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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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High-copy expression of α-synuclein-GFP variants in yeast.A. Yeast cells expressing GAL1-driven ASYN-GFP variants from 2 µ plasmids were spotted in 10-fold dilutions on selection plates containing 2% glucose (control) or 2% galactose. After incubation for 3 days at 30°C the plates were photographed. Expression of GFP from the same promoter was used as a control. B. Live-cell fluorescence microscopy of yeast cells expressing ASYN-GFP. Yeast cells, pre-grown to mid-log phase, were induced in galactose-containing medium and examined for aggregates at 6 hours of induction. GFP-expressing cells were used as control. Scale bar: 1 µM. C. Aggregate quantification of yeast cells, expressing ASYN-GFP. For each strain, the number of cells displaying cytoplasmic foci is presented as percent of the total number of cells counted. For quantification of aggregation at least 300 cells were counted per strain and per experiment. GFP-expressing cells were used as a control. Student's t test (*p<0.05, **p<0.01, ***p<0.001). D. Protein levels of ASYN-GFP variants. Expression of ASYN-GFP variants was induced for 6 hours in galactose-containing medium. Equal amounts of crude protein extracts were used for Western analysis with anti-ASYN antibody and anti-cdc28 antibody as a loading control. n = 2.
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pgen-1004741-g005: High-copy expression of α-synuclein-GFP variants in yeast.A. Yeast cells expressing GAL1-driven ASYN-GFP variants from 2 µ plasmids were spotted in 10-fold dilutions on selection plates containing 2% glucose (control) or 2% galactose. After incubation for 3 days at 30°C the plates were photographed. Expression of GFP from the same promoter was used as a control. B. Live-cell fluorescence microscopy of yeast cells expressing ASYN-GFP. Yeast cells, pre-grown to mid-log phase, were induced in galactose-containing medium and examined for aggregates at 6 hours of induction. GFP-expressing cells were used as control. Scale bar: 1 µM. C. Aggregate quantification of yeast cells, expressing ASYN-GFP. For each strain, the number of cells displaying cytoplasmic foci is presented as percent of the total number of cells counted. For quantification of aggregation at least 300 cells were counted per strain and per experiment. GFP-expressing cells were used as a control. Student's t test (*p<0.05, **p<0.01, ***p<0.001). D. Protein levels of ASYN-GFP variants. Expression of ASYN-GFP variants was induced for 6 hours in galactose-containing medium. Equal amounts of crude protein extracts were used for Western analysis with anti-ASYN antibody and anti-cdc28 antibody as a loading control. n = 2.

Mentions: We started by investigating the toxicity of the selected variants of ASYN by taking advantage of the budding yeast as a model of synucleinopathies, as previously described [23], [44]. In conditions where the expression of ASYN was induced, using galactose-containing media, we found that almost all mutations induced toxicity similar to WT ASYN (Fig. 5A). In line with the observation in H4 cells, the TP mutant did not form inclusions and the A30P mutant strongly impaired inclusion formation (Fig. 3B-C and 5B-C). Neither of these mutants was toxic in yeast (Fig. 5A) [23], [44]. Importantly, we found no significant differences in the levels of expression of all variants tested, ruling out the possibility that toxicity and/or inclusion formation were due to differences in the levels of expression of ASYN (Fig. 5D).


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)

High-copy expression of α-synuclein-GFP variants in yeast.A. Yeast cells expressing GAL1-driven ASYN-GFP variants from 2 µ plasmids were spotted in 10-fold dilutions on selection plates containing 2% glucose (control) or 2% galactose. After incubation for 3 days at 30°C the plates were photographed. Expression of GFP from the same promoter was used as a control. B. Live-cell fluorescence microscopy of yeast cells expressing ASYN-GFP. Yeast cells, pre-grown to mid-log phase, were induced in galactose-containing medium and examined for aggregates at 6 hours of induction. GFP-expressing cells were used as control. Scale bar: 1 µM. C. Aggregate quantification of yeast cells, expressing ASYN-GFP. For each strain, the number of cells displaying cytoplasmic foci is presented as percent of the total number of cells counted. For quantification of aggregation at least 300 cells were counted per strain and per experiment. GFP-expressing cells were used as a control. Student's t test (*p<0.05, **p<0.01, ***p<0.001). D. Protein levels of ASYN-GFP variants. Expression of ASYN-GFP variants was induced for 6 hours in galactose-containing medium. Equal amounts of crude protein extracts were used for Western analysis with anti-ASYN antibody and anti-cdc28 antibody as a loading control. n = 2.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004741-g005: High-copy expression of α-synuclein-GFP variants in yeast.A. Yeast cells expressing GAL1-driven ASYN-GFP variants from 2 µ plasmids were spotted in 10-fold dilutions on selection plates containing 2% glucose (control) or 2% galactose. After incubation for 3 days at 30°C the plates were photographed. Expression of GFP from the same promoter was used as a control. B. Live-cell fluorescence microscopy of yeast cells expressing ASYN-GFP. Yeast cells, pre-grown to mid-log phase, were induced in galactose-containing medium and examined for aggregates at 6 hours of induction. GFP-expressing cells were used as control. Scale bar: 1 µM. C. Aggregate quantification of yeast cells, expressing ASYN-GFP. For each strain, the number of cells displaying cytoplasmic foci is presented as percent of the total number of cells counted. For quantification of aggregation at least 300 cells were counted per strain and per experiment. GFP-expressing cells were used as a control. Student's t test (*p<0.05, **p<0.01, ***p<0.001). D. Protein levels of ASYN-GFP variants. Expression of ASYN-GFP variants was induced for 6 hours in galactose-containing medium. Equal amounts of crude protein extracts were used for Western analysis with anti-ASYN antibody and anti-cdc28 antibody as a loading control. n = 2.
Mentions: We started by investigating the toxicity of the selected variants of ASYN by taking advantage of the budding yeast as a model of synucleinopathies, as previously described [23], [44]. In conditions where the expression of ASYN was induced, using galactose-containing media, we found that almost all mutations induced toxicity similar to WT ASYN (Fig. 5A). In line with the observation in H4 cells, the TP mutant did not form inclusions and the A30P mutant strongly impaired inclusion formation (Fig. 3B-C and 5B-C). Neither of these mutants was toxic in yeast (Fig. 5A) [23], [44]. Importantly, we found no significant differences in the levels of expression of all variants tested, ruling out the possibility that toxicity and/or inclusion formation were due to differences in the levels of expression of ASYN (Fig. 5D).

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