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

Oliveras-Salvá M, Van der Perren A, Casadei N, Stroobants S, Nuber S, D'Hooge R, Van den Haute C, Baekelandt V - Mol Neurodegener (2013)

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurosciences and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, Laboratory for Neurobiology and Gene Therapy, Kapucijnenvoer 33, box 7001, 3000 Leuven, Belgium. veerle.baekelandt@med.kuleuven.be.

ABSTRACT

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.

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Phosphorylation of α-synuclein at S129 increases over time in a dose-dependent manner after rAAV2/7-α-synuclein delivery. (A) Representative images of P-S129 α-synuclein expression in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose show that overexpression of WT α-synuclein induces a progressive and dose-dependent increase over time in P-S129 α-synuclein. Lack of immunoreactivity in the contralateral SN at 8 weeks after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. (B) Stereological quantification of the number of P-S129 α-synuclein positive cells in the injected SN after 5 days, 4 weeks and 8 weeks. (C) Percentage of P-S129 α-synuclein positive cells in the SN at 5 days, 4 weeks and 8 weeks post-injection. Asterisks (*) depict significant increase respective to 5 days, unless specified otherwise. 5 days 4,0E + 11 GC/ml WT/A53T: n = 3; 5 days 2,6/8,0E + 11 GC/ml WT: n = 4; 4 weeks: n = 4; 8 weeks: n = 4.
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Figure 4: Phosphorylation of α-synuclein at S129 increases over time in a dose-dependent manner after rAAV2/7-α-synuclein delivery. (A) Representative images of P-S129 α-synuclein expression in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose show that overexpression of WT α-synuclein induces a progressive and dose-dependent increase over time in P-S129 α-synuclein. Lack of immunoreactivity in the contralateral SN at 8 weeks after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. (B) Stereological quantification of the number of P-S129 α-synuclein positive cells in the injected SN after 5 days, 4 weeks and 8 weeks. (C) Percentage of P-S129 α-synuclein positive cells in the SN at 5 days, 4 weeks and 8 weeks post-injection. Asterisks (*) depict significant increase respective to 5 days, unless specified otherwise. 5 days 4,0E + 11 GC/ml WT/A53T: n = 3; 5 days 2,6/8,0E + 11 GC/ml WT: n = 4; 4 weeks: n = 4; 8 weeks: n = 4.

Mentions: Previous studies have shown that approximately 90% of the α-synuclein accumulated in the LBs is phosphorylated at serine residue at position 129 (P-S129) [30,31]. Therefore, it is considered a marker of α-synuclein neuropathology [49]. In order to further characterize the neuropathology induced by the overexpression of α-synuclein in mouse SN, we performed immunohistochemical stainings for P-S129 α-synuclein (Figure 4A). Overall, we observed a net increase over time in the number of P-S129 α-synuclein-positive cells after injection of either rAAV2/7-α-synuclein WT or A53T viral vectors while the contralateral side showed no apparent immunoreactivity. A maximum of P-S129 α-synuclein-positive cells was reached at 4 weeks post-injection by WT α-synuclein vector delivered at 4,0E + 11 or 8,0E + 11 GC/ml (Figure 4B). At 8 weeks after injection, the number of positive cells at the intermediate dose remained invariable, while a trend to a decrease was observed for the highest dose, suggesting that the vast cell loss observed at this time point affects the P-S129 α-synuclein counts. For the lowest α-synuclein vector dose (2,6E + 11 GC/ml), a more moderate and delayed increase in the number of nigral P-S129 α-synuclein-positive cells was observed over time, in accordance with the lower rate of neurodegeneration achieved by this dose. Interestingly, the A53T α-synuclein condition exhibited a somewhat lower number of nigral P-S129 α-synuclein-positive cells at 5 days and 4 weeks when compared to the WT α-synuclein viral vector.


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

Oliveras-Salvá M, Van der Perren A, Casadei N, Stroobants S, Nuber S, D'Hooge R, Van den Haute C, Baekelandt V - Mol Neurodegener (2013)

Phosphorylation of α-synuclein at S129 increases over time in a dose-dependent manner after rAAV2/7-α-synuclein delivery. (A) Representative images of P-S129 α-synuclein expression in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose show that overexpression of WT α-synuclein induces a progressive and dose-dependent increase over time in P-S129 α-synuclein. Lack of immunoreactivity in the contralateral SN at 8 weeks after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. (B) Stereological quantification of the number of P-S129 α-synuclein positive cells in the injected SN after 5 days, 4 weeks and 8 weeks. (C) Percentage of P-S129 α-synuclein positive cells in the SN at 5 days, 4 weeks and 8 weeks post-injection. Asterisks (*) depict significant increase respective to 5 days, unless specified otherwise. 5 days 4,0E + 11 GC/ml WT/A53T: n = 3; 5 days 2,6/8,0E + 11 GC/ml WT: n = 4; 4 weeks: n = 4; 8 weeks: n = 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Phosphorylation of α-synuclein at S129 increases over time in a dose-dependent manner after rAAV2/7-α-synuclein delivery. (A) Representative images of P-S129 α-synuclein expression in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose show that overexpression of WT α-synuclein induces a progressive and dose-dependent increase over time in P-S129 α-synuclein. Lack of immunoreactivity in the contralateral SN at 8 weeks after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. (B) Stereological quantification of the number of P-S129 α-synuclein positive cells in the injected SN after 5 days, 4 weeks and 8 weeks. (C) Percentage of P-S129 α-synuclein positive cells in the SN at 5 days, 4 weeks and 8 weeks post-injection. Asterisks (*) depict significant increase respective to 5 days, unless specified otherwise. 5 days 4,0E + 11 GC/ml WT/A53T: n = 3; 5 days 2,6/8,0E + 11 GC/ml WT: n = 4; 4 weeks: n = 4; 8 weeks: n = 4.
Mentions: Previous studies have shown that approximately 90% of the α-synuclein accumulated in the LBs is phosphorylated at serine residue at position 129 (P-S129) [30,31]. Therefore, it is considered a marker of α-synuclein neuropathology [49]. In order to further characterize the neuropathology induced by the overexpression of α-synuclein in mouse SN, we performed immunohistochemical stainings for P-S129 α-synuclein (Figure 4A). Overall, we observed a net increase over time in the number of P-S129 α-synuclein-positive cells after injection of either rAAV2/7-α-synuclein WT or A53T viral vectors while the contralateral side showed no apparent immunoreactivity. A maximum of P-S129 α-synuclein-positive cells was reached at 4 weeks post-injection by WT α-synuclein vector delivered at 4,0E + 11 or 8,0E + 11 GC/ml (Figure 4B). At 8 weeks after injection, the number of positive cells at the intermediate dose remained invariable, while a trend to a decrease was observed for the highest dose, suggesting that the vast cell loss observed at this time point affects the P-S129 α-synuclein counts. For the lowest α-synuclein vector dose (2,6E + 11 GC/ml), a more moderate and delayed increase in the number of nigral P-S129 α-synuclein-positive cells was observed over time, in accordance with the lower rate of neurodegeneration achieved by this dose. Interestingly, the A53T α-synuclein condition exhibited a somewhat lower number of nigral P-S129 α-synuclein-positive cells at 5 days and 4 weeks when compared to the WT α-synuclein viral vector.

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurosciences and Leuven Research Institute for Neuroscience and Disease (LIND), KU Leuven, Laboratory for Neurobiology and Gene Therapy, Kapucijnenvoer 33, box 7001, 3000 Leuven, Belgium. veerle.baekelandt@med.kuleuven.be.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus