Detection of α-syn pores.

<p><b>A</b>) Pore formation results in increased current-flows over the membrane (black trace) compared to intact bilayers (grey trace) when a voltage ramp is applied. <b>B</b>) Pore detection rate for oligomers obtained with 2.1 µM α-syn with 1% DMSO and 20 µM Fe³⁺ at RT. 4 and 24 h of incubation did not lead to any pore detections (N = 8, each). In 33% of all 48 h-samples (N = 9) pore formations could be detected, increasing to a maximum at 72 h (N = 10). Further incubation results in a decrease of pore detection (N = 10). Non-incubated α-syn monomers did not lead to pore detections (N = 4), as well as DMSO and Fe³⁺ (“buffer”) after all tested incubation times (N = 4, each). α-hemolysin was used as a positive control (α-HL; N = 4). <b>C</b>) In a further set of experiments, the effects of different α-syn concentrations and the effect of the aggregation inhibitor baicalein were investigated after incubation of α-syn for 72 h with DMSO and Fe³⁺. Shown is the probability of pore detection following sequential application of aliquots of the respective samples to the bilayer. α-syn was used at 7.0 µM (N = 69), 2.1 µM (N = 35) and 0.7 µM (N = 8) with up to 9 aliquots applied per sample. With decreasing α-syn concentration, cumulative pore detection rate decreases significantly (p<0.001). Co-incubation of 2.1 µM of α-syn with 50 µM of baicalein (N = 8) significantly decreases pore detection compared to 2.1 µM control condition (p<0.005). <b>D</b>) When different voltages were clamped to membranes with inserted pores, step-like changes in conductivity were consistently observed during the duration of the voltage-pulse.</p

Schematic illustration of different models for increased membrane permeability caused by α-syn oligomers.

<p>In principle, α-syn oligomers could cause increased conductance of lipid membranes by different modes of action. <b>A</b>) A diffuse damage of the bilayer could lead to an unspecific increase in transmembrane current flow. <b>B</b>) Distinct pores could be formed in the bilayer that switch between two or more different conformational states, resulting in corresponding changes in conductivity. <b>C</b>) Different numbers of uniform pores could spontaneously insert and de-insert into the membrane leading to step-like changes in conductivity. <b>D</b>) The number of “open” pores could fluctuate due to open and closure events of permanently inserted pore complexes.</p

Schematic illustrating the influence of mimicking Ser129 phosphorylation on asyn membrane interactions.

<p>A. For stressed gel-state membranes (DPPC-SUV), mimicking phosphorylation at Ser129 mildly reduces asyn binding affinity. B. Both pseudophosphorylated and unphosphorylated asyn monomers show no affinity to membranes in the liquid-crystalline state (POPC). C. Fe³⁺ induced asyn oligomers show a high membrane affinity and potentially act as membrane pores. Pseudophosphorylation at Ser129 shows a differential influence on asyn aggregation and binding behaviour. While increasing oligomer formation in presence of trivalent metal-ions, Ser129 pseudophosphorylation inhibits membrane binding and may thus allow oligomer sequestration into larger aggregates such as fibrils.</p

Influence of metal-ion induced asyn oligomer formation and pseudophosphorylation on membrane binding.

<p>A. FIDA analysis (upper panel) and 2D FIDA histograms (lower panel) demonstrate that Fe³⁺/Al³⁺ induced asyn⁶⁴⁷ oligomers show a high affinity to POPC-SUV⁴⁸⁸. Conversely, membrane binding of asyn129E⁶⁴⁷ oligomers is nearly abolished for Fe³⁺ and markedly reduced for Al³⁺ induced oligomers. B. SIFT segment analysis also demonstrates an increased mixed particle signal for asyn⁶⁴⁷ as compared to asyn129E⁶⁴⁷ (“bound oligomer”, corresponding to SIFT segments 4–15; also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098906#pone-0098906-g001" target="_blank">figure 1A</a>.), while levels of free oligomers are higher in asyn129E⁶⁴⁷ (“oligo”, corresponding to segments 16–18). Levels of significance are depicted as * = p<0.05, ** = p<0.01; n = 3.</p

Effect of Ser129 pseudophosphorylation on asyn monomer binding to lipid vesicles.

<p>aSyn⁶⁴⁷ and asyn129E⁶⁴⁷ were coincubated with POPC- and DPPC-SUV⁴⁸⁸. Schematic A. demonstrates the appearance of vesicles, protein monomers and oligomers in 2D FIDA histograms (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098906#pone.0098906-Hogen1" target="_blank">[7]</a>). B. aSyn⁶⁴⁷ and asyn129E⁶⁴⁷ monomers show no interactions with POPC-SUV. Conversely, extensive monomer binding to DPPC-SUV is seen irrespective of pseudophosphorylation status. C. Quantitative FIDA analysis shows a mild tendency towards reduced vesicle binding of asyn129E⁶⁴⁷. D. Quantitative 2D-FIDA analysis of monomers bound to DPPC-SUV demonstrates an overall higher amount of bound monomers (total brightness), determined through brightness and concentration of bicolored particles. Furthermore, the higher fluorescence intensity of the bicolored particles (single vesicle brightness) indicates that more monomers are bound per vesicle. E. In a control experiment, asyn⁶⁴⁷ and asyn129E⁶⁴⁷ particle brightness (Q) as determined by 1D FIDA analysis (one component fit) is unchanged in presence of POPC-SUV as compared to control measurements in the absence of vesicles, confirming that no membrane binding takes place. However, Q increases in presence of DPPC-SUV. Dissolution of DPPC-SUV by SDS yields an asyn⁶⁴⁷ particle brightness similar to monomeric asyn⁶⁴⁷, indicating that oligomer formation is not induced by binding to DPPC-SUV. Levels of significance are depicted as * = p<0.05; n = 3.</p

Conformational changes measured by <i>in vitro</i> conversion reactions.

