RESUMO
Multiple system atrophy is a progressive neurodegenerative disease with prominent autonomic and motor features. During early stages, different subtypes of the disease are distinguished by their predominant parkinsonian or cerebellar symptoms, reflecting its heterogeneous nature. The pathognomonic feature of multiple system atrophy is the presence of α-synuclein (αSyn) protein deposits in oligodendroglial cells. αSyn can assemble in specific cellular or disease environments and form αSyn strains with unique structural features, but the ability of αSyn strains to propagate in oligodendrocytes remains elusive. Recently, it was shown that αSyn strains with related conformations exist in the brains of patients. Here, we investigated whether different αSyn strains can influence multiple system atrophy progression in a strain-dependent manner. To this aim, we injected two recombinant αSyn strains (fibrils and ribbons) in multiple system atrophy transgenic mice and found that they determined disease severity in multiple system atrophy via host-restricted and cell-specific pathology in vivo. αSyn strains significantly impact disease progression in a strain-dependent way via oligodendroglial, neurotoxic and immune-related mechanisms. Neurodegeneration and brain atrophy were accompanied by unique microglial and astroglial responses and the recruitment of central and peripheral immune cells. The differential activation of microglial cells correlated with the structural features of αSyn strains both in vitro and in vivo. Spectral analysis showed that ribbons propagated oligodendroglial inclusions that were structurally distinct from those of fibrils, with resemblance to oligodendroglial inclusions, in the brains of patients with multiple system atrophy. This study, therefore, shows that the multiple system atrophy phenotype is governed by both the nature of the αSyn strain and the host environment and that by injecting αSyn strains into an animal model of the disease, a more comprehensive phenotype can be established.
Assuntos
Atrofia de Múltiplos Sistemas , alfa-Sinucleína , Camundongos , Animais , alfa-Sinucleína/metabolismo , Atrofia de Múltiplos Sistemas/patologia , Modelos Animais de Doenças , Camundongos Transgênicos , Gravidade do Paciente , Encéfalo/patologiaRESUMO
Synucleinopathies, such as Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB), are defined by the presence of α-synuclein (αSYN) aggregates throughout the nervous system but diverge from one another with regard to their clinical and pathological phenotype. The recent generation of pure fibrillar αSYN polymorphs with noticeable differences in structural and phenotypic traits has led to the hypothesis that different αSYN strains may be in part responsible for the heterogeneous nature of synucleinopathies. To further characterize distinct αSYN strains in the human brain, and establish a structure-pathology relationship, we pursued a detailed comparison of αSYN assemblies derived from well-stratified patients with distinct synucleinopathies. We exploited the capacity of αSYN aggregates found in the brain of patients suffering from PD, MSA or DLB to seed and template monomeric human αSYN in vitro via a protein misfolding cyclic amplification assay. A careful comparison of the properties of total brain homogenates and pure in vitro amplified αSYN fibrillar assemblies upon inoculation in cells and in the rat brain demonstrates that the intrinsic structure of αSYN fibrils dictates synucleinopathies characteristics. We report that MSA strains show several similarities with PD strains, but are significantly more potent in inducing motor deficits, nigrostriatal neurodegeneration, αSYN pathology, spreading, and inflammation, reflecting the aggressive nature of this disease. In contrast, DLB strains display no or only very modest neuropathological features under our experimental conditions. Collectively, our data demonstrate a specific signature for PD, MSA, and DLB-derived strains that differs from previously described recombinant strains, with MSA strains provoking the most aggressive phenotype and more similarities with PD compared to DLB strains.
Assuntos
Demência/patologia , Doença por Corpos de Lewy/patologia , Atrofia de Múltiplos Sistemas/patologia , Doença de Parkinson/patologia , alfa-Sinucleína/metabolismo , Idoso , Idoso de 80 Anos ou mais , Encéfalo/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.
