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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 phosphorylation of α-SYN at S129 in parkin−/− mice compared to parkin+/+ mice. (A) Representative images of P-S129 α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week and 4 weeks after injection with a high titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overviews 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 = 4) and 4 weeks (n = 15-16) after injection with a high titer of rAAV2/7-WT α-SYN. (Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test, # p < 0.05 versus parkin+/+, **p < 0.01 versus 1 week, ***p < 0.001 versus 1 week). (C) 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 = 4) and 4 weeks (n = 15-16) after injection with a high titer of rAAV2/7-WT α-SYN. (Mean ± SEM). (D) Quantification of the number of P-S129 α-SYN positive neuritic inclusions in the striatum at 4 weeks (n = 14–15, Mean ± SEM). (E) Picture of the P-S129 α-SYN staining in the striatum (left: 10 x) and magnification (right: 40 x) of neuritic inclusions. Scale bar = 50 μm.
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Fig4: Increased phosphorylation of α-SYN at S129 in parkin−/− mice compared to parkin+/+ mice. (A) Representative images of P-S129 α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week and 4 weeks after injection with a high titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overviews 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 = 4) and 4 weeks (n = 15-16) after injection with a high titer of rAAV2/7-WT α-SYN. (Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test, # p < 0.05 versus parkin+/+, **p < 0.01 versus 1 week, ***p < 0.001 versus 1 week). (C) 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 = 4) and 4 weeks (n = 15-16) after injection with a high titer of rAAV2/7-WT α-SYN. (Mean ± SEM). (D) Quantification of the number of P-S129 α-SYN positive neuritic inclusions in the striatum at 4 weeks (n = 14–15, Mean ± SEM). (E) Picture of the P-S129 α-SYN staining in the striatum (left: 10 x) and magnification (right: 40 x) of neuritic inclusions. Scale bar = 50 μm.

Mentions: In a next step, we determined the number of cells in the SN that were positive for α-SYN phosphorylated at serine residue 129 (P-S129), a form of α-SYN which is considered to be the pathological form of α-SYN and the most abundant modification of α-SYN in LBs [34,35]. Therefore, we performed an immunohistochemical staining with an antibody specifically recognizing this phosphorylated form of α-SYN [34] and stereologically quantified the number of positive cells in the injected side of the whole SN. No P-S129 positive cells were observed in the non-injected side of the SN, indicating that only phosphorylation of the overexpressed human α-SYN is within the limits of detection. At 1 week after injection, no difference was observed between parkin−/− mice (3563 ± 259 cells) and parkin+/+ mice (3371 ± 416 cells). At 4 weeks after injection, the number of cells positive for P-S129 α-SYN was increased in both groups when compared to 1 week. Interestingly, this number was significantly higher in the parkin−/− mice (7632 ± 291 cells) than in the parkin+/+ mice (6288 ± 495 cells) (p = 0,027) (Figure 4A-B). This higher number of P-S129 α-SYN positive cells in the parkin−/− mice was not caused by higher expression levels of α-SYN, since the total number of α-SYN positive cells in the SN was similar in the parkin+/+ and parkin−/− mice at both time points (Figure 4C). Differences in the affinities of the P-S129 α-SYN antibody and α-SYN antibody explain the apparent lower number of α-SYN positive cells compared to the number of P-S129 α-SYN positive cells. We also stained the striatum for P-S129 α-SYN to check if the dopaminergic terminals also contain phosphorylated α-SYN. We detected P-S129 α-SYN-positive neuritic inclusions (Figure 4E) but no difference in the number of these inclusions was observed between parkin+/+ and parkin−/− mice at 4 weeks after injection (Figure 4D).Figure 4


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 phosphorylation of α-SYN at S129 in parkin−/− mice compared to parkin+/+ mice. (A) Representative images of P-S129 α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week and 4 weeks after injection with a high titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overviews 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 = 4) and 4 weeks (n = 15-16) after injection with a high titer of rAAV2/7-WT α-SYN. (Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test, # p < 0.05 versus parkin+/+, **p < 0.01 versus 1 week, ***p < 0.001 versus 1 week). (C) 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 = 4) and 4 weeks (n = 15-16) after injection with a high titer of rAAV2/7-WT α-SYN. (Mean ± SEM). (D) Quantification of the number of P-S129 α-SYN positive neuritic inclusions in the striatum at 4 weeks (n = 14–15, Mean ± SEM). (E) Picture of the P-S129 α-SYN staining in the striatum (left: 10 x) and magnification (right: 40 x) of neuritic inclusions. Scale bar = 50 μm.
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Fig4: Increased phosphorylation of α-SYN at S129 in parkin−/− mice compared to parkin+/+ mice. (A) Representative images of P-S129 α-SYN expression in the SN of parkin−/− and parkin+/+ mice at 1 week and 4 weeks after injection with a high titer of rAAV2/7-WT α-SYN. Right panels are magnifications of the overviews 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 = 4) and 4 weeks (n = 15-16) after injection with a high titer of rAAV2/7-WT α-SYN. (Mean ± SEM, two-way ANOVA followed by Bonferroni post-hoc test, # p < 0.05 versus parkin+/+, **p < 0.01 versus 1 week, ***p < 0.001 versus 1 week). (C) 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 = 4) and 4 weeks (n = 15-16) after injection with a high titer of rAAV2/7-WT α-SYN. (Mean ± SEM). (D) Quantification of the number of P-S129 α-SYN positive neuritic inclusions in the striatum at 4 weeks (n = 14–15, Mean ± SEM). (E) Picture of the P-S129 α-SYN staining in the striatum (left: 10 x) and magnification (right: 40 x) of neuritic inclusions. Scale bar = 50 μm.
Mentions: In a next step, we determined the number of cells in the SN that were positive for α-SYN phosphorylated at serine residue 129 (P-S129), a form of α-SYN which is considered to be the pathological form of α-SYN and the most abundant modification of α-SYN in LBs [34,35]. Therefore, we performed an immunohistochemical staining with an antibody specifically recognizing this phosphorylated form of α-SYN [34] and stereologically quantified the number of positive cells in the injected side of the whole SN. No P-S129 positive cells were observed in the non-injected side of the SN, indicating that only phosphorylation of the overexpressed human α-SYN is within the limits of detection. At 1 week after injection, no difference was observed between parkin−/− mice (3563 ± 259 cells) and parkin+/+ mice (3371 ± 416 cells). At 4 weeks after injection, the number of cells positive for P-S129 α-SYN was increased in both groups when compared to 1 week. Interestingly, this number was significantly higher in the parkin−/− mice (7632 ± 291 cells) than in the parkin+/+ mice (6288 ± 495 cells) (p = 0,027) (Figure 4A-B). This higher number of P-S129 α-SYN positive cells in the parkin−/− mice was not caused by higher expression levels of α-SYN, since the total number of α-SYN positive cells in the SN was similar in the parkin+/+ and parkin−/− mice at both time points (Figure 4C). Differences in the affinities of the P-S129 α-SYN antibody and α-SYN antibody explain the apparent lower number of α-SYN positive cells compared to the number of P-S129 α-SYN positive cells. We also stained the striatum for P-S129 α-SYN to check if the dopaminergic terminals also contain phosphorylated α-SYN. We detected P-S129 α-SYN-positive neuritic inclusions (Figure 4E) but no difference in the number of these inclusions was observed between parkin+/+ and parkin−/− mice at 4 weeks after injection (Figure 4D).Figure 4

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