RESUMEN
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
BACKGROUND: Multiple system atrophy (MSA) is a neurodegenerative disease characterized by parkinsonism, ataxia and dysautonomia. Histopathologically, the hallmark of MSA is the abnormal accumulation of alpha-synuclein (α-syn) within oligodendroglial cells, leading to neuroinflammation, demyelination and neuronal death. Currently, there is no disease-modifying treatment for MSA. In this sense, we have previously shown that next-generation active vaccination technology with short peptides, AFFITOPEs®, was effective in two transgenic models of synucleinopathies at reducing behavioral deficits, α-syn accumulation and inflammation. RESULTS: In this manuscript, we used the most effective AFFITOPE® (AFF 1) for immunizing MBP-α-syn transgenic mice, a model of MSA that expresses α-syn in oligodendrocytes. Vaccination with AFF 1 resulted in the production of specific anti-α-syn antibodies that crossed into the central nervous system and recognized α-syn aggregates within glial cells. Active vaccination with AFF 1 resulted in decreased accumulation of α-syn, reduced demyelination in neocortex, striatum and corpus callosum, and reduced neurodegeneration. Clearance of α-syn involved activation of microglia and reduced spreading of α-syn to astroglial cells. CONCLUSIONS: This study further validates the efficacy of vaccination with AFFITOPEs® for ameliorating the neurodegenerative pathology in synucleinopathies.
Asunto(s)
Enfermedades Desmielinizantes/prevención & control , Atrofia de Múltiples Sistemas/patología , Atrofia de Múltiples Sistemas/prevención & control , Trastornos Parkinsonianos/patología , alfa-Sinucleína/inmunología , Animales , Astrocitos/citología , Astrocitos/inmunología , Astrocitos/metabolismo , Enfermedades Desmielinizantes/inmunología , Modelos Animales de Enfermedad , Ratones Transgénicos , Microglía/citología , Microglía/inmunología , Atrofia de Múltiples Sistemas/inmunología , Neuronas/citología , Neuronas/inmunología , Oligodendroglía/citología , Oligodendroglía/inmunología , Trastornos Parkinsonianos/inmunología , Vacunación/métodosRESUMEN
Tauopathies are a group of disorders leading to cognitive and behavioral impairment in the aging population. While four-repeat (4R) Tau is more abundant in corticobasal degeneration, progressive supranuclear palsy, and Alzheimer's disease, three-repeat (3R) Tau is the most abundant splice, in Pick's disease. A number of transgenic models expressing wild-type and mutant forms of the 4R Tau have been developed. However, few models of three-repeat Tau are available. A transgenic mouse model expressing three-repeat Tau was developed bearing the mutations associated with familial forms of Pick's disease (L266V and G272V mutations). Two lines expressing high (Line 13) and low (Line 2) levels of the three-repeat mutant Tau were analyzed. By Western blot, using antibodies specific to three-repeat Tau, Line 13 expressed 5-times more Tau than Line 2. The Tau expressed by these mice was most abundant in the frontal-temporal cortex and limbic system and was phosphorylated at residues detected by the PHF-1, AT8, CP9 and CP13 antibodies. The higher-expressing mice displayed hyperactivity, memory deficits in the water maze and alterations in the round beam. The behavioral deficits started at 6-8 months of age and were associated with a progressive increase in the accumulation of 3R Tau. By immunocytochemistry, mice from Line 13 displayed extensive accumulation of 3R Tau in neuronal cells bodies in the pyramidal neurons of the neocortex, CA1-3 regions, and dentate gyrus of the hippocampus. Aggregates in the granular cells had a globus appearance and mimic Pick's-like inclusions. There were abundant dystrophic neurites, astrogliosis and synapto-dendritic damage in the neocortex and hippocampus of the higher expresser line. The hippocampal lesions were moderately argyrophilic and Thioflavin-S negative. By electron microscopy, discrete straight filament aggregates were detected in some neurons in the hippocampus. This model holds promise for better understanding the natural history and progression of 3R tauopathies and their relationship with mitochondrial alterations and might be suitable for therapeutical testing.