Journal: JACS Au

Article Title: Early-Onset Parkinson Mutation Remodels Monomer–Fibril Interactions to Allosterically Amplify Synuclein’s Amyloid Cascade

doi: 10.1021/jacsau.3c00655

Figure Lengend Snippet: (A) Domain architecture of αS and location of the PD-related early onset mutation E46K. The net charges of the N-, NAC and C-terminal regions of αS are +4 for wt (+6 for E46K), −1 and −12, respectively. (B) Mechanism of αS fibril formation. ‘Closed’ monomer conformers are stabilized by electrostatic interactions between the N- and C-terminal regions, which shield the NAC region. In “open” monomers, the NAC region is available for self-association into primary nuclei, oligomers, and eventually fibrils. The latter recruit monomers to further accelerate fibril elongation and secondary nucleation. (C) SEC-MALS measurements for αS monomers. Size exclusion chromatography profiles and molecular weights are shown for wt and E46K αS monomers. E46K enhances the electrostatic attraction between the N- and C-terminal regions of αS, resulting in a more compact ensemble. (D) Time-dependent Thioflavin T (ThT) fluorescence for wt and E46K αS, showing that E46K amplifies αS aggregation. (E) DLS measurements of wt and E46K αS fibrils after ultracentrifugation. (F) TEM images of preformed αS fibrils after ultracentrifugation. (G) Fluorescence of ANS bound to 250 μM wt and E46K fibrils. (H) Fluorescence of ANS at emission wavelengths of 465 and 485 nm. Two-way ANOVA was used to determine the statistical significance for the wt vs E46K comparisons with **** representing p -values <0.0001.

Article Snippet: The plasmid for E46K mutation was purchased from Addgene (#105728), while the S129C and S129C/E46K plasmids were generated through site-directed mutagenesis.

Techniques: Mutagenesis, Size-exclusion Chromatography, Fluorescence

Journal: JACS Au

Article Title: Early-Onset Parkinson Mutation Remodels Monomer–Fibril Interactions to Allosterically Amplify Synuclein’s Amyloid Cascade

doi: 10.1021/jacsau.3c00655

Figure Lengend Snippet: Effect of E46K on transient αS monomer–monomer and monomer-fibril interactions. (A) NMR intermolecular PRE quantified through intensity ratios of oxidized and reduced intensities for 300 μM spin-labeled 14 N-labeled αS monomers mixed with 300 μM nonspin-labeled 15 N-labeled αS monomers. Black: wt αS; Purple: E46K αS. The site where the MTSL is located is labeled with a red arrow, and the site of the mutation is labeled as a red star. (B) Difference between the PRE ratios of E46K and wt αS in panel A. (C) Differences between the residue-specific 15 N- R 2 of 250 μM αS in the presence and absence of 3-fold molar-excess of αS fibrils. Black: wt αS monomers and wt fibrils; Purple: E46K αS monomers and E46K fibrils. (D) Difference between the Δ R 2 profiles in C of E46K and wt. The charge of each amino acid along the sequence is listed on the top with a red (blue, gray) bar representing a negative (positive, neutral) charge. Light gray regions highlight E46K vs wt differences.

Article Snippet: The plasmid for E46K mutation was purchased from Addgene (#105728), while the S129C and S129C/E46K plasmids were generated through site-directed mutagenesis.

Techniques: Labeling, Mutagenesis, Residue, Sequencing

Journal: JACS Au

Article Title: Early-Onset Parkinson Mutation Remodels Monomer–Fibril Interactions to Allosterically Amplify Synuclein’s Amyloid Cascade

doi: 10.1021/jacsau.3c00655

Figure Lengend Snippet: E46K mutation amplifies the closed-to-open shift induced by αS fibril in αS monomers. (A) Intramolecular PREs as quantified by the ratios of oxidized and reduced intensities for 120 μM spin-labeled 15 N wt αS monomer in the absence and presence of 3-fold excess unlabeled wt αS fibrils without spin-label. (B) As A but for αS E46K. (C) Difference between the PRE profiles in A. (D) As C but for E46K. (E) Difference between the PRE differentials in C and D. The light gray background region highlights PRE changes occurring in the NAC region. (F) Scheme illustrating fibril-induced closed-to-open monomer shifts.

Article Snippet: The plasmid for E46K mutation was purchased from Addgene (#105728), while the S129C and S129C/E46K plasmids were generated through site-directed mutagenesis.

Techniques: Mutagenesis, Labeling

Journal: JACS Au

Article Title: Early-Onset Parkinson Mutation Remodels Monomer–Fibril Interactions to Allosterically Amplify Synuclein’s Amyloid Cascade

doi: 10.1021/jacsau.3c00655

Figure Lengend Snippet: Transient αS monomer-fibril heterointeractions. (A) Residue-specific 15 N- R 2 relaxation rates of 250 μM wt αS monomers in the presence of 3-fold excess E46K αS fibrils. 15 N- R 2 data from homomixtures is included for the convenience of comparison. (B) As A after subtracting monomer rates. (C, D) As panels A and B, but swapping wt and E46K, i.e. , E46K monomer in the presence of 3-fold excess wt αS fibrils. Light gray background highlights key differences between line and bar plots in parts (C) and (D).

