RESUMEN
Neuroinflammation plays a key role in exacerbating dopaminergic neuron (DAN) loss in Parkinson's disease (PD). However, it remains unresolved how to effectively normalize this immune response given the complex interplay between the innate and adaptive immune responses occurring within a scarcely accessible organ like the brain. In this study, we uncovered a consistent correlation between neuroinflammation, brain parenchymal lymphocytes, and DAN loss among several commonly used mouse models of PD generated by a variety of pathological triggers. We validated a viral therapeutic approach for the microglia-specific expression of interleukin 10 (IL-10) to selectively mitigate the excessive inflammatory response. We found that this approach induced a local nigral IL-10 release that alleviated DAN loss in mice overexpressing the human SNCA gene in the substantia nigra. Single-cell transcriptomics revealed that IL-10 induced the emergence of a molecularly distinct microglial cell state, enriched in markers of cell activation with enhanced expression of prophagocytic pathways. IL-10 promoted microglial phagocytotic and clearance activities in vitro and reduced αSYN aggregate burden in the nigral area in mice overexpressing SNCA. Furthermore, IL-10 stimulated the differentiation of CD4+ T lymphocytes into active T regulatory cells and promoted inhibitory characteristics in CD8+ T cells. In summary, our results show that local and microglia-specific IL-10 transduction elicited strong immunomodulation in the nigral tissue with enhanced suppression of lymphocyte toxicity that was associated with DAN survival. These results offer insights into the therapeutic benefits of IL-10 and showcase a promising gene delivery approach that could minimize undesired side effects.
Asunto(s)
Modelos Animales de Enfermedad , Neuronas Dopaminérgicas , Interleucina-10 , Microglía , Enfermedad de Parkinson , alfa-Sinucleína , Animales , Interleucina-10/metabolismo , Interleucina-10/genética , Microglía/metabolismo , Microglía/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/terapia , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Técnicas de Transferencia de Gen , Ratones , Enfermedades Neuroinflamatorias/metabolismo , Sustancia Negra/metabolismo , Sustancia Negra/patología , Ratones Endogámicos C57BL , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/inmunología , FagocitosisRESUMEN
Although adeno-associated virus 9 (AAV9) has been highly exploited as delivery platform for gene-based therapies, its efficacy is hampered by low efficiency in crossing the adult blood-brain barrier (BBB) and pronounced targeting to the liver upon intravenous delivery. We generated a new galactose binding-deficient AAV9 peptide display library and selected two new AAV9 engineered capsids with enhanced targeting in mouse and marmoset brains after intravenous delivery. Interestingly, the loss of galactose binding greatly reduced undesired targeting to peripheral organs, particularly the liver, while not compromising transduction of the brain vasculature. However, the galactose binding was necessary to efficiently infect non-endothelial brain cells. Thus, the combinatorial actions of the galactose-binding domain and the incorporated displayed peptide are crucial to enhance BBB crossing along with brain cell transduction. This study describes two novel capsids with high brain endothelial infectivity and extremely low liver targeting based on manipulating the AAV9 galactose-binding domain.
RESUMEN
BACKGROUND: Neurodevelopmental disorders (NDDs) are heterogeneous conditions due to alterations of a variety of molecular mechanisms and cell dysfunctions. SETD5 haploinsufficiency leads to NDDs due to chromatin defects. Epigenetic basis of NDDs has been reported in an increasing number of cases while mitochondrial dysfunctions are more common within NDD patients than in the general population. METHODS: We investigated in vitro neural stem cells as well as the brain of the Setd5 haploinsufficiency mouse model interrogating its transcriptome, analyzing mitochondrial structure, biochemical composition, and dynamics, as well as mitochondrial functionality. RESULTS: Mitochondrial impairment is facilitated by transcriptional aberrations originated by the decrease of the SETD5 enzyme. Low levels of SETD5 resulted in fragmented mitochondria, reduced mitochondrial membrane potential, and ATP production both in neural precursors and neurons. Mitochondria were also mislocalized in mutant neurons, with reduced organelles within neurites and synapses. LIMITATIONS: We found several defects in the mitochondrial compartment; however, we can only speculate about their position in the hierarchy of the pathological mechanisms at the basis of the disease. CONCLUSIONS: Our study explores the interplay between chromatin regulation and mitochondria functions as a possible important aspect of SETD5-associated NDD pathophysiology. Our data, if confirmed in patient context, suggest that the mitochondrial activity and dynamics may represent new therapeutic targets for disorders associated with the loss of SETD5.