<p><b>(A)</b><i>In vitro</i> conversion reactions have been performed with radiolabeled wild-type and PrP^C(TetraH>G) purified from RK13 cells and PrP^Sc purified from brains of RML-infected Tga20 mice as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188989#pone.0188989.ref004" target="_blank">4</a>]. Samples were analyzed by SDS-PAGE-fluorography, and relative conversion efficiencies (CVE) were calculated from band intensities before and after digestion with proteinase K using the formula CVE [%] = [I°_+PK / (I°_-PK*10)]*100. PrP^C with substituted OR histidines (PrP^C(TetraH>G)) is only half as efficient in converting to the misfolded, PK-resistant conformer than wt PrP^C. Mean values ± standard error (SEM) were determined from 11 independent experiments for each PrP^C type. P-values (p (two sided) = 0.07, p (one sided) = 0.036) were obtained by T-Test calculation. (<b>B</b>) Control reactions performed in the absence of PrP^Sc seed.</p

Western blot analysis of brain lysates from RML-infected wt and transgenic mice for total PrP^C and PrP^Sc using monoclonal antibody 4H11.

<p>(<b>A</b>) Brain homogenates from terminally ill wt and PrP(TetraH>G) line 34 mice were either left untreated (- PK) or subjected to digestion with proteinase K (+ PK). Blots were reprobed for β-actin to control for equal loading. Bands corresponding to total PrP are marked on the left. Irrelevant lanes have been excised at two positions. Molecular weight standards are given on the right (in kDa). (<b>B</b>) Corresponding immunoblot analysis of brain homogenates extracted from RML-infected PrP(H95G) mice from the three different lines 11, 13, and 4, respectively, and corresponding wt control (lanes 1 and 2) before (-) and after (+) treatment with PK. Molecular weight standards are given on the right (in kDa).</p

Lesion profiles induced by mouse-adapted scrapie isolate RML in wild-type and transgenic mice expressing mutant PrP.

<p>The extent of spongiform change (<b>A</b>) and reactive gliosis (<b>C</b>) in brain sections of terminally ill wt and PrP(TetraH>G) was assessed semi-quantitatively in a blinded fashion in nine areas of grey matter and three areas of white matter by lesion profiling. Animals were scored on a scale of 0–5 in each specific area, and mean scores (n = 6 (C57/129Sv), versus n = 7 (PrP(TetraH>G), line 34), respectively) are shown graphically (error bars plus SD). Blue diamonds: C57/129Sv. Red squares: PrP(TetraH>G). Analogous data from PrP(H95G) mice are shown in panels <b>B</b> and <b>D</b> (n = 4 (PrP(H95G), line 13), n = 5 (PrP(H95G), line 4) and n = 7 (PrP(H95G), line 11), respectively); data for C57/129Sv wt animals has been re-plotted for comparative purposes. Blue diamonds: C57/129Sv. Red squares: PrP(H95G), line 4. Green triangles: PrP(H95G), line 11. Grey circles: PrP(H95G), line 13. Scoring areas as follows: Grey matter: 1, dorsal medulla, 2, cerebellar cortex, 3, superior colliculus, 4, hypothalamus, 5, medial thalamus, 6, hippocampus, 7, septum, 8, medial cerebral cortex at septum level, 9, medial cerebral cortex at thalamus level. White matter: 1*, cerebellar white matter, 2*, mesencephalic tegmentum, 3* pyramidal tract.</p

Neuropathological changes and PrP^Sc distribution pattern in brain sections of secondary passage transgenic mice and corresponding wt controls.

<p>(<b>A</b>) First column: Paraffin-embedded tissue (PET) blots demonstrating PrP^Sc deposits in hippocampus (upper panels) and cerebellum (lower panels) of wt (C57/129Sv) and PrP(TetraH>G), line 34 inoculated with PrP(TetraH>G)-passaged prions. Second column: Corresponding PrP^Sc immunhistochemistry (mAb CDC-1). Third column: Hematoxylin and eosin stainings (H&E) of hippocampal and cerebellar sections demonstrating spongiform changes. Fourth column: GFAP immunostaining demonstrating gliosis. (<b>B</b>) Corresponding hippocampal brain sections from terminally ill PrP(H95G) mice (line 13) and corresponding wt controls (C57/129Sv) challenged with PrP(H95G)-passaged prions.</p

PrP^C levels and glycosylation profile in healthy wild-type and transgenic mice expressing mutant PrP.

<p><b>(A)</b> Brain homogenates (10 μg per lane) derived from mice expressing full length mouse wild-type (wt), PrP(H95G) lines 4, 11 and 13, PrP(TetraH>G) line 34 and PrP null controls (<i>Prnp</i>^0/0) were subjected to immunoblot analysis using monoclonal antibody SHA31. Blots were reprobed for β-actin to control for equal loading. Molecular weight is indicated on the left (in kDa). (<b>B</b>) Removal of N-linked glycans on PrP^C encoded by the PrP(H95G), lines 4, 11, and 13 as well as PrP(TetraH>G), line 34 using peptide N-glycosidase F (PNGase F) and probing of the treated samples with monoclonal antibody SHA31. fl = full-length PrP.</p