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Nigral overexpression of alpha-synuclein in the absence of parkin enhances alpha-synuclein phosphorylation but does not modulate dopaminergic neurodegeneration.

Van Rompuy AS, Oliveras-Salvá M, Van der Perren A, Corti O, Van den Haute C, Baekelandt V - Mol Neurodegener (2015)

Bottom Line: Conflicting results were reported about the effect of the absence of parkin on alpha-synuclein-mediated neurotoxicity using a transgenic approach.The increased alpha-synuclein phosphorylation was confirmed in a parkin knockdown cell line.These findings support a functional relationship between parkin and alpha-synuclein phosphorylation in rodent brain.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven, Flanders, Belgium. annexsophie.vanrompuy@uzleuven.be.

ABSTRACT

Background: Alpha-synuclein is a key protein in the pathogenesis of Parkinson's disease. Mutations in the parkin gene are the most common cause of early-onset autosomal recessive Parkinson's disease, probably through a loss-of-function mechanism. However, the molecular mechanism by which loss of parkin function leads to the development of the disease and the role of alpha-synuclein in parkin-associated Parkinson's disease is still not elucidated. Conflicting results were reported about the effect of the absence of parkin on alpha-synuclein-mediated neurotoxicity using a transgenic approach. In this study, we investigated the effect of loss of parkin on alpha-synuclein neuropathology and toxicity in adult rodent brain using viral vectors. Therefore, we overexpressed human wild type alpha-synuclein in the substantia nigra of parkin knockout and wild type mice using two different doses of recombinant adeno-associated viral vectors.

Results: No difference was observed in nigral dopaminergic cell loss between the parkin knockout mice and wild type mice up to 16 weeks after viral vector injection. However, the level of alpha-synuclein phosphorylated at serine residue 129 in the substantia nigra was significantly increased in the parkin knockout mice compared to the wild type mice while the total expression level of alpha-synuclein was similar in both groups. The increased alpha-synuclein phosphorylation was confirmed in a parkin knockdown cell line.

Conclusions: These findings support a functional relationship between parkin and alpha-synuclein phosphorylation in rodent brain.

No MeSH data available.


Related in: MedlinePlus

Increased P-S129 α-SYN in parkin−/− mice after injection with a low titer of rAAV2/7-WT α-SYN. (A) Representative images of P-S129 α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week, 4 weeks, 8 weeks and 16 weeks after injection with a low titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overview of the injected side (middle panels). Scale bar overviews = 400 μm and magnifications = 50 μm. (B) Stereological quantification of the number of P-S129 α-SYN positive cells in the injected side of the SN of parkin+/+ and parkin−/− mice at 1 week (n = 2-3), 4 weeks (n = 10-13), 8 weeks (n = 5-6) and 16 weeks (n = 6) after injection. Asterisks depict significant increase respective to 1 week, unless specified otherwise. (Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test, student’s T-test followed by Benjamini-Hochberg to compare parkin+/+ and parkin−/− separately at the different time points, *p < 0.05, **p < 0.01, ***p < 0.001). (C) Representative images of α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week, 4 weeks, 8 weeks and 16 weeks after injection with a low titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overview of the injected side (middle panels). Scale bar overviews = 400 μm and magnifications = 50 μm. (D) Stereological quantification of the number of α-SYN positive cells in the injected side of the SN of parkin+/+ and parkin−/− mice at 1 week (n = 2-3), 4 weeks (n = 10-13), 8 weeks (n = 5-6) and 16 weeks (n = 6) after injection with a low titer of rAAV2/7-WT α-SYN. (Mean ± SEM).
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Fig6: Increased P-S129 α-SYN in parkin−/− mice after injection with a low titer of rAAV2/7-WT α-SYN. (A) Representative images of P-S129 α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week, 4 weeks, 8 weeks and 16 weeks after injection with a low titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overview of the injected side (middle panels). Scale bar overviews = 400 μm and magnifications = 50 μm. (B) Stereological quantification of the number of P-S129 α-SYN positive cells in the injected side of the SN of parkin+/+ and parkin−/− mice at 1 week (n = 2-3), 4 weeks (n = 10-13), 8 weeks (n = 5-6) and 16 weeks (n = 6) after injection. Asterisks depict significant increase respective to 1 week, unless specified otherwise. (Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test, student’s T-test followed by Benjamini-Hochberg to compare parkin+/+ and parkin−/− separately at the different time points, *p < 0.05, **p < 0.01, ***p < 0.001). (C) Representative images of α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week, 4 weeks, 8 weeks and 16 weeks after injection with a low titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overview of the injected side (middle panels). Scale bar overviews = 400 μm and magnifications = 50 μm. (D) Stereological quantification of the number of α-SYN positive cells in the injected side of the SN of parkin+/+ and parkin−/− mice at 1 week (n = 2-3), 4 weeks (n = 10-13), 8 weeks (n = 5-6) and 16 weeks (n = 6) after injection with a low titer of rAAV2/7-WT α-SYN. (Mean ± SEM).