Asunto(s)
Haploinsuficiencia , Células-Madre Neurales , Ratones , Animales , Humanos , Neuronas/metabolismo , Mitocondrias/metabolismo , Células-Madre Neurales/metabolismo , Cromatina/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismoRESUMEN
Acute myeloid leukemia (AML) is a hematological malignancy derived from neoplastic myeloid progenitor cells characterized by abnormal clonal proliferation and differentiation. Although novel therapeutic strategies have recently been introduced, the prognosis of AML is still unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)-T-cell therapy in AML has been hampered by several factors, including the poor accumulation of the blood-injected cells in the leukemia bone marrow (BM) niche in which chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within this niche, could improve T-cell homing to the BM and consequently enhance their intimate contact with BM-resident AML cells, facilitating disease eradication. Specifically, we engineered conventional CD33.CAR-cytokine-induced killer cells (CIKs) with the wild-type (wt) CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of patients with warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis syndrome. Overexpression of both CXCR4wt and CXCR4mut in CD33.CAR-CIKs resulted in significant improvement of chemotaxis toward recombinant CXCL12 or BM stromal cell-conditioned medium, with no observed impairment of cytotoxic potential in vitro. Moreover, CXCR4-overexpressing CD33.CAR-CIKs showed enhanced in vivo BM homing, associated with a prolonged retention for the CXCR4R334X variant. However, only CD33.CAR-CIKs coexpressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a noncanonical role for CXCR4 in modulating CAR-CIK functions independent of BM homing. Taken together, these data suggest that arming CAR-CIKs with CXCR4 may represent a promising strategy for increasing their therapeutic potential for AML.
Asunto(s)
Antineoplásicos , Células Asesinas Inducidas por Citocinas , Leucemia Mieloide Aguda , Animales , Médula Ósea/patología , Células Asesinas Inducidas por Citocinas/patología , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Linfocitos T , Células de la Médula Ósea/patologíaRESUMEN
Current therapies remain unsatisfactory in preventing the recurrence of glioblastoma multiforme (GBM), which leads to poor patient survival. By rational engineering of the transcription factor SOX2, a key promoter of GBM malignancy, together with the Kruppel-associated box and DNA methyltransferase3A/L catalytic domains, we generated a synthetic repressor named SOX2 epigenetic silencer (SES), which induces the transcriptional silencing of its original targets. By doing so, SES kills both glioma cell lines and patient-derived cancer stem cells in vitro and in vivo. SES expression, through local viral delivery in mouse xenografts, induces strong regression of human tumors and survival rescue. Conversely, SES is not harmful to neurons and glia, also thanks to a minimal promoter that restricts its expression in mitotically active cells, rarely present in the brain parenchyma. Collectively, SES produces a significant silencing of a large fraction of the SOX2 transcriptional network, achieving high levels of efficacy in repressing aggressive brain tumors.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioma , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Epigénesis Genética , Glioblastoma/metabolismo , Glioma/patología , Humanos , Ratones , Células Madre Neoplásicas/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Dravet syndrome is a severe epileptic encephalopathy caused primarily by haploinsufficiency of the SCN1A gene. Repetitive seizures can lead to endurable and untreatable neurological deficits. Whether this severe pathology is reversible after symptom onset remains unknown. To address this question, we generated a Scn1a conditional knock-in mouse model (Scn1a Stop/+) in which Scn1a expression can be re-activated on-demand during the mouse lifetime. Scn1a gene disruption leads to the development of seizures, often associated with sudden unexpected death in epilepsy (SUDEP) and behavioral alterations including hyperactivity, social interaction deficits and cognitive impairment starting from the second/third week of age. However, we showed that Scn1a gene re-activation when symptoms were already manifested (P30) led to a complete rescue of both spontaneous and thermic inducible seizures, marked amelioration of behavioral abnormalities and normalization of hippocampal fast-spiking interneuron firing. We also identified dramatic gene expression alterations, including those associated with astrogliosis in Dravet syndrome mice, that, accordingly, were rescued by Scn1a gene expression normalization at P30. Interestingly, regaining of Nav1.1 physiological level rescued seizures also in adult Dravet syndrome mice (P90) after months of repetitive attacks. Overall, these findings represent a solid proof-of-concept highlighting that disease phenotype reversibility can be achieved when Scn1a gene activity is efficiently reconstituted in brain cells.
