Title: Development, characterization and validation of a rat model for Parkinson's disease based on viral vector mediated overexpression of A53T alpha-synuclein
Other Titles: Ontwikkeling, karakterisatie en validatie van een ratmodel voor de ziekte van Parkinson gebaseerd op overexpressie van A53T alfa-synucleïne via virale vectoren.
Authors: Van der Perren, Anke
Issue Date: 5-Jun-2012
Abstract: Parkinson’s disease (PD) is a progressive, age-related neurodegenerative movement disorder affecting about 1–2% of people above 60 years. Prominent neuropathological hallmarks of PD are the loss of the dopaminergic neurons in the substantia nigra and the presence of α-synuclein containing intracellular inclusions: Lewy bodies and Lewy neurietes. Alpha-synuclein, a protein of 140 amino acids localized in nerve terminals, is the major fibrillar protein in Lewy bodies and Lewy neurietes in sporadic and inherited PD. Moreover, point mutations (A53T, A30P, E46K) and multiplications of the α-synuclein gene cause a rare early-onset familial form of PD, suggesting that gene dosage and aberrant protein structure may accelerate disease onset and progression. Experimentalmodels of PD are indispensable tools in preclinical research both for the elucidation of the disease mechanisms and for the development of novel therapeutics. The toxin-based models utilizing 6-OHDA, MPTP or rotenone have been extremely useful for the development of symptomatic therapies, but these models have limitations: they do not fully address the underlying neuropathological changes and they are not really progressive. A large number of transgenic mice overexpressing mutant or wild type α-synuclein have been generated during the last decade, but none ofthem have so far reproduced the prominent, progressive neurodegenerative changes that are the hallmarks of human PD. In this regard, overexpression of α-synuclein by viral vector-mediated gene delivery offers a valuable alternative approach. Recombinant adeno-associated viral (rAAV) vectors are particularly attractive for targeting the SN because of their high titers and tropism for neurons while inducing minimal immune responses in the brain. First, in light of the further development of a viral vector-mediated rat model for PD, we performed a comprehensive comparison of seven rAAV vector serotypes (rAAV 2/1, 2/2, 2/5, 2/6.2, 2/7, 2/8 and 2/9) for tropism and transduction efficiency of dopaminergic neurons in the substantia nigra of adult rats. Our results show that various rAAV serotypes efficiently transduce nigraldopaminergic neurons, but significant differences in transgene expression pattern and level were observed. For targeting the dopaminergic neurons of the substantia nigra pars compacta rAAV 2/7, 2/9, 2/1 and 2/8performed best, with rAAV 2/7 being superior in terms of overall transgene expression levels. Next, we aimed to generate a robust rat model for PD by rAAV 2/7-mediated overexpression of A53T α-synuclein in the rat substantia nigra. Nigral delivery of A53T α-synuclein by rAAV2/7 resulted in high α-synuclein expression and concomitant loss of the nigral neurons and projecting fibers in the striatum in a time- and dose-dependent manner. In addition the consistent and reproducible levels of neurodegeneration achieved in our model were accompanied by dose-dependent motor impairments in all animals which could be completely reversed by L-DOPA administration. Progressive dopaminergic dysfunction was corroborated by non-invasive imaging and microdialysis. Furthermore, we observed both α-synuclein-positive cytoplasmic aggregates and dystrophic neurites. Using a combination of immunohistochemical characterization and western blotanalysis of tissue homogenates, we could confirm that α-synuclein becomes phosphorylated, ubiquitinated and insoluble over time, indicative of a pathological aggregation process. To summarize, we successfully developed, characterized and validated a rat PD model based on the rAAV2/7-mediated expression of human A53T α-synuclein in the substantia nigra. Finally, we studied the neuroprotective effect of FK506 on α-synuclein induced neurodegeneration in our rat model. The survival rate of dopaminergic neurons was significantly higher in rats treated with FK506 compared to placebo controls four weeks after A53T α-synuclein rAAV2/7 injection. Moreover, this neuroprotection resulted in a positive trend towards behavioral improvement. To understand the mechanism by which FK506prevents α-synuclein-induced degeneration, we investigated whether FK506 affects α-synuclein aggregation or α-synuclein-induced neuroinflammation in vivo. Based on our data, we hypothesize that in our fast A53T α-synuclein rAAV2/7 based rat model the immunosuppressive properties of FK506 may have been the predominant factor to determine the neuroprotective effects and may have masked a potential more subtle and slower effect on α-synuclein aggregation.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Research Group for Neurobiology and Gene Therapy
Facility for Surgery and Anaesthesiology
Department of Human Genetics - miscellaneous

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