RESUMO
Lysosomal impairment is strongly implicated in Parkinson's disease (PD). Among the several PD-linked genes, the ATP13A2 gene, associated with the PARK9 locus, encodes a transmembrane lysosomal P5-type ATPase. Mutations in the ATP13A2 gene were primarily identified as the cause of Kufor-Rakeb syndrome (KRS), a juvenile-onset form of PD. Subsequently, an increasing list of several mutations has been described. These mutations result in truncation of the ATP13A2 protein, leading to a loss of function but surprisingly causing heterogeneity and variability in the clinical symptoms associated with different brain pathologies. In vitro studies show that its loss compromises lysosomal function, contributing to cell death. To understand the role of ATP13A2 dysfunction in disease, we disrupted its expression through a viral vector-based approach in nonhuman primates. Here, in this pilot study, we injected bilaterally into the substantia nigra of macaques, a lentiviral vector expressing an ATP13A2 small hairpin RNA. Animals were terminated five months later, and brains were harvested and compared with historical non-injected control brains to evaluate cerebral pathological markers known to be affected in KRS and PD. We characterised the pattern of dopaminergic loss in the striatum and the substantia nigra, the regional distribution of α-synuclein immunoreactivity in several brain structures, and its pathological status (i.e., S129 phosphorylation), the accumulation of heavy metals in nigral sections and occurrence of lysosomal dysfunction. This proof-of-concept experiment highlights the potential value of lentivirus-mediated ATP13A2 silencing to induce significant and ongoing degeneration in the nigrostriatal pathway, α-synuclein pathology, and iron accumulation in nonhuman primates.
RESUMO
Aggregation of α-synuclein (α-syn) is the cornerstone of neurodegenerative diseases termed synucleinopathies, which include Parkinson's Disease (PD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA). These synucleinopathies are characterized by the deposit of aggregated α-syn in intracellular inclusions observable in neurons and glial cells. In PD and DLB, these aggregates, predominantly located in neurons, are called Lewy Bodies (LBs). These LBs are one of the pathological hallmarks of PD and DLB, alongside dopaminergic neuron loss in the substantia nigra. Previous studies have demonstrated the ability of PD patient-derived LB fractions to induce nigrostriatal neurodegeneration and α-syn pathology when injected into the striatum or the enteric nervous system of non-human primates. Here, we report the pathological consequences of injecting these LB fractions into the cortex of non-human primates. To this end, we inoculated mesencephalic PD patient-derived LB fractions into the prefrontal cortex of baboon monkeys terminated one year later. Extensive analyses were performed to evaluate pathological markers known to be affected in LB pathologies. We first assessed the hypothesized presence of phosphorylated α-syn at S129 (pSyn) in the prefrontal cortices. Second, we quantified the neuronal, microglial, and astrocytic cell survival in the same cortices. Third, we characterized these cortical LB injections' putative impact on the integrity of the nigrostriatal system. Overall, we observed pSyn accumulation around the injection site in the dorsal prefrontal cortex, in connected cortical regions, and further towards the striatum, suggesting α-syn pathological propagation. The pathology was also accompanied by neuronal loss in these prefrontal cortical regions and the caudate nucleus, without, however, loss of nigral dopamine neurons. In conclusion, this pilot study provides novel data demonstrating the toxicity of patient-derived extracts, their potential to propagate from the cortex to the striatum in non-human primates, and a possible primate model of DLB.
RESUMO
The role of alpha-synuclein in Parkinson's disease has been heavily investigated since its discovery as a component of Lewy bodies. Recent rodent data demonstrate that alpha-synuclein strain structure is critical for differential propagation and toxicity. Based on these findings, we have compared, for the first time, in this pilot study, the capacity of two alpha-synuclein strains and patient-derived Lewy body extracts to model synucleinopathies after intra-putaminal injection in the non-human primate brain. Functional alterations triggered by these injections were evaluated in vivo using glucose positron emission tomography imaging. Post-mortem immunohistochemical and biochemical analyses were used to detect neuropathological alterations in the dopaminergic system and alpha-synuclein pathology propagation. In vivo results revealed a decrease in glucose metabolism more pronounced in alpha-synuclein strain-injected animals. Histology showed a decreased number of dopaminergic tyrosine hydroxylase-positive cells in the substantia nigra to different extents according to the inoculum used. Biochemistry revealed that alpha-synuclein-induced aggregation, phosphorylation, and propagation in different brain regions are strain-specific. Our findings show that distinct alpha-synuclein strains can induce specific patterns of synucleinopathy in the non-human primate, changes in the nigrostriatal pathway, and functional alterations that resemble early-stage Parkinson's disease.
