recombinant α-synuclein monomer Search Results


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  • 99
    Millipore α synuclein
    Association of exogenous fibrillated <t>α-synuclein</t> with SN membranous structures including synaptic terminals Immuno-gold TEM detected fibrillated α-Syn ( A ) in pre-incubated SNs (Brain-Pre-treated) as gold particles in multiple synaptic terminals (small arrows) as well as intact extrasynaptic fibrils (large arrow). Labeled monomeric forms ( B ) were also found in synaptic terminals when pre-incubated with SNs. C . Gold-labeling was detected in synaptic terminals in mature SNs exposed to exogenous fibrillated α-Syn (small arrows). The exogenous monomeric form was found mainly outside synaptic terminals (large arrow)( D ) Endogenous labeling in WT SNs ( E ) and KO SNs ( F ) served as positive and negative controls respectively (panels A–D, F , scale bar = 0.5 µm; panel E , scale bar = 0.25µm).
    α Synuclein, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α synuclein/product/Millipore
    Average 99 stars, based on 539 article reviews
    Price from $9.99 to $1999.99
    α synuclein - by Bioz Stars, 2020-03
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    95
    rPeptide recombinant α synuclein monomer
    Cross-binding competition ELISA assays. a , b , and c : The two site inhibition curves show distinct high and low binding components in plasma from Parkinson’s Disease ( PD-blue, squares ), Multiple System Atrophy ( MSA-green, triangles ) patients and normal control ( NC-black, circles ) subjects. Ten plasma samples from each group were pooled and incubated in 1:400 dilution with increasing concentration of α-, β-, and <t>γ-synuclein</t> monomers in combinations as indicated, with subsequent measurement of free NAbs by ELISA on plates coated with 10 μg/ml of <t>α-synuclein.</t> d - i : Percentage of inhibition (PI) of individual plasma samples with free d , e : α−/β-synuclein monomers, F , G : α−/γ-synuclein monomers; h , i : β−/γ-synuclein monomer on plates coated with 10 μg/mL of α-synuclein monomer. Plasma samples from Parkinson’s Disease (PD), Multiple System Atrophy (MSA) patients and normal controls (NC) were tested at 1:400 dilution in the presence of ( d , f , h ) 100 nM or ( e , g , i ) 10 nM α-, β-, and γ-synuclein monomers in combinations. Horizontal bars represent the mean values +/− SEM. Significance was tested using Man–Whitney’s U test ( P
    Recombinant α Synuclein Monomer, supplied by rPeptide, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant α synuclein monomer/product/rPeptide
    Average 95 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    recombinant α synuclein monomer - by Bioz Stars, 2020-03
    95/100 stars
      Buy from Supplier

    83
    Millipore recombinant α synuclein monomers
    The E46K mutation in <t>α-synuclein</t> ablates replication of MSA prions. α-Synuclein prions were isolated from three MSA patient samples and one control sample by phosphotungstic acid precipitation and were incubated with HEK cells expressing mutated and truncated α-synuclein–YFP fusion proteins. ( A ) MSA prions were unable to replicate in cells expressing the E46K mutation. Coexpression with the A53T mutation marginally improved infection. However, coexpression of the A30P and A53T mutations resulted in robust replication of MSA prions. ( B ) Truncation of α-syn*A53T at residue 95, but not at residue 97, hindered MSA prion infection in the HEK cells. Data are shown as mean ± SD. * P
    Recombinant α Synuclein Monomers, supplied by Millipore, used in various techniques. Bioz Stars score: 83/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant α synuclein monomers/product/Millipore
    Average 83 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    recombinant α synuclein monomers - by Bioz Stars, 2020-03
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    Image Search Results


    Association of exogenous fibrillated α-synuclein with SN membranous structures including synaptic terminals Immuno-gold TEM detected fibrillated α-Syn ( A ) in pre-incubated SNs (Brain-Pre-treated) as gold particles in multiple synaptic terminals (small arrows) as well as intact extrasynaptic fibrils (large arrow). Labeled monomeric forms ( B ) were also found in synaptic terminals when pre-incubated with SNs. C . Gold-labeling was detected in synaptic terminals in mature SNs exposed to exogenous fibrillated α-Syn (small arrows). The exogenous monomeric form was found mainly outside synaptic terminals (large arrow)( D ) Endogenous labeling in WT SNs ( E ) and KO SNs ( F ) served as positive and negative controls respectively (panels A–D, F , scale bar = 0.5 µm; panel E , scale bar = 0.25µm).