Assuntos
Apoptose , Endodesoxirribonucleases/metabolismo , Neuroblastoma/patologia , Neurônios/metabolismo , Doença de Parkinson/patologia , Substância Negra/patologia , alfa-Sinucleína/metabolismo , Idoso , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Dano ao DNA/genética , Dopamina/farmacologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Endodesoxirribonucleases/genética , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neurônios/citologia , Estresse Oxidativo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Substância Negra/metabolismo , Células Tumorais Cultivadas , alfa-Sinucleína/genéticaRESUMO
Intra-arterial injection of mesenchymal stem cells has been proven to result in a superior nephroprotection compared to intravenous injection. This avoids initial passage through filter organs such as the lung, liver and spleen. The aim of the present study was to investigate whether suprarenal aortic delivery results in a homogenous distribution to both kidneys. Chinese ink was used to evaluate the renal distribution pattern for the comparison of two retrograde intra-aortic injection methods. In the first, the aorta caudal to the renal branches was temporarily clamped and Chinese ink was injected at the level of the renal arteries. In the second, a distal aortic clamp was combined with alternated clamping of the contralateral arteries. Immediately after injection, kidneys were harvested for histological analysis. Amniotic fluid stem cells labeled with LacZ were injected in the aorta by alternated clamping of the renal arteries in order to track the cells in a rat ischemia/reperfusion model. Without renal artery clamping, intra-aortic administration resulted in a delivery of the ink into the right kidney, whereas administration with alternated clamping of the contralateral renal artery, together with distal aortic artery clamping, resulted in a more homogenous distribution of the ink in both kidneys. Moreover, LacZ-positive cells were found in both kidneys after 6 h of injection. In conclusion, the retrograde administration of Chinese ink in two steps is a fast and reproducible technique, which results in a more homogenous distribution of the stain in both kidneys than a single administration combined by only clamping the aorta.
Assuntos
Rim/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Animais , Aorta Abdominal , Constrição , Rim/metabolismo , Masculino , Ratos , Ratos Wistar , Artéria RenalRESUMO
During the pathogenesis of Parkinson's disease (PD), aggregation of alpha-synuclein (αSyn) induces a vicious cycle of cellular impairments that lead to neurodegeneration. Consequently, removing toxic αSyn aggregates constitutes a plausible strategy against PD. In this work, we tested whether stimulating the autolysosomal degradation of αSyn aggregates through the Ras-related in brain 7 (Rab7) pathway can reverse αSyn-induced cellular impairment and prevent neurodegeneration in vivo. The disease-related A53T mutant of αSyn was expressed in primary neurons and in dopaminergic neurons of the rat brain simultaneously with wild type (WT) Rab7 or the T22N mutant as negative control. The cellular integrity was quantified by morphological and biochemical analyses. In primary neurons, WT Rab7 rescued the αSyn-induced loss of neurons and neurites. Furthermore, Rab7 decreased the amount of reactive oxygen species and the amount of Triton X-100 insoluble αSyn. In rat brain, WT Rab7 reduced αSyn-induced loss of dopaminergic axon terminals in the striatum and the loss of dopaminergic dendrites in the substantia nigra pars reticulata. Further, WT Rab7 lowered αSyn pathology as quantified by phosphorylated αSyn staining. Finally, WT Rab7 attenuated αSyn-induced DNA damage in primary neurons and rat brain. In brief, Rab7 reduced αSyn-induced pathology, ameliorated αSyn-induced neuronal degeneration, oxidative stress and DNA damage. These findings indicate that Rab7 is able to disrupt the vicious cycle of cellular impairment, αSyn pathology and neurodegeneration present in PD. Stimulation of Rab7 and the autolysosomal degradation pathway could therefore constitute a beneficial strategy for PD.
