RESUMO
Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to increase the lesion in mice and follow up in vivo expression. To this end, we developed a bioluminescent expression reporter of the human A53T-α-synuclein gene using the NanoLuc system into an AAV2/9, embedded or not in a fibroin solution to stabilise its expression in space and time. We first verified the expression of the fused protein in vitro on transfected cells by bioluminescence and Western blotting. Next, two groups of C57Bl6Jr mice were unilaterally injected with the AAV-NanoLuc-human-A53T-α-synuclein above the substantia nigra combined (or not) with fibroin. We first show that the in vivo cerebral bioluminescence signal was more intense in the presence of fibroin. Using immunohistochemistry, we find that the human-A53T-α-synuclein protein is more restricted to the ipsilateral side with an overall greater magnitude of the lesion when fibroin was added. However, we also detected a bioluminescence signal in peripheral organs in both conditions, confirmed by the presence of viral DNA corresponding to the injected AAV in the liver using qPCR.
Assuntos
Dependovirus , Fibroínas , Vetores Genéticos , Medições Luminescentes , Camundongos Endogâmicos C57BL , alfa-Sinucleína , Animais , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Dependovirus/genética , Humanos , Camundongos , Medições Luminescentes/métodos , Vetores Genéticos/genética , Fibroínas/metabolismo , Sistema Nervoso Central/metabolismo , Masculino , Luciferases/metabolismo , Luciferases/genéticaRESUMO
Loss of dopaminergic midbrain neurons perturbs l-serine and d-serine homeostasis in the post-mortem caudate putamen (CPu) of Parkinson's disease (PD) patients. However, it is unclear whether the severity of dopaminergic nigrostriatal degeneration plays a role in deregulating serine enantiomers' metabolism. Here, through high-performance liquid chromatography (HPLC), we measured the levels of these amino acids in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys and MPTP-plus-probenecid (MPTPp)-treated mice to determine whether and how dopaminergic midbrain degeneration affects the levels of serine enantiomers in various basal ganglia subregions. In addition, in the same brain regions, we measured the levels of key neuroactive amino acids modulating glutamatergic neurotransmission, including l-glutamate, glycine, l-aspartate, d-aspartate, and their precursors l-glutamine, l-asparagine. In monkeys, MPTP treatment produced severe denervation of nigrostriatal dopaminergic fibers (â75%) and increased the levels of serine enantiomers in the rostral putamen (rPut), but not in the subthalamic nucleus, and the lateral and medial portion of the globus pallidus. Moreover, this neurotoxin significantly reduced the protein expression of the astrocytic serine transporter ASCT1 and the glycolytic enzyme GAPDH in the rPut of monkeys. Conversely, concentrations of d-serine and l-serine, as well as ASCT1 and GAPDH expression were unaffected in the striatum of MPTPp-treated mice, which showed only mild dopaminergic degeneration (â30%). These findings unveil a link between the severity of dopaminergic nigrostriatal degeneration and striatal serine enantiomers concentration, ASCT1 and GAPDH expression. We hypothesize that the up-regulation of d-serine and l-serine levels occurs as a secondary response within a homeostatic loop to support the metabolic and neurotransmission demands imposed by the degeneration of dopaminergic neurons.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Serina , Camundongos , Animais , Serina/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Dopamina/metabolismo , Corpo Estriado/metabolismo , Mesencéfalo/metabolismo , Aminoácidos/metabolismo , Putamen/metabolismo , HomeostaseRESUMO
Adeno-associated virus (AAV) vectors are increasingly used as an effective and safe approach to deliver genetic material to the central nervous system (CNS). The AAV9-derived variants, AAV-PHP. B and AAV-PHP.eB, reportedly broadly transduce cells throughout the CNS compared to the original serotype 9, AAV9. As non-human primate data are scarce, we here evaluated the CNS transduction efficiencies after lumbar intrathecal bolus delivery of identical doses of either AAV-PHP. B:CAG-EGFP or AAV-PHP. eB:CAG-EGFP in rhesus macaque monkeys. AAV-PHP.eB achieved a more efficient and widespread CNS transduction compared to AAV-PHP.B. We report a strong neuronal and oligodendroglial tropism for both variants in the putamen and in the hippocampus. This proof-of-concept experiment highlights the potential value of intrathecal infusions of AAV-PHP.eB to distribute genetic material in the CNS with cell-type specificity and introduces a new opportunity to model brain diseases in rhesus macaque monkeys and further develop gene therapies targeting the CNS in humans.
