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E46K mutant ?-synuclein is more degradation resistant and exhibits greater toxic effects than wild-type ?-synuclein in Drosophila models of Parkinson's disease.

ABSTRACT: Parkinson's disease (PD) is one of the most common neurodegenerative diseases, which is characterized by progressive motor dysfunction as well as non-motor symptoms. Pathological and genetic studies have demonstrated that ?-synuclein (?Syn) plays key roles in the pathogenesis of PD. Although several missense mutations in the ?Syn gene have been identified as causes of familial PD, the mechanisms underlying the variance in the clinical phenotypes of familial PD caused by different mutations remain elusive. Here, we established novel Drosophila models expressing either wild-type (WT) ?Syn or one of five ?Syn mutants (A30P, E46K, H50Q, G51D, and A53T) using site-specific transgenesis, which express transgenes at equivalent levels. Expression of either WT or mutant ?Syn in the compound eyes by the GMR-GAL4 driver caused mild rough eye phenotypes with no obvious difference among the mutants. Upon pan-neuronal expression by the nSyb-GAL4 driver, these ?Syn-expressing flies showed a progressive decline in locomotor function. Notably, we found that E46K, H50Q, G51D, and A53T ?Syn-expressing flies showed earlier onset of locomotor dysfunction than WT ?Syn-expressing flies, suggesting their enhanced toxic effects. Whereas mRNA levels of WT and mutant ?Syn were almost equivalent, we found that protein expression levels of E46K ?Syn were higher than those of WT ?Syn. In vivo chase experiments using the drug-inducible GMR-GeneSwitch driver demonstrated that degradation of E46K ?Syn protein was significantly slower than WT ?Syn protein, indicating that the E46K ?Syn mutant gains resistance to degradation in vivo. We therefore conclude that our novel site-specific transgenic fly models expressing either WT or mutant ?Syn are useful to explore the mechanisms by which different ?Syn mutants gain toxic functions in vivo.

SUBMITTER: Sakai R

PROVIDER: S-EPMC6594597 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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E46K mutant α-synuclein is more degradation resistant and exhibits greater toxic effects than wild-type α-synuclein in Drosophila models of Parkinson's disease.

Sakai Ryusuke R Suzuki Mari M Ueyama Morio M Takeuchi Toshihide T Minakawa Eiko N EN Hayakawa Hideki H Baba Kousuke K Mochizuki Hideki H Nagai Yoshitaka Y

PloS one 20190626 6

Parkinson's disease (PD) is one of the most common neurodegenerative diseases, which is characterized by progressive motor dysfunction as well as non-motor symptoms. Pathological and genetic studies have demonstrated that α-synuclein (αSyn) plays key roles in the pathogenesis of PD. Although several missense mutations in the αSyn gene have been identified as causes of familial PD, the mechanisms underlying the variance in the clinical phenotypes of familial PD caused by different mutations remai ...[more]

PMID: 31242217

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Project description:The E46K genetic missense mutation of the wild-type ?-synuclein protein was recently identified in a family of Spanish origin with hereditary Parkinson's disease. Detailed understanding of the structures of the monomeric E46K mutant-type ?-synuclein protein as well as the impact of the E46K missense mutation on the conformations and free energy landscapes of the wild-type ?-synuclein are required for gaining insights into the pathogenic mechanism of Parkinson's disease. In this study, we use extensive parallel tempering molecular dynamics simulations along with thermodynamic calculations to assess the secondary and tertiary structural properties as well as the conformational preferences of the monomeric wild-type and E46K mutant-type ?-synuclein proteins in an aqueous solution environment. We also present the residual secondary structure component conversion stabilities with dynamics using a theoretical strategy, which we most recently developed. To the best of our knowledge, this study presents the first detailed comparison of the structural and thermodynamic properties of the wild-type and E46K mutant-type ?-synuclein proteins in an aqueous solution environment at the atomic level with dynamics. We find that the E46K mutation results not only in local but also in long-range changes in the structural properties of the wild-type ?-synuclein protein. The mutation site shows a significant decrease in helical content as well as a large increase in ?-sheet structure formation upon E46K mutation. In addition, the ?-sheet content of the C-terminal region increases significantly in the E46K mutant-type ?S in comparison to the wild-type ?S. Our theoretical strategy developed to assess the thermodynamic preference of secondary structure transitions indicates that this shift in secondary structure is the result of a decrease in the thermodynamic preference of turn to helix conversions while the coil to ?-sheet preference increases for these residues. Long-range intramolecular protein interactions of the C-terminal with the N-terminal and NAC regions increase upon E46K mutation, resulting in more compact structures for the E46K mutant-type rather than wild-type ?S. However, the E46K mutant-type ?S structures are less stable than the wild-type ?S. Overall, our results show that the E46K mutant-type ?S has a higher propensity to aggregate than the wild-type ?S and that the N-terminal and C-terminal regions are reactive toward fibrillization and aggregation upon E46K mutation and we explain the associated reasons based on the structural properties herein. Small molecules or drugs that can block the specific residues forming abundant ?-sheet structure, which we report here, might help to reduce the reactivity of these intrinsically disordered fibrillogenic proteins toward aggregation and their toxicity.

Project description:Parkinson's Disease (PD) is a neurodegenerative and progressive disorder characterised by intracytoplasmic inclusions called Lewy bodies (LB) and degeneration of dopaminergic neurons in the substantia nigra (SN). Aggregated α-synuclein (αSYN) is known to be the main component of the LB. It has also been reported to interact with several proteins and organelles. Galectin-3 (GAL3) is known to have a detrimental function in neurodegenerative diseases. It is a galactose-binding protein without known catalytic activity and is expressed mainly by activated microglial cells in the central nervous system (CNS). GAL3 has been previously found in the outer layer of the LB in post-mortem brains. However, the role of GAL3 in PD is yet to be elucidated. In post-mortem samples, we identified an association between GAL3 and LB in all the PD subjects studied. GAL3 was linked to less αSYN in the LB outer layer and other αSYN deposits, including pale bodies. GAL3 was also associated with disrupted lysosomes. In vitro studies demonstrate that exogenous recombinant Gal3 is internalised by neuronal cell lines and primary neurons where it interacts with endogenous αSyn fibrils. In addition, aggregation experiments show that Gal3 affects spatial propagation and the stability of pre-formed αSyn fibrils resulting in short, amorphous toxic strains. To further investigate these observations in vivo, we take advantage of WT and Gal3KO mice subjected to intranigral injection of adenovirus overexpressing human αSyn as a PD model. In line with our in vitro studies, under these conditions, genetic deletion of GAL3 leads to increased intracellular αSyn accumulation within dopaminergic neurons and remarkably preserved dopaminergic integrity and motor function. Overall, our data suggest a prominent role for GAL3 in the aggregation process of αSYN and LB formation, leading to the production of short species to the detriment of larger strains which triggers neuronal degeneration in a mouse model of PD.

Dataset Information

E46K mutant ?-synuclein is more degradation resistant and exhibits greater toxic effects than wild-type ?-synuclein in Drosophila models of Parkinson's disease.

Publications

E46K mutant α-synuclein is more degradation resistant and exhibits greater toxic effects than wild-type α-synuclein in Drosophila models of Parkinson's disease.

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