Asunto(s)
Disfunción Cognitiva/genética , Epilepsias Mioclónicas/genética , Hipocampo/metabolismo , Interneuronas/metabolismo , Canal de Sodio Activado por Voltaje NAV1.1/genética , Muerte Súbita e Inesperada en la Epilepsia/prevención & control , Potenciales de Acción/fisiología , Animales , Cerebelo/metabolismo , Cerebelo/fisiopatología , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/fisiopatología , Disfunción Cognitiva/prevención & control , Cuerpo Estriado/metabolismo , Cuerpo Estriado/fisiopatología , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Epilepsias Mioclónicas/metabolismo , Epilepsias Mioclónicas/fisiopatología , Epilepsias Mioclónicas/prevención & control , Técnicas de Sustitución del Gen , Terapia Genética/métodos , Hipocampo/fisiopatología , Humanos , Interneuronas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Canal de Sodio Activado por Voltaje NAV1.1/deficiencia , Muerte Súbita e Inesperada en la Epilepsia/patologíaRESUMEN
Recent findings in human samples and animal models support the involvement of inflammation in the development of Parkinson's disease. Nevertheless, it is currently unknown whether microglial activation constitutes a primary event in neurodegeneration. We generated a new mouse model by lentiviral-mediated selective α-synuclein (αSYN) accumulation in microglial cells. Surprisingly, these mice developed progressive degeneration of dopaminergic (DA) neurons without endogenous αSYN aggregation. Transcriptomics and functional assessment revealed that αSYN-accumulating microglial cells developed a strong reactive state with phagocytic exhaustion and excessive production of oxidative and proinflammatory molecules. This inflammatory state created a molecular feed-forward vicious cycle between microglia and IFNγ-secreting immune cells infiltrating the brain parenchyma. Pharmacological inhibition of oxidative and nitrosative molecule production was sufficient to attenuate neurodegeneration. These results suggest that αSYN accumulation in microglia induces selective DA neuronal degeneration by promoting phagocytic exhaustion, an excessively toxic environment and the selective recruitment of peripheral immune cells.
Asunto(s)
Neuronas Dopaminérgicas/patología , Microglía/metabolismo , Degeneración Nerviosa/patología , Fagocitosis/fisiología , alfa-Sinucleína/metabolismo , Inmunidad Adaptativa/fisiología , Animales , Receptor 1 de Quimiocinas CX3C/genética , Receptor 1 de Quimiocinas CX3C/metabolismo , Encefalitis/metabolismo , Encefalitis/patología , Expresión Génica , Inmunidad Innata/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/patología , Óxido Nítrico/metabolismo , Óxido Nítrico/toxicidad , Enfermedad de Parkinson/patología , Especies Reactivas de Oxígeno/metabolismo , Sustancia Negra/metabolismo , Sustancia Negra/patología , alfa-Sinucleína/genéticaRESUMEN
Parkinson's disease is a neurodegenerative disorder partly caused by the loss of the dopamine neurons of the nigrostriatal pathway. It is accompanied by motor as well as non-motor symptoms, including pain and depression. The tail of the ventral tegmental area (tVTA) or rostromedial tegmental nucleus (RMTg) is a GABAergic mesopontine structure that acts as a major inhibitory brake for the substantia nigra pars compacta (SNc) dopamine cells, thus controlling their neuronal activity and related motor functions. The present study tested the influence of suppressing this tVTA brake on motor and non-motor symptoms in a rat model of Parkinson's disease. Using behavioral approaches, we showed that male Sprague-Dawley rats with bilateral and partial 6-hydroxydopamine SNc lesion displayed motor impairments in the rotarod test, impairments that were no more present following a co-lesion of the tVTA. Using a larger set of behavioral tests, we then showed that such SNc lesion also led to non-motor symptoms, including lower body weight, lower mechanical nociceptive thresholds in the forceps test and lower thermal nociceptive thresholds in the incremented hot-plate test, and a decreased sucrose preference in a 2-bottle choice paradigm. The excitotoxic co-lesion of the tVTA led to compensation of body weight, mechanical nociceptive thresholds and anhedonia-like behavior. These findings illustrate the major influence that the tVTA exerts on the dopamine system, modulating the motor and non-motor symptoms related to a partial loss of dopamine cells.
