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1.
Proc Natl Acad Sci U S A ; 117(26): 15209-15220, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32541058

RESUMO

Preclinical assessment of the therapeutic potential of dopamine (DA) neuron replacement in Parkinson's disease (PD) has primarily been performed in the 6-hydroxydopamine toxin model. While this is a good model to assess graft function, it does not reflect the pathological features or progressive nature of the disease. In this study, we establish a humanized transplantation model of PD that better recapitulates the main disease features, obtained by coinjection of preformed human α-synuclein (α-syn) fibrils and adeno-associated virus (AAV) expressing human wild-type α-syn unilaterally into the rat substantia nigra (SN). This model gives rise to DA neuron dysfunction and progressive loss of DA neurons from the SN and terminals in the striatum, accompanied by extensive α-syn pathology and a prominent inflammatory response, making it an interesting and relevant model in which to examine long-term function and integrity of transplanted neurons in a PD-like brain. We transplanted DA neurons derived from human embryonic stem cells (hESCs) into the striatum and assessed their survival, growth, and function over 6 to 18 wk. We show that the transplanted cells, even in the presence of ongoing pathology, are capable of innervating the DA-depleted striatum. However, on closer examination of the grafts, we found evidence of α-syn pathology in the form of inclusions of phosphorylated α-syn in a small fraction of the grafted DA neurons, indicating host-to-graft transfer of α-syn pathology, a phenomenon that has previously been observed in PD patients receiving fetal tissue grafts but has not been possible to demonstrate and study in toxin-based animal models.


Assuntos
Células-Tronco Embrionárias/fisiologia , Transplante de Células-Tronco , Sinucleinopatias , alfa-Sinucleína/metabolismo , Animais , Sobrevivência Celular , Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo , Feminino , Humanos , Inflamação , Degeneração Neural , Ratos , Ratos Sprague-Dawley , Substância Negra/citologia
2.
Neurobiol Dis ; 150: 105255, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33421564

RESUMO

In Parkinson's disease, synucleinopathy is hypothesized to spread from the enteric nervous system, via the vagus nerve, to the central nervous system. Recent evidences collected in non-human primates challenge however the hypothesis of a transmission of α-synuclein (α-syn) pathology through the vagus nerve. Would the hypothesis whereby the bloodstream acts as a route for long-distance transmission of pathological α-syn hold true, an inter-individual transmission of synucleinopathy could occur via blood contact. Here, we used a parabiosis approach to join the circulatory systems of wild type and GFP transgenic C57BL/6 J mice, for which one of the partners parabiont received a stereotaxic intranigral injection of patient-derived α-syn aggregates. While the Lewy Body-receiving mice exhibited a loss of dopamine neurons and an increase in nigral S129 phosphorylated α-syn immunoreactivity, their parabiotic bloodstream-sharing partners did not show any trend for a lesion or change in S129 phosphorylated-α-syn levels. Altogether, our study suggests that, in the patient-derived α-synuclein aggregates-injected mouse model and within the selected time frame, the disease is not "transmitted" through the bloodstream.


Assuntos
Corpos de Lewy/transplante , Neostriado/patologia , Neurônios/patologia , Parabiose , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Substância Negra/patologia , alfa-Sinucleína/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Técnicas Estereotáxicas , alfa-Sinucleína/sangue
3.
Proc Natl Acad Sci U S A ; 114(39): E8284-E8293, 2017 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-28900002

RESUMO

Although a causative role of α-synuclein (α-syn) is well established in Parkinson's disease pathogenesis, available animal models of synucleinopathy do not replicate the full range of cellular and behavioral changes characteristic of the human disease. This study was designed to generate a more faithful model of Parkinson's disease by injecting human α-syn fibril seeds into the rat substantia nigra (SN), in combination with adenoassociated virus (AAV)-mediated overexpression of human α-syn, at levels that, by themselves, are unable to induce acute dopamine (DA) neurodegeneration. We show that the ability of human α-syn fibrils to trigger Lewy-like α-synuclein pathology in the affected DA neurons is dramatically enhanced in the presence of elevated levels of human α-syn. This synucleinopathy was fully developed already 10 days after fibril injection, accompanied by progressive degeneration of dopaminergic neurons in SN, neuritic swelling, reduced striatal DA release, and impaired motor behavior. Moreover, a prominent inflammatory response involving both activation of resident microglia and infiltration of CD4+ and CD8+ T lymphocytes was observed. Hypertrophic microglia were found to enclose or engulf cells and processes containing Lewy-like α-syn aggregates. α-Syn aggregates were also observed inside these cells, suggesting transfer of phosphorylated α-syn from the affected nigral neurons. The nigral pathology triggered by fibrils in combination with AAV-mediated overexpression of α-syn reproduced many of the cardinal features of the human disease. The short time span and the distinct sequence of pathological and degenerative changes make this combined approach attractive as an experimental model for the assessment of neuroprotective and disease-modifying strategies.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Neurônios Dopaminérgicos/metabolismo , Microglia/metabolismo , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , alfa-Sinucleína/toxicidade , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/patologia , Humanos , Microglia/patologia , Doença de Parkinson/patologia , Ratos , Substância Negra/patologia
4.
Gene Ther ; 26(1-2): 57-64, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30531868