Assuntos
Neurônios Dopaminérgicos/metabolismo , alfa-Sinucleína/biossíntese , alfa-Sinucleína/toxicidade , proteínas de unión al GTP Rab7/biossíntese , proteínas de unión al GTP Rab7/farmacologia , Animais , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Fetal gene therapy is one of the possible new therapeutic strategies for congenital or perinatal diseases with high mortality or morbidity. We developed a novel delivery strategy to inject directly into the fetal mouse trachea. Intratracheal (i.t.) injection at embryonic day 18 (E18) was more efficient in targeting the fetal lung than conventional intra-amniotic (i.a.) delivery. Viral vectors derived from adeno-associated virus serotype 6.2, with tropism for the airway epithelium and not earlier tested in the fetal mouse lung, were injected into the fetal trachea. Bioluminescence (BL) imaging (BLI) was combined with magnetic resonance (MR) imaging (MRI) for noninvasive and accurate localization of transgene expression in vivo. Histological analysis for ß-galactosidase (ß-gal) revealed 17.5% of epithelial cells transduced in the conducting airways and 1.5% in the alveolar cells. Stable gene expression was observed up to 1 month after injection. This study demonstrates that direct injection of rAAV2/6.2 in the fetal mouse trachea is superior to i.a. delivery for transducing the lung. Second, as stable gene transfer was detected up to 1 postnatal month, this approach may be useful to evaluate fetal gene therapy for pulmonary diseases such as cystic fibrosis, requiring both substantial numbers of transduced cells as well as prolonged gene expression to obtain a stable phenotypic effect.
Assuntos
Dependovirus , Feto , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Pulmão , Traqueia , Líquido Amniótico , Animais , Dependovirus/genética , Camundongos , Modelos AnimaisRESUMO
The development of disease-modifying therapies for Parkinson's disease is a major challenge which would be facilitated by a better understanding of the pathogenesis. Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein are key players in Parkinson's disease, but their relationship remains incompletely resolved. Previous studies investigating the effect of LRRK2 on α-synuclein-induced neurotoxicity and neuroinflammation in preclinical Parkinson's disease models have reported conflicting results. Here, we aimed to further explore the functional interaction between α-synuclein and LRRK2 and to evaluate the therapeutic potential of targeting physiological LRRK2 levels. We studied the effects of total LRRK2 protein loss as well as pharmacological LRRK2 kinase inhibition in viral vector-mediated α-synuclein-based Parkinson's disease models developing early- and late-stage neurodegeneration. Surprisingly, total LRRK2 ablation or in-diet treatment with the LRRK2 kinase inhibitor MLi-2 did not significantly modify α-synuclein-induced motor deficits, dopaminergic cell loss, or α-synuclein pathology. Interestingly, we found a significant effect on α-synuclein-induced neuroinflammatory changes in the absence of LRRK2, with a reduced microglial activation and CD4+ and CD8+ T cell infiltration. This observed lack of protection against α-synuclein-induced toxicity should be well considered in light of the ongoing therapeutic development of LRRK2 kinase inhibitors for idiopathic Parkinson's disease. Future studies will be crucial to understand the link between these neuroinflammatory processes and disease progression as well as the role of α-synuclein and LRRK2 in these pathological events.
Assuntos
Indazóis/administração & dosagem , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doenças Neurodegenerativas/enzimologia , Doenças Neuroinflamatórias/enzimologia , Pirimidinas/administração & dosagem , alfa-Sinucleína/toxicidade , Animais , Masculino , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/patologia , Doenças Neuroinflamatórias/induzido quimicamente , Doenças Neuroinflamatórias/patologia , Ratos , Ratos Long-Evans , Ratos TransgênicosRESUMO
We investigated the glucose metabolism in an adeno-associated viral vector based alpha-synuclein rat model for Parkinson's disease (PD) using longitudinal 18F-FDG PET imaging, which resulted in an improved characterization of this animal model. We generated a PD specific pattern (PDSP) based on a multivariate classification approach to differentiate between a PD and control group at a late disease stage, where the neurodegeneration is considered nearly complete. In particular, we applied a principal component analysis prior to classification by a support vector machine (SVM). Moreover, by using a SVM for regression to predict corresponding motor scores, a PD motor pattern (PDMP) was derived as well. The PDSP mainly corresponds to the PDMP and overlaps to a large extent with the human pattern. We were able to quantify disease expression at previous time points by projecting onto the PDSP and PDMP. While a univariate analysis indicated metabolic changes which did not persist through time, both PDSP and PDMP were able to differentiate significantly (p-value < 0.05) between the PD and control group at week 4, 6 and 9 post injection, while no significant differences were obtained at baseline and at week 3, which is in accordance with the animal model.
