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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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ASYN bPCA.A. Schematic representation of the ASYN bPCA constructs. Non-bioluminescent halves of humanized Gaussia luciferase (hGLuc) were fused to ASYN monomers. B-C. Intact cells (intracellular) and medium (extracellular) from H4 cells co-transfected with S1 and S2 were assayed for luciferase activity 48 hours post-transfection. Intracellular (B) and extracellular (C) TP displayed a 3-fold increase in luciferase activity compared to WT. n = 12. Student's t test (*p<0.05, **p<0.01, ***p<0.001) D. Ratio of luciferase activity in media compared to cells was expressed. n = 12, Student's t test (*p<0.05, **p<0.01, ***p<0.001) E-F. Levels of ASYN. Immunoblot analysis of the expression levels of ASYN showing similar levels. n = 3.
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pgen-1004741-g007: ASYN bPCA.A. Schematic representation of the ASYN bPCA constructs. Non-bioluminescent halves of humanized Gaussia luciferase (hGLuc) were fused to ASYN monomers. B-C. Intact cells (intracellular) and medium (extracellular) from H4 cells co-transfected with S1 and S2 were assayed for luciferase activity 48 hours post-transfection. Intracellular (B) and extracellular (C) TP displayed a 3-fold increase in luciferase activity compared to WT. n = 12. Student's t test (*p<0.05, **p<0.01, ***p<0.001) D. Ratio of luciferase activity in media compared to cells was expressed. n = 12, Student's t test (*p<0.05, **p<0.01, ***p<0.001) E-F. Levels of ASYN. Immunoblot analysis of the expression levels of ASYN showing similar levels. n = 3.

Mentions: To assess the effect of the selected mutations on the distribution of ASYN oligomers inside and outside cells, we used a previously described bioluminescent protein complementation assay (bPCA) that enables the detection of oligomeric species with great sensitivity [46]. In this assay, reconstitution of Gaussia princeps luciferase activity upon ASYN oligomerization was used as a readout [47] (Fig. 7A). Consistent with the results obtained with the Venus-based BiFC assay (Fig. 2A), we detected reconstitution of luciferase activity with all mutants tested. However, we observed a strong increase in intracellular (Fig. 7B) and extracellular (Fig. 7C) luciferase activity with the TP and Y125F ASYN mutants when compared to WT ASYN. This indicates that not only these mutations are able to promote increased formation of oligomers inside cells, but also in the extracellular space.


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 bPCA.A. Schematic representation of the ASYN bPCA constructs. Non-bioluminescent halves of humanized Gaussia luciferase (hGLuc) were fused to ASYN monomers. B-C. Intact cells (intracellular) and medium (extracellular) from H4 cells co-transfected with S1 and S2 were assayed for luciferase activity 48 hours post-transfection. Intracellular (B) and extracellular (C) TP displayed a 3-fold increase in luciferase activity compared to WT. n = 12. Student's t test (*p<0.05, **p<0.01, ***p<0.001) D. Ratio of luciferase activity in media compared to cells was expressed. n = 12, Student's t test (*p<0.05, **p<0.01, ***p<0.001) E-F. Levels of ASYN. Immunoblot analysis of the expression levels of ASYN showing similar levels. n = 3.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004741-g007: ASYN bPCA.A. Schematic representation of the ASYN bPCA constructs. Non-bioluminescent halves of humanized Gaussia luciferase (hGLuc) were fused to ASYN monomers. B-C. Intact cells (intracellular) and medium (extracellular) from H4 cells co-transfected with S1 and S2 were assayed for luciferase activity 48 hours post-transfection. Intracellular (B) and extracellular (C) TP displayed a 3-fold increase in luciferase activity compared to WT. n = 12. Student's t test (*p<0.05, **p<0.01, ***p<0.001) D. Ratio of luciferase activity in media compared to cells was expressed. n = 12, Student's t test (*p<0.05, **p<0.01, ***p<0.001) E-F. Levels of ASYN. Immunoblot analysis of the expression levels of ASYN showing similar levels. n = 3.
Mentions: To assess the effect of the selected mutations on the distribution of ASYN oligomers inside and outside cells, we used a previously described bioluminescent protein complementation assay (bPCA) that enables the detection of oligomeric species with great sensitivity [46]. In this assay, reconstitution of Gaussia princeps luciferase activity upon ASYN oligomerization was used as a readout [47] (Fig. 7A). Consistent with the results obtained with the Venus-based BiFC assay (Fig. 2A), we detected reconstitution of luciferase activity with all mutants tested. However, we observed a strong increase in intracellular (Fig. 7B) and extracellular (Fig. 7C) luciferase activity with the TP and Y125F ASYN mutants when compared to WT ASYN. This indicates that not only these mutations are able to promote increased formation of oligomers inside cells, but also in the extracellular space.

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