Article Snippet: The plasmid for E46K mutation was purchased from Addgene (#105728), while the S129C and S129C/E46K plasmids were generated through site-directed mutagenesis.

Techniques: Residue, Comparison

Journal: JACS Au

Article Title: Early-Onset Parkinson Mutation Remodels Monomer–Fibril Interactions to Allosterically Amplify Synuclein’s Amyloid Cascade

doi: 10.1021/jacsau.3c00655

Figure Lengend Snippet: Proposed multipronged mechanism of action for the early onset PD mutation E46K. Key self-association equilibria and interactions perturbed by E46K are illustrated for both wt and E46K αS. The mutation acts at multiple levels (purple arrows and lines) and results in new types of monomer-fibril recognition modes denoted as 1–3 and involving monomers’ NTR, CTR and NAC regions, respectively. Modes 1 and 3 apply to both wt and mutant fibrils, as indicated by the curved arrows. The wt mechanism agrees with previous findings. , Fibril structures are adapted from PDB structures 6CU7 and 6UFR for WT and E46K fibrils, , respectively. N- and C- termini are depicted as disordered lines.

Article Snippet: The plasmid for E46K mutation was purchased from Addgene (#105728), while the S129C and S129C/E46K plasmids were generated through site-directed mutagenesis.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Systematic identification of structure-specific protein–protein interactions

doi: 10.1101/2023.02.01.522707

Figure Lengend Snippet: Quality control experiments for aSyn monomer and amyloid fibrils. (a) SEC analysis of purified aSyn monomer. (b) SDS PAGE and Native PAGE of monomeric aSyn. (c) TEM image of aSyn amyloid fibrils. (d) ThT intensity of PBS control, aSyn monomer, and aSyn amyloid fibrils.

Article Snippet: For the formation of mature aSyn amyloid fibrils, Eppendorf LoBind microcentrifuge tubes (1.5 mL) containing 0.75 mL of 5 mg/mL monomeric aSyn in PBS buffer, pH 7.4, 150 mM NaCl were incubated at 37 °C on a thermomixer under agitation at 800 rpm for 1–2 weeks.

Techniques: Control, Purification, SDS Page, Clear Native PAGE

Journal: Nature Communications

Article Title: The extracellular chaperone Clusterin enhances Tau aggregate seeding in a cellular model

doi: 10.1038/s41467-021-25060-1

Figure Lengend Snippet: a Aggregation of α-Syn (300 μM) without (black) or with Clu (0.3 μM and 3 μM in cyan and red, respectively) was monitored by ThT fluorescence. Molar ratios of α-Syn:Clu are indicated. arb.units, arbitrary units. Averages ± SEM ( n = 4 independent experiments). b Effect of Clu on seeding potency of α-Syn aggregation reactions as in ( a ). Seeding was measured by quantifying the fraction of HEK293T cells stably expressing GFP-α-Syn(A53T) that contained aggregates after 24 h of seeding (200 ng α-Syn) with lipofectamine. Molar ratios of α-Syn:Clu are indicated. Significance is represented relative to α-Syn alone at each time point. Averages ± SEM ( n = at least 100 cells examined over three independent experiments). *** p < 0.001 by two-way ANOVA with Sidak post hoc test. (α-Syn 24 h vs. α-Syn/Clu 1000:1 24 h p = 1.9 × 10 −8 ; α-Syn 24 h vs. α-Syn/Clu 100:1 24 h p = 8.4 × 10 −13 ; α-Syn 36 h vs. α-Syn/Clu 1000:1 36 h p = 4.5 × 10 −7 ; α-Syn 36 h vs. α-Syn/Clu 100:1 36 h p = 1.3 × 10 −11 ; α-Syn 72 h vs. α-Syn/Clu 1000:1 72 h p = 1.8 × 10 −7 ; α-Syn 72 h vs. α-Syn/Clu 100:1 72 h p = 2.3 × 10 −13 ). c Representative images of HEK 293 T GFP-α-Syn(A53T) cells seeded with aggregation reactions (200 ng α-Syn after 0 h, 24 h and 72 h aggregation ( a )) with or without Clu. GFP-α-Syn(A53T) and DAPI nuclear staining are shown in green and blue, respectively. Arrowheads indicate aggregates. Scale bar, 30 μm. d Heparan sulfate proteoglycan (HSPG) mediated internalization of α-Syn and α-Syn/Clu aggregates. Seeding was measured by quantifying the fraction of GFP-α-Syn(A53T) cells that contained aggregates after 72 h of seeding (50 µg α-Syn after 72 h aggregation ( a )) without lipofectamine (−Heparin). GFP-α-Syn(A53T) cells were also treated with α-Syn and α-Syn/Clu aggregates (50 µg) in combination with heparin (200 µg/ml). Molar ratios of α-Syn:Clu are indicated. Data represent the mean ± SEM ( n = at least 1000 cells examined over three independent experiments). ** p < 0.01, *** p < 0.001 by two-way ANOVA with Sidak post hoc test (−Heparin α-Syn vs. −Heparin α-Syn/Clu 100:1 p = 0.003; −Heparin α-Syn vs. +Heparin α-Syn p = 4.8 × 10 −5 ; −Heparin α-Syn/Clu 1000:1 vs. +Heparin α-Syn/Clu 1000:1 p = 0.007). The significance of +Heparin reactions is relative to the respective −Heparin control.