RESUMO

Glial cell-line derived neurotrophic factor (GDNF) is a promising therapeutic molecule to treat Parkinson's disease. Despite an excellent profile in experimental settings, clinical trials testing GDNF have failed. One of the theories to explain these negative outcomes is that the clinical trials were done in late-stage patients that have advanced nigrostriatal degeneration and may therefore not respond to a neurotrophic factor therapy. Based on this idea, we tested if the stage of nigrostriatal degeneration is important for GDNF-based therapies. Lentiviral vectors expressing regulated GDNF were delivered to the striatum of rats to allow GDNF expression to be turned on either while the nigrostriatal system was degenerating or after the nigrostriatal system had been fully lesioned by 6-OHDA. In the group of animals where GDNF expression was on during degeneration, neurons were rescued and there was a reversal of motor deficits. Turning GDNF expression on after the nigrostriatal system was lesioned did not rescue neurons or reverse motor deficits. In fact, these animals were indistinguishable from the control groups. Our results suggest that GDNF can reverse motor deficits and nigrostriatal pathology despite an ongoing nigrostriatal degeneration, if there is still a sufficient number of remaining neurons to respond to therapy.


Assuntos
Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Doença de Parkinson/terapia , Degeneração Estriatonigral/terapia , Animais , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Lentivirus/genética , Oxidopamina/toxicidade , Doença de Parkinson/etiologia , Ratos , Ratos Sprague-Dawley , Degeneração Estriatonigral/etiologia , Substância Negra/metabolismo , Substância Negra/patologia
5.
Eur J Neurosci ; 49(4): 533-560, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30552719

RESUMO

Parkinson's disease (PD) is a progressive neurological disorder characterised by aberrant accumulation of insoluble proteins, including alpha-synuclein, and a loss of dopaminergic neurons in the substantia nigra. The extended neurodegeneration leads to a drop of striatal dopamine levels responsible for disabling motor and non-motor impairments. Although the causes of the disease remain unclear, it is well accepted among the scientific community that the disorder may also have a genetic component. For that reason, the number of genetically engineered animal models has greatly increased over the past two decades, ranging from invertebrates to more complex organisms such as mice and rats. This trend is growing as new genetic variants associated with the disease are discovered. The EU Joint Programme - Neurodegenerative Disease Research (JPND) has promoted the creation of an online database aiming at summarising the different features of experimental models of Parkinson's disease. This review discusses available genetic models of PD and the extent to which they adequately mirror the human pathology and reflects on future development and uses of genetically engineered experimental models for the study of PD.


Assuntos
Animais Geneticamente Modificados , Modelos Animais de Doenças , Engenharia Genética , Doença de Parkinson , Animais , Humanos
6.
Brain Behav Immun ; 61: 155-164, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27864045

RESUMO

Although intrastriatal transplantation of fetal cells for the treatment of Parkinson's disease had shown encouraging results in initial open-label clinical trials, subsequent double-blind studies reported more debatable outcomes. These studies highlighted the need for greater preclinical analysis of the parameters that may influence the success of cell therapy. While much of this has focused on the cells and location of the transplants, few have attempted to replicate potentially critical patient centered factors. Of particular relevance is that patients will be under continued L-DOPA treatment prior to and following transplantation, and that typically the grafts will not be immunologically compatible with the host. The aim of this study was therefore to determine the effect of chronic L-DOPA administered during different phases of the transplantation process on the survival and function of grafts with differing degrees of immunological compatibility. To that end, unilaterally 6-OHDA lesioned rats received sham surgery, allogeneic or xenogeneic transplants, while being treated with L-DOPA before and/or after transplantation. Irrespective of the L-DOPA treatment, dopaminergic grafts improved function and reduced the onset of L-DOPA induced dyskinesia. Importantly, although L-DOPA administered post transplantation was found to have no detrimental effect on graft survival, it did significantly promote the immune response around xenogeneic transplants, despite the administration of immunosuppressive treatment (cyclosporine). This study is the first to systematically examine the effect of L-DOPA on graft tolerance, which is dependent on the donor-host compatibility. These findings emphasize the importance of using animal models that adequately represent the patient paradigm.