Asunto(s)
Enfermedad de Parkinson/metabolismo , Área Tegmental Ventral/metabolismo , Anhedonia , Animales , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Masculino , Modelos Teóricos , Vías Nerviosas/metabolismo , Oxidopamina/farmacología , Porción Compacta de la Sustancia Negra/metabolismo , Ratas , Ratas Sprague-Dawley , Prueba de Desempeño de Rotación con Aceleración Constante , Sustancia Negra/metabolismoRESUMEN
Dravet syndrome (DS) is a severe epileptic encephalopathy caused mainly by heterozygous loss-of-function mutations of the SCN1A gene, indicating haploinsufficiency as the pathogenic mechanism. Here we tested whether catalytically dead Cas9 (dCas9)-mediated Scn1a gene activation can rescue Scn1a haploinsufficiency in a mouse DS model and restore physiological levels of its gene product, the Nav1.1 voltage-gated sodium channel. We screened single guide RNAs (sgRNAs) for their ability to stimulate Scn1a transcription in association with the dCas9 activation system. We identified a specific sgRNA that increases Scn1a gene expression levels in cell lines and primary neurons with high specificity. Nav1.1 protein levels were augmented, as was the ability of wild-type immature GABAergic interneurons to fire action potentials. A similar enhancement of Scn1a transcription was achieved in mature DS interneurons, rescuing their ability to fire. To test the therapeutic potential of this approach, we delivered the Scn1a-dCas9 activation system to DS pups using adeno-associated viruses. Parvalbumin interneurons recovered their firing ability, and febrile seizures were significantly attenuated. Our results pave the way for exploiting dCas9-based gene activation as an effective and targeted approach to DS and other disorders resulting from altered gene dosage.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Epilepsias Mioclónicas/terapia , Terapia Genética/métodos , Interneuronas/metabolismo , Canal de Sodio Activado por Voltaje NAV1.1/genética , Convulsiones/terapia , Activación Transcripcional , Potenciales de Acción , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Neuronas GABAérgicas/metabolismo , Hipocampo/citología , Hipocampo/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Canal de Sodio Activado por Voltaje NAV1.1/metabolismo , Resultado del TratamientoRESUMEN
Stem cell-derived neurons are generally obtained in mass cultures that lack both spatial organization and any meaningful connectivity. We implement a microfluidic system for long-term culture of human neurons with patterned projections and synaptic terminals. Co-culture of human midbrain dopaminergic and striatal medium spiny neurons on the microchip establishes an orchestrated nigro-striatal circuitry with functional dopaminergic synapses. We use this platform to dissect the mitochondrial dysfunctions associated with a genetic form of Parkinson's disease (PD) with OPA1 mutations. Remarkably, we find that axons of OPA1 mutant dopaminergic neurons exhibit a significant reduction of mitochondrial mass. This defect causes a significant loss of dopaminergic synapses, which worsens in long-term cultures. Therefore, PD-associated depletion of mitochondria at synapses might precede loss of neuronal connectivity and neurodegeneration. In vitro reconstitution of human circuitries by microfluidic technology offers a powerful system to study brain networks by establishing ordered neuronal compartments and correct synapse identity.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , GTP Fosfohidrolasas/metabolismo , Dispositivos Laboratorio en un Chip , Mitocondrias/metabolismo , Neostriado/metabolismo , Sustancia Negra/metabolismo , Sinapsis/metabolismo , Axones/metabolismo , Células Cultivadas , GTP Fosfohidrolasas/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Red Nerviosa/metabolismo , Neuritas/metabolismo , Enfermedad de Parkinson/metabolismoRESUMEN
