuclein (αSyn) is a neuropathologic hallmark of Parkinson's disease and other synucleinopathies. In Lewy bodies, αSyn is extensively phosphorylated, predominantly at serine 129 (S129). Recent studies in yeast have shown that, at toxic levels, αSyn disrupts Rab homeostasis, causing an initial endoplasmic reticulum-to-Golgi block that precedes a generalized trafficking collapse. However, whether αSyn phosphorylation modulates trafficking defects has not been evaluated. Here, we show that constitutive expression of αSyn in yeast impairs late-exocytic, early-endocytic and/or recycling trafficking. Although members of the casein kinase I (CKI) family phosphorylate αSyn at S129, they attenuate αSyn toxicity and trafficking defects by an S129 phosphorylation-independent mechanism. Surprisingly, phosphorylation of S129 modulates αSyn toxicity and trafficking defects in a manner strictly determined by genetic background. Abnormal endosome morphology, increased levels of the endosome marker Rab5 and co-localization of mammalian CKI with αSyn aggregates are observed in brain sections from αSyn-overexpressing mice and human synucleinopathies. Our results contribute to evidence that suggests αSyn-induced defects in endocytosis, exocytosis and/or recycling of vesicles involved in these cellular processes might contribute to the pathogenesis of synucleinopathies.
Asunto(s)
Levaduras/metabolismo , alfa-Sinucleína/genética , Animales , Quinasa de la Caseína I/genética , Quinasa de la Caseína I/metabolismo , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Fosforilación , Transporte de Proteínas , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND: Alzheimer's Disease (AD) and Fronto temporal lobar dementia (FTLD) are common causes of dementia in the aging population for which limited therapeutical options are available. These disorders are associated with Tau accumulation. We have previously shown that Cerebrolysin™ (CBL), a neuropeptide mixture with neurotrophic effects, ameliorates the behavioral deficits and neuropathological alterations in amyloid precursor protein (APP) transgenic (tg) mouse model of AD by reducing hyper-phosphorylated Tau. CBL has been tested in clinical trials for AD, however it's potential beneficial effects in FTLD are unknown. For this purpose we sought to investigate the effects of CBL in a tg model of tauopathy. Accordingly, double tg mice expressing mutant Tau under the mThy-1 promoter and GSK3ß (to enhance Tau phosphorylation) were treated with CBL and evaluated neuropathologically. RESULTS: Compared to single Tau tg mice the Tau/GSK3ß double tg model displayed elevated levels of Tau phosphorylation and neurodegeneration in the hippocampus. CBL treatment reduced the levels of Tau phosphorylation in the dentate gyrus and the degeneration of pyramidal neurons in the temporal cortex and hippocampus of the Tau/GSK3ß double tg mice. Interestingly, the Tau/GSK3ß double tg mice also displayed elevated levels of Dynamin-related protein-1 (Drp-1), a protein that hydrolyzes GTP and is required for mitochondrial division. Ultrastructural analysis of the mitochondria in the Tau/GSK3ß double tg mice demonstrated increased numbers and fragmentation of mitochondria in comparison to non-tg mice. CBL treatment normalized levels of Drp-1 and restored mitochondrial structure. CONCLUSIONS: These results suggest that the ability of CBL to ameliorate neurodegenerative pathology in the tauopathy model may involve reducing accumulation of hyper-phosphorylated Tau and reducing alterations in mitochondrial biogenesis associated with Tau.