Assuntos
Antiparkinsonianos/administração & dosagem , Transplante de Células , Sobrevivência de Enxerto/efeitos dos fármacos , Imunidade Ativa/efeitos dos fármacos , Levodopa/administração & dosagem , Doença de Parkinson Secundária/terapia , Animais , Antiparkinsonianos/uso terapêutico , Terapia Combinada , Feminino , Sobrevivência de Enxerto/imunologia , Levodopa/uso terapêutico , Doença de Parkinson Secundária/tratamento farmacológico , Ratos , Ratos Sprague-Dawley
7.
Neurobiol Dis ; 50: 142-50, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23072976

RESUMO

Abnormal involuntary movement (AIM) rating scales are frequently used to study the mechanisms underlying L-DOPA-induced dyskinesia (LID) in 6-OHDA lesioned rodents and the propensity of novel treatments for Parkinson's disease to induce or alleviate similar abnormal behaviours. Despite the existence of at least one well validated method, other AIM scales are also in use. Moreover, there have been developments and variations in the original scales and their methods of use, without re-validation. In this study, 6-OHDA medial forebrain bundle lesioned Sprague-Dawley rats were treated with chronic L-DOPA 6 mg/kg/day for 5 weeks followed by 12 mg/kg/day for another 5 weeks. Rats were assessed weekly by simultaneous ratings on four published AIM and stereotypy scales with concurrent recording of rotation, over 3 hours following L-DOPA injection. Three contemporary AIM scales have then been validated pharmacologically using agents that are known to reduce LID clinically and in primates (amantadine) or to interfere with the activity of L-DOPA (the D(1) and D(2) dopamine receptor antagonists, SCH-23390 and raclopride) respectively. We also demonstrate that AIM, stereotypic and rotational behaviour are distinct motor dysfunctions induced by chronic and acute treatment of L-DOPA, and should be assessed separately. The undertaking of assessments at multiple time points is essential especially when testing the efficacy of new potential anti-dyskinetic treatments. Importantly critical to all AIM and rotation testing is the internal validation of both the scale being used and the environment being used.


Assuntos
Agonistas de Dopamina/efeitos adversos , Discinesia Induzida por Medicamentos/diagnóstico , Levodopa/efeitos adversos , Índice de Gravidade de Doença , Adrenérgicos/toxicidade , Amantadina/farmacologia , Animais , Antiparkinsonianos/farmacologia , Oxidopamina/toxicidade , Transtornos Parkinsonianos/tratamento farmacológico , Ratos , Ratos Sprague-Dawley
8.
Heliyon ; 7(2): e06302, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33665452

RESUMO

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.

9.
Mol Ther Methods Clin Dev ; 11: 29-39, 2018 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-30324128

RESUMO

Regulation of therapeutic transgene expression can increase the safety of gene therapy interventions, especially when targeting critical organs such as the brain. Although several gene expression systems have been described, none of the current systems has the required safety profile for clinical applications. Our group has previously adapted a system for novel gene regulation based on the destabilizing domain degron technology to successfully regulate glial cell-line derived neurotrophic factor in the brain (GDNF-F-DD). In the present study, we used GDNF-F-DD as a proof-of-principle molecule to fully characterize DD regulation in the brain. Our results indicate that DD could be regulated in a dose-dependent manner. In addition, GDNF-F-DD could also be induced in vivo repeatedly, without loss of activity or efficacy in vivo. Finally, DD regulation was able to be sustained for 24 weeks without loss of expression or any overt toxicity. The present study shows that DD has great potential to regulate gene expression in the brain.

10.
Mol Ther Methods Clin Dev ; 5: 16027, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27069954

RESUMO

Preclinical and clinical data stress the importance of pharmacologically-controlling glial cell line-derived neurotrophic factor (GDNF) intracerebral administration to treat PD. The main challenge is finding a combination of a genetic switch and a drug which, when administered at a clinically-approved dose, reaches the brain in sufficient amounts to induce a therapeutic effect. We describe a highly-sensitive doxycycline-inducible adeno-associated virus (AAV) vector. This vector allowed for the first time a longitudinal analysis of inducible transgene expression in the brain using bioluminescence imaging. To evaluate the dose range of GDNF biological activity, the inducible AAV vector (8.0 × 10(9) viral genomes) was injected in the rat striatum at four delivery sites and increasing doxycycline doses administered orally. ERK/Akt signaling activation as well as tyrosine hydroxylase downregulation, a consequence of long-term GDNF treatment, were induced at plasmatic doxycycline concentrations of 140 and 320 ng/ml respectively, which are known not to increase antibiotic-resistant microorganisms in patients. In these conditions, GDNF covered the majority of the striatum. No behavioral abnormalities or weight loss were observed. Motor asymmetry resulting from unilateral GDNF treatment only appeared with a 2.5-fold higher vector and a 13-fold higher inducer doses. Our data suggest that using the herein-described inducible AAV vector, biological effects of GDNF can be obtained in response to sub-antimicrobial doxycycline doses.

