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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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Related in: MedlinePlus

ASYN partially co-localizes with endosomes/lysosomes.A. Immunocytochemistry analysis of H4 cells expressing selected ASYN mutants. Partial co-localization of ASYN and LAMP1 suggests interplay between lysosomal degradation and ASYN inclusion formation. B. E57K and Y125F inclusions co-localize with lysosomal marker LAMP-1. We detected the presence of endosomes/lysosomes surrounding the aggregates in E57K and Y125F. This indicates that, maybe this could be the preferential via for degradation for these mutations. Scale bar: 10 µm.
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pgen-1004741-g009: ASYN partially co-localizes with endosomes/lysosomes.A. Immunocytochemistry analysis of H4 cells expressing selected ASYN mutants. Partial co-localization of ASYN and LAMP1 suggests interplay between lysosomal degradation and ASYN inclusion formation. B. E57K and Y125F inclusions co-localize with lysosomal marker LAMP-1. We detected the presence of endosomes/lysosomes surrounding the aggregates in E57K and Y125F. This indicates that, maybe this could be the preferential via for degradation for these mutations. Scale bar: 10 µm.

Mentions: Knowing that ASYN is predominantly degraded by lysosomal pathways and, therefore, requires intact lysosomal function, we used lysosomal associated membrane protein 1 (LAMP-1) as a marker to establish the relationship with aggregation formation. We observed that LAMP-1 partially co-localized with ASYN inclusions (Fig. 9A), suggesting that at least some types of inclusions might be degraded in lysosomes. Interestingly, we observed that some inclusions formed by two of the mutations that primarily accumulated thioS-negative inclusions (E57K and Y125F, Fig. 6B) stained positive for LAMP-1 at the periphery (Fig. 9B), reinforcing the idea that specific types of ASYN inclusions are degraded in lysosomes.


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 partially co-localizes with endosomes/lysosomes.A. Immunocytochemistry analysis of H4 cells expressing selected ASYN mutants. Partial co-localization of ASYN and LAMP1 suggests interplay between lysosomal degradation and ASYN inclusion formation. B. E57K and Y125F inclusions co-localize with lysosomal marker LAMP-1. We detected the presence of endosomes/lysosomes surrounding the aggregates in E57K and Y125F. This indicates that, maybe this could be the preferential via for degradation for these mutations. Scale bar: 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004741-g009: ASYN partially co-localizes with endosomes/lysosomes.A. Immunocytochemistry analysis of H4 cells expressing selected ASYN mutants. Partial co-localization of ASYN and LAMP1 suggests interplay between lysosomal degradation and ASYN inclusion formation. B. E57K and Y125F inclusions co-localize with lysosomal marker LAMP-1. We detected the presence of endosomes/lysosomes surrounding the aggregates in E57K and Y125F. This indicates that, maybe this could be the preferential via for degradation for these mutations. Scale bar: 10 µm.
Mentions: Knowing that ASYN is predominantly degraded by lysosomal pathways and, therefore, requires intact lysosomal function, we used lysosomal associated membrane protein 1 (LAMP-1) as a marker to establish the relationship with aggregation formation. We observed that LAMP-1 partially co-localized with ASYN inclusions (Fig. 9A), suggesting that at least some types of inclusions might be degraded in lysosomes. Interestingly, we observed that some inclusions formed by two of the mutations that primarily accumulated thioS-negative inclusions (E57K and Y125F, Fig. 6B) stained positive for LAMP-1 at the periphery (Fig. 9B), reinforcing the idea that specific types of ASYN inclusions are degraded in lysosomes.

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