primary antibodies against ps129-α-syn clone 81a  (Millipore)

Journal: Nature Communications

Article Title: Phenotypic manifestation of α-synuclein strains derived from Parkinson’s disease and multiple system atrophy in human dopaminergic neurons

doi: 10.1038/s41467-021-23682-z

Figure Lengend Snippet: a PMCA was performed on PD (top panels, in red and corresponding controls, blue) and MSA (bottom panels, in green) and the corresponding controls (in blue). Patient brain homogenates (2% w:v) for the first cycle, in PMCA buffer (150 mM KCl, 50 mM Tris-HCl, pH 7.5) containing monomeric α-synuclein (100 µM). The following amplification cycles were seeded by 2 or 5% (v:v) of the preceding amplification cycles for PD and MSA, respectively. The amounts of brain homogenates and PMCA-amplified assemblies used in each amplification reaction were defined through an optimisation study aimed at maintaining high stringency by minimising the de novo aggregation of α-synuclein under the experimental conditions we used. The time at which an aliquot from a given amplification cycle was withdrawn for a subsequent amplification reaction (last timepoint for each amplification cycle) was also defined through an optimisation study aimed at avoiding the formation of de novo α-synuclein fibrillar assemblies. The curves represent an average of n = 4 replicates ± SD. b Electron micrographs of α-synuclein assemblies obtained after the third cycle of amplification by PMCA from each of the three PD, five MSA cases and de novo-generated α-synuclein fibrils and ribbons. For PD3, we examined the temporal gyrus (PD 3G) as well as the cingulate cortex (PD 3C). Scale bar: 200 nm. c Limited proteolytic patterns of α-synuclein assemblies obtained after the third cycle of amplification by PMCA from PD-, MSA- and de novo-generated α-synuclein fibrils and ribbons. Monomeric α-synuclein concentration is 100 µM. Proteinase K concentration is 3.8 µg/ml. Samples were withdrawn from the reaction, immediately after PK addition (lane most to the left, labelled 0) and at time 1, 5, 15, 30 and 60 min. PAGE analysis was performed and the gels were stained with Coomassie blue. The intensity of the proteolytic band generated at 15 min and indicated by the arrowhead was normalised to that of α-synuclein at time 0 for each PD (red) and MSA (green) patient sample ( n = 4 PD and n = 5 MSA). PD patient-derived assemblies exhibited higher resistance to proteolysis than MSA patient-derived assemblies. In panel c , *** P < 0.001, two-sided unpaired Student’s t test. Source data for a , c are provided as a Source Data file.

Article Snippet: The membranes were blocked in 5% dried skimmed milk and probed with the anti-α-synuclein antibody 4B12 (Biolegend, cat # 807801) and anti-P-S129 α-synuclein antibody 81A (Millipore, cat# MABN826).

Techniques: Amplification, Generated, Concentration Assay, Staining, Derivative Assay

Journal: Nature Communications

Article Title: Phenotypic manifestation of α-synuclein strains derived from Parkinson’s disease and multiple system atrophy in human dopaminergic neurons