RESUMO
With the emergence of disease-modifying therapies for Parkinson's disease, reliable longitudinal markers are needed to quantify pathology and demonstrate disease progression. We developed the A53T-AAV rat model of synucleinopathy by combining longitudinal measures over 12 weeks. We first characterized the progression of the motor and dopaminergic deficits. Then, we monitored the disease progression using the [18F]FMT Positron Emission Tomography (PET) radiotracer. The nigral injection of A53T-AAV led to an increase in phosphorylated α-synuclein on S129, a progressive accumulation of α-synuclein aggregates, and a decrease of dopaminergic function associated with a deterioration of motor activity. The longitudinal monitoring of A53T-AAV rats with [18F]FMT PET showed a progressive reduction of the Kc outcome parameter in the caudate putamen from the lesioned side. Interestingly, the progressive reduction in the [18F]FMT PET signal correlated with defects in the stepping test. In conclusion, we established a progressive rat model of α-synuclein pathology which monitors the deficit longitudinally using both the [18F]FMT PET tracer and behavioral parameters, 2 features that have strong relevance for translational approaches.
Assuntos
Dependovirus , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/fisiologia , Atividade Motora , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/fisiopatologia , Sinucleinopatias/diagnóstico por imagem , Sinucleinopatias/fisiopatologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Radioisótopos de Flúor , Masculino , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação , Tomografia por Emissão de Pósitrons , Agregados Proteicos , Ratos Sprague-Dawley , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , Tirosina , alfa-Sinucleína/metabolismoRESUMO
Adenosine A1/A2A receptors (A1R/A2AR) represent targets in nondopaminergic treatment of motor disorders such as Parkinson's disease (PD). As an innovative strategy, multitargeting ligands (MTLs) were developed to achieve comprehensive PD therapies simultaneously addressing comorbid symptoms such as sleep disruption. Recognizing the wake-promoting capacity of histamine H3 receptor (H3R) antagonists in combination with the "caffeine-like effects" of A1R/A2AR antagonists, we designed A1R/A2AR/H3R MTLs, where a piperidino-/pyrrolidino(propyloxy)phenyl H3R pharmacophore was introduced with overlap into an adenosine antagonist arylindenopyrimidine core. These MTLs showed distinct receptor binding profiles with overall nanomolar H3R affinities (Ki < 55 nM). Compound 4 (ST-2001, Ki (A1R) = 11.5 nM, Ki (A2AR) = 7.25 nM) and 12 (ST-1992, Ki (A1R) = 11.2 nM, Ki (A2AR) = 4.01 nM) were evaluated in vivo. l-DOPA-induced dyskinesia was improved after administration of compound 4 (1 mg kg-1, i.p. rats). Compound 12 (2 mg kg-1, p.o. mice) increased wakefulness representing novel pharmacological tools for PD therapy.
Assuntos
Antagonistas do Receptor A1 de Adenosina/uso terapêutico , Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Antagonistas dos Receptores Histamínicos H3/uso terapêutico , Doença de Parkinson Secundária/tratamento farmacológico , Antagonistas do Receptor A1 de Adenosina/síntese química , Antagonistas do Receptor A1 de Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/síntese química , Antagonistas do Receptor A2 de Adenosina/metabolismo , Animais , Discinesias/tratamento farmacológico , Antagonistas dos Receptores Histamínicos H3/síntese química , Antagonistas dos Receptores Histamínicos H3/metabolismo , Humanos , Levodopa/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Oxidopamina , Doença de Parkinson Secundária/induzido quimicamente , Piperidinas/síntese química , Piperidinas/metabolismo , Piperidinas/uso terapêutico , Pirimidinas/síntese química , Pirimidinas/metabolismo , Pirimidinas/uso terapêutico , Pirrolidinas/síntese química , Pirrolidinas/metabolismo , Pirrolidinas/uso terapêutico , Ratos Sprague-Dawley , Receptor A2A de Adenosina/metabolismo , Receptores Histamínicos H3/metabolismo , Vigília/efeitos dos fármacosRESUMO
Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) is thought to enhance transgene expression of target genes delivered by adeno-associated viral (AAV) vectors. This study assessed the protein expression of α-synuclein, phosphorylated α-synuclein at Serine 129, extent of nigrostriatal degeneration as well as subsequent behavioral deficits induced by unilateral intranigral stereotactic injection in male adult C57BL/6J mice of an AAV2/9 expressing A53T human α-synuclein under the control of the synapsin promoter in presence or absence of the WPRE. The presence of WPRE enabled to achieve greater nigrostriatal degeneration and synucleinopathy which was concomitant with worsened forelimb use asymmetry. This work refines a mouse Parkinson's disease model in which anatomo-pathology is related to behavioral deficits.