Asunto(s)
Aminoácidos/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Fármacos Neuroprotectores/farmacología , Tauopatías/tratamiento farmacológico , Tauopatías/patología , Envejecimiento , Animales , Modelos Animales de Enfermedad , Dinaminas/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hipocampo/efectos de los fármacos , Hipocampo/patología , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Fosforilación/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Células Piramidales/patología , Lóbulo Temporal/efectos de los fármacos , Lóbulo Temporal/patología , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Progressive accumulation of α-synuclein (α-syn) in limbic and striatonigral systems is associated with the neurodegenerative processes in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). The murine Thy-1 (mThy1)-α-syn transgenic (tg) model recapitulates aspects of degenerative processes associated with α-syn accumulation in these disorders. Given that axonal and synaptic pathologies are important features of DLB and PD, we sought to investigate the extent and characteristics of these alterations in mThy1-α-syn tg mice and to determine the contribution of α-syn c-terminally cleaved at amino acid 122 (CT α-syn) to these abnormalities. We generated a novel polyclonal antibody (SYN105) against the c-terminally truncated sequence (amino acids 121 to 123) of α-syn (CT α-syn) and performed immunocytochemical and ultrastructural analyses in mThy1-α-syn tg mice. We found abundant clusters of dystrophic neurites in layers 2 to 3 of the neocortex, the stratum lacunosum, the dentate gyrus, and cornu ammonis 3 of the hippocampus, striatum, thalamus, midbrain, and pons. Dystrophic neurites displayed intense immunoreactivity detected with the SYN105 antibody. Double-labeling studies with antibodies to phosphorylated neurofilaments confirmed the axonal location of full-length and CT α-syn. α-Syn immunoreactive dystrophic neurites contained numerous electrodense laminated structures. These results show that neuritic dystrophy is a prominent pathologic feature of the mThy1-α-syn tg model and suggest that CT α-syn might play an important role in the process of axonal damage in these mice as well as in DLB and PD.
Asunto(s)
Axones/patología , Enfermedad por Cuerpos de Lewy/patología , Proteínas Mutantes/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Axones/metabolismo , Axones/ultraestructura , Biomarcadores/metabolismo , Demografía , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Neuritas/metabolismo , Neuritas/patología , Neuritas/ultraestructura , Transporte de Proteínas , Sinapsis/metabolismo , Sinapsis/patología , Sinapsis/ultraestructura , Antígenos Thy-1/metabolismo , alfa-Sinucleína/inmunologíaRESUMEN
Immunotherapeutic approaches are currently in the spotlight for their potential as disease-modifying treatments for neurodegenerative disorders. The discovery that α-synuclein (α-syn) can transmit from cell to cell in a prion-like fashion suggests that immunization might be a viable option for the treatment of synucleinopathies. This possibility has been bolstered by the development of next-generation active vaccination technology with short peptides-AFFITOPEs(®) (AFF)- that do not elicit an α-syn-specific T cell response. This approach allows for the production of long term, sustained, more specific, non-cross reacting antibodies suitable for the treatment of synucleinopathies, such as Parkinson's disease (PD). In this context, we screened a large library of peptides that mimic the C-terminus region of α-syn and discovered a novel set of AFF that identified α-syn oligomers. Next, the peptide that elicited the most specific response against α-syn (AFF 1) was selected for immunizing two different transgenic (tg) mouse models of PD and Dementia with Lewy bodies, the PDGF- and the mThy1-α-syn tg mice. Vaccination with AFF 1 resulted in high antibody titers in CSF and plasma, which crossed into the CNS and recognized α-syn aggregates. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn oligomers in axons and synapses, accompanied by reduced degeneration of TH fibers in the caudo-putamen nucleus and by improvements in motor and memory deficits in both in vivo models. Clearance of α-syn involved activation of microglia and increased anti-inflammatory cytokine expression, further supporting the efficacy of this novel active vaccination approach for synucleinopathies.
Asunto(s)
Enfermedad por Cuerpos de Lewy/terapia , Enfermedad de Parkinson/terapia , Vacunación/métodos , Animales , Anticuerpos/sangre , Anticuerpos/líquido cefalorraquídeo , Axones/patología , Axones/fisiología , Núcleo Caudado/patología , Núcleo Caudado/fisiopatología , Ensayos Clínicos como Asunto , Modelos Animales de Enfermedad , Humanos , Enfermedad por Cuerpos de Lewy/patología , Enfermedad por Cuerpos de Lewy/fisiopatología , Trastornos de la Memoria/patología , Trastornos de la Memoria/fisiopatología , Trastornos de la Memoria/terapia , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/patología , Microglía/fisiología , Actividad Motora/fisiología , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/terapia , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/fisiopatología , Putamen/patología , Putamen/fisiopatología , Sinapsis/patología , Sinapsis/fisiología , Linfocitos T/patología , Linfocitos T/fisiología , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Neurosin is a predominant serine protease in the central nervous system (CNS) and has been shown to play a role in the clearance of α-synuclein (α-syn) which is centrally involved in the pathogenesis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Although it has been previously shown that neurosin and α-syn colocalize and that neurosin degrades α-syn aggregates in vitro, it is not clear if neurosin is dysregulated in the brains of patients with PD/DLB and to what extent delivery of neurosin into the CNS might ameliorate the deficits associated with α-syn accumulation in vivo. We analyzed the levels of neurosin in the brains of patients with PD/DLB and in α-syn transgenic (tg) models. With increased accumulation of α-syn, we observed decreased neurosin expression. Lentiviral vector (LV) driven expression of neurosin in neuronal cell cultures reduced the accumulation of wild type but not A53T α-syn and prevented α-syn associated toxicity. Neuropathological analysis following delivery of LV-Neurosin to α-syn tg mice resulted in reduced accumulation of α-syn and reversal of neurodegenerative alterations in wild type but not A53T α-syn tg mice. Therefore, viral vector driven expression of neurosin may warrant further investigation as a potential therapeutic tool for DLB.
Asunto(s)
Modelos Animales de Enfermedad , Calicreínas/genética , Lentivirus/genética , Enfermedad por Cuerpos de Lewy/metabolismo , alfa-Sinucleína/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Humanos , Inmunohistoquímica , Enfermedad por Cuerpos de Lewy/patología , Ratones , Ratones Transgénicos , Microscopía Confocal , Neuronas/metabolismoRESUMEN
Abnormal accumulation of α-synuclein is centrally involved in the pathogenesis of many disorders with Parkinsonism and dementia. Previous in vitro studies suggest that α-synuclein dysregulates intracellular calcium. However, it is unclear whether these alterations occur in vivo. For this reason, we investigated calcium dynamics in transgenic mice expressing human WT α-synuclein using two-photon microscopy. We imaged spontaneous and stimulus-induced neuronal activity in the barrel cortex. Transgenic mice exhibited augmented, long-lasting calcium transients characterized by considerable deviation from the exponential decay. The most evident pathology was observed in response to a repetitive stimulation in which subsequent stimuli were presented before relaxation of calcium signal to the baseline. These alterations were detected in the absence of significant increase in neuronal spiking response compared with age-matched controls, supporting the possibility that α-synuclein promoted alterations in calcium dynamics via interference with intracellular buffering mechanisms. The characteristic shape of calcium decay and augmented response during repetitive stimulation can serve as in vivo imaging biomarkers in this model of neurodegeneration, to monitor progression of the disease and screen candidate treatment strategies.
Asunto(s)
Encefalopatías/metabolismo , Encéfalo/metabolismo , Calcio/metabolismo , Neuronas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/patología , Encefalopatías/patología , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Transgénicos , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/patologíaRESUMEN
Parkinson disease is characterized by the loss of dopaminergic neurons mainly in the substantia nigra. Accumulation of α-synuclein and cell loss has been also reported in many other brain regions including the hippocampus, where it might impair adult neurogenesis, contributing to nonmotor symptoms. However, the molecular mechanisms of these alterations are still unknown. In this report we show that α-synuclein-accumulating adult rat hippocampus neural progenitors present aberrant neuronal differentiation, with reduction of Notch1 expression and downstream signaling targets. We characterized a Notch1 proximal promoter that contains p53 canonical response elements. In vivo binding of p53 represses the transcription of Notch1 in neurons. Moreover, we demonstrated that α-synuclein directly binds to the DNA at Notch1 promoter vicinity and also interacts with p53 protein, facilitating or increasing Notch1 signaling repression, which interferes with maturation and survival of neural progenitors cells. This study provides a molecular basis for α-synuclein-mediated disruption of adult neurogenesis in Parkinson disease.