    Journal: Journal of neuroscience research

    Article Title: Stimulation of Synaptoneurosome Glutamate Release by Monomeric and Fibrillated α-Synuclein

    doi: 10.1002/jnr.24024

    Figure Lengend Snippet: Association of exogenous fibrillated α-synuclein with SN membranous structures including synaptic terminals Immuno-gold TEM detected fibrillated α-Syn ( A ) in pre-incubated SNs (Brain-Pre-treated) as gold particles in multiple synaptic terminals (small arrows) as well as intact extrasynaptic fibrils (large arrow). Labeled monomeric forms ( B ) were also found in synaptic terminals when pre-incubated with SNs. C . Gold-labeling was detected in synaptic terminals in mature SNs exposed to exogenous fibrillated α-Syn (small arrows). The exogenous monomeric form was found mainly outside synaptic terminals (large arrow)( D ) Endogenous labeling in WT SNs ( E ) and KO SNs ( F ) served as positive and negative controls respectively (panels A–D, F , scale bar = 0.5 µm; panel E , scale bar = 0.25µm).

    Article Snippet: Both a primary rabbit polyclonal antibody raised against human α-synuclein (aa 111–131immunogen, 1:10,000 dilution, AB5308, EMD/Millipore, Temecula, CA)(RRID:AB_91785) and a mouse monoclonal antibody raised against rat α-synuclein (aa 15–123 immunogen, 1:2000 dilution, Clone 42, BD Transduction Laboratories™, San Jose, CA)(RRID: AB_398107) were used.

    Techniques: Transmission Electron Microscopy, Incubation, Labeling

    Trypsin-resistance of SN-internalized α-synuclein Full-length, native monomeric α-synuclein protein (Monomer lanes) were either added exogenously after the final SN fraction (SN Treated, top panels) or added to the initial homogenization prior to SN fractionation (Brain-Pre-treated, middle panels). The use of SNs prepared from KO mice (control lanes, Ctrl) eliminated interference from endogenous mouse synuclein. SNs were either untreated (left panels) or treated with trypsin (right panels) and resolved by SDS-PAGE and Western immunoblotting (mouse monoclonal anti-α-synuclein, 1:2000). Full-length monomers were resolved as single 15–16 kDa immunoreactive bands in untreated SNs. There were relative degrees of α-synuclein (αSyn) protease digestion in trypsin-treated samples for each SN condition (SN Treated vs Brain Pre-Treated). Solid arrows denote protein bands due to trypsin digestion; dashed arrow denotes partial digestion. The α-synuclein protein alone served as a positive control for maximal trypsin digestion (bottom panel). Similar qualitative protein banding-patterns of digestion were observed in a second set of experiments. Molecular weight standards (15 kDa, 20 kDa) are denoted.

    Journal: Journal of neuroscience research

    Article Title: Stimulation of Synaptoneurosome Glutamate Release by Monomeric and Fibrillated α-Synuclein

    doi: 10.1002/jnr.24024

    Figure Lengend Snippet: Trypsin-resistance of SN-internalized α-synuclein Full-length, native monomeric α-synuclein protein (Monomer lanes) were either added exogenously after the final SN fraction (SN Treated, top panels) or added to the initial homogenization prior to SN fractionation (Brain-Pre-treated, middle panels). The use of SNs prepared from KO mice (control lanes, Ctrl) eliminated interference from endogenous mouse synuclein. SNs were either untreated (left panels) or treated with trypsin (right panels) and resolved by SDS-PAGE and Western immunoblotting (mouse monoclonal anti-α-synuclein, 1:2000). Full-length monomers were resolved as single 15–16 kDa immunoreactive bands in untreated SNs. There were relative degrees of α-synuclein (αSyn) protease digestion in trypsin-treated samples for each SN condition (SN Treated vs Brain Pre-Treated). Solid arrows denote protein bands due to trypsin digestion; dashed arrow denotes partial digestion. The α-synuclein protein alone served as a positive control for maximal trypsin digestion (bottom panel). Similar qualitative protein banding-patterns of digestion were observed in a second set of experiments. Molecular weight standards (15 kDa, 20 kDa) are denoted.

    Article Snippet: Both a primary rabbit polyclonal antibody raised against human α-synuclein (aa 111–131immunogen, 1:10,000 dilution, AB5308, EMD/Millipore, Temecula, CA)(RRID:AB_91785) and a mouse monoclonal antibody raised against rat α-synuclein (aa 15–123 immunogen, 1:2000 dilution, Clone 42, BD Transduction Laboratories™, San Jose, CA)(RRID: AB_398107) were used.

    Techniques: Homogenization, Fractionation, Mouse Assay, SDS Page, Western Blot, Positive Control, Molecular Weight

    Fibrillation of native, full-length α-Syn A . Fibrillation of human native, full-length α-Syn was monitored over a 72 hour time course by TFT fluorescence relative to non-aggregated control proteins (histone, BSA, IgG). B . Fibrillation of full-length α-Syn was performed with either NaCl (100 mM) or GdnCl (100 mM) and monitored with TFT, DCVJ, or ANS as fluorescent probes (top right, bottom panels). Differential fibrillation patterns raised the possibility of preferential binding of NaCl-derived fibrils to TFT, while GdnCl-derived fibrils showed preferential binding to DCVJ and ANS possibly due to oligomer-enrichment. C . Native-PAGE/immunoblotting: Native, full-length monomers and large aggregates (likely fibrils in wells) and possible oligomers (just below wells) of α-Syn obtained using GdnCl were detected by Native-PAGE using a rabbit polyclonal anti α-synuclein antibody (1:10,000). D . TEM micrographs show monomers alone, NaCl-derived fibrils ( E ) or GdnCl-derived fibrils ( F ) in the absence of fluorescent probes. Additional TEM micrographs show fibrillated α-Syn using TFT, DCVJ, and ANS as fluorescent probes (G-I). Fibrils were represented by strands while structures reminiscent of oligomers were represented as circular globules (scale bars = 0.5 µm).

    Journal: Journal of neuroscience research

    Article Title: Stimulation of Synaptoneurosome Glutamate Release by Monomeric and Fibrillated α-Synuclein

    doi: 10.1002/jnr.24024

    Figure Lengend Snippet: Fibrillation of native, full-length α-Syn A . Fibrillation of human native, full-length α-Syn was monitored over a 72 hour time course by TFT fluorescence relative to non-aggregated control proteins (histone, BSA, IgG). B . Fibrillation of full-length α-Syn was performed with either NaCl (100 mM) or GdnCl (100 mM) and monitored with TFT, DCVJ, or ANS as fluorescent probes (top right, bottom panels). Differential fibrillation patterns raised the possibility of preferential binding of NaCl-derived fibrils to TFT, while GdnCl-derived fibrils showed preferential binding to DCVJ and ANS possibly due to oligomer-enrichment. C . Native-PAGE/immunoblotting: Native, full-length monomers and large aggregates (likely fibrils in wells) and possible oligomers (just below wells) of α-Syn obtained using GdnCl were detected by Native-PAGE using a rabbit polyclonal anti α-synuclein antibody (1:10,000). D . TEM micrographs show monomers alone, NaCl-derived fibrils ( E ) or GdnCl-derived fibrils ( F ) in the absence of fluorescent probes. Additional TEM micrographs show fibrillated α-Syn using TFT, DCVJ, and ANS as fluorescent probes (G-I). Fibrils were represented by strands while structures reminiscent of oligomers were represented as circular globules (scale bars = 0.5 µm).