RESUMO
BACKGROUND: Autophagy is intensively studied in cancer, metabolic and neurodegenerative diseases, but little is known about its role in pathological conditions linked to altered neurotransmission. We examined the involvement of autophagy in levodopa (l-dopa)-induced dyskinesia, a frequent motor complication developed in response to standard dopamine replacement therapy in parkinsonian patients. METHODS: We used mouse and non-human primate models of Parkinson's disease to examine changes in autophagy associated with chronic l-dopa administration and to establish a causative link between impaired autophagy and dyskinesia. RESULTS: We found that l-dopa-induced dyskinesia is associated with accumulation of the autophagy-specific substrate p62, a marker of autophagy deficiency. Increased p62 was observed in a subset of projection neurons located in the striatum and depended on l-dopa-mediated activation of dopamine D1 receptors, and mammalian target of rapamycin. Inhibition of mammalian target of rapamycin complex 1 with rapamycin counteracted the impairment of autophagy produced by l-dopa, and reduced dyskinesia. The anti-dyskinetic effect of rapamycin was lost when autophagy was constitutively suppressed in D1 receptor-expressing striatal neurons, through inactivation of the autophagy-related gene protein 7. CONCLUSIONS: These findings indicate that augmented responsiveness at D1 receptors leads to dysregulated autophagy, and results in the emergence of l-dopa-induced dyskinesia. They further suggest the enhancement of autophagy as a therapeutic strategy against dyskinesia. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Discinesia Induzida por Medicamentos , Transtornos Parkinsonianos , Animais , Antiparkinsonianos/toxicidade , Autofagia , Corpo Estriado , Modelos Animais de Doenças , Discinesia Induzida por Medicamentos/tratamento farmacológico , Discinesia Induzida por Medicamentos/etiologia , Humanos , Levodopa/toxicidade , Camundongos , OxidopaminaRESUMO
Multiple system atrophy (MSA) is a rare and extremely debilitating progressive neurodegenerative disease characterized by variable combinations of parkinsonism, cerebellar ataxia, dysautonomia, and pyramidal dysfunction. MSA is a unique synucleinopathy, in which alpha synuclein-rich aggregates are present in the cytoplasm of oligodendroglia. The precise origin of the alpha synuclein (aSyn) found in the glial cytoplasmic inclusions (GCIs) as well the mechanisms of neurodegeneration in MSA remain unclear. Despite this fact, cell and animal models of MSA rely on oligodendroglial overexpression of aSyn. In the present study, we utilized a novel oligotrophic AAV, Olig001, to overexpress aSyn specifically in striatal oligodendrocytes of rats and nonhuman primates in an effort to further characterize our novel viral vector-mediated MSA animal models. Using two cohorts of animals with 10-fold differences in Olig001 vector titers, we show a dose-dependent formation of MSA-like pathology in rats. High titer of Olig001-aSyn in these animals were required to produce the formation of pS129+ and proteinase K resistant aSyn-rich GCIs, demyelination, and neurodegeneration. Using this knowledge, we injected high titer Olig001 in the putamen of cynomolgus macaques. After six months, histological analysis showed that oligodendroglial overexpression of aSyn resulted in the formation of hallmark GCIs throughout the putamen, demyelination, a 44% reduction of striatal neurons and a 12% loss of nigral neurons. Furthermore, a robust inflammatory response similar to MSA was produced in Olig001-aSyn NHPs, including microglial activation, astrogliosis, and a robust infiltration of T cells into the CNS. Taken together, oligodendroglial-specific viral vector-mediated overexpression of aSyn in rats and nonhuman primates faithfully reproduces many of the pathological disease hallmarks found in MSA. Future studies utilizing these large animal models of MSA would prove extremely valuable as a pre-clinical platform to test novel therapeutics that are so desperately needed for MSA.
Assuntos
Modelos Animais de Doenças , Atrofia de Múltiplos Sistemas/genética , Neostriado/patologia , Neurônios/patologia , Oligodendroglia/patologia , Putamen/patologia , alfa-Sinucleína/genética , Animais , Dependovirus , Vetores Genéticos , Humanos , Macaca fascicularis , Atrofia de Múltiplos Sistemas/metabolismo , Atrofia de Múltiplos Sistemas/patologia , Oligodendroglia/metabolismo , Ratos , Técnicas Estereotáxicas , alfa-Sinucleína/metabolismoRESUMO
In order to model various aspects of Huntington's disease (HD) pathology, in particular protein spread, we administered adeno-associated virus (AAV) expressing green fluorescent protein (GFP) or GFP coupled to HTT-Exon1 (19Q or 103Q) to the central nervous system of adult wild-type (WT) mice and non-human primates. All animals underwent behavioral testing and post-mortem analyses to determine the long-term consequences of AAV injection. Both mice and non-human primates demonstrated behavioral changes at 2-3 weeks post-surgery. In mice, these changes were absent after 3 months while in non-human primates, they persisted in the majority of tested animals. Post-mortem analysis revealed that spreading of the aggregates was limited, although the virus did spread between synaptically-connected brain regions. Despite circumscribed spreading, the presence of mHTT generated changes in endogenous huntingtin (HTT) levels in both models. Together, these results suggest that viral expression of mHTTExon1 can induce spreading and seeding of HTT in both mice and non-human primates.
Assuntos
Dependovirus/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Agregação Patológica de Proteínas , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Humanos , Macaca mulatta , Masculino , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Multiple system atrophy (MSA) is a rare, untreatable neurodegenerative disorder characterized by accumulation of α-synuclein in oligodendroglial inclusions. As such, MSA is a synucleinopathy along with Parkinson's disease (PD) and dementia with Lewy bodies. Activation of the abelson tyrosine kinase c-Abl leads to phosphorylation of α-synuclein at tyrosine 39, thereby promoting its aggregation and subsequent neurodegeneration. The c-Abl inhibitor nilotinib used for the treatment of chronic myeloid leukemia based on data collected in preclinical models of PD might interfere with pathogenic mechanisms that are relevant to PD and dementia with Lewy bodies, which motivated its assessment in an open-label clinical trial in PD and dementia with Lewy bodies patients. The objective of this study was to assess the preclinical efficacy of nilotinib in the specific context of MSA. METHODS: Mice expressing human wild-type α-synuclein in oligodendrocytes received daily injection of nilotinib (1 or 10 mg/kg) over 12 weeks. Postmortem analysis included the assessment of c-Abl activation, α-synuclein burden, and dopaminergic neurodegeneration. RESULTS: α-Synuclein phosphorylated at tyrosine 39 was detected in glial cytoplasmic inclusions in MSA patients. Increased activation of c-Abl and α-synuclein phosphorylation at tyrosine 39 were found in transgenic mice. Despite significant inhibition of c-Abl and associated reduction of α-synuclein phosphorylation at tyrosine 39 by 40%, nilotinib failed to reduce α-synuclein aggregate burden (including phosphorylation at serine 129) in the striatum and cortex or to lessen neurodegeneration in the substantia nigra. CONCLUSIONS: This preclinical study suggests that partial inhibition of c-Abl and reduction of α-synuclein phosphorylation at tyrosine 39 may not be a relevant target for MSA. © 2020 International Parkinson and Movement Disorder Society.
Assuntos
Atrofia de Múltiplos Sistemas , Sinucleinopatias , Animais , Encéfalo/metabolismo , Humanos , Camundongos , Atrofia de Múltiplos Sistemas/tratamento farmacológico , Pirimidinas , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
The synucleinopathies Parkinson's disease (PD) and Multiple system atrophy (MSA) - characterized by α-synuclein intracytoplasmic inclusions into, respectively, neurons and oligodendrocytes - are associated with impairment of the autophagy-lysosomal pathways (ALP). Increased expression of the master regulator of ALP, transcription factor EB (TFEB), is hypothesized to promote the clearance of WT α-synuclein and survival of dopaminergic neurons. Here, we explore the efficacy of targeted TFEB overexpression either in neurons or oligodendrocytes to reduce the pathological burden of α-synuclein in a PD rat model and a MSA mouse model. While TFEB neuronal expression was sufficient to prevent neurodegeneration in the PD model, we show that only TFEB oligodendroglial overexpression leads to neuroprotective effects in the MSA model. These beneficial effects were associated with a decreased accumulation of α-synuclein into oligodendrocytes through recovery of the ALP machinery. Our study demonstrates that the cell type where α-synuclein aggregates dictates the target of TFEB overexpression in order to be protective, paving the way for adapted therapies.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Atrofia de Múltiplos Sistemas/patologia , Doença de Parkinson/patologia , Idoso , Animais , Autofagia , Encéfalo/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atrofia de Múltiplos Sistemas/metabolismo , Oligodendroglia/metabolismo , Doença de Parkinson/metabolismo , Ratos , Ratos Sprague-Dawley , alfa-Sinucleína/metabolismoRESUMO
Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These side effects, which are caused by the excessive swings in striatal dopamine caused by intermittent oral delivery, can be avoided by delivering l-DOPA in a more continuous manner. Local gene delivery of the l-DOPA synthesizing enzymes, tyrosine hydroxylase and guanosine-tri-phosphate-cyclohydrolase-1, offers a new approach to a more refined dopaminergic therapy where l-DOPA is delivered continuously at the site where it is needed i.e. the striatum. In this study we have explored the therapeutic efficacy of adeno-associated viral vector-mediated l-DOPA delivery to the putamen in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys, the standard non-human primate model of Parkinson's disease. Viral vector delivery of the two enzymes, tyrosine hydroxylase and guanosine-5'-tri-phosphate-cyclohydrolase-1, bilaterally into the dopamine-depleted putamen, induced a significant, dose-dependent improvement of motor behaviour up to a level identical to that obtained with the optimal dose of peripheral l-DOPA. Importantly, this improvement in motor function was obtained without any adverse dyskinetic effects. These results provide proof-of-principle for continuous vector-mediated l-DOPA synthesis as a novel therapeutic strategy for Parkinson's disease. The constant, local supply of l-DOPA obtained with this approach holds promise as an efficient one-time treatment that can provide long-lasting clinical improvement and at the same time prevent the appearance of motor fluctuations and dyskinetic side effects associated with standard oral dopaminergic medication.
Assuntos
Antiparkinsonianos/administração & dosagem , GTP Cicloidrolase/administração & dosagem , Vetores Genéticos/uso terapêutico , Levodopa/biossíntese , Transtornos Parkinsonianos/terapia , Putamen/metabolismo , Tirosina 3-Mono-Oxigenase/administração & dosagem , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/análogos & derivados , Animais , Antiparkinsonianos/uso terapêutico , Dependovirus/genética , Avaliação Pré-Clínica de Medicamentos , Feminino , GTP Cicloidrolase/análise , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Genes Reporter , Genes Sintéticos , Vetores Genéticos/administração & dosagem , Humanos , Macaca mulatta , Masculino , Atividade Motora/efeitos dos fármacos , Transtornos Parkinsonianos/induzido quimicamente , Parte Compacta da Substância Negra/química , Parte Compacta da Substância Negra/patologia , Estudo de Prova de Conceito , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/análise , Proteínas Recombinantes/uso terapêutico , Tirosina 3-Mono-Oxigenase/análise , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Most neurodegenerative disorders are characterized by deposits of misfolded proteins and neuronal degeneration in specific brain regions. Growing evidence indicates that lysosomal impairment plays a primary pathogenic role in these diseases, in particular, the occurrence of increased lysosomal pH. Thus, therapeutic development aiming at restoring lysosomal function represents a novel, precise, and promising strategy for the treatment of these pathologies. Herein we demonstrate that acidic oil-in-water nanoemulsions loaded with poly(dl-lactide- co-glycolide) (PLGA) are able to rescue impaired lysosomal pH in genetic cellular models of Parkinson's disease. For in vivo assays, nanoemulsions were labeled with an original synthetic hydrophobic far red-emitting dye to allow fluorescence monitoring. Following stereotaxic injection in the mouse brain, widespread diffusion of the nanocarrier was observed, up to 500 µm from the injection site, as well as internalization into the lysosomal compartment in brain cells. Finally, promising preliminary assays of systemic administration demonstrate that a fraction of the formulation crosses the blood brain barrier, penetrates the brain parenchyma, is internalized by cells, and colocalizes with lysosomal markers. Overall, these results suggest the feasibility and the therapeutic potential of this new nanoformulation as an effective drug delivery tool to the brain, with the potential to rescue pathological lysosomal deficits.
