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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.

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Mutations effect on ASYN oligomerization.A. Schematic representation of Bimolecular Fluorescence Complementation assay (BiFC). ASYN BiFC constructs in anti-parallel orientation. B. Representative pictures of ASYN oligomerization. HEK-293 cells overexpressing VN-ASYN and ASYN-VC constructs. The green fluorescence results from the reconstitution of the Venus fluorophore, promoted by the interaction of the proteins of interest. Scale bar: 10 µm. C. Oligomerization efficiency. Mean fluorescence intensity of cells expressing different ASYN mutants was assessed 24 hours post-transfection, using a microcapillary system (GuavaeasyCyte HT system). For each sample 25,000 events were counted. D. Intracellular distribution of oligomeric ASYN. Nuclear and cytoplasmic venus fluorescence intensities in HEK-293 cells were quantified using ImageJ. The graph demonstrates an increase in nuclear fluorescence in cells expressing ASYN mutants. For each experiment>25 cells were analysed. E-F. Levels of ASYN.E. Representative immunoblot showing the expression levels of ASYN. F. Immunoblot analysis of the expression levels of VN-ASYN and ASYN-VC from all the mutations studied in HEK-293 cells. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 3.
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pgen-1004741-g002: Mutations effect on ASYN oligomerization.A. Schematic representation of Bimolecular Fluorescence Complementation assay (BiFC). ASYN BiFC constructs in anti-parallel orientation. B. Representative pictures of ASYN oligomerization. HEK-293 cells overexpressing VN-ASYN and ASYN-VC constructs. The green fluorescence results from the reconstitution of the Venus fluorophore, promoted by the interaction of the proteins of interest. Scale bar: 10 µm. C. Oligomerization efficiency. Mean fluorescence intensity of cells expressing different ASYN mutants was assessed 24 hours post-transfection, using a microcapillary system (GuavaeasyCyte HT system). For each sample 25,000 events were counted. D. Intracellular distribution of oligomeric ASYN. Nuclear and cytoplasmic venus fluorescence intensities in HEK-293 cells were quantified using ImageJ. The graph demonstrates an increase in nuclear fluorescence in cells expressing ASYN mutants. For each experiment>25 cells were analysed. E-F. Levels of ASYN.E. Representative immunoblot showing the expression levels of ASYN. F. Immunoblot analysis of the expression levels of VN-ASYN and ASYN-VC from all the mutations studied in HEK-293 cells. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 3.

Mentions: To investigate the molecular determinants of ASYN oligomerization and aggregation in a cellular context, we used site-directed mutagenesis to generate a panel of 19 ASYN point mutants including five mutations associated with familial PD (A30P, E46K, H50Q, G51D and A53T) and others known to interfere with different aspects of ASYN biology (Fig. 1 and Table S1). Then, we analysed the behaviour of each mutant in established paradigms of ASYN oligomerization (Fig. 2A) or aggregation (Fig. 3A).


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)

Mutations effect on ASYN oligomerization.A. Schematic representation of Bimolecular Fluorescence Complementation assay (BiFC). ASYN BiFC constructs in anti-parallel orientation. B. Representative pictures of ASYN oligomerization. HEK-293 cells overexpressing VN-ASYN and ASYN-VC constructs. The green fluorescence results from the reconstitution of the Venus fluorophore, promoted by the interaction of the proteins of interest. Scale bar: 10 µm. C. Oligomerization efficiency. Mean fluorescence intensity of cells expressing different ASYN mutants was assessed 24 hours post-transfection, using a microcapillary system (GuavaeasyCyte HT system). For each sample 25,000 events were counted. D. Intracellular distribution of oligomeric ASYN. Nuclear and cytoplasmic venus fluorescence intensities in HEK-293 cells were quantified using ImageJ. The graph demonstrates an increase in nuclear fluorescence in cells expressing ASYN mutants. For each experiment>25 cells were analysed. E-F. Levels of ASYN.E. Representative immunoblot showing the expression levels of ASYN. F. Immunoblot analysis of the expression levels of VN-ASYN and ASYN-VC from all the mutations studied in HEK-293 cells. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004741-g002: Mutations effect on ASYN oligomerization.A. Schematic representation of Bimolecular Fluorescence Complementation assay (BiFC). ASYN BiFC constructs in anti-parallel orientation. B. Representative pictures of ASYN oligomerization. HEK-293 cells overexpressing VN-ASYN and ASYN-VC constructs. The green fluorescence results from the reconstitution of the Venus fluorophore, promoted by the interaction of the proteins of interest. Scale bar: 10 µm. C. Oligomerization efficiency. Mean fluorescence intensity of cells expressing different ASYN mutants was assessed 24 hours post-transfection, using a microcapillary system (GuavaeasyCyte HT system). For each sample 25,000 events were counted. D. Intracellular distribution of oligomeric ASYN. Nuclear and cytoplasmic venus fluorescence intensities in HEK-293 cells were quantified using ImageJ. The graph demonstrates an increase in nuclear fluorescence in cells expressing ASYN mutants. For each experiment>25 cells were analysed. E-F. Levels of ASYN.E. Representative immunoblot showing the expression levels of ASYN. F. Immunoblot analysis of the expression levels of VN-ASYN and ASYN-VC from all the mutations studied in HEK-293 cells. Student's t test (*p<0.05, **p<0.01, ***p<0.001). n = 3.
Mentions: To investigate the molecular determinants of ASYN oligomerization and aggregation in a cellular context, we used site-directed mutagenesis to generate a panel of 19 ASYN point mutants including five mutations associated with familial PD (A30P, E46K, H50Q, G51D and A53T) and others known to interfere with different aspects of ASYN biology (Fig. 1 and Table S1). Then, we analysed the behaviour of each mutant in established paradigms of ASYN oligomerization (Fig. 2A) or aggregation (Fig. 3A).

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