Dysfunctions in mitochondrial dynamics and metabolism are common pathological processes associated with Parkinson's disease (PD). It was recently shown that an inherited form of PD and dementia is caused by mutations in the OPA1 gene, which encodes for a key player in mitochondrial fusion and structure. iPSC-derived neural cells from these patients exhibited severe mitochondrial fragmentation, respiration impairment, ATP deficits, and heightened oxidative stress. Reconstitution of normal levels of OPA1 in PD-derived neural cells normalized mitochondria morphology and function. OPA1-mutated neuronal cultures showed reduced survival in vitro. Intriguingly, selective inhibition of necroptosis effectively rescued this survival deficit. Additionally, dampening necroptosis in MPTP-treated mice protected from DA neuronal cell loss. This human iPSC-based model captures both early pathological events in OPA1 mutant neural cells and the beneficial effects of blocking necroptosis, highlighting this cell death process as a potential therapeutic target for PD.
Asunto(s)
Apoptosis/efectos de los fármacos , Neuronas Dopaminérgicas/patología , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/patología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/efectos de los fármacos , GTP Fosfohidrolasas/genética , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutación/genética , Necrosis , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Estrés Oxidativo/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The lack of technology for direct global-scale targeting of the adult mouse nervous system has hindered research on brain processing and dysfunctions. Currently, gene transfer is normally achieved by intraparenchymal viral injections, but these injections target a restricted brain area. Herein, we demonstrated that intravenous delivery of adeno-associated virus (AAV)-PHP.B viral particles permeated and diffused throughout the neural parenchyma, targeting both the central and the peripheral nervous system in a global pattern. We then established multiple procedures of viral transduction to control gene expression or inactivate gene function exclusively in the adult nervous system and assessed the underlying behavioral effects. Building on these results, we established an effective gene therapy strategy to counteract the widespread accumulation of α-synuclein deposits throughout the forebrain in a mouse model of synucleinopathy. Transduction of A53T-SCNA transgenic mice with AAV-PHP.B-GBA1 restored physiological levels of the enzyme, reduced α-synuclein pathology, and produced significant behavioral recovery. Finally, we provided evidence that AAV-PHP.B brain penetration does not lead to evident dysfunctions in blood-brain barrier integrity or permeability. Altogether, the AAV-PHP.B viral platform enables non-invasive, widespread, and long-lasting global neural expression of therapeutic genes, such as GBA1, providing an invaluable approach to treat neurodegenerative diseases with diffuse brain pathology such as synucleinopathies.
Asunto(s)
Dependovirus/genética , Expresión Génica , Vectores Genéticos/genética , beta-Glucosidasa/metabolismo , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Electroencefalografía , Activación Enzimática , Orden Génico , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/administración & dosificación , Humanos , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Transducción Genética , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
Alpha-synuclein (α-syn) is involved in both familial and sporadic Parkinson's disease (PD). One of the proposed pathogenic mechanisms of α-syn mutations is mitochondrial dysfunction. However, it is not entirely clear the impact of impaired mitochondrial dynamics induced by α-syn on neurodegeneration and whether targeting this pathway has therapeutic potential. In this study we evaluated whether inhibition of mitochondrial fission is neuroprotective against α-syn overexpression in vivo. To accomplish this goal, we overexpressed human A53T-α- synuclein (hA53T-α-syn) in the rat nigrostriatal pathway, with or without treatment using the small molecule Mitochondrial Division Inhibitor-1 (mdivi-1), a putative inhibitor of the mitochondrial fission Dynamin-Related Protein-1 (Drp1). We show here that mdivi-1 reduced neurodegeneration, α-syn aggregates and normalized motor function. Mechanistically, mdivi-1 reduced mitochondrial fragmentation, mitochondrial dysfunction and oxidative stress. These in vivo results support the negative role of mutant α-syn in mitochondrial function and indicate that mdivi-1 has a high therapeutic potential for PD.