Assuntos
Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Nanopartículas , Doenças Neurodegenerativas/tratamento farmacológico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/uso terapêutico , Animais , Barreira Hematoencefálica , Linhagem Celular Tumoral , Portadores de Fármacos , Emulsões , Endocitose , Humanos , Camundongos , Doenças Neurodegenerativas/metabolismo , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/farmacocinéticaRESUMO
Fibrillization of α-synuclein is instrumental for the development of Parkinson's disease (PD), thus modulating this process can have profound impact on disease initiation/progression. Here, the impact of the p.G2019S mutation of leucine-rich repeat kinase 2 (LRRK2), which is most frequently associated with familial and sporadic PD, on α-synuclein pathology was investigated. G2019S knock-in mice and wild-type controls were injected with a recombinant adeno-associated viral vector serotype 2/9 (AAV2/9) overexpressing human mutant p.A53T α-synuclein (AAV2/9-hα-syn). Control animals were injected with AAV2/9 carrying green fluorescent protein. Motor behavior, transgene expression, α-syn and pSer129 α-syn load, number of nigral dopamine neurons and density of striatal dopaminergic terminals were evaluated. To investigate the effect of aging, experiments were performed in 3- and 12-month-old mice, evaluated 20 and 12â¯weeks after virus injection, respectively. hα-syn overexpression induced progressive motor deficits, loss of nigral dopaminergic neurons and striatal terminals, and appearance of proteinase K-resistant aggregates of pSer129 α-syn in both young and old mice. Although no genotype difference was observed in 3-month-old mice, degeneration of nigral dopaminergic neurons was higher in 12-month-old G2019S knock-in mice compared with age-matched wild-type controls (-55% vs -39%, respectively). Consistently, a two-fold higher load of pSer129 α-syn aggregates was found in 12-month-old G2019S knock-in mice. We conclude that G2019S LRRK2 facilitates α-synucleinopathy and degeneration of nigral dopaminergic neurons, and that aging is a major determinant of this effect.
Assuntos
Envelhecimento/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Mutação/genética , alfa-Sinucleína/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Técnicas de Introdução de Genes/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/metabolismoRESUMO
Recombinant adeno-associated virus serotype 9 (rAAV2/9) and pseudotype rhesus-10 (rAAV2/rh10) are used for gene delivery, especially into the central nervous system. Both serotypes cross the blood-brain barrier and mediate stable long-term transduction in dividing and nondividing cells. Among possible routes of administration, intracardiac injection holds the potential for widespread vector diffusion associated with a relatively simple approach. In this study adopting the intracardiac route, we compare the cell-specific tropism and transfection efficacy of a panel of engineered rAAV2/9 and rAAV2/rh10 vectors encoding the enhanced green fluorescent protein. We observed transduction in the brain and peripherally, with a predominant neuronal tropism while the various serotypes achieved different expression patterns.
Assuntos
Técnicas de Transferência de Genes/normas , Terapia Genética/métodos , Vetores Genéticos/genética , Miocárdio/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Técnicas de Transferência de Genes/efeitos adversos , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Ratos , Ratos Sprague-Dawley , SorogrupoRESUMO
Eltoprazine, a serotonergic (5-HT)1A/B receptor agonist, is a potential treatment for L-DOPA-induced dyskinesia (LID) in Parkinson's disease (PD) but notably compromises the anti-parkinsonian effects of L-DOPA, as seen in rodent and monkey models of PD. Preladenant, a selective adenosine A2a receptor antagonist, mediates modest anti-parkinsonian effects in parkinsonian monkeys. In a recent investigation, combined eltoprazine and preladenant treatment with a sub-threshold dose of L-DOPA acutely attenuated dyskinesia without exacerbating PD disability in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated macaques. The aim of this study was to investigate the daily repeated treatment effects of eltoprazine (1mg/kg) alone, and in combination with preladenant (5mg/kg), on the motor symptoms of PD and LID in MPTP-treated macaques. The anti-dyskinetic and -parkinsonian effects of combinative drug administration with a sub-threshold dose of L-DOPA were measured over 14 days. Eltoprazine treatment alone produced a near-complete suppression of dyskinesia but consistently increased parkinsonism. The administration of preladenant with eltoprazine prevented the increased severity of parkinsonian motor symptoms but was unable to maintain a reduced expression of dyskinesia with repeated administration. These data demonstrate the clinical utility of the modulation of the serotonergic and adenosine neurotransmitter systems with selective pharmacological agents for only acute treatment of LID. This multi-targeted approach is unsuitable as a long-term treatment regimen due to unsustainable therapeutic effects on dyskinesia.