Assuntos
Encéfalo/metabolismo , Dependovirus/metabolismo , Fluordesoxiglucose F18/metabolismo , Vetores Genéticos/metabolismo , Glucose/metabolismo , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/metabolismo , Tomografia por Emissão de Pósitrons , Animais , Modelos Animais de Doenças , Feminino , Atividade Motora , Doença de Parkinson/fisiopatologia , Ratos Wistar , Máquina de Vetores de SuporteRESUMO
Innate immune activation and chronic neuroinflammation are characteristic features of many neurodegenerative diseases including Parkinson's disease (PD) and may contribute to the pathophysiology of the disease. The discovery of misfolded alpha-synuclein (αSYN) protein aggregates, which amplify in a "prion-like" fashion, has led us to consider that pathogenic αSYN might be hijacking the activation and mobilization mechanism of the peripheral immune system to reach and disseminate within the CNS. Furthermore, our lab and other groups have recently shown that αSYN can adopt distinct fibril conformations or "strains" with varying levels of pathogenic impact. Therefore, the aim of this study was to assess the impact of peripheral inflammation on αSYN spreading in order to better understand the participation of the immune system in the progression of PD. The results presented here show that intraperitoneal LPS injection prior to systemic intravenous recombinant administration of two different αSYN pathogenic strains (fibrils or ribbons) in wild type mice, induces an increase in brain resident microglia and promotes the recruitment of leukocytes toward the brain and the spinal cord. Our findings show for the first time that αSYN can be internalized by LPS-primed inflammatory monocytes, which in turn favors the dissemination from the periphery toward the brain and spinal cord. Further, we found a differential recruitment of CD4+ and CD8+ T cells after LPS priming and subsequent administration of the αSYN ribbons strain. Together, these data argue for a role of the peripheral immune system in αSYN pathology.
Assuntos
Encéfalo/imunologia , Vigilância Imunológica , Inflamação/imunologia , Monócitos/metabolismo , Medula Espinal/imunologia , alfa-Sinucleína/metabolismo , alfa-Sinucleína/farmacologia , Administração Intravenosa , Animais , Encéfalo/patologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Feminino , Injeções Intraperitoneais , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Doença de Parkinson/imunologia , Agregados Proteicos , Medula Espinal/patologia , alfa-Sinucleína/administração & dosagemRESUMO
Rat models based on viral vector-mediated overexpression of α-synuclein are regarded as highly valuable models that closely mimic cardinal features of human Parkinson's disease (PD) such as L-DOPA-dependent motor impairment, dopaminergic neurodegeneration and α-synuclein inclusions. To date, the downstream effects of dopaminergic cell loss on brain glucose metabolism, including the neuroinflammation component, have not been phenotyped in detail for this model. Cerebral glucose metabolism was monitored throughout different stages of the disease using in vivo 2-[18F]-fluoro-2-deoxy-d-glucose ([18F]FDG) positron emission tomography (PET) and was combined with in vitro [18F]DPA-714 autoradiography to assess concomitant inflammation. Rats were unilaterally injected with recombinant adeno-associated viral vector serotype 2/7 (rAAV2/7) encoding either A53T α-synuclein or eGFP. Brain [18F]FDG microPET was performed at baseline, 1, 2, 3, 4, 6, and 9â¯weeks post-surgery, in combination with behavioral tests. As a second experiment, [18F]DPA-714 autoradiography was executed across the same timeline. Voxel-based analysis of relative [18F]FDG uptake showed a dynamic pattern of PD-related metabolic changes throughout the disease progression (weeks 2-9). Glucose hypermetabolism covering a large bilateral area reaching from the insular, motor- and somatosensory cortex to the striatum was observed at week 2. At week 4, hypermetabolism presented in a cluster covering the ipsilateral nigra-thalamic region, whereas hypometabolism was noted in the ipsilateral striatum at week 6. Elevated [18F]FDG uptake was seen in a cluster extending across the contralateral striatum, motor- and somatosensory cortex at week 9. Increased [18F]FDG in the region of the substantia nigra was associated with increased [18F]DPA-714 binding, and correlated significantly with motor symptoms. These findings point to disease-associated metabolic and neuroinflammatory changes taking place in the primary area of dopaminergic neurodegeneration but also closely interconnected motor and somatosensory brain regions.