    Article Snippet: Both a primary rabbit polyclonal antibody raised against human α-synuclein (aa 111–131immunogen, 1:10,000 dilution, AB5308, EMD/Millipore, Temecula, CA)(RRID:AB_91785) and a mouse monoclonal antibody raised against rat α-synuclein (aa 15–123 immunogen, 1:2000 dilution, Clone 42, BD Transduction Laboratories™, San Jose, CA)(RRID: AB_398107) were used.

    Techniques: Fluorescence, Binding Assay, Derivative Assay, Clear Native PAGE, Transmission Electron Microscopy

    Synaptoneurosomes (SNs) fractionation and glutamate assay ) from forebrains of different genotypic mice (WT, ASOTg, KO). For reconstitution experiments, recombinant human α-synuclein (α-Syn) proteoforms (monomers, fibrillated) were either pre-incubated with WT brain tissue (Brain Pre-treated) prior to SN fractionation or post-incubated (SN treated) after the final SN preparation. Glutamate oxidase activity coupled to horseradish peroxidase (HRP) mediated Amplex Red fluorescence (Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit, ThermoFisher) was used to measure either basal (H 2 O ± EGTA) or stimulated (40 mM KCl/0.5 mM CaCl 2 ) glutamate release from isolated SNs.

    Journal: Journal of neuroscience research

    Article Title: Stimulation of Synaptoneurosome Glutamate Release by Monomeric and Fibrillated α-Synuclein

    doi: 10.1002/jnr.24024

    Figure Lengend Snippet: Synaptoneurosomes (SNs) fractionation and glutamate assay ) from forebrains of different genotypic mice (WT, ASOTg, KO). For reconstitution experiments, recombinant human α-synuclein (α-Syn) proteoforms (monomers, fibrillated) were either pre-incubated with WT brain tissue (Brain Pre-treated) prior to SN fractionation or post-incubated (SN treated) after the final SN preparation. Glutamate oxidase activity coupled to horseradish peroxidase (HRP) mediated Amplex Red fluorescence (Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit, ThermoFisher) was used to measure either basal (H 2 O ± EGTA) or stimulated (40 mM KCl/0.5 mM CaCl 2 ) glutamate release from isolated SNs.

    Article Snippet: Both a primary rabbit polyclonal antibody raised against human α-synuclein (aa 111–131immunogen, 1:10,000 dilution, AB5308, EMD/Millipore, Temecula, CA)(RRID:AB_91785) and a mouse monoclonal antibody raised against rat α-synuclein (aa 15–123 immunogen, 1:2000 dilution, Clone 42, BD Transduction Laboratories™, San Jose, CA)(RRID: AB_398107) were used.

    Techniques: Fractionation, Glutamate Assay, Mouse Assay, Recombinant, Incubation, Activity Assay, Fluorescence, Isolation

    Comparison of the patterns of α-synuclein distribution in the brains of the α-synuclein E57K and α-synuclein wild-type transgenic mice. Vibratome sections were immunostained with the rabbit polyclonal antibody (Millipore) against full-length total α-synuclein. ( A ) Low-magnification photomicrographs of regions of interest, including the neocortex (1), hippocampus (2), striatum (3) and (4) thalamus in the non-transgenic α-synuclein E57K and α-synuclein wild-type transgenic mice. ( B ) Staining against α-synuclein was present in the neuropil of the neocortex in Lines 9 and 16, and to a lesser extent in Line 54, whereas in the α-synuclein wild-type transgenic Line 61 mouse model, α-synuclein immunoreactivity was present in the neuropil and neuronal cell bodies (arrow). The inset box illustrates a detailed area at higher magnification. ( C ) Immunoreactive α-synuclein punctae were also present in the neuropil of the hippocampus and striatum in Lines 9 and 16, and to a lesser extent in Line 54, whereas in the α-synuclein wild-type transgenic Line 61 mice, α-synuclein immunostaining was also present in neuronal cell bodies (N) and dystrophic neurites (arrows). ( D ) α-Synuclein was present in the neuropil of the thalamus in Lines 9 and 16, and to a lesser extent in Line 54, and occasionally in the cell bodies in the substantia nigra pars compacta (insets) whereas in the α-synuclein wild-type transgenic Line 61 tissue, α-synuclein immunostaining was present in the neuropil and dystrophic neurites. In the substantia nigra pars compacta, α-synuclein accumulated in the neuronal cell bodies (arrows). For analysis, six non-transgenic, n = 6 α-synuclein E57K transgenic mice from each line and six α-synuclein wild-type transgenic Line 61 mice (8–10 months old) were used. Scale bars: A = 150 µm; B = 25 µm; C = 50; D = 25 µm.