Assuntos
Doença de Parkinson , Sinucleinopatias , Animais , alfa-Sinucleína/metabolismo , Doença de Parkinson/metabolismo , Projetos Piloto , Corpos de Lewy/metabolismo , Sinucleinopatias/patologia , Substância Negra/metabolismo , Dopamina/metabolismo , Primatas/metabolismoRESUMO
Synucleinopathies encompass several neurodegenerative diseases, which include Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. These diseases are characterized by the deposit of α-synuclein aggregates in intracellular inclusions in neurons and glial cells. Unlike Parkinson's disease and dementia with Lewy bodies, where aggregates are predominantly neuronal, multiple system atrophy is associated with α-synuclein cytoplasmic inclusions in oligodendrocytes. Glial cytoplasmic inclusions are the pathological hallmark of multiple system atrophy and are associated with neuroinflammation, modest demyelination and, ultimately, neurodegeneration. To evaluate the possible pathogenic role of glial cytoplasmic inclusions, we inoculated glial cytoplasmic inclusion-containing brain fractions obtained from multiple system atrophy patients into the striatum of non-human primates. After a 2-year in vivo phase, extensive histochemical and biochemical analyses were performed on the whole brain. We found loss of both nigral dopamine neurons and striatal medium spiny neurons, as well as loss of oligodendrocytes in the same regions, which are characteristics of multiple system atrophy. Furthermore, demyelination, neuroinflammation and α-synuclein pathology were also observed. These results show that the α-synuclein species in multiple system atrophy-derived glial cytoplasmic inclusions can induce a pathological process in non-human primates, including nigrostriatal and striatofugal neurodegeneration, oligodendroglial cell loss, synucleinopathy and gliosis. The present data pave the way for using this experimental model for MSA research and therapeutic development.
Assuntos
Doenças Desmielinizantes , Doença por Corpos de Lewy , Atrofia de Múltiplos Sistemas , Doença de Parkinson , Sinucleinopatias , Animais , Encéfalo/patologia , Doenças Desmielinizantes/patologia , Humanos , Corpos de Inclusão/metabolismo , Doença por Corpos de Lewy/patologia , Atrofia de Múltiplos Sistemas/patologia , Doença de Parkinson/patologia , alfa-Sinucleína/metabolismoRESUMO
The G2019S mutation of LRRK2 represents a risk factor for idiopathic Parkinson's disease. Here, we investigate whether LRRK2 kinase activity regulates susceptibility to the environmental toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). G2019S knock-in mice (bearing enhanced kinase activity) showed greater nigro-striatal degeneration compared to LRRK2 knock-out, LRRK2 kinase-dead and wild-type mice following subacute MPTP treatment. LRRK2 kinase inhibitors PF-06447475 and MLi-2, tested under preventive or therapeutic treatments, protected against nigral dopamine cell loss in G2019S knock-in mice. MLi-2 also rescued striatal dopaminergic terminal degeneration in both G2019S knock-in and wild-type mice. Immunoblot analysis of LRRK2 Serine935 phosphorylation levels confirmed target engagement of LRRK2 inhibitors. However, MLi-2 abolished phosphoSerine935 levels in the striatum and midbrain of both wild-type and G2019S knock-in mice whereas PF-06447475 partly reduced phosphoSerine935 levels in the midbrain of both genotypes. In vivo and ex vivo uptake of the 18-kDa translocator protein (TSPO) ligand [18F]-VC701 revealed a similar TSPO binding in MPTP-treated wild-type and G2019S knock-in mice which was consistent with an increased GFAP striatal expression as revealed by Real Time PCR. We conclude that LRRK2 G2019S, likely through enhanced kinase activity, confers greater susceptibility to mitochondrial toxin-induced parkinsonism. LRRK2 kinase inhibitors are neuroprotective in this model.
Assuntos
Doença de Parkinson , Transtornos Parkinsonianos , Animais , Corpo Estriado/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Camundongos , Mutação , Doença de Parkinson/metabolismo , Transtornos Parkinsonianos/metabolismo , FosforilaçãoRESUMO
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
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
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/sangueRESUMO
Designer receptors exclusively activated by designer drugs (DREADDs) are widely used in rodents to manipulate neuronal activity and establish causal links between structure and function. Their utilization in non-human primates (NHPs) is, however, limited and their efficacy still debated. Here, we recorded and examined the neuronal activity in the hM4Di DREADD-transduced and hM4Di DREADD-free GPe of two anesthetized animals following local intra-GPe microinjection of clozapine-N-oxide (CNO). Our results revealed that the neuronal activity of the well-isolated units recorded in the hM4Di DREADD-transduced GPe exhibited diverse patterns in timing and polarity (increase/decrease) of firing rate modulations following CNO injection. Nevertheless, significant decreases in activity were more frequent (and more pronounced) than significant increases in activity during CNO injection (6/18 vs. 3/18 units) and were exclusive after CNO Injection (8/18 units). In contrast, only one of the 8 well-isolated units recorded in hM4Di DREADD-free GPe exhibited a significant increase in activity after CNO injection. Overall, the number of units exhibiting a significant period-related decrease following CNO injection was significantly larger in hM4Di DREADD-transduced GPe than in the hM4Di DREADD-free GPe (8/18 [44.4%] vs. 0/8 [0%]). Moreover, postmortem histochemical analysis revealed that hM4Di DREADDs were expressed at high level in the GPe neurons located in the vicinity of the viral vector injection sites. Our results therefore show in vivo hM4Di DREADD-based inhibition of pallidal neurons in the NHP model and reinforce the view that DREADD technology can be effective in NHPs.