Assuntos
Antiparkinsonianos/efeitos adversos , Discinesia Induzida por Medicamentos/tratamento farmacológico , Levodopa/efeitos adversos , Doença de Parkinson/tratamento farmacológico , Piperazinas/farmacologia , Animais , Antiparkinsonianos/uso terapêutico , Comportamento Animal/efeitos dos fármacos , Interações Medicamentosas , Discinesia Induzida por Medicamentos/metabolismo , Discinesia Induzida por Medicamentos/fisiopatologia , Feminino , Levodopa/uso terapêutico , Macaca fascicularis , Atividade Motora/efeitos dos fármacos , Doença de Parkinson/complicações , Piperazinas/uso terapêutico , Fatores de TempoRESUMO
BACKGROUND: MSA is a fatal neurodegenerative disorder characterized by a combination of autonomic dysfunction, cerebellar ataxia, and l-dopa unresponsive parkinsonism. The hallmark of MSA is the accumulation of α-synuclein, forming cytoplasmic inclusions in oligodendrocytes. Adeno-associated viruses allow efficient targeting of disease-associated genes in selected cellular ensembles and have proven efficient for the neuronal overexpression of α-synuclein in the substantia nigra in the context of PD. OBJECTIVES: We aimed to develop viral-based models of MSA. METHODS: Chimeric viral vectors expressing either human wild-type α-synuclein or green fluorescent protein under the control of mouse myelin basic protein were injected in the striatum of rats and monkeys. Rats underwent a longitudinal motor assessment before histopathological analysis at 3 and 6 months. RESULTS: Injection of vectors expressing α-synuclein in the striatum resulted in >80% oligodendroglial selectivity in rats and >60% in monkeys. Rats developed progressive motor deficits that were l-dopa unresponsive when assessed at 6 months. Significant loss of dopaminergic neurons occurred at 3 months, further progressing at 6 months, together with a loss of striatal neurons. Prominent α-synuclein accumulation, including phosphorylated and proteinase-K-resistant α-synuclein, was detected in the striatum and substantia nigra. CONCLUSIONS: Viral-mediated oligodendroglial expression of α-synuclein allows replicating some of the key features of MSA. This flexible strategy can be used to investigate, in several species, how α-synuclein accumulation in selected oligodendroglial populations contributes to the pathophysiology of MSA and offers a new framework for preclinical validation of therapeutic strategies. © 2017 International Parkinson and Movement Disorder Society.
Assuntos
Dependovirus/genética , Regulação da Expressão Gênica/genética , Atrofia de Múltiplos Sistemas/genética , Atrofia de Múltiplos Sistemas/patologia , Oligodendroglia/metabolismo , alfa-Sinucleína/metabolismo , Animais , Animais Geneticamente Modificados , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Dopaminérgicos/uso terapêutico , Haplorrinos , Humanos , Levodopa/uso terapêutico , Masculino , Atrofia de Múltiplos Sistemas/etiologia , Proteína Básica da Mielina/imunologia , Proteínas do Tecido Nervoso/metabolismo , Fosforilação/genética , Desempenho Psicomotor/fisiologia , Ratos , Ratos Sprague-Dawley , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/genéticaRESUMO
A whole brain immediate early gene mapping highlighted the dorsolateral bed nucleus of the stria terminalis (dlBST) as a structure putatively involved in L-3,4-dihydroxyphenylalanine (L-Dopa)-induced dyskinesia (LID), the debilitating side-effects of chronic dopamine replacement therapy in Parkinson's disease (PD). dlBST indeed displayed an overexpression of ∆FosB, ARC, Zif268 and FRA2 only in dyskinetic rats. We thus hypothesized that dlBST could play a role in LID hyperkinetic manifestations. To assess the causal role of the dlBST in LID, we used Daun02 inactivation to selectively inhibit the electrical activity of dlBST ΔFosB-expressing neurons. Daun02 is a prodrug converted into Daunorubicin by ß-galactosidase. Then, the newly synthesized Daunorubicin is an inhibitor of neuronal excitability. Therefore, following induction of abnormal involuntary movements (AIMs), 6-OHDA rats were injected with Daun02 in the dlBST previously expressing ß-galactosidase under control of the FosB/ΔFosB promoter. Three days after Daun02 administration, the rats were tested daily with L-Dopa to assess LID. Pharmacogenetic inactivation of ∆FosB-expressing neuron electrophysiological activity significantly reduced AIM severity. The present study highlights the role of dlBST in the rodent analog of LID, offering a new target to investigate LID pathophysiology.
Assuntos
Discinesia Induzida por Medicamentos/genética , Levodopa/toxicidade , Núcleos Septais/metabolismo , Animais , Antiparkinsonianos/uso terapêutico , Antiparkinsonianos/toxicidade , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Daunorrubicina/análogos & derivados , Daunorrubicina/farmacologia , Modelos Animais de Doenças , Discinesia Induzida por Medicamentos/etiologia , Discinesia Induzida por Medicamentos/prevenção & controle , Expressão Gênica/efeitos dos fármacos , Levodopa/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Proteínas Proto-Oncogênicas c-fos/genética , Ratos Sprague-DawleyRESUMO
The therapeutic use of statins has been associated to a reduced risk of Parkinson's disease (PD) and may hold neuroprotective potential by counteracting the degeneration of dopaminergic neurons. Transcriptional activation of the sterol regulatory element-binding protein (SREBP) is one of the major downstream signaling pathways triggered by the cholesterol-lowering effect of statins. In a previous study in neuroblastoma cells, we have shown that statins consistently induce the upregulation of presynaptic dopaminergic proteins and changes of their function and these effects were accompanied by downstream activation of SREBP. In this study, we aimed to determine the direct role of SREBP pathway in the modulation of dopaminergic phenotype. We demonstrate that treatment of SH-SY5Y cells with U18666A, an SREBP activator, increases the translocation of SREBPs into the nucleus, increases the expression of SREBP-1, SREBP-2, and of the presynaptic dopaminergic markers such as vesicular monoamine transporter 2, synaptic vesicle glycoprotein 2 A and 2 C, synaptogyrin-3, and tyrosine hydroxylase. The addition of SREBP inhibitor, PF-429242, blocks the increase of U18666A-induced expression of SREBPs and presynaptic markers. Our results, in line with previously reported effects of statins, demonstrate that direct stimulation of SREBP translocation is associated to differentiation toward a dopaminergic-like phenotype and suggest that SREBP-mediated transcriptional activity may lead to the restoration of the presynaptic dopamine markers and may contribute to neuroprotection of dopaminergic neurons. These findings further support the potential protective role of statin in PD and shed light upon SREBP as a potential new target for developing disease-modifying treatment in PD.
Assuntos
Androstenos/farmacologia , Dopaminérgicos/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Terminações Pré-Sinápticas/efeitos dos fármacos , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Anticolesterolemiantes/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/fisiologia , Pirrolidinas/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Ligação a Elemento Regulador de Esterol/antagonistas & inibidores , Sinaptogirinas/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
See Stayte and Vissel (doi:10.1093/awx064) for a scientific commentary on this article. Multiple system atrophy is a fatal sporadic adult-onset neurodegenerative disorder with no symptomatic or disease-modifying treatment available. The cytopathological hallmark of multiple system atrophy is the accumulation of α-synuclein aggregates in oligodendrocytes, forming glial cytoplasmic inclusions. Impaired insulin/insulin-like growth factor-1 signalling (IGF-1) and insulin resistance (i.e. decreased insulin/IGF-1) have been reported in other neurodegenerative disorders such as Alzheimer's disease. Increasing evidence also suggests impaired insulin/IGF-1 signalling in multiple system atrophy, as corroborated by increased insulin and IGF-1 plasma concentrations in multiple system atrophy patients and reduced IGF-1 brain levels in a transgenic mouse model of multiple system atrophy. We here tested the hypothesis that multiple system atrophy is associated with brain insulin resistance and showed increased expression of the key downstream messenger insulin receptor substrate-1 phosphorylated at serine residue 312 in neurons and oligodendrocytes in the putamen of patients with multiple system atrophy. Furthermore, the expression of insulin receptor substrate 1 (IRS-1) phosphorylated at serine residue 312 was more apparent in inclusion bearing oligodendrocytes in the putamen. By contrast, it was not different between both groups in the temporal cortex, a less vulnerable structure compared to the putamen. These findings suggest that insulin resistance may occur in multiple system atrophy in regions where the neurodegenerative process is most severe and point to a possible relation between α-synuclein aggregates and insulin resistance. We also observed insulin resistance in the striatum of transgenic multiple system atrophy mice and further demonstrate that the glucagon-like peptide-1 analogue exendin-4, a well-tolerated and Federal Drug Agency-approved antidiabetic drug, has positive effects on insulin resistance and monomeric α-synuclein load in the striatum, as well as survival of nigral dopamine neurons. Additionally, plasma levels of exosomal neural-derived IRS-1 phosphorylated at serine residue 307 (corresponding to serine residue 312 in humans) negatively correlated with survival of nigral dopamine neurons in multiple system atrophy mice treated with exendin-4. This finding suggests the potential for developing this peripheral biomarker candidate as an objective outcome measure of target engagement for clinical trials with glucagon-like peptide-1 analogues in multiple system atrophy. In conclusion, our observation of brain insulin resistance in multiple system atrophy patients and transgenic mice together with the beneficial effects of the glucagon-like peptide-1 agonist exendin-4 in transgenic mice paves the way for translating this innovative treatment into a clinical trial.