Systematic comparison of the effects of Alpha-synuclein mutations on its oligomerization and aggregation

Copyright: © 2014 Lázaro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.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.This work was supported by the DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).info:eu-repo/semantics/publishedVersio

Fault-kinematic and geomorphic observations along the North Tehran Thrust and Mosha Fasham Fault, Alborz mountains Iran: Implications for fault-system evolution and interaction in a changing tectonic regime

Neighbouring faults can interact, potentially link up and grow, and consequently increase the seismic and related natural hazards in their vicinity. Despite evidence of Quaternary faulting, the kinematic relationships between the neighbouring Mosha Fasham Fault (MFF) and the North Tehran Thrust (NTT) and their temporal evolution in the Alborz mountains are not well understood. The ENE-striking NTT is a frontal thrust that delimits the Alborz mountains to the south with a 2000 m topographic front with respect to the proximal Tehran plain. However, no large instrumentally recorded earthquakes have been attributed to that fault. In contrast, the sigmoidally shaped MFF is a major strike-slip fault, located within the Alborz Mountains. Sinistral motion along the eastern part of the MFF is corroborated by microseismicity and fault kinematic analysis, which documents recent transtensional deformation associated with NNE-SSW oriented shortening. To better understand the activity of these faults on different timescales, we combined fault-kinematic analysis and geomorphic observations, to infer the kinematic history of these structures. Our fault kinematic study reveals an early dextral shear for the NTT and the central MFF, responsible for dextral strike-slip and oblique reverse faulting during NW-oriented shortening. This deformation regime was superseded by NE-oriented shortening, associated with sinistral-oblique thrusting along the NTT and the central-western MFF, sinistral strike-slip motion along subsidiary faults in the central MFF segment, and folding and tilting of Eocene to Miocene units in the MFF footwall. Continued thrusting along the NTT took place during the Quaternary. However, folding in the hanging wall and sinistral stream-offsets indicate a left-oblique component and Quaternary strike-slip reactivation of the eastern NTT-segment, close to its termination. This complex history of faulting under different stress directions has resulted in a composite landscape with inherited topographic signatures. Our study shows that the topography of the hanging wall of the NTT reflects a segmentation into sectors with semi-independent uplift histories. Areas of high topographic residuals and apparent high uplift underscore the fault kinematics. Combined, our data suggest an early mechanical linkage of the NTT and MFF fault systems during a former dextral transpressional stage, caused by NW-compression. During NE-oriented shortening, the NTT and MFF were reactivated and incorporated into a nascent transpressional duplex. The youngest manifestation of motion in this system is sinistral transtension. However, this deformation is not observed everywhere and has not yet resulted in topographic inversion. © 2009 The Authors Journal compilation © 2009 RAS

Correlation between the effects of ASYN mutations on oligomerization and inclusion formation.

<p>The graph depicts how mutations affect oligomerization and inclusion formation, enabling the selection of mutants with different effects. Values were attributed to ASYN mutations according to the results from the two models (oligomerization and inclusion formation) using WT ASYN as reference (center of the graph).</p

ASYN biochemical state.

<p><b>A. Native Gels.</b> Immunoblot analysis of native PAGE of cells transfected with the BiFC constructs in HEK 293 cells. Smears indicate the presence of oligomeric species of ASYN with different sizes. n = 2. <b>B. STED microscopy.</b> Selected mutants were imaged in order to characterize the fine structure of the inclusions. <b>C. Thioflavin S staining.</b> H4 cells expressing selected SynT mutants were incubated with ThioS in order to reveal beta sheet-rich structures. Some of the inclusions display amyloid-like properties, with increased staining in the inner part of the inclusions, indicated with arrow heads (▸). Scale bar: 10 µm. <b>D-E</b>. <b>Triton X-100 solubility assay and quantification.</b> H4 cells show that all mutants form detergent insoluble species. Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001). n = 2. Quantification of insoluble fraction shows a decrease in TP and E57K mutants.</p

ASYN mutation effects in the inclusion formation.

<p><b>A. Constructs used in the aggregation model.</b> This model consists of co-expressing SynT together with synphilin-1. <b>B. Inclusion pattern in H4 cells.</b> Different SynT mutants resulted in the formation of distinct inclusion formation in human H4 cells. Scale bar: 10 µm. <b>C. Inclusion quantification.</b>>50 cells were scored per experiment and classified in different groups according to the pattern of inclusions. Representative cells were drawn to show type of inclusions present in each categories. Lysine mutants (E35K, E57K) increase the percentage of cells with inclusions and the number of inclusions per cell, whereas A30P and proline mutants reduce percentage of cells with inclusions and also the number of inclusions per cell. <b>D-E. Levels of ASYN.</b> Immunoblot analysis of the expression levels of ASYN. Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001). n = 3.</p

ASYN bPCA.

<p><b>A. Schematic representation of the ASYN bPCA constructs.</b> Non-bioluminescent halves of humanized Gaussia luciferase (hGLuc) were fused to ASYN monomers. <b>B-C</b>. 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>B</b>) and extracellular (<b>C</b>) TP displayed a 3-fold increase in luciferase activity compared to WT. n = 12. Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001) <b>D</b>. Ratio of luciferase activity in media compared to cells was expressed. n = 12, Student's <i>t</i> test (*p<0.05, **p<0.01, ***p<0.001) <b>E-F. Levels of ASYN.</b> Immunoblot analysis of the expression levels of ASYN showing similar levels. n = 3.</p