Assuntos
Clozapina , Neurônios , Animais , Fenômenos Eletrofisiológicos , Globo Pálido , PrimatasRESUMO
OBJECTIVE: Whether the basal ganglia are involved in the cortical synchronization during focal seizures is still an open question. In the present study, we proposed to synchronize cortico-striatal activities acutely inducing striatal disinhibition, performing GABA-antagonist injections within the putamen in primates. METHOD: Experiments were performed on three fascicularis monkeys. During each experimental session, low volumes of bicuculline (0.5-4 µL) were injected at a slow rate of 1 µL/min. Spontaneous behavioral changes were classified according to Racine's scale modified for primates. These induced motor behaviors were correlated with electromyographic, electroencephalographic, and putaminal and pallidal local field potentials changes in activity. RESULTS: acute striatal desinhibition induced focal motor seizures. Seizures were closely linked to cortical epileptic activity synchronized with a striatal paroxysmal activity. These changes in striatal activity preceded the cortical epileptic activity and the induced myoclonia, and both cortical and subcortical activities were coherently synchronized during generalized seizures. INTERPRETATION: Our results strongly suggest the role of the sensorimotor striatum in the regulation and synchronization of cortical excitability. These dramatic changes in the activity of this "gating" pathway might influence seizure susceptibility by modulating the threshold for the initiation of focal motor seizures.
Assuntos
Córtex Cerebral/fisiopatologia , Sincronização Cortical , Putamen/fisiopatologia , Convulsões/fisiopatologia , Animais , Bicuculina/administração & dosagem , Feminino , Antagonistas de Receptores de GABA-A/administração & dosagem , Macaca fascicularis , Masculino , Vias Neurais/fisiopatologia , Putamen/efeitos dos fármacos , Ratos Sprague-Dawley , Convulsões/etiologiaRESUMO
In Parkinson's disease, synucleinopathy is hypothesized to spread from the enteric nervous system, via the vagus nerve, to the CNS. Here, we compare, in baboon monkeys, the pathological consequences of either intrastriatal or enteric injection of α-synuclein-containing Lewy body extracts from patients with Parkinson's disease. This study shows that patient-derived α-synuclein aggregates are able to induce nigrostriatal lesions and enteric nervous system pathology after either enteric or striatal injection in a non-human primate model. This finding suggests that the progression of α-synuclein pathology might be either caudo-rostral or rostro-caudal, varying between patients and disease subtypes. In addition, we report that α-synuclein pathological lesions were not found in the vagal nerve in our experimental setting. This study does not support the hypothesis of a transmission of α-synuclein pathology through the vagus nerve and the dorsal motor nucleus of the vagus. Instead, our results suggest a possible systemic mechanism in which the general circulation would act as a route for long-distance bidirectional transmission of endogenous α-synuclein between the enteric and the central nervous systems. Taken together, our study provides invaluable primate data exploring the role of the gut-brain axis in the initiation and propagation of Parkinson's disease pathology and should open the door to the development and testing of new therapeutic approaches aimed at interfering with the development of sporadic Parkinson's disease.
Assuntos
Encéfalo/patologia , Neuroimunomodulação/fisiologia , Doença de Parkinson/fisiopatologia , Nervo Vago/patologia , alfa-Sinucleína/toxicidade , Idoso , Animais , Sistema Nervoso Entérico/efeitos dos fármacos , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/patologia , Feminino , Humanos , Corpos de Lewy/metabolismo , Corpos de Lewy/patologia , Masculino , Papio , alfa-Sinucleína/administração & dosagemRESUMO
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.