11.
Exp Biol Med (Maywood) ; 238(7): 725-32, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23828589

RESUMO

One of the well-recognized problems of long-term L-3,4-dihydroxyphenylalanine (L-DOPA) therapy in the treatment of Parkinson's disease is the development of L-DOPA induced dyskinesia. These abnormal movements cause significant disability and narrow the therapeutic window of L-DOPA. Cell transplantation is one of the most promising upcoming therapies for the treatment of Parkinson's disease, and may help alleviate or avoid L-DOPA-induced dyskinesia. However, the more recently acknowledged phenomenon of graft-induced dyskinesia is posing a major obstacle to the success of this treatment. This motor side-effect closely resembles abnormal movements induced by chronic L-DOPA treatment, yet they remain after withdrawal of the medication indicating their origins lie in the transplant. In this review, we compare these two therapy-induced adverse effects, from the way they manifest in patients to the possible mechanisms underlying their development.


Assuntos
Transplante de Células/efeitos adversos , Discinesias/etiologia , Levodopa/efeitos adversos , Animais , Modelos Animais de Doenças , Discinesias/patologia , Humanos
12.
Neuropharmacology ; 63(5): 818-28, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22722025

RESUMO

Foetal cell transplantation in patients with Parkinson's disease can induce motor complications independent of L-DOPA administration, known as graft-induced dyskinesia. In the 6-OHDA lesioned rat model of Parkinson's disease, post-transplantation abnormal movements can develop in response to an amphetamine challenge, a behaviour which is used to model graft-induced dyskinesia. Although L-DOPA-induced dyskinesia has been well characterised pharmacologically, we lack knowledge on the modulation of post-transplantation amphetamine-induced dyskinesia which may shed light on the mechanisms underlying graft-induced dyskinesia. We assessed a series of drugs effective at reducing L-DOPA-induced dyskinesia against post-transplantation amphetamine-induced dyskinesia. Agents include: dopaminergic antagonists (D1: CP94253; D2: SCH-22390; D3: nafadotride), serotonergic agonists (5-HT(1A): 8-OH-DPAT; 5-HT(1B): CP94253), opioid antagonist (µ: naloxone), cannabinoid agonist (CB1: WIN55, 212-2), adrenergic antagonist (α1 and α2: yohimbine) and glutamatergic antagonists (NMDA: amantadine and MK-801; mGluR5: MTEP; AMPA: IEM1460). Abnormal involuntary movements in response to amphetamine were decreased by SCH-22390, raclopride, CP94253 and 8-OH-DPAT, yet were unaltered by naloxone, WIN55, 212-2, yohimbine, amantadine, MTEP and IEM1460. Unusually, MK-801 increased the appearance of amphetamine-induced dyskinesia. The results suggest that dopaminergic, serotoninergic and glutamatergic systems are likely to have a fundamental role in the development of graft-induced dyskinesias, which are mechanistically distinct from L-DOPA-induced behvaviours. Importantly, the expression of D1 and D2 receptors was unrelated to the severity of AIMs.


Assuntos
Antidiscinéticos/uso terapêutico , Transplante de Células/efeitos adversos , Discinesia Induzida por Medicamentos/tratamento farmacológico , Discinesias/tratamento farmacológico , Neostriado/efeitos dos fármacos , Neurônios/transplante , Doença de Parkinson/terapia , Animais , Antidiscinéticos/administração & dosagem , Antidiscinéticos/efeitos adversos , Antidiscinéticos/metabolismo , Modelos Animais de Doenças , Antagonistas de Dopamina/administração & dosagem , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/uso terapêutico , Inibidores da Captação de Dopamina/administração & dosagem , Inibidores da Captação de Dopamina/toxicidade , Relação Dose-Resposta a Droga , Discinesia Induzida por Medicamentos/metabolismo , Discinesia Induzida por Medicamentos/patologia , Discinesias/etiologia , Discinesias/metabolismo , Discinesias/patologia , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/efeitos adversos , Antagonistas de Aminoácidos Excitatórios/metabolismo , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Feminino , Mesencéfalo/citologia , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Metanfetamina/administração & dosagem , Metanfetamina/toxicidade , Neostriado/metabolismo , Neostriado/patologia , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Receptores de Neurotransmissores/agonistas , Receptores de Neurotransmissores/antagonistas & inibidores , Receptores de Neurotransmissores/metabolismo , Agonistas do Receptor de Serotonina/administração & dosagem , Agonistas do Receptor de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/uso terapêutico
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