Asunto(s)
Dinámicas Mitocondriales/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Quinazolinonas/farmacología , Degeneración Estriatonigral/tratamiento farmacológico , alfa-Sinucleína/genética , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Dinaminas/antagonistas & inhibidores , Dinaminas/genética , Dinaminas/metabolismo , Expresión Génica , Inyecciones Intraperitoneales , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Actividad Motora/efectos de los fármacos , Mutación , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson Secundaria/genética , Enfermedad de Parkinson Secundaria/metabolismo , Enfermedad de Parkinson Secundaria/patología , Porción Compacta de la Sustancia Negra/efectos de los fármacos , Porción Compacta de la Sustancia Negra/metabolismo , Porción Compacta de la Sustancia Negra/patología , Agregado de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Degeneración Estriatonigral/genética , Degeneración Estriatonigral/metabolismo , Degeneración Estriatonigral/patología , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
To investigate whether the endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) contributes to the death of dopamine neurons in Parkinson's disease, we undertook a genetic and a pharmacological approach using NOP receptor knockout (NOP(-/-)) mice, and the selective and potent small molecule NOP receptor antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). Stereological unbiased methods were used to estimate the total number of dopamine neurons in the substantia nigra of i) NOP(-/-) mice acutely treated with the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), ii) naïve mice subacutely treated with MPTP, alone or in combination with SB-612111, iii) rats injected with a recombinant adeno-associated viral (AAV) vector overexpressing human mutant p.A53T α-synuclein, treated with vehicle or SB-612111. NOP(-/-) mice showed a 50% greater amount of nigral dopamine neurons spared in response to acute MPTP compared to controls, which was associated with a milder motor impairment. SB-612111, given 4 days after MPTP treatment to mimic the clinical condition, prevented the loss of nigral dopamine neurons and striatal dopaminergic terminals caused by subacute MPTP. SB-612111, administered a week after the AAV injections in a clinically-driven protocol, also increased by 50% both the number of spared nigral dopamine neurons and striatal dopamine terminals, and prevented accompanying motor deficits induced by α-synuclein. We conclude that endogenous N/OFQ contributes to dopamine neuron loss in pathogenic and etiologic models of Parkinson's disease through NOP receptor-mediated mechanisms. NOP receptor antagonists might prove effective as disease-modifying agents in Parkinson's disease, through the rescue of degenerating nigral dopamine neurons and/or the protection of the healthy ones.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Receptores Opioides/metabolismo , Sustancia Negra/metabolismo , Sustancia Negra/patología , Animales , Cicloheptanos/administración & dosificación , Neuronas Dopaminérgicas/efectos de los fármacos , Eliminación de Gen , Locomoción/efectos de los fármacos , Intoxicación por MPTP , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Antagonistas de Narcóticos/administración & dosificación , Trastornos Parkinsonianos/genética , Piperidinas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptores Opioides/genética , Sustancia Negra/efectos de los fármacos , Receptor de NociceptinaRESUMEN
Long-term l-3,4-dihydroxyphenylalanine (L-Dopa) treatment in Parkinson's disease leads to involuntary movements called dyskinesia, notably through an overexpression of immediate-early genes (IEG). Their rapid transcription involves the stalling of RNA polymerase II on IEG promoters, a mechanism that critically depends on the presence of the negative elongation factor (NELF) protein complex. We here down-regulated the key NELF-E subunit using lentiviral vector delivery of a short hairpin RNA in the striatum of 6-hydroxydopamine lesioned rats. Such NELF-E reduced expression significantly attenuated the development of abnormal involuntary movements in response to chronic L-Dopa treatment. Effectiveness of silencing was demonstrated by the significant decrease in striatal ∆FosB, ARC and Zif268 IEG expression. Repression of NELF-mediating RNA polymerase II stalling thus achieves both antidyskinetic and potentiation of antiparkinsonian L-Dopa effect, highlighting the role of transcriptional events in dyskinesia establishment, acute dyskinetic manifestation and in the therapeutic response to L-Dopa.