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedad de Pick , Secuencias Repetitivas de Aminoácido , Tauopatías , Proteínas tau/química , Proteínas tau/genética , Animales , Axones/patología , Conducta Animal , Regulación de la Expresión Génica , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Filamentos Intermedios/patología , Memoria , Ratones , Ratones Transgénicos , Mitocondrias/patología , Neocórtex/metabolismo , Neocórtex/patología , Enfermedad de Pick/genética , Enfermedad de Pick/metabolismo , Enfermedad de Pick/patología , Enfermedad de Pick/fisiopatología , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tauopatías/genética , Tauopatías/metabolismo , Tauopatías/patología , Tauopatías/fisiopatología , Factores de Tiempo , Proteínas tau/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Anti-retrovirals have improved and extended the life expectancy of patients with HIV. However, as this population ages, the prevalence of cognitive changes is increasing. Aberrant activation of kinases, such as receptor tyrosine kinases (RTKs) and cyclin-dependent kinase 5 (CDK5), play a role in the mechanisms of HIV neurotoxicity. Inhibitors of CDK5, such as roscovitine, have neuroprotective effects; however, CNS penetration is low. Interestingly, tyrosine kinase inhibitors (TKIs) display some CDK inhibitory activity and ability to cross the blood-brain barrier. EXPERIMENTAL APPROACH: We screened a small group of known TKIs for a candidate with additional CDK5 inhibitory activity and tested the efficacy of the candidate in in vitro and in vivo models of HIV-gp120 neurotoxicity. KEY RESULTS: Among 12 different compounds, sunitinib inhibited CDK5 with an IC50 of 4.2 µM. In silico analysis revealed that, similarly to roscovitine, sunitinib fitted 6 of 10 features of the CDK5 pharmacophore. In a cell-based model, sunitinib reduced CDK5 phosphorylation (pCDK5), calpain-dependent p35/p25 conversion and protected neuronal cells from the toxic effects of gp120. In glial fibrillary acidic protein-gp120 transgenic (tg) mice, sunitinib reduced levels of pCDK5, p35/p25 and phosphorylated tau protein, along with amelioration of the neurodegenerative pathology. CONCLUSIONS AND IMPLICATIONS: Compounds such as sunitinib with dual kinase inhibitory activity could ameliorate the cognitive impairment associated with chronic HIV infection of the CNS. Moreover, repositioning existing low MW compounds holds promise for the treatment of patients with neurodegenerative disorders.
Asunto(s)
Complejo SIDA Demencia , Antineoplásicos/farmacología , Quinasa 5 Dependiente de la Ciclina/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/toxicidad , Indoles/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Animales , Dasatinib , Clorhidrato de Erlotinib , Flavonoides/farmacología , Proteína gp120 de Envoltorio del VIH/genética , Técnicas In Vitro , Lapatinib , Ratones , Ratones Transgénicos , Enfermedades Neurodegenerativas , Purinas/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Ratas , Roscovitina , Sunitinib , Tiazoles/farmacologíaRESUMEN
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
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
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
BACKGROUND: In dementia with Lewy bodies (DLB) abnormal interactions between α-synuclein (α-syn) and beta amyloid (Aß) result in selective degeneration of neurons in the neocortex, limbic system and striatum. However, factors rendering these neurons selectively vulnerable have not been fully investigated. The metabotropic glutamate receptor 5 (mGluR5) has been shown to be up regulated in DLB and might play a role as a mediator of the neurotoxic effects of Aß and α-syn in vulnerable neuronal populations. In this context, the main objective of the present study was to investigate the role of mGluR5 as a mediator of the neurotoxic effects of α-syn and Aß in the hippocampus. RESULTS: We generated double transgenic mice over-expressing amyloid precursor protein (APP) and α-syn under the mThy1 cassette and investigated the relationship between α-syn cleavage, Aß, mGluR5 and neurodegeneration in the hippocampus. We found that compared to the single tg mice, the α-syn/APP tg mice displayed greater accumulation of α-syn and mGluR5 in the CA3 region of the hippocampus compared to the CA1 and other regions. This was accompanied by loss of CA3 (but not CA1) neurons in the single and α-syn/APP tg mice and greater loss of MAP 2 and synaptophysin in the CA3 in the α-syn/APP tg. mGluR5 gene transfer using a lentiviral vector into the hippocampus CA1 region resulted in greater α-syn accumulation and neurodegeneration in the single and α-syn/APP tg mice. In contrast, silencing mGluR5 with a lenti-shRNA protected neurons in the CA3 region of tg mice. In vitro, greater toxicity was observed in primary hippocampal neuronal cultures treated with Aß oligomers and over-expressing α-syn; this effect was attenuated by down-regulating mGluR5 with an shRNA lentiviral vector. In α-syn-expressing neuronal cells lines, Aß oligomers promoted increased intracellular calcium levels, calpain activation and α-syn cleavage resulting in caspase-3-dependent cell death. Treatment with pharmacological mGluR5 inhibitors such as 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) attenuated the toxic effects of Aß in α-syn-expressing neuronal cells. CONCLUSIONS: Together, these results support the possibility that vulnerability of hippocampal neurons to α-syn and Aß might be mediated via mGluR5. Moreover, therapeutical interventions targeting mGluR5 might have a role in DLB.