rAAV2/7 vector-mediated overexpression of alpha-synuclein in mouse substantia nigra induces protein aggregation and progressive dose-dependent neurodegeneration

Background Alpha-synuclein is a key protein implicated in the pathogenesis of Parkinson's disease (PD). It is the main component of the Lewy bodies, a cardinal neuropathological feature in the disease. In addition, whole locus multiplications and point mutations in the gene coding for alpha-synuclein lead to autosomal dominant monogenic PD. Over the past decade, research on PD has impelled the development of new animal models based on alpha-synuclein. In this context, transgenic mouse lines have failed to reproduce several hallmarks of PD, especially the strong and progressive dopaminergic neurodegeneration over time that occurs in the patients. In contrast, viral vector-based models in rats and non-human primates display prominent, although highly variable, nigral dopaminergic neuron loss. However, the few studies available on viral vector-mediated overexpression of alpha-synuclein in mice report a weak neurodegenerative process and no clear Lewy body-like pathology. To address this issue, we performed a comprehensive comparative study of alpha-synuclein overexpression by means of recombinant adeno-associated viral vectors serotype 2/7 (rAAV2/7) at different doses in adult mouse substantia nigra. Results We noted a significant and dose-dependent alpha-synucleinopathy over time upon nigral viral vector-mediated alpha-synuclein overexpression. We obtained a strong, progressive and dose-dependent loss of dopaminergic neurons in the substantia nigra, reaching a maximum of 82% after 8 weeks. This effect correlated with a reduction in tyrosine hydroxylase immunoreactivity in the striatum. Moreover, behavioural analysis revealed significant motor impairments from 12 weeks after injection on. In addition, we detected the presence of alpha-synuclein-positive aggregates in the remaining surviving neurons. When comparing wild-type to mutant A53T alpha-synuclein at the same vector dose, both induced a similar degree of cell death. These data were supported by a biochemical analysis that showed a net increase in soluble and insoluble alpha-synuclein expression over time to the same extent for both alpha-synuclein variants. Conclusions In conclusion, our in vivo data provide evidence that strong and significant alpha-synuclein-induced neuropathology and progressive dopaminergic neurodegeneration can be achieved in mouse brain by means of rAAV2/7

Peripheral Inflammation Regulates CNS Immune Surveillance Through the Recruitment of Inflammatory Monocytes Upon Systemic α-Synuclein Administration

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

Linking Neuroinflammation and Neurodegeneration in Parkinson’s Disease

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

Viral-vector based models of Parkinson's disease

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.status: publishe

Long-term overexpression of human wild-type and T240R mutant parkin in rat substantia nigra induces progressive dopaminergic neurodegeneration

Mutations in the parkin gene are the most common cause of early-onset autosomal recessive Parkinson disease (PD). The pathogenic mechanisms of how parkin mutations lead to the development of PD are not fully understood. Studies of cell cultures and of Drosophila have suggested a dominant negative effect for the clinical parkin mutant T240R. Conversely, the neuroprotective capacity of parkin has been widely reported; this suggests that the parkin protein may have a potential therapeutic role in PD. Here, we aimed to develop a novel genetic rodent model of PD by overexpression of T240R-parkin and human wild-type parkin as a control in the dopaminergic neurons of adult rats using adeno-associated viral vectors (rAAV2/8). Surprisingly, we found that overexpression not only of T240R-parkin but also of human wild-type parkin induced progressive and dose-dependent dopaminergic cell death in rats, starting from 8 weeks after injection. This degeneration was specific for parkin because similar overexpressionof enhanced green fluorescent protein did not lead to nigral degeneration. Our results warrant caution to the development of therapeutic strategies for PD based on overexpression of parkin or enhancing parkin activity because this might be deleterious for dopaminergic neurons in the long-term.status: publishe

Identifying a glucose metabolic brain pattern in an adeno-associated viral vector based rat model for Parkinson's disease using F-18-FDG PET imaging

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.status: publishe

Comparison of different rAAV serotypes for transduction of dopaminergic neurons in rat substantia nigra

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Unraveling the role of FK506 binding proteins in the aggregation and neurotoxicity of α-synuclein, a key player in Parkinson’s disease

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