doi: 10.1038/s41467-021-23682-z

Figure Lengend Snippet: Similar levels of pSYN were detected by HTRF in neuronal lysates seeded with a 0.1 μM or b 1 μM fibrils when comparing de novo fibrils to each case of PD- or MSA-amplified strains ( n = 9 for panels a and b ). c Confocal images of SNCA TRIP dopaminergic neurons treated with de novo or brain-amplified fibrils. pSYN (upper), tyrosine hydroxylase (TH, lower). Images are representative of three independent differentiations (scale bar: 50 µm) and quantified in d , pSYN-positive aggregates induced by MSA-amplified fibrils were shorter and more speckled, whereas pSYN-positive aggregates induced by PD-amplified fibrils were neuritic and longer in length compared to ones induced by de novo-generated fibrils ( n = 9). e Proteinase K digestion of cell lysates followed by immunoblotting with a mixture of anti-α-synuclein antibodies (ASyM, 4B12 and 10D2) revealed different fragments and resistance to proteolysis in PD-seeded versus MSA-seeded neuronal aggregates (immunoblot representative of n = 2). f Representative confocal images from three independent differentiations depicting nuclear fragmentation (DAPI) and axonal degeneration (TUJ1), which were especially prominent in SNCA TRIP neurons treated with PD- or MSA-amplified fibrils. Healthy control (upper) and SNCA TRIP (lower) lines were treated with de novo fibrils, PD- or MSA-amplified fibrils (scale bar: 30 µm). g Viability was similar across conditions at 2 weeks post seeding ( n = 9). h Increased nuclear fragmentation was detected in SNCA TRIP lines seeded with MSA-amplified fibrils followed by PD-amplified fibrils at 3 weeks post seeding ( n = 9). Healthy control lines did not exhibit a similarly progressive phenotype ( n = 9). i α-Syn monomer and homogenates (hm) from each PD and MSA cases did not cause toxicity in contrast to corresponding amplified fibrils ( n = 3). NSC = non-seeded control, which means untreated neurons, Mono = neurons treated with monomeric α-Syn. Each dot corresponds to one clone differentiated once and data are mean ± s.e.m from at least n = 3 differentiations per clone. In panels d , g , h , i * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test. Source data for a , b , d , e , g , h , i are provided as a Source Data file.

Article Snippet: The membranes were blocked in 5% dried skimmed milk and probed with the anti-α-synuclein antibody 4B12 (Biolegend, cat # 807801) and anti-P-S129 α-synuclein antibody 81A (Millipore, cat# MABN826).

Techniques: Amplification, Generated, Western Blot

Journal: Nature Communications

Article Title: Phenotypic manifestation of α-synuclein strains derived from Parkinson’s disease and multiple system atrophy in human dopaminergic neurons

doi: 10.1038/s41467-021-23682-z

Figure Lengend Snippet: a α-Synuclein-BirA* expressing clonal cells were exposed to fibrils and biotin, resulting in biotinylation of proteins in proximity to aggregating α-synuclein inside cells. Biotinylated proteins were captured using streptavidin beads. Cells were treated with fibrils and biotin and stained for pSyn (green) and streptavidin (red) to determine biotinylated proteins. Co-localisation indicates that aggregates contain biotinylated proteins. Images are representative of three independent experiments (scale bar: 10 µm). b Mass spectrometry of biotinylated proteins (1002 identified in total shown as circles) from cells seeded with de novo-generated or brain-amplified fibrils identified 56 differentially interacting proteins (blue dots), including DJ-1 (in red); SNCA is shown for comparison (black dot). c DJ-1 immunocapture followed by streptavidin immunoblotting confirmed the enhanced interaction between DJ-1 and intracellular α-synuclein assemblies seeded with de novo-generated fibrils compared to PD- or MSA-amplified fibrils ( n = 5). d Pathway of MGO detoxification involving Glo-1 and DJ-1. e , f CRISPR/Cas9 knockout of the deglycase DJ-1, increased Ser129 phosphorylation of α-synuclein-venus (pSYN) induced by de novo fibrils, PD- or MSA-amplified fibrils compared to the non-target control and g , aggregate-induced toxicity as measured by LDH release ( n = 3 per sgRNA) . h , i CRISPR/Cas9 knockout of the glyoxalase Glo1, similarly increased Ser129 phosphorylation of α-synuclein-venus (pSYN) and j , aggregate-induced toxicity as measured by LDH release ( n = 3 per sgRNA). Two sgRNAs (sg1, sg2) were used per target for the knockout experiments (total n = 6 per target) . k pSyn-positive aggregated α-synuclein was immunoreactive for advanced glycation end-products. Immunoblot is representative of two independent experiments. l Illustrated chromatograms for each monoisotopic SILAC precursor ion that are shown in Supplementary Fig. . The highest abundance of GlyGly-modified K12 α-synuclein peptide was seen in de novo-generated fibril-treated samples with lower abundance in the PD- and MSA-amplified fibril-treated samples ( n = 2). m MGO increased fibril-induced aggregation as measured by pSYN in iPSC-derived dopaminergic neurons ( n = 6). n Exposure to MGO increased fibril-induced death of dopaminergic neurons as quantified by nuclear fragmentation ( n = 6). NSC = non-seeded control, which means untreated cells. Each dot corresponds to one biological replicate ( c , f , g , i , j ) or one clone differentiated once ( m , n ) and data are mean ± s.e.m. * P < 0.05, ** P < 0.01, by one-way ANOVA followed by Tukey’s multiple comparison test ( c , f , g , i , j , n ) or two-sided unpaired Student’s t test ( m ). Source data for c , e , f , g , h , i , j , k , m , n are provided as a Source Data file.