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Discinesia Inducida por Medicamentos/metabolismo , Levodopa/toxicidad , ARN Polimerasa II/metabolismo , Factores de Transcripción/metabolismo , Animales , Antiparkinsonianos/toxicidad , Técnicas de Cultivo de Célula , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ratas Sprague-Dawley , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: Recent findings have shown that pharmacogenetic manipulations of the Ras-ERK pathway provide a therapeutic means to tackle l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID). First, we investigated whether a prolonged l-DOPA treatment differentially affected ERK signaling in medium spiny neurons of the direct pathway (dMSNs) and in cholinergic aspiny interneurons (ChIs) and assessed the role of Ras-GRF1 in both subpopulations. Second, using viral-assisted technology, we probed Ras-GRF1 and Ras-GRF2 as potential targets in this pathway. We investigated how selective blockade of striatal Ras-GRF1 or Ras-GRF2 expression impacted on LID (induction, maintenance, and reversion) and its neurochemical correlates. METHODS: We used both Ras-GRF1 knockout mice and lentiviral vectors (LVs) delivering short-hairpin RNA sequences (shRNAs) to obtain striatum-specific gene knockdown of Ras-GRF1 and Ras-GRF2. The consequences of these genetic manipulations were evaluated in the 6-hydroxydopamine mouse model of Parkinson's disease. Escalating doses of l-DOPA were administered and then behavioral analysis with immunohistochemical assays and in vivo microdialysis were performed. RESULTS: Ras-GRF1 was found essential in controlling ERK signaling in dMSNs, but its ablation did not prevent ERK activation in ChIs. Moreover, striatal injection of LV-shRNA/Ras-GRF1 attenuated dyskinesia development and ERK-dependent signaling, whereas LV-shRNA/Ras-GRF2 was without effect, ruling out the involvement of Ras-GRF2 in LID expression. Accordingly, Ras-GRF1 but not Ras-GRF2 striatal gene-knockdown reduced l-DOPA-induced GABA and glutamate release in the substantia nigra pars reticulata, a neurochemical correlate of dyskinesia. Finally, inactivation of Ras-GRF1 provided a prolonged anti-dyskinetic effect for up to 7 weeks and significantly attenuated symptoms in animals with established LID. INTERPRETATION: Our results suggest that Ras-GRF1 is a promising target for LID therapy based on Ras-ERK signaling inhibition in the striatum.
RESUMEN
INTRODUCTION: Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons as well as the presence of proteinaceous inclusions named Lewy bodies. α-synuclein (α-syn) is a major constituent of Lewy bodies, and the first disease-causing protein characterized in PD. Several α-syn-based animal models of PD have been developed to investigate the pathophysiology of PD, but none of them recapitulate the full picture of the disease. Ageing is the most compelling and major risk factor for developing PD but its impact on α-syn toxicity remains however unexplored. In this study, we developed and exploited a recombinant adeno-associated viral (AAV) vector of serotype 9 overexpressing mutated α-syn to elucidate the influence of ageing on the dynamics of PD-related neurodegeneration associated with α-syn pathology in different mammalian species. RESULTS: Identical AAV pseudotype 2/9 vectors carrying the DNA for human mutant p.A53T α-syn were injected into the substantia nigra to induce neurodegeneration and synucleinopathy in mice, rats and monkeys. Rats were used first to validate the ability of this serotype to replicate α-syn pathology and second to investigate the relationship between the kinetics of α-syn-induced nigrostriatal degeneration and the progressive onset of motor dysfunctions, strikingly reminiscent of the impairments observed in PD patients. In mice, AAV2/9-hα-syn injection into the substantia nigra was associated with accumulation of α-syn and phosphorylated hα-syn, regardless of mouse strain. However, phenotypic mutants with either accelerated senescence or resistance to senescence did not display differential susceptibility to hα-syn overexpression. Of note, p-α-syn levels correlated with nigrostriatal degeneration in mice. In monkeys, hα-syn-induced degeneration of the nigrostriatal pathway was not affected by the age of the animals. Unlike mice, monkeys did not exhibit correlations between levels of phosphorylated α-syn and neurodegeneration. CONCLUSIONS: In conclusion, AAV2/9-mediated hα-syn induces robust nigrostriatal neurodegeneration in mice, rats and monkeys, allowing translational comparisons among species. Ageing, however, neither exacerbated nigrostriatal neurodegeneration nor α-syn pathology per se. Our unprecedented multi-species investigation thus favours the multiple-hit hypothesis for PD wherein ageing would merely be an aggravating, additive, factor superimposed upon an independent disease process.