Mentions: We also investigated the effect of absence of parkin on α-SYN phosphorylation in the low dose α-SYN set-up. The number of P-S129 α-SYN positive cells in the injected side progressively increased over time until 8 weeks and remained stable at 16 weeks after viral vector delivery to the SN (Figure 6A-B). Here again, we observed a significantly higher level of α-SYN phosphorylation in the parkin−/− mice compared to the parkin+/+ mice at 8 weeks and 16 weeks after injection (respectively 9389 ± 337 cells versus 7263 ± 532 cells at 8 weeks, p = 0,0179; 7954 ± 518 cells versus 6000 ± 313 cells at 16 weeks, p = 0,009). Immunohistochemical staining for α-SYN confirmed expression of α-SYN up to 16 weeks after injection (Figure 6C). As seen before, the total number of α-SYN positive cells was similar in the parkin−/− mice and the parkin+/+ mice at the 4 different time points (Figure 6D).Figure 6


Nigral overexpression of alpha-synuclein in the absence of parkin enhances alpha-synuclein phosphorylation but does not modulate dopaminergic neurodegeneration.

Van Rompuy AS, Oliveras-Salvá M, Van der Perren A, Corti O, Van den Haute C, Baekelandt V - Mol Neurodegener (2015)

Increased P-S129 α-SYN in parkin−/− mice after injection with a low titer of rAAV2/7-WT α-SYN. (A) Representative images of P-S129 α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week, 4 weeks, 8 weeks and 16 weeks after injection with a low titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overview of the injected side (middle panels). Scale bar overviews = 400 μm and magnifications = 50 μm. (B) Stereological quantification of the number of P-S129 α-SYN positive cells in the injected side of the SN of parkin+/+ and parkin−/− mice at 1 week (n = 2-3), 4 weeks (n = 10-13), 8 weeks (n = 5-6) and 16 weeks (n = 6) after injection. Asterisks depict significant increase respective to 1 week, unless specified otherwise. (Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test, student’s T-test followed by Benjamini-Hochberg to compare parkin+/+ and parkin−/− separately at the different time points, *p < 0.05, **p < 0.01, ***p < 0.001). (C) Representative images of α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week, 4 weeks, 8 weeks and 16 weeks after injection with a low titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overview of the injected side (middle panels). Scale bar overviews = 400 μm and magnifications = 50 μm. (D) Stereological quantification of the number of α-SYN positive cells in the injected side of the SN of parkin+/+ and parkin−/− mice at 1 week (n = 2-3), 4 weeks (n = 10-13), 8 weeks (n = 5-6) and 16 weeks (n = 6) after injection with a low titer of rAAV2/7-WT α-SYN. (Mean ± SEM).
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Fig6: Increased P-S129 α-SYN in parkin−/− mice after injection with a low titer of rAAV2/7-WT α-SYN. (A) Representative images of P-S129 α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week, 4 weeks, 8 weeks and 16 weeks after injection with a low titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overview of the injected side (middle panels). Scale bar overviews = 400 μm and magnifications = 50 μm. (B) Stereological quantification of the number of P-S129 α-SYN positive cells in the injected side of the SN of parkin+/+ and parkin−/− mice at 1 week (n = 2-3), 4 weeks (n = 10-13), 8 weeks (n = 5-6) and 16 weeks (n = 6) after injection. Asterisks depict significant increase respective to 1 week, unless specified otherwise. (Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test, student’s T-test followed by Benjamini-Hochberg to compare parkin+/+ and parkin−/− separately at the