Article Snippet: Plasmid pT7-7 α-Syn A53T for the expression and purification of recombinant α-Syn was a gift from Hilal Lashuel (Addgene plasmid #105727 ) and EGFP-α-SynA53T plasmid for α-Syn expression in HEK293T and SH-SY5Y cells were a gift from David Rubinsztein (Addgene plasmid #40823 ).

Techniques: Fluorescence, Stable Transfection, Expressing, Staining, Control

Journal: NPJ Parkinson's disease

Article Title: Dynamic physiological α-synuclein S129 phosphorylation is driven by neuronal activity.

doi: 10.1038/s41531-023-00444-w

Figure Lengend Snippet: Fig. 7 Synaptic plasticity is impaired in S129AKI mouse. a–f Generation of the S129A knock-in mutation in mice. a Genomic structure of the mouse SNCA gene. Exons are depicted after transcript variant SNCA-201 (ENSMUST00000114268.5), with coding and non-coding regions shown as filled or open boxes, respectively. Exon 5 is boxed with red dashed line. b A knock-in strategy using CRISPR-Cas9 and ssODN was employed to generate the S129A mutation in Exon 5 of the endogenous SNCA gene. Relative positions of sgRNA (orange horizontal line), ssODN (blue horizontal line), and the S129A mutation (red vertical line) are indicated. c–f ES cell clone screening and mouse genotyping. A 3-primer PCR strategy (primers indicated with black and red arrows) was designed to distinguish WT and mutant alleles (c, d). For genotyping, a common pair of primers (indicated with green arrows) was used for PCR followed by sequencing to distinguish different genotypes (c, e, f). g Total mouse brain homogenates from indicated genotypes. WB for total αS, pS129 (D1R1R) and GAPDH. h Input and out current curve from hippocampal slices (CA1 region) of indicated mouse genotypes. N = 3 animals each, n = 16 (WT) or 17 (S129AKI) individual slices. i Paired- pulse facilitation of WT and S129AKI hippocampal slices. Inter-stimulation intervals as indicated. N = 3 animals each, n = 16 (WT) or 17 (S129AKI) individual slices. Unpaired t-tests for 20, 40, 60, 100, 200, and 500 ms with Welch’s correction; two-tailed; mean ± SD; ns not significant; *p < 0.05; **p < 0.01. j Short-term plasticity assessed by multi-pulse events. Values normalized to first excitatory post synaptic current (EPSP). N = 3 animals each, n = 16 (WT) or 17 (S129AKI) individual slices. Unpaired t-tests for pulses 2, 3, 4, 5, 6, 7, and 8 with Welch’s correction; two-tailed; mean ± SD; ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001. k, l Long Term Potentiation (LTP) of WT and S129AKI. LTP induced by standard 100 Hz stimulation. N = 3 animals each, n = 8 (WT) or 10 (S129AKI) individual slices. Unpaired t-tests for panel l (values at 60 min from K) with Welch’s correction; two-tailed; mean ± SD; **p < 0.01.

Article Snippet: Briefly, 293-T cells were transfected with αS WT or S129A plasmids along with pMD2.G and psPAX2 (packaging plasmids: Addgene #12259 and #12260, respectively).

Techniques: Knock-In, Mutagenesis, Variant Assay, CRISPR, Sequencing, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Temperature is a key determinant of alpha- and beta-synuclein membrane interactions in neurons

doi: 10.1016/j.jbc.2021.100271

Figure Lengend Snippet: The human αS A30P mutant is predominantly cytosolic. Cytosolic and membrane fractions were sequentially extracted from cortical neurons expressing human wt or A30P αS at 37 °C ( A ). The solubility of control proteins GAPDH and calnexin is depicted in panel B . Representative western blot images to the quantification are shown in panel C . Digitonin concentration, 800 or 900 μg/ml. (C, cytosol and M, membrane). N = 3 independent experiments, performed on different days in n = 12 + 4 + 4 independent wells (total n = 20). ∗∗∗∗ p < 0.0001. ns, not significant.

Article Snippet: 293-T cells were transfected with αS wt or A30P plasmids along with pMD2.G and psPAX2 (packaging plasmids: Addgene #12259 and #12260, respectively).

Techniques: Mutagenesis, Membrane, Expressing, Solubility, Control, Western Blot, Concentration Assay