Article Snippet: The membranes were blocked in 5% dried skimmed milk and probed with the anti-α-synuclein antibody 4B12 (Biolegend, cat # 807801) and anti-P-S129 α-synuclein antibody 81A (Millipore, cat# MABN826).

Techniques: Expressing, Staining, Mass Spectrometry, Generated, Amplification, Western Blot, CRISPR, Knock-Out, Modification, Derivative Assay

Journal: Nature Communications

Article Title: Phenotypic manifestation of α-synuclein strains derived from Parkinson’s disease and multiple system atrophy in human dopaminergic neurons

doi: 10.1038/s41467-021-23682-z

Figure Lengend Snippet: a Approach used to knock out DJ-1 in the SNCA TRIP line. b Loss of DJ-1 protein in selected iPSC clones. Immunoblot is representative of n = 3 biological replicates . c SNCA TRIP/DJ-1KO clones differentiated into dopaminergic neurons. Images are representative of three independent differentiations (scale bar: 30 μm). d Quantification of the percentage of TH- and TUJ1-positive neurons in DJ-1 knockout clones was similar to the corresponding SNCA TRIP iPSC line of origin ( n = 3). e Immunoblotting at DIV60 showed increased α-synuclein levels at baseline in SNCA TRIP/DJ-1KO neurons compared to the SNCA TRIP neurons derived from the iPSC line of origin and quantified in panel f ( n = 9 for SNCA TRIP/DJ-1KO and n = 3 for SNCA TRIP ). g Reduced OCR, maximal respiration and ATP production in SNCA TRIP/DJ-1KO neurons at baseline DIV60 relative to the SNCA TRIP neurons and healthy control lines ( n = 9). h Accelerated neuronal loss was detected already at 1 week post seeding in SNCA TRIP/DJ-1KO clones irrespective of the strain used when compared to seeded neurons from the SNCA TRIP iPSC line of origin. Images are representative of three independent differentiations (scale bar: 30 μm). i Quantification of nuclear fragmentation in the SNCA TRIP/DJ-1KO clones seeded with de novo-generated fibrils, PD-amplified fibrils or MSA-amplified fibrils at 1 week post seeding ( n = 9 for SNCA TRIP/DJ-1KO and n = 3 for SNCA TRIP ). At this timepoint, neurons from the SNCA TRIP iPSC line of origin did not exhibit a similarly severe phenotype. j Increased LDH release was detected in seeded SNCA TRIP/DJ-1KO clones ( n = 9 for SNCA TRIP/DJ-1KO and n = 3 for SNCA TRIP ). k Despite the progressive cell loss, enhanced pSyn was detected in surviving SNCA TRIP/DJ-1KO neurons at 2 weeks post seeding as quantified by HTRF ( n = 9 for SNCA TRIP/DJ-1KO and n = 4 for SNCA TRIP ). NSC = non-seeded control, which means untreated neurons. Each dot corresponds to one clone differentiated once and data are mean ± s.e.m from at least n = 3 differentiations per clone. # P = 0.06, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test ( g , i ) or two-sided unpaired Student’s t test ( f , j , k ). Source data for b , d , e , f , g , I , j , k are provided as a Source Data file.

Article Snippet: The membranes were blocked in 5% dried skimmed milk and probed with the anti-α-synuclein antibody 4B12 (Biolegend, cat # 807801) and anti-P-S129 α-synuclein antibody 81A (Millipore, cat# MABN826).

Techniques: Knock-Out, Clone Assay, Western Blot, Derivative Assay, Generated, Amplification