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Comparison of the patterns of α-synuclein distribution in the brains of the α-synuclein E57K and α-synuclein wild-type transgenic mice. Vibratome sections were immunostained with the rabbit polyclonal antibody (Millipore) against full-length total α-synuclein. ( A ) Low-magnification photomicrographs of regions of interest, including the neocortex (1), hippocampus (2), striatum (3) and (4) thalamus in the non-transgenic α-synuclein E57K and α-synuclein wild-type transgenic mice. ( B ) Staining against α-synuclein was present in the neuropil of the neocortex in Lines 9 and 16, and to a lesser extent in Line 54, whereas in the α-synuclein wild-type transgenic Line 61 mouse model, α-synuclein immunoreactivity was present in the neuropil and neuronal cell bodies (arrow). The inset box illustrates a detailed area at higher magnification. ( C ) Immunoreactive α-synuclein punctae were also present in the neuropil of the hippocampus and striatum in Lines 9 and 16, and to a lesser extent in Line 54, whereas in the α-synuclein wild-type transgenic Line 61 mice, α-synuclein immunostaining was also present in neuronal cell bodies (N) and dystrophic neurites (arrows). ( D ) α-Synuclein was present in the neuropil of the thalamus in Lines 9 and 16, and to a lesser extent in Line 54, and occasionally in the cell bodies in the substantia nigra pars compacta (insets) whereas in the α-synuclein wild-type transgenic Line 61 tissue, α-synuclein immunostaining was present in the neuropil and dystrophic neurites. In the substantia nigra pars compacta, α-synuclein accumulated in the neuronal cell bodies (arrows). For analysis, six non-transgenic, n = 6 α-synuclein E57K transgenic mice from each line and six α-synuclein wild-type transgenic Line 61 mice (8–10 months old) were used. Scale bars: A = 150 µm; B = 25 µm; C = 50; D = 25 µm.

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Staining, Immunostaining

    Ultrastructural and immunogold analyses of the synaptic terminals in the α-synuclein E57K and α-synuclein wild-type transgenic mice. Vibratome sections were post-fixed with glutaraldehyde and embedded in Epon® araldite, and ultra-thin sections from the neocortex were prepared for transmitted electron microscopy (TEM) and immunogold analysis. ( A ) Representative electron micrographs from the neuropil of non-transgenic mice displaying normal characteristics for presynaptic terminals (PST), post-synaptic densities, and dendrites (d). In the neuropil of the higher expressing α-synuclein E57K transgenic Lines 9 and 16, and in the α-synuclein wild-type Line 61, the synapses showed depletion of vesicles in the nerve terminals. Electrodense bodies were seen in Line 16 (arrow). ( B ) Image analysis of serial micrographs to estimate the numbers of synaptic vesicles per terminal. Compared with non-transgenic controls, the higher-expressing α-synuclein E57K transgenic mouse Lines 9 and 16, and α-synuclein wild-type Line 61, showed a significant reduction in the number of vesicles. No alterations were observed in mice from Line 54 (lower expressing). ( C ) Representative electron micrographs from the neuropil of non-transgenic mice immunostained with the LB509 antibody against pathological α-synuclein. Only a few scattered gold particles were detected in the neuropil. In the neuropil of the α-synuclein E57K transgenic and α-synuclein wild-type lines, the synapses showed abundant gold particles clustering at the nerve terminals and mitochondria. ( D ) Image analysis of serial micrographs to estimate the numbers of gold particles (LB509 reactive) per terminal. Compared with non-transgenic controls the higher-expressing α-synuclein E57K transgenic mouse Lines 9 and 16 showed a significant increase, as did α-synuclein wild-type Line 61. To a lesser extent, mice from Line 54 (lower expressing) also displayed accumulation of gold particles in synapses. n = 4 mice per group, age 8–10 months. * P