Assuntos
Encéfalo/metabolismo , Encéfalo/patologia , Glucose/metabolismo , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , Animais , Dependovirus , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Inflamação/metabolismo , Inflamação/patologia , Ratos , Ratos Wistar , alfa-Sinucleína/genéticaRESUMO
Aggregation of alpha-synuclein (α-SYN) is the pathological hallmark of several diseases named synucleinopathies, including Parkinson's disease (PD), which is the most common neurodegenerative motor disorder. Alpha-SYN has been linked to synaptic function both in physiological and pathological conditions. However, the exact link between neuronal activity, α-SYN toxicity and disease progression in PD is not clear. In this study, we aimed to investigate the effect of chronic neuromodulation in an α-SYN-based rat model for PD using chemogenetics. To do this, we expressed excitatory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) combined with mutant A53T α-SYN, using two different recombinant adeno-associated viral (rAAV) vectors (serotypes 2/7 and 2/8) in rat substantia nigra (SN) and investigated the effect on motor behavior, synapses and neuropathology. We found that chronic neuromodulation aggravates motor deficits induced by α-SYN, without altering dopaminergic neurodegeneration. In addition, neuronal activation led to changes in post-translational modification and subcellular localization of α-SYN, linking neuronal activity to the pathophysiological role of α-SYN in PD.
Assuntos
Neurônios/fisiologia , Doença de Parkinson/fisiopatologia , Substância Negra/fisiopatologia , Sinapses/fisiologia , alfa-Sinucleína/metabolismo , Potenciais de Ação , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/fisiologia , Feminino , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Ratos Wistar , Substância Negra/metabolismo , Sinapses/metabolismoRESUMO
The purinergic P2X7 receptor is a key mediator in (neuro)inflammation, a process that is associated with neurodegeneration and excitotoxicity in Parkinson's disease (PD). Recently, P2X7 imaging has become possible with [11C]JNJ-(54173)717. We investigated P2X7 availability, in comparison with availability of the translocator protein (TSPO), in two well-characterized rat models of PD using in vitro autoradiography at multiple time points throughout the disease progression. Rats received either a unilateral injection with 6-hydroxydopamine (6-OHDA) in the striatum, or with recombinant adeno-associated viral vector overexpressing human A53T alpha-synuclein (α-SYN) in the substantia nigra. Transverse cryosections were incubated with [11C]JNJ-717 for P2X7 or [18F]DPA-714 for TSPO. [11C]JNJ-717 binding ratios were transiently elevated in the striatum of 6-OHDA rats at day 14-28 post-injection, with peak P2X7 binding at day 14. This largely coincided with the time course of striatal [18F]DPA-714 binding which was elevated at day 7-21, with peak TSPO binding at day 7. Increased P2X7 availability co-localized with microglial, but not astrocyte or neuronal markers. In the chronic α-SYN model, no significant differences were found in P2X7 binding, although in vitro TSPO overexpression was reported previously. This first study showed an increased P2X7 availability in the acute PD model in a time window corresponding with elevated TSPO binding and motor behavior changes. In contrast, the dynamics of TSPO and P2X7 were divergent in the chronic α-SYN model where no P2X7 changes were detectable. Overall, extended P2X7 phenotyping is warranted prior to implementation of P2X7 imaging for monitoring of neuroinflammation.