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Enfermedad por Cuerpos de Lewy/metabolismo , Neuronas/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , alfa-Sinucleína/metabolismo , Péptidos beta-Amiloides/toxicidad , Precursor de Proteína beta-Amiloide/genética , Animales , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Enfermedad por Cuerpos de Lewy/patología , Ratones , Ratones Transgénicos , Neuronas/patología , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , alfa-Sinucleína/genéticaRESUMEN
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
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
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
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
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
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.
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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
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.
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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
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.
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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
BACKGROUND: Parkinson's disease (PD) is a multifactorial disease where environmental factors act on genetically predisposed individuals. Although only 5% of PD manifestations are associated with specific mutations, majority of PD cases are of idiopathic origin, where environment plays a prominent role. Concurrent exposure to Paraquat (PQ) and Maneb (MB) in rural workers increases the risk for PD and exposure of adult mice to MB/PQ results in dopamine fiber loss and decreased locomotor activity. While PD is characterized by neuronal loss in the substantia nigra, we previously showed that accumulation of α-synuclein in the limbic system contributes to neurodegeneration by interfering with adult neurogenesis. RESULTS: We investigated the effect of pesticides on adult hippocampal neurogenesis in two transgenic models: Line 61, expressing the human wild type SNCA gene and Line LRRK2(G2019S), expressing the human LRRK2 gene with the mutation G2019S. Combined exposure to MB/PQ resulted in significant reduction of neuronal precursors and proliferating cells in non-transgenic animals, and this effect was increased in transgenic mice, in particular for Line 61, suggesting that α-synuclein accumulation and environmental toxins have a synergistic effect. We further investigated the transcription of 84 genes with direct function on neurogenesis. Overexpresion of α-synuclein resulted in the downregulation of 12% of target genes, most of which were functionally related to cell differentiation, while LRRK2 mutation had a minor impact on gene expression. MB/PQ also affected transcription in non-transgenic backgrounds, but when transgenic mice were exposed to the pesticides, profound alterations in gene expression affecting 27% of the studied targets were observed in both transgenic lines. Gene enrichment analysis showed that 1:3 of those genes were under the regulation of FoxF2 and FoxO3A, suggesting a primary role of these proteins in the response to genetic and environmental cues. CONCLUSIONS: We report that adult neurogenesis is highly susceptible to multiple "risk factors" for PD, including α-synuclein accumulation, LRRK2 G2019 mutation and exposure to environmental toxins. We identified specific groups of genes that are responsive to each stressor, while uncovering a novel function for Fox transcription factors in PD.
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Interacción Gen-Ambiente , Hipocampo/efectos de los fármacos , Maneb/toxicidad , Neurogénesis/efectos de los fármacos , Paraquat/toxicidad , Plaguicidas/toxicidad , Transcripción Genética/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/patología , Humanos , Inmunohistoquímica , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Ratones , Ratones Transgénicos , Enfermedad de Parkinson/genética , Proteínas Serina-Treonina Quinasas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , alfa-Sinucleína/genéticaRESUMEN
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.
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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
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.