different time points, *p < 0.05, **p < 0.01, ***p < 0.001). (C) Representative images of α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week, 4 weeks, 8 weeks and 16 weeks after injection with a low titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overview of the injected side (middle panels). Scale bar overviews = 400 μm and magnifications = 50 μm. (D) Stereological quantification of the number of α-SYN positive cells in the injected side of the SN of parkin+/+ and parkin−/− mice at 1 week (n = 2-3), 4 weeks (n = 10-13), 8 weeks (n = 5-6) and 16 weeks (n = 6) after injection with a low titer of rAAV2/7-WT α-SYN. (Mean ± SEM).
Mentions: We also investigated the effect of absence of parkin on α-SYN phosphorylation in the low dose α-SYN set-up. The number of P-S129 α-SYN positive cells in the injected side progressively increased over time until 8 weeks and remained stable at 16 weeks after viral vector delivery to the SN (Figure 6A-B). Here again, we observed a significantly higher level of α-SYN phosphorylation in the parkin−/− mice compared to the parkin+/+ mice at 8 weeks and 16 weeks after injection (respectively 9389 ± 337 cells versus 7263 ± 532 cells at 8 weeks, p = 0,0179; 7954 ± 518 cells versus 6000 ± 313 cells at 16 weeks, p = 0,009). Immunohistochemical staining for α-SYN confirmed expression of α-SYN up to 16 weeks after injection (Figure 6C). As seen before, the total number of α-SYN positive cells was similar in the parkin−/− mice and the parkin+/+ mice at the 4 different time points (Figure 6D).Figure 6

Bottom Line: Conflicting results were reported about the effect of the absence of parkin on alpha-synuclein-mediated neurotoxicity using a transgenic approach.The increased alpha-synuclein phosphorylation was confirmed in a parkin knockdown cell line.These findings support a functional relationship between parkin and alpha-synuclein phosphorylation in rodent brain.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven, Flanders, Belgium. annexsophie.vanrompuy@uzleuven.be.

ABSTRACT

Background: Alpha-synuclein is a key protein in the pathogenesis of Parkinson's disease. Mutations in the parkin gene are the most common cause of early-onset autosomal recessive Parkinson's disease, probably through a loss-of-function mechanism. However, the molecular mechanism by which loss of parkin function leads to the development of the disease and the role of alpha-synuclein in parkin-associated Parkinson's disease is still not elucidated. Conflicting results were reported about the effect of the absence of parkin on alpha-synuclein-mediated neurotoxicity using a transgenic approach. In this study, we investigated the effect of loss of parkin on alpha-synuclein neuropathology and toxicity in adult rodent brain using viral vectors. Therefore, we overexpressed human wild type alpha-synuclein in the substantia nigra of parkin knockout and wild type mice using two different doses of recombinant adeno-associated viral vectors.

Results: No difference was observed in nigral dopaminergic cell loss between the parkin knockout mice and wild type mice up to 16 weeks after viral vector injection. However, the level of alpha-synuclein phosphorylated at serine residue 129 in the substantia nigra was significantly increased in the parkin knockout mice compared to the wild type mice while the total expression level of alpha-synuclein was similar in both groups. The increased alpha-synuclein phosphorylation was confirmed in a parkin knockdown cell line.

Conclusions: These findings support a functional relationship between parkin and alpha-synuclein phosphorylation in rodent brain.

No MeSH data available.


Related in: MedlinePlus