Limits...
Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation.

Lázaro DF, Rodrigues EF, Langohr R, Shahpasandzadeh H, Ribeiro T, Guerreiro P, Gerhardt E, Kröhnert K, Klucken J, Pereira MD, Popova B, Kruse N, Mollenhauer B, Rizzoli SO, Braus GH, Danzer KM, Outeiro TF - PLoS Genet. (2014)

Bottom Line: In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour.While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect.Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

View Article: PubMed Central - PubMed

Affiliation: Department of NeuroDegeneration and Restorative Research, Center for Nanoscale Microscopy and Molecular Physiology of the Brain University Medical Goettingen, Goettingen, Germany.

ABSTRACT
Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

Show MeSH

Related in: MedlinePlus

ASYN biochemical state.A. Native Gels. Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. B. STED microscopy. Selected mutants were imaged in order to characterize the fine structure of the inclusions. C. Thioflavin S staining. H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. D-E. Triton X-100 solubility assay and quantification. H4 cells show that all mutants form detergent insoluble species. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 2. Quantification of insoluble fraction shows a decrease in TP and E57K mutants.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4230739&req=5

pgen-1004741-g006: ASYN biochemical state.A. Native Gels. Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. B. STED microscopy. Selected mutants were imaged in order to characterize the fine structure of the inclusions. C. Thioflavin S staining. H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. D-E. Triton X-100 solubility assay and quantification. H4 cells show that all mutants form detergent insoluble species. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 2. Quantification of insoluble fraction shows a decrease in TP and E57K mutants.

Mentions: To further assess the biochemical nature of the ASYN species visualized by the BiFC assay, we employed non-denaturing polyacrylamide gel electrophoresis (native-PAGE). Immunoblot analysis showed a smear, which is indicative of the accumulation of oligomeric species of various sizes (Fig. 6A).


Systematic comparison of the effects of alpha-synuclein mutations on its oligomerization and aggregation.

Lázaro DF, Rodrigues EF, Langohr R, Shahpasandzadeh H, Ribeiro T, Guerreiro P, Gerhardt E, Kröhnert K, Klucken J, Pereira MD, Popova B, Kruse N, Mollenhauer B, Rizzoli SO, Braus GH, Danzer KM, Outeiro TF - PLoS Genet. (2014)

ASYN biochemical state.A. Native Gels. Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. B. STED microscopy. Selected mutants were imaged in order to characterize the fine structure of the inclusions. C. Thioflavin S staining. H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. D-E. Triton X-100 solubility assay and quantification. H4 cells show that all mutants form detergent insoluble species. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 2. Quantification of insoluble fraction shows a decrease in TP and E57K mutants.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230739&req=5

pgen-1004741-g006: ASYN biochemical state.A. Native Gels. Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. B. STED microscopy. Selected mutants were imaged in order to characterize the fine structure of the inclusions. C. Thioflavin S staining. H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. D-E. Triton X-100 solubility assay and quantification. H4 cells show that all mutants form detergent insoluble species. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 2. Quantification of insoluble fraction shows a decrease in TP and E57K mutants.
Mentions: To further assess the biochemical nature of the ASYN species visualized by the BiFC assay, we employed non-denaturing polyacrylamide gel electrophoresis (native-PAGE). Immunoblot analysis showed a smear, which is indicative of the accumulation of oligomeric species of various sizes (Fig. 6A).

Bottom Line: In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour.While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect.Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

View Article: PubMed Central - PubMed

Affiliation: Department of NeuroDegeneration and Restorative Research, Center for Nanoscale Microscopy and Molecular Physiology of the Brain University Medical Goettingen, Goettingen, Germany.

ABSTRACT
Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

Show MeSH
Related in: MedlinePlus