RESUMO
Neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD) impose a pressing burden on our developed and consequently aging society. Misfolded protein aggregates are a critical aspect of several neurodegenerative diseases. Nevertheless, several questions remain unanswered regarding the role of misfolded protein aggregates and the cause of neuronal cell death. Recently, it has been postulated that neuroinflammatory processes might play a crucial role in the pathogenesis of PD. Numerous postmortem, brain imaging, epidemiological, and animal studies have documented the involvement of the innate and adaptive immunity in neurodegeneration. Whether these inflammatory processes are directly involved in the etiology of PD or represent secondary consequences of nigrostriatal pathway injury is the subject of intensive research. Immune alterations in response to extracellular α-synuclein may play a critical role in modulating Parkinson's disease progression. In this review, we address the current concept of neuroinflammation and its involvement in PD-associated neurodegeneration.
Assuntos
Doenças Neurodegenerativas/imunologia , Neurônios/fisiologia , Doença de Parkinson/imunologia , Deficiências na Proteostase/imunologia , alfa-Sinucleína/imunologia , Animais , Apoptose , Humanos , Inflamação , Inflamação NeurogênicaRESUMO
Several lines of evidence point to alterations in glutamatergic signaling in Parkinson's disease (PD) and levodopa-induced dyskinesia (LID), involving the metabotropic glutamate receptor type 5 (mGluR5). Using small-animal positron emission tomography (PET) with [18F]FPEB and proton magnetic resonance spectroscopy, we investigated cerebral changes in the mGluR5 and glutamate/glutamine availability in vivo in PD rats and following onset of LIDs. In parallel, behavioral tests were performed. Comparing PD to control rats, mGluR5 binding potential was decreased in a cluster comprising the bilateral caudate-putamen (CP), ipsilateral motor cortex and somatosensory cortex, and the contralateral somatosensory cortex and parietal association cortex, with the most pronounced reduction in the ipsilateral CP. mGluR5 binding potentials were not significantly altered upon levodopa (L-DOPA) treatment. However, following L-DOPA, an increase in relative mGluR5 uptake was present in the contralateral motor cortex and somatosensory cortex. Glutamate and glutamine concentrations did not differ between control and untreated PD rats or between hemispheres. Though, glutamine levels were higher in the contralateral CP of saline- and L-DOPA-treated rats as compared to the ipsilateral side. Relative mGluR5 uptake in the CP of levodopa-treated rats was also found positively correlated with abnormal involuntary movement scores. Conclusively, mGluR5 availability and glutamine concentrations in the CP are involved in PD, whereas mGluR5 availability in cortical regions may be involved in LID pathology.
Assuntos
Córtex Cerebral/metabolismo , Discinesias , Glutamina/metabolismo , Levodopa , Oxidopamina , Doença de Parkinson , Putamen/metabolismo , Receptor de Glutamato Metabotrópico 5/metabolismo , Doença Aguda , Animais , Córtex Cerebral/diagnóstico por imagem , Modelos Animais de Doenças , Discinesias/etiologia , Discinesias/metabolismo , Feminino , Levodopa/efeitos adversos , Oxidopamina/efeitos adversos , Doença de Parkinson/etiologia , Doença de Parkinson/metabolismo , Tomografia por Emissão de Pósitrons , Espectroscopia de Prótons por Ressonância Magnética , Putamen/diagnóstico por imagem , Ratos WistarRESUMO
The P2X7 receptor plays a significant role in microglial activation, and as a potential drug target, the P2X7 receptor is also an interesting target in positron emission tomography. The current study aimed at the development and evaluation of a potent tracer targeting the P2X7 receptor, to which end four adamantanyl benzamide analogues with high affinity for the human P2X7 receptor were labelled with carbon-11. All four analogues could be obtained in excellent radiochemical yield and high radiochemical purity and molar activity, and all analogues entered the rat brain. [11C]SMW139 showed the highest metabolic stability in rat plasma, and showed high binding to the hP2X7 receptor in vivo in a hP2X7 receptor overexpressing rat model. Although no significant difference in binding of [11C]SMW139 was observed between post mortem brain tissue of Alzheimer's disease patients and that of healthy controls in in vitro autoradiography experiments, [11C]SMW139 could be a promising tracer for P2X7 receptor imaging using positron emission tomography, due to high receptor binding in vivo in the hP2X7 receptor overexpressing rat model. However, further investigation of both P2X7 receptor expression and binding of [11C]SMW139 in other neurological diseases involving microglial activation is warranted.