Asunto(s)
Regulación de la Expresión Génica , Neurogénesis , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Receptor Notch1/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , alfa-Sinucleína/metabolismo , Animales , Apoptosis , Linaje de la Célula , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Lentivirus/genética , Regiones Promotoras Genéticas , Ratas , Transducción de SeñalRESUMEN
In Parkinson's disease (PD) patients, alpha-synuclein (α-syn) pathology advances in form of Lewy bodies and Lewy neurites throughout the brain. Clinically, PD is defined by motor symptoms that are predominantly attributed to the dopaminergic cell loss in the substantia nigra. However, motor deficits are frequently preceded by smell deficiency or neuropsychological symptoms, including increased anxiety and cognitive dysfunction. Accumulating evidence indicates that aggregation of α-syn impairs synaptic function and neurogenic capacity that may be associated with deficits in memory, learning and mood. Whether and how α-syn accumulation contributes to neuropathological events defining these earliest signs of PD is presently poorly understood. We used a tetracycline-suppressive (tet-off) transgenic mouse model that restricts overexpression of human A30P α-syn to neurons owing to usage of the neuron-specific CaMKIIα promoter. Abnormal accumulation of A30P correlated with a decreased survival of newly generated neurons in the hippocampus and olfactory bulb. Furthermore, when A30P α-syn expression was suppressed, we observed reduction of the human protein in neuronal soma. However, residual dox resistant A30P α-syn was detected in glial cells within the hippocampal neurogenic niche, concomitant with the failure to fully restore hippocampal neurogenesis. This finding is indicative to a potential spread of pathology from neuron to glia. In addition, mice expressing A30P α-syn show increased anxiety-related behavior that was reversed after dox treatment. This implies that glial A30P α-synucleinopathy within the dentate gyrus is part of a process leading to impaired hippocampal neuroplasticity, which is, however, not a sole critical event for circuits implicated in anxiety-related behavior.
Asunto(s)
Alanina/genética , Ansiedad , Neurogénesis/genética , Neuroglía/patología , Prolina/genética , alfa-Sinucleína/genética , Animales , Ansiedad/genética , Ansiedad/patología , Ansiedad/fisiopatología , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Recuento de Células , Modelos Animales de Enfermedad , Proteínas de Dominio Doblecortina , Doxiciclina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuropéptidos/metabolismo , Bulbo Olfatorio/metabolismo , Bulbo Olfatorio/patologíaRESUMEN
Alzheimer's disease (AD) is characterized by degeneration of neocortex, limbic system, and basal forebrain, accompanied by accumulation of amyloid-ß and tangle formation. Cerebrolysin (CBL), a peptide mixture with neurotrophic-like effects, is reported to improve cognition and activities of daily living in patients with AD. Likewise, CBL reduces synaptic and behavioral deficits in transgenic (tg) mice overexpressing the human amyloid precursor protein (hAPP). The neuroprotective effects of CBL may involve multiple mechanisms, including signaling regulation, control of APP metabolism, and expression of neurotrophic factors. We investigate the effects of CBL in the hAPP tg model of AD on levels of neurotrophic factors, including pro-nerve growth factor (NGF), NGF, brain-derived neurotrophic factor (BDNF), neurotropin (NT)-3, NT4, and ciliary neurotrophic factor (CNTF). Immunoblot analysis demonstrated that levels of pro-NGF were increased in saline-treated hAPP tg mice. In contrast, CBL-treated hAPP tg mice showed levels of pro-NGF comparable to control and increased levels of mature NGF. Consistently with these results, immunohistochemical analysis demonstrated increased NGF immunoreactivity in the hippocampus of CBL-treated hAPP tg mice. Protein levels of other neurotrophic factors, including BDNF, NT3, NT4, and CNTF, were unchanged. mRNA levels of NGF and other neurotrophins were also unchanged. Analysis of neurotrophin receptors showed preservation of the levels of TrKA and p75(NTR) immunoreactivity per cell in the nucleus basalis. Cholinergic cells in the nucleus basalis were reduced in the saline-treated hAPP tg mice, and treatment with CBL reduced these cholinergic deficits. These results suggest that the neurotrophic effects of CBL might involve modulation of the pro-NGF/NGF balance and a concomitant protection of cholinergic neurons.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Aminoácidos/uso terapéutico , Encéfalo/metabolismo , Colinérgicos/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/efectos de los fármacos , Colina O-Acetiltransferasa/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Ratones , Ratones Transgénicos , Mutación/genética , Factores de Crecimiento Nervioso/genética , ARN Mensajero/metabolismo , Receptor trkA/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismoRESUMEN
Aged (>50 years old) human immunodeficiency virus (HIV) patients are the fastest-growing segment of the HIV-infected population in the USA and despite antiretroviral therapy, HIV-associated neurocognitive disorder (HAND) prevalence has increased or remained the same among this group. Autophagy is an intracellular clearance pathway for aggregated proteins and aged organelles; dysregulation of autophagy is implicated in the pathogenesis of Parkinson's disease, Alzheimer's disease, and HAND. Here, we hypothesized that dysregulated autophagy may contribute to aging-related neuropathology in HIV-infected individuals. To explore this possibility, we surveyed autophagy marker levels in postmortem brain samples from a cohort of well-characterized <50 years old (young) and >50 years old (aged) HIV+ and HIV encephalitis (HIVE) patients. Detailed clinical and neuropathological data showed the young and aged HIVE patients had higher viral load, increased neuroinflammation and elevated neurodegeneration; however, aged HIVE postmortem brain tissues showed the most severe neurodegenerative pathology. Interestingly, young HIVE patients displayed an increase in beclin-1, cathepsin-D and light chain (LC)3, but these autophagy markers were reduced in aged HIVE cases compared to age-matched HIV+ donors. Similar alterations in autophagy markers were observed in aged gp120 transgenic (tg) mice; beclin-1 and LC3 were decreased in aged gp120 tg mice while mTor levels were increased. Lentivirus-mediated beclin-1 gene transfer, that is known to activate autophagy pathways, increased beclin-1, LC3, and microtubule-associated protein 2 expression while reducing glial fibrillary acidic protein and Iba1 expression in aged gp120 tg mice. These data indicate differential alterations in the autophagy pathway in young versus aged HIVE patients and that autophagy reactivation may ameliorate the neurodegenerative phenotype in these patients.
Asunto(s)
Complejo SIDA Demencia/metabolismo , Proteínas Reguladoras de la Apoptosis/biosíntesis , Autofagia/fisiología , Terapia Genética/métodos , Proteínas de la Membrana/biosíntesis , Complejo SIDA Demencia/genética , Complejo SIDA Demencia/patología , Adulto , Factores de Edad , Animales , Proteínas Reguladoras de la Apoptosis/análisis , Beclina-1 , Western Blotting , Femenino , Técnicas de Transferencia de Gen , Proteína gp120 de Envoltorio del VIH , Humanos , Inmunohistoquímica , Masculino , Proteínas de la Membrana/análisis , Ratones , Ratones Transgénicos , Microscopía Confocal , Persona de Mediana EdadRESUMEN
DNA methylation is a major epigenetic modification that regulates gene expression. Dnmt1, the maintenance DNA methylation enzyme, is abundantly expressed in the adult brain and is mainly located in the nuclear compartment, where it has access to chromatin. Hypomethylation of CpG islands at intron 1 of the SNCA gene has recently been reported to result in overexpression of α-synuclein in Parkinson disease (PD) and related disorders. We therefore investigated the mechanisms underlying altered DNA methylation in PD and dementia with Lewy bodies (DLB). We present evidence of reduction of nuclear Dnmt1 levels in human postmortem brain samples from PD and DLB patients as well as in the brains of α-synuclein transgenic mice models. Furthermore, sequestration of Dnmt1 in the cytoplasm results in global DNA hypomethylation in human and mouse brains, involving CpG islands upstream of SNCA, SEPW1, and PRKAR2A genes. We report that association of Dnmt1 and α-synuclein might mediate aberrant subcellular localization of Dnmt1. Nuclear Dnmt1 levels were partially rescued by overexpression of Dnmt1 in neuronal cell cultures and in α-synuclein transgenic mice brains. Our results underscore a novel mechanism for epigenetic dysregulation in Lewy body diseases, which might underlie the decrease in DNA methylation reported for PD and DLB.
Asunto(s)
Encéfalo/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Epigénesis Genética , Enfermedad por Cuerpos de Lewy/metabolismo , alfa-Sinucleína/metabolismo , Adulto , Animales , Islas de CpG , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , Humanos , Enfermedad por Cuerpos de Lewy/genética , Ratones , Ratones Noqueados , Selenoproteína W/genética , Selenoproteína W/metabolismo , alfa-Sinucleína/genéticaRESUMEN
Recent treatments with highly active antiretroviral therapy (HAART) regimens have been shown to improve general clinical status in patients with human immunodeficiency virus (HIV) infection; however, the prevalence of cognitive alterations and neurodegeneration has remained the same or has increased. These deficits are more pronounced in the subset of HIV patients with the inflammatory condition known as HIV encephalitis (HIVE). Activation of signaling pathways such as GSK3ß and CDK5 has been implicated in the mechanisms of HIV neurotoxicity; however, the downstream mediators of these effects are unclear. The present study investigated the involvement of CDK5 and tau phosphorylation in the mechanisms of neurodegeneration in HIVE. In the frontal cortex of patients with HIVE, increased levels of CDK5 and p35 expression were associated with abnormal tau phosphorylation. Similarly, transgenic mice engineered to express the HIV protein gp120 exhibited increased brain levels of CDK5 and p35, alterations in tau phosphorylation, and dendritic degeneration. In contrast, genetic knockdown of CDK5 or treatment with the CDK5 inhibitor roscovitine improved behavioral performance in the water maze test and reduced neurodegeneration, abnormal tau phosphorylation, and astrogliosis in gp120 transgenic mice. These findings indicate that abnormal CDK5 activation contributes to the neurodegenerative process in HIVE via abnormal tau phosphorylation; thus, reducing CDK5 might ameliorate the cognitive impairments associated with HIVE.