Assuntos
Doença de Parkinson/metabolismo , Área Tegmentar Ventral/metabolismo , Anedonia , Animais , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Masculino , Modelos Teóricos , Vias Neurais/metabolismo , Oxidopamina/farmacologia , Parte Compacta da Substância Negra/metabolismo , Ratos , Ratos Sprague-Dawley , Teste de Desempenho do Rota-Rod , Substância Negra/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
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
Mutations in the GBA1 gene encoding the lysosomal enzyme glucocerebrosidase (GBA1) are important risk factors for Parkinson's disease (PD). In vitro, altered GBA1 activity promotes alpha-synuclein accumulation whereas elevated levels of alpha-synuclein compromise GBA1 function, thus supporting a pathogenic mechanism in PD. However, the mechanisms by which GBA1 deficiency is linked to increased risk of PD remain elusive, partially because of lack of aged models of GBA1 deficiency. As knocking-out GBA1 in the entire brain induces massive neurodegeneration and early death, we generated a mouse model of GBA1 deficiency amenable to investigate the long-term consequences of compromised GBA1 function in dopaminergic neurons. DAT-Cre and GBA1-floxed mice were bred to obtain selective homozygous disruption of GBA1 in midbrain dopamine neurons (DAT-GBA1-KO). Mice were followed for motor function, neuronal survival, alpha-synuclein phosphorylation and glial activation. Susceptibility to nigral viral vector-mediated overexpression of mutated (A53T) alpha-synuclein was assessed. Despite loss of GBA1 and substrate accumulation, DAT-GBA1-KO mice displayed normal motor performances and preserved dopaminergic neurons despite robust microglial activation in the substantia nigra, without accumulation of endogenous alpha-synuclein with respect to wild-type mice. Lysosomal function was only marginally affected. Screening of micro-RNAs linked to the regulation of GBA1, alpha-synuclein or neuroinflammation did not reveal significant alterations. Viral-mediated overexpression of A53T-alpha-synuclein yielded similar neurodegeneration in DAT-GBA1-KO mice and wild-type mice. These results indicate that loss of GBA1 function in mouse dopaminergic neurons is not critical for alpha-synuclein accumulation or neurodegeneration and suggest the involvement of GBA1 deficiency in other cell types as a potential mechanism.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Animais , Encéfalo/metabolismo , Doença de Gaucher/genética , Doença de Gaucher/metabolismo , Vetores Genéticos , Mesencéfalo/metabolismo , Camundongos , Camundongos Knockout , Microglia/metabolismo , Modelos Animais , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Neurodegenerative diseases are characterized by the degeneration of specific brain areas associated with accumulation of disease-related protein in extra- or intra-cellular deposits. Their preclinical investigations are mostly based on genetically-engineered animals. Despite their interest, these models are often based on high level of disease-related protein expression, thus questioning their relevance to human pathology and calling for the alternate use of ecological models. In the past few years, Octodon degus has emerged as a promising animal model displaying age-dependent Alzheimer's disease-related pathology. As neurodegenerative-related proteins often co-deposit in the brain of patients, we assessed the occurrence of α-synuclein-related pathology in this model using state-of-the-art immunohistochemistry and biochemistry. Despite our efforts and in contrast with previously published results, our study argues against the use of Octodon degus as a suitable natural model of neurodegenerative disorder as we failed to identify either Parkinson's disease- or Alzheimer's disease-related brain pathologies.
Assuntos
Encéfalo/patologia , Modelos Animais de Doenças , Octodon/metabolismo , Envelhecimento/patologia , Doença de Alzheimer , Animais , Encéfalo/metabolismo , Doença de Parkinson , alfa-SinucleínaRESUMO
Although much progress have been made in recent years, the etiology of idiopathic Parkinson's disease remains obscure. The chance discovery that injection of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a syndrome very similar to parkinsonism introduced the "environmental toxin" hypothesis but no toxin was ever found in any quantity in patients' brains. We have unexpectedly now found, however, that, in mice, very low doses of MPTP induce as much dopaminergic neuronal death as far higher doses. Cellular detoxification mechanisms would appear to be incapacitated at such low doses. This could infer that the barely discernible presence of an unidentified neurotoxin may be responsible for the onset of Parkinson's disease.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Intoxicação por MPTP/metabolismo , Doença de Parkinson/etiologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , Animais , Neurônios Dopaminérgicos/efeitos dos fármacos , Relação Dose-Resposta a Droga , CamundongosRESUMO
The discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that induces parkinsonism in both human and primate, has prompted the search for environmental toxins potentially responsible for idiopathic Parkinson's disease (PD). The present study reports the ultimate effects of MPTP intoxication of a female macaque monkey, which unraveled to be pregnant after parkinsonism had developed, upon its fetus. Detailed examination of the offpsring nigrostriatal pathway showed that tyrosine hydroxylase immunoreactivity in caudate-putamen nuclei and substantia nigra compacta (SNc) was not different from an age-matched control. Biochemical analysis of the tissue content of dopaminergic markers further suggested modification of metabolism in the MPTP-exposed monkey. These data suggest that early prenatal intoxication does not destroy nigrostriatal neurons, most likely because dopamine neurons had not developed yet when exposed to MPTP.