Assuntos
Microglia/metabolismo , Imagem Molecular , Tomografia por Emissão de Pósitrons , Antagonistas do Receptor Purinérgico P2X/química , Compostos Radiofarmacêuticos/química , Receptores Purinérgicos P2X7/química , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Humanos , Microglia/efeitos dos fármacos , Estrutura Molecular , Tomografia por Emissão de Pósitrons/métodos , Ligação Proteica , Antagonistas do Receptor Purinérgico P2X/farmacocinética , Radioquímica , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Coloração e Rotulagem , Distribuição TecidualRESUMO
In order to study the molecular pathways of Parkinson's disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic α-SYN mouse models develop gradual α-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson's disease.
Assuntos
Técnicas de Transferência de Genes , Doença de Parkinson/genética , alfa-Sinucleína/genética , Animais , Dependovirus/genética , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Feminino , Vetores Genéticos/administração & dosagem , Injeções , Camundongos Transgênicos , Ratos Wistar , Substância Negra/metabolismo , Transgenes/genéticaRESUMO
Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recent successes in recombinant adeno-associated viral vector (rAAV)-based gene therapy in the clinic for hereditary disorders such as Leber's congenital amaurosis and hemophilia B encouraged us to reexplore an rAAV approach for pulmonary gene transfer. Only limited clinical successes have been achieved for airway gene transfer so far, underscoring the need for further preclinical development of rAAV-based gene therapy for pulmonary disorders. We sought to determine the preclinical potential of an airway-tropic serotype, rAAV2/5, encoding reporter genes when delivered to mouse airways. Although several groups have assessed the stability of gene transfer using a nonintegrating rAAV in mouse airways, long-term stability for more than a year has not been reported. Additionally, an extensive quantitative analysis of the specific cell types targeted by rAAV2/5 using cell-specific markers is lacking. We obtained sustained gene expression in upper and lower airways up to 15 months after vector administration, a substantial proportion of the lifespan of a laboratory mouse. In addition, we demonstrated that readministration of rAAV2/5 to the airways is feasible and increases gene expression 14 months after primary vector administration, despite the presence of circulating neutralizing antibodies. Finally, identification of transduced cell types revealed different subpopulations being targeted by rAAV2/5, with 64% of ß-galactosidase-positive cells being ciliated cells, 34% club cells in the conducting airways, and 75% alveolar type II cells in the alveoli at 1 month postinjection. This underscores the therapeutic potential of a nonintegrating rAAV vector to develop a gene therapeutic drug for a variety of pulmonary disorders, such as cystic fibrosis, primary ciliary dyskinesia, and surfactant deficiencies.
Assuntos
Dependovirus/genética , Terapia Genética , Pneumopatias , Pulmão/metabolismo , Animais , Dependovirus/isolamento & purificação , Regulação da Expressão Gênica , Vetores Genéticos/genética , Humanos , Pulmão/patologia , Pneumopatias/genética , Pneumopatias/terapia , Camundongos , Imagem Molecular , Transgenes/genéticaRESUMO
In order to study the molecular pathways of Parkinson's disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models as an alternative to toxin-based models. Viral vector-mediated loco-regional gene delivery provides an attractive way to express transgenes in the central nervous system. Several vector systems based on various viruses have been developed. In this chapter, we give an overview of the different viral vector systems used for targeting the CNS. Further, we describe the different viral vector-based PD models currently available based on overexpression strategies for autosomal dominant genes such as α-synuclein and LRRK2, and knockout or knockdown strategies for autosomal recessive genes, such as parkin, DJ-1, and PINK1. Models based on overexpression of α-synuclein are the most prevalent and extensively studied, and therefore the main focus of this chapter. Many efforts have been made to increase the expression levels of α-synuclein in the dopaminergic neurons. The best α-synuclein models currently available have been developed from a combined approach using newer AAV serotypes and optimized vector constructs, production, and purification methods. These third-generation α-synuclein models show improved face and predictive validity, and therefore offer the possibility to reliably test novel therapeutics.
