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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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A-B morphology analysis of Golgi apparatus.The morphology of the Golgi apparatus in>50 cells was analysed and quantified. We observed that, in the BiFC assay, E35K and E57K mutants displayed increased Golgi fragmentation (A). In the aggregation model, Golgi morphology appeared normal, displaying a compact appearance near the nucleus (B). Levels of BiP in the oligomerization assay (C) and in the aggregation model (E), assessed by immunoblot analysis and respective quantifications (D and E). n = 3. Student's t test (*p<0.05, **p<0.01, ***p<0.001).
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pgen-1004741-g010: A-B morphology analysis of Golgi apparatus.The morphology of the Golgi apparatus in>50 cells was analysed and quantified. We observed that, in the BiFC assay, E35K and E57K mutants displayed increased Golgi fragmentation (A). In the aggregation model, Golgi morphology appeared normal, displaying a compact appearance near the nucleus (B). Levels of BiP in the oligomerization assay (C) and in the aggregation model (E), assessed by immunoblot analysis and respective quantifications (D and E). n = 3. Student's t test (*p<0.05, **p<0.01, ***p<0.001).

Mentions: Given that fragmentation of Golgi apparatus (GA) has been described in several neurodegenerative diseases [51], [52], [53], we next investigated the cellular consequences of the accumulation of ASYN oligomers or inclusions on this organelle. For this, we examined the morphological integrity of the GA using fluorescence microscopy of cells immunostained for Giantin, an endogenous transmembrane protein of the cis and medial Golgi complex (Fig. 10). We defined three types of Golgi structures (non-fragmented, diffuse and fragmented). In general, we observed that in the ASYN oligomerization model there was an increased percentage of cells displaying fragmented Golgi, in comparison to what was observed in the aggregation model (Fig.10 A-B and Fig. S2). In particular, we found a statistically significant increase in the percentage of cells displaying fragmentation of the GA for the E35K and E57K mutants (Fig. 10A and Fig. S2A). In the ASYN aggregation paradigm, the GA displayed normal compact morphology near the nucleus (Fig. 10B and Fig. S2B).


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)

A-B morphology analysis of Golgi apparatus.The morphology of the Golgi apparatus in>50 cells was analysed and quantified. We observed that, in the BiFC assay, E35K and E57K mutants displayed increased Golgi fragmentation (A). In the aggregation model, Golgi morphology appeared normal, displaying a compact appearance near the nucleus (B). Levels of BiP in the oligomerization assay (C) and in the aggregation model (E), assessed by immunoblot analysis and respective quantifications (D and E). n = 3. Student's t test (*p<0.05, **p<0.01, ***p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004741-g010: A-B morphology analysis of Golgi apparatus.The morphology of the Golgi apparatus in>50 cells was analysed and quantified. We observed that, in the BiFC assay, E35K and E57K mutants displayed increased Golgi fragmentation (A). In the aggregation model, Golgi morphology appeared normal, displaying a compact appearance near the nucleus (B). Levels of BiP in the oligomerization assay (C) and in the aggregation model (E), assessed by immunoblot analysis and respective quantifications (D and E). n = 3. Student's t test (*p<0.05, **p<0.01, ***p<0.001).
Mentions: Given that fragmentation of Golgi apparatus (GA) has been described in several neurodegenerative diseases [51], [52], [53], we next investigated the cellular consequences of the accumulation of ASYN oligomers or inclusions on this organelle. For this, we examined the morphological integrity of the GA using fluorescence microscopy of cells immunostained for Giantin, an endogenous transmembrane protein of the cis and medial Golgi complex (Fig. 10). We defined three types of Golgi structures (non-fragmented, diffuse and fragmented). In general, we observed that in the ASYN oligomerization model there was an increased percentage of cells displaying fragmented Golgi, in comparison to what was observed in the aggregation model (Fig.10 A-B and Fig. S2). In particular, we found a statistically significant increase in the percentage of cells displaying fragmentation of the GA for the E35K and E57K mutants (Fig. 10A and Fig. S2A). In the ASYN aggregation paradigm, the GA displayed normal compact morphology near the nucleus (Fig. 10B and Fig. S2B).

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