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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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rAAV2/7 vector-mediated overexpression of α-synuclein in mouse SN induces progressive cell loss and α-synuclein aggregation. (A) Images of immunohistochemical stainings for α-synuclein showing the whole area of transduction 5 days after rAAV2/7-α-synuclein injection. Scale bar = 500 μm. (B) Overexpression of WT α-synuclein induces a progressive and dose-dependent α-synuclein-positive cell loss over time in the injected SN. Absence of immunoreactivity in the contralateral SN at 5 days after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. N-inj: non-injected side. (C) High magnification pictures demonstrate the presence of α-synuclein-positive aggregates (arrows) in the SN for each vector dose at 4 weeks post-injection. (D) High magnification picture of an α-synuclein-positive cell without aggregates. Scale bars (C-D) = 5 μm. (E) Stereological quantification of the α-synuclein-positive volume in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose after 5 days, 4 weeks and 8 weeks. (F) Stereological quantification of the number of α-synuclein-positive cells in the SN of mice after 5 days, 4 weeks and 8 weeks. Asterisks (*) depict significant decrease respective to 4 weeks, 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 2: rAAV2/7 vector-mediated overexpression of α-synuclein in mouse SN induces progressive cell loss and α-synuclein aggregation. (A) Images of immunohistochemical stainings for α-synuclein showing the whole area of transduction 5 days after rAAV2/7-α-synuclein injection. Scale bar = 500 μm. (B) Overexpression of WT α-synuclein induces a progressive and dose-dependent α-synuclein-positive cell loss over time in the injected SN. Absence of immunoreactivity in the contralateral SN at 5 days after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. N-inj: non-injected side. (C) High magnification pictures demonstrate the presence of α-synuclein-positive aggregates (arrows) in the SN for each vector dose at 4 weeks post-injection. (D) High magnification picture of an α-synuclein-positive cell without aggregates. Scale bars (C-D) = 5 μm. (E) Stereological quantification of the α-synuclein-positive volume in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose after 5 days, 4 weeks and 8 weeks. (F) Stereological quantification of the number of α-synuclein-positive cells in the SN of mice after 5 days, 4 weeks and 8 weeks. Asterisks (*) depict significant decrease respective to 4 weeks, 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: In order to evaluate the dose- and time-dependent effects of α-synuclein overexpression, rAAV2/7-α-synuclein WT vector injections were unilaterally performed in adult mouse SN at 3 different vector titers: 2,6E + 11 GC/ml; 4,0E + 11 GC/ml and 8,0E + 11 GC/ml. Mice were perfused for immunohistochemical analysis at 5 days, 4 weeks and 8 weeks after surgery. Staining of the nigral sections for human α-synuclein revealed widespread expression of the transgene in the injected area after 5 days (Figure 2A-B). In addition, we observed a progressive reduction in α-synuclein expression over time in the injected region for all vector doses tested, and the loss was faster and stronger with increasing viral vector titer (Figure 2B).


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)

rAAV2/7 vector-mediated overexpression of α-synuclein in mouse SN induces progressive cell loss and α-synuclein aggregation. (A) Images of immunohistochemical stainings for α-synuclein showing the whole area of transduction 5 days after rAAV2/7-α-synuclein injection. Scale bar = 500 μm. (B) Overexpression of WT α-synuclein induces a progressive and dose-dependent α-synuclein-positive cell loss over time in the injected SN. Absence of immunoreactivity in the contralateral SN at 5 days after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. N-inj: non-injected side. (C) High magnification pictures demonstrate the presence of α-synuclein-positive aggregates (arrows) in the SN for each vector dose at 4 weeks post-injection. (D) High magnification picture of an α-synuclein-positive cell without aggregates. Scale bars (C-D) = 5 μm. (E) Stereological quantification of the α-synuclein-positive volume in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose after 5 days, 4 weeks and 8 weeks. (F) Stereological quantification of the number of α-synuclein-positive cells in the SN of mice after 5 days, 4 weeks and 8 weeks. Asterisks (*) depict significant decrease respective to 4 weeks, 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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Show All Figures
getmorefigures.php?uid=PMC4222878&req=5

Figure 2: rAAV2/7 vector-mediated overexpression of α-synuclein in mouse SN induces progressive cell loss and α-synuclein aggregation. (A) Images of immunohistochemical stainings for α-synuclein showing the whole area of transduction 5 days after rAAV2/7-α-synuclein injection. Scale bar = 500 μm. (B) Overexpression of WT α-synuclein induces a progressive and dose-dependent α-synuclein-positive cell loss over time in the injected SN. Absence of immunoreactivity in the contralateral SN at 5 days after injection when expression was maximal. Right panels are magnifications of the overview (left panels). Scale bars = 200 μm. N-inj: non-injected side. (C) High magnification pictures demonstrate the presence of α-synuclein-positive aggregates (arrows) in the SN for each vector dose at 4 weeks post-injection. (D) High magnification picture of an α-synuclein-positive cell without aggregates. Scale bars (C-D) = 5 μm. (E) Stereological quantification of the α-synuclein-positive volume in the SN of mice injected with 3 different WT α-synuclein vector doses and a unique A53T α-synuclein vector dose after 5 days, 4 weeks and 8 weeks. (F) Stereological quantification of the number of α-synuclein-positive cells in the SN of mice after 5 days, 4 weeks and 8 weeks. Asterisks (*) depict significant decrease respective to 4 weeks, 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: In order to evaluate the dose- and time-dependent effects of α-synuclein overexpression, rAAV2/7-α-synuclein WT vector injections were unilaterally performed in adult mouse SN at 3 different vector titers: 2,6E + 11 GC/ml; 4,0E + 11 GC/ml and 8,0E + 11 GC/ml. Mice were perfused for immunohistochemical analysis at 5 days, 4 weeks and 8 weeks after surgery. Staining of the nigral sections for human α-synuclein revealed widespread expression of the transgene in the injected area after 5 days (Figure 2A-B). In addition, we observed a progressive reduction in α-synuclein expression over time in the injected region for all vector doses tested, and the loss was faster and stronger with increasing viral vector titer (Figure 2B).

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