Assuntos
Dependovirus , Modelos Animais de Doenças , Vetores Genéticos , Lentivirus , Doença de Parkinson/genética , alfa-Sinucleína/metabolismo , AnimaisRESUMO
Parkinson's disease is a neurodegenerative disorder characterized by motor and non-motor disturbances. Various pathogenic pathways drive disease progression including oxidative stress, mitochondrial dysfunction, α-synuclein aggregation and impairment of protein degradation systems. Dysfunction of the ubiquitin-proteasome system in the substantia nigra of Parkinson's disease patients is believed to be one of the causes of protein aggregation and cell death associated with this disorder. Lactacystin, a potent inhibitor of the proteasome, was previously delivered to the nigrostriatal pathway of rodents to model nigrostriatal degeneration. Although lactacystin-treated animals develop parkinsonian motor impairment, it is currently unknown whether they also develop non-motor symptoms characteristic of this disorder. In order to further describe the proteasome inhibition model of Parkinson's disease, we characterized the unilateral lactacystin model, performed by stereotaxic injection of the toxin in the substantia nigra of mice. We studied the degree of neurodegeneration and the behavioral phenotype 1 and 3 weeks after lactacystin lesion both in terms of motor impairment, as well as non-motor symptoms. We report that unilateral administration of 3 µg lactacystin to the substantia nigra of mice leads to partial (~40%) dopaminergic cell loss and concurrent striatal dopamine depletion, accompanied by increased expression of Ser129-phosphorylated α-synuclein. Behavioral characterization of the model revealed parkinsonian motor impairment, as well as signs of non-motor disturbances resembling early stage Parkinson's disease including sensitive and somatosensory deficits, anxiety-like behavior, and perseverative behavior. The consistent finding of good face validity, together with relevant construct validity, warrant a further evaluation of proteasome inhibition models of Parkinson's disease in pre-clinical research and validation of therapeutic targets.
RESUMO
BACKGROUND: Alpha-synuclein is a key protein in the pathogenesis of Parkinson's disease. Mutations in the parkin gene are the most common cause of early-onset autosomal recessive Parkinson's disease, probably through a loss-of-function mechanism. However, the molecular mechanism by which loss of parkin function leads to the development of the disease and the role of alpha-synuclein in parkin-associated Parkinson's disease is still not elucidated. Conflicting results were reported about the effect of the absence of parkin on alpha-synuclein-mediated neurotoxicity using a transgenic approach. In this study, we investigated the effect of loss of parkin on alpha-synuclein neuropathology and toxicity in adult rodent brain using viral vectors. Therefore, we overexpressed human wild type alpha-synuclein in the substantia nigra of parkin knockout and wild type mice using two different doses of recombinant adeno-associated viral vectors. RESULTS: No difference was observed in nigral dopaminergic cell loss between the parkin knockout mice and wild type mice up to 16 weeks after viral vector injection. However, the level of alpha-synuclein phosphorylated at serine residue 129 in the substantia nigra was significantly increased in the parkin knockout mice compared to the wild type mice while the total expression level of alpha-synuclein was similar in both groups. The increased alpha-synuclein phosphorylation was confirmed in a parkin knockdown cell line. CONCLUSIONS: These findings support a functional relationship between parkin and alpha-synuclein phosphorylation in rodent brain.