Asunto(s)
Envejecimiento , Intoxicación por MPTP/patología , Degeneración Estriatonigral/patología , Sustancia Negra/metabolismo , alfa-Sinucleína/metabolismo , Animales , Fenómenos Biomecánicos , Callithrix , Modelos Animales de Enfermedad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Intoxicación por MPTP/inducido químicamente , Ratones , Actividad Motora , Análisis de Componente Principal , Desempeño Psicomotor/fisiología , Ratas , Degeneración Estriatonigral/etiología , Factores de Tiempo , Transducción Genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Parkinson's disease (PD) is characterized by severe locomotor deficits and is commonly treated with the dopamine (DA) precursor l-3,4-dihydroxyphenylalanine (L-DOPA), but its prolonged use causes dyskinesias referred to as L-DOPA-induced dyskinesias (LIDs). Recent studies in animal models of PD have suggested that dyskinesias are associated with the overactivation of G protein-mediated signaling through DA receptors. ß-Arrestins desensitize G protein signaling at DA receptors (D1R and D2R) in addition to activating their own G protein-independent signaling events, which have been shown to mediate locomotion. Therefore, targeting ß-arrestins in PD L-DOPA therapy might prove to be a desirable approach. Here we show in a bilateral DA-depletion mouse model of Parkinson's symptoms that genetic deletion of ß-arrestin2 significantly limits the beneficial locomotor effects while markedly enhancing the dyskinesia-like effects of acute or chronic L-DOPA treatment. Viral rescue or overexpression of ß-arrestin2 in knockout or control mice either reverses or protects against LIDs and its key biochemical markers. In other more conventional animal models of DA neuron loss and PD, such as 6-hydroxydopamine-treated mice or rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated nonhuman primates, ß-arrestin2 overexpression significantly reduced dyskinesias while maintaining the therapeutic effect of L-DOPA. Considerable efforts are being spent in the pharmaceutical industry to identify therapeutic approaches to block LIDs in patients with PD. Our results point to a potential therapeutic approach, whereby development of either a genetic or pharmacological intervention to enhance ß-arrestin2- or limit G protein-dependent D1/D2R signaling could represent a more mechanistically informed strategy.
Asunto(s)
Arrestinas/metabolismo , Discinesias/metabolismo , Levodopa/química , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/química , Animales , Arrestinas/genética , Conducta Animal , Modelos Animales de Enfermedad , Dopamina/metabolismo , Discinesia Inducida por Medicamentos/metabolismo , Eliminación de Gen , Macaca , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Oxidopamina/química , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Regulación hacia Arriba , beta-ArrestinasRESUMEN
Among the mechanisms underlying the development of L-dopa-induced dyskinesia (LID) in Parkinson's disease, complex alterations in dopamine signaling in D1 receptor (D1R)-expressing medium spiny striatal neurons have been unraveled such as, but not limited to, dysregulation of D1R expression, lateral diffusion, intraneuronal trafficking, subcellular localization and desensitization, leading to a pathological anchorage of D1R at the plasma membrane. Such anchorage is partly due to a decreased proteasomal activity that is specific of the L-dopa-exposed dopamine-depleted striatum, results from D1R activation and feeds-back the D1R exaggerated cell surface abundance. The precise mechanisms by which L-dopa affects striatal proteasome activity remained however unknown. We here show, in a series of in vitro ex vivo and in vivo models, that such rapid modulation of striatal proteasome activity intervenes through D1R-mediated disassembly of the 26S proteasome rather than change in transcription or translation of proteasome or proteasome subunits intraneuronal relocalization.