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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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Efficient dopaminergic neuron transduction upon rAAV2/7 vector delivery in mouse SN. (A-B) Representative confocal images of fluorescent double immunostainings for α-synuclein (green) and TH (red) at (A) 5 days and at (B) 8 weeks after injection of rAAV2/7-α-synuclein WT at 8,0E + 11 GC/ml. (A) At 5 days post-injection, pictures reveal extensive co-localization (merge) in the transduced region. Bottom panels are magnifications of the overviews (upper panels). Scale bar upper panel = 200 μm and bottom panel = 50 μm. Confocal images of double immunostainings for α-synuclein (green) and NeuN or GFAP (red) show an almost exclusive neuronal transduction. Scale bar = 100 μm. (B) At 8 weeks after injection, a clear degeneration of the nigral dopaminergic neurons is observed upon rAAV2/7 vector-mediated α-synuclein overexpression. Dopaminergic neurons remain widely present in the contralateral side. (C-D) Fluorescent double stainings for GFP (green) and TH (red) at (C) 5 days and at (D) 8 weeks post-injection show that rAAV2/7-eGFP at 8,0E + 11 GC/ml does not induce any dopaminergic cell death over time. Inj: injected side; N-inj: non-injected side.
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Figure 1: Efficient dopaminergic neuron transduction upon rAAV2/7 vector delivery in mouse SN. (A-B) Representative confocal images of fluorescent double immunostainings for α-synuclein (green) and TH (red) at (A) 5 days and at (B) 8 weeks after injection of rAAV2/7-α-synuclein WT at 8,0E + 11 GC/ml. (A) At 5 days post-injection, pictures reveal extensive co-localization (merge) in the transduced region. Bottom panels are magnifications of the overviews (upper panels). Scale bar upper panel = 200 μm and bottom panel = 50 μm. Confocal images of double immunostainings for α-synuclein (green) and NeuN or GFAP (red) show an almost exclusive neuronal transduction. Scale bar = 100 μm. (B) At 8 weeks after injection, a clear degeneration of the nigral dopaminergic neurons is observed upon rAAV2/7 vector-mediated α-synuclein overexpression. Dopaminergic neurons remain widely present in the contralateral side. (C-D) Fluorescent double stainings for GFP (green) and TH (red) at (C) 5 days and at (D) 8 weeks post-injection show that rAAV2/7-eGFP at 8,0E + 11 GC/ml does not induce any dopaminergic cell death over time. Inj: injected side; N-inj: non-injected side.

Mentions: rAAV2/7-α-synuclein WT and rAAV2/7-eGFP vectors were stereotactically injected in mouse SN (2 μl of 8,0E + 11 GC/ml). We performed fluorescent double immunostainings to assess transgene expression (Figure 1). At 5 days post-injection, widespread transgene expression was evident in the injected side for both α-synuclein and eGFP viral vectors (Figure 1A, C). Transgene expression of eGFP appeared stronger than α-synuclein expression at comparable vector doses, although this might be due to differential sensitivities of the respective antibodies. Co-localization of α-synuclein or GFP with the dopaminergic marker tyrosine hydroxylase (TH) indicated efficient transduction of the nigral dopaminergic neurons distributed throughout the SN pars compacta. Approximately 85% of the dopaminergic neurons in SN were transduced by the viral vectors as assessed by high magnification confocal imaging (Figure 1A). In addition, double immunostaining for α-synuclein and the neuron-specific nuclear marker NeuN clearly indicated efficient transduction of neurons in the injected area, around 95% of double-positive cells, whereas double immunostainings for α-synuclein and the astrocytic marker GFAP showed an absence of co-localization, pointing to an almost exclusive neuronal transduction.


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)

Efficient dopaminergic neuron transduction upon rAAV2/7 vector delivery in mouse SN. (A-B) Representative confocal images of fluorescent double immunostainings for α-synuclein (green) and TH (red) at (A) 5 days and at (B) 8 weeks after injection of rAAV2/7-α-synuclein WT at 8,0E + 11 GC/ml. (A) At 5 days post-injection, pictures reveal extensive co-localization (merge) in the transduced region. Bottom panels are magnifications of the overviews (upper panels). Scale bar upper panel = 200 μm and bottom panel = 50 μm. Confocal images of double immunostainings for α-synuclein (green) and NeuN or GFAP (red) show an almost exclusive neuronal transduction. Scale bar = 100 μm. (B) At 8 weeks after injection, a clear degeneration of the nigral dopaminergic neurons is observed upon rAAV2/7 vector-mediated α-synuclein overexpression. Dopaminergic neurons remain widely present in the contralateral side. (C-D) Fluorescent double stainings for GFP (green) and TH (red) at (C) 5 days and at (D) 8 weeks post-injection show that rAAV2/7-eGFP at 8,0E + 11 GC/ml does not induce any dopaminergic cell death over time. Inj: injected side; N-inj: non-injected side.
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Related In: Results  -  Collection

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Figure 1: Efficient dopaminergic neuron transduction upon rAAV2/7 vector delivery in mouse SN. (A-B) Representative confocal images of fluorescent double immunostainings for α-synuclein (green) and TH (red) at (A) 5 days and at (B) 8 weeks after injection of rAAV2/7-α-synuclein WT at 8,0E + 11 GC/ml. (A) At 5 days post-injection, pictures reveal extensive co-localization (merge) in the transduced region. Bottom panels are magnifications of the overviews (upper panels). Scale bar upper panel = 200 μm and bottom panel = 50 μm. Confocal images of double immunostainings for α-synuclein (green) and NeuN or GFAP (red) show an almost exclusive neuronal transduction. Scale bar = 100 μm. (B) At 8 weeks after injection, a clear degeneration of the nigral dopaminergic neurons is observed upon rAAV2/7 vector-mediated α-synuclein overexpression. Dopaminergic neurons remain widely present in the contralateral side. (C-D) Fluorescent double stainings for GFP (green) and TH (red) at (C) 5 days and at (D) 8 weeks post-injection show that rAAV2/7-eGFP at 8,0E + 11 GC/ml does not induce any dopaminergic cell death over time. Inj: injected side; N-inj: non-injected side.
Mentions: rAAV2/7-α-synuclein WT and rAAV2/7-eGFP vectors were stereotactically injected in mouse SN (2 μl of 8,0E + 11 GC/ml). We performed fluorescent double immunostainings to assess transgene expression (Figure 1). At 5 days post-injection, widespread transgene expression was evident in the injected side for both α-synuclein and eGFP viral vectors (Figure 1A, C). Transgene expression of eGFP appeared stronger than α-synuclein expression at comparable vector doses, although this might be due to differential sensitivities of the respective antibodies. Co-localization of α-synuclein or GFP with the dopaminergic marker tyrosine hydroxylase (TH) indicated efficient transduction of the nigral dopaminergic neurons distributed throughout the SN pars compacta. Approximately 85% of the dopaminergic neurons in SN were transduced by the viral vectors as assessed by high magnification confocal imaging (Figure 1A). In addition, double immunostaining for α-synuclein and the neuron-specific nuclear marker NeuN clearly indicated efficient transduction of neurons in the injected area, around 95% of double-positive cells, whereas double immunostainings for α-synuclein and the astrocytic marker GFAP showed an absence of co-localization, pointing to an almost exclusive neuronal transduction.

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