Asunto(s)
Quinasa 5 Dependiente de la Ciclina/metabolismo , Regulación de la Expresión Génica , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas tau/metabolismo , Adulto , Animales , Encéfalo/metabolismo , Encéfalo/virología , Femenino , Lóbulo Frontal/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Masculino , Ratones , Persona de Mediana Edad , Enfermedades Neurodegenerativas/virología , Fosfotransferasas/metabolismo , Purinas/farmacología , Roscovitina , Transducción de Señal , Proteínas tau/químicaRESUMEN
Neuronal accumulation of alpha-synuclein and Lewy body formation are characteristic to many neurodegenerative diseases, including Parkinson's disease (PD). This Lewy pathology appears to spread throughout the brain as the disease progresses. Furthermore, recent studies showed the occurrence of Lewy pathology in neurons grafted into the brains of PD patients, suggesting the spread of pathology from the host tissues to the grafts. The mechanism underlying this propagation is unknown. Here, we show that alpha-synuclein is transmitted via endocytosis to neighboring neurons and neuronal precursor cells, forming Lewy-like inclusions. Moreover, alpha-synuclein was transmitted from the affected neurons to engrafted neuronal precursor cells in a transgenic model of PD-like pathology. Failure of the protein quality control systems, especially lysosomes, promoted the accumulation of transmitted alpha-synuclein and inclusion formation. Cells exposed to neuron-derived alpha-synuclein showed signs of apoptosis, such as nuclear fragmentation and caspase 3 activation, both in vitro and in vivo. These findings demonstrate the cell-to-cell transmission of alpha-synuclein aggregates and provide critical insights into the mechanism of pathological progression in PD and other proteinopathies.
Asunto(s)
Apoptosis , Cuerpos de Lewy/fisiología , Neuronas/fisiología , Transmisión Sináptica , alfa-Sinucleína/metabolismo , Animales , Transporte Biológico , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ratas , Trasplante de Células Madre , alfa-Sinucleína/toxicidadRESUMEN
During aging and in the progression of Alzheimer's disease (AD), synaptic plasticity and neuronal integrity are disturbed. In addition to the alterations in plasticity in mature neurons, the neurodegenerative process in AD has been shown to be accompanied by alterations in neurogenesis. Members of the bone morphogenetic protein (BMP) family of growth factors have been implicated as important regulators of neurogenesis and neuronal cell fate determination during development; however, their role in adult neurogenesis and in AD is less clear. We show here by qRT-PCR analysis that BMP6 mRNA levels were significantly increased in the hippocampus of human patients with AD and in APP transgenic mice compared to controls. Immunoblot and immunohistochemical analyses confirmed that BMP6 protein levels were increased in human AD brains and APP transgenic mouse brains compared to controls and accumulated around hippocampal plaques. The increased levels of BMP6 were accompanied by defects in hippocampal neurogenesis in AD patients and APP transgenic mice. In support of a role for BMP6 in defective neurogenesis in AD, we show in an in vitro model of adult neurogenesis that treatment with amyloid-ß(1-42) protein (Aß) resulted in increased expression of BMP6, and that exposure to recombinant BMP6 resulted in reduced proliferation with no toxic effects. Together, these results suggest that Aß-associated increases in BMP6 expression in AD may have deleterious effects on neurogenesis in the hippocampus, and therapeutic approaches could focus on normalization of BMP6 levels to protect against AD-related neurogenic deficits.