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Ultrastructural and immunogold analyses of the synaptic terminals in the α-synuclein E57K and α-synuclein wild-type transgenic mice. Vibratome sections were post-fixed with glutaraldehyde and embedded in Epon® araldite, and ultra-thin sections from the neocortex were prepared for transmitted electron microscopy (TEM) and immunogold analysis. ( A ) Representative electron micrographs from the neuropil of non-transgenic mice displaying normal characteristics for presynaptic terminals (PST), post-synaptic densities, and dendrites (d). In the neuropil of the higher expressing α-synuclein E57K transgenic Lines 9 and 16, and in the α-synuclein wild-type Line 61, the synapses showed depletion of vesicles in the nerve terminals. Electrodense bodies were seen in Line 16 (arrow). ( B ) Image analysis of serial micrographs to estimate the numbers of synaptic vesicles per terminal. Compared with non-transgenic controls, the higher-expressing α-synuclein E57K transgenic mouse Lines 9 and 16, and α-synuclein wild-type Line 61, showed a significant reduction in the number of vesicles. No alterations were observed in mice from Line 54 (lower expressing). ( C ) Representative electron micrographs from the neuropil of non-transgenic mice immunostained with the LB509 antibody against pathological α-synuclein. Only a few scattered gold particles were detected in the neuropil. In the neuropil of the α-synuclein E57K transgenic and α-synuclein wild-type lines, the synapses showed abundant gold particles clustering at the nerve terminals and mitochondria. ( D ) Image analysis of serial micrographs to estimate the numbers of gold particles (LB509 reactive) per terminal. Compared with non-transgenic controls the higher-expressing α-synuclein E57K transgenic mouse Lines 9 and 16 showed a significant increase, as did α-synuclein wild-type Line 61. To a lesser extent, mice from Line 54 (lower expressing) also displayed accumulation of gold particles in synapses. n = 4 mice per group, age 8–10 months. * P

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Electron Microscopy, Transmission Electron Microscopy, Expressing

    Generation of transgenic mice expressing the synthetic α-synuclein mutation E57K under the mThy-1 promoter. ( A ) Diagram of the mThy-1 construct. ( B ) Levels of α-synuclein messenger RNA expressed as a ratio to the housekeeping gene Gadph in non-transgenic and α-synuclein E57K Lines 9, 16, and 54, showing that Line 54 had 0.2 of the expression compared to α-synuclein E57K Line 9 and 16 and α-synuclein wild-type Line 61 transgenic mice. ( C ) Representative western blot (SDS) and ( D ) analysis of the levels of α-synuclein in the cytosolic fractions showing that Lines 9 and 16 were higher expressers of α-synuclein monomers and dimers, which was to a similar extent as Line 61. (E) Representative western blot (SDS) and ( F ) analysis of levels of monomer, dimer, and oligomer α-synuclein immunoreactivity in the membrane-bound fractions. Across all lines monomeric α-synuclein (14 kDa) was present to a greater extent in the cytosolic fraction compared to the membrane fraction. For analysis, six non-transgenic and six mThy-1 α-synuclein E57K transgenic mice (3–4 months old) from each line were used. * P

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Generation of transgenic mice expressing the synthetic α-synuclein mutation E57K under the mThy-1 promoter. ( A ) Diagram of the mThy-1 construct. ( B ) Levels of α-synuclein messenger RNA expressed as a ratio to the housekeeping gene Gadph in non-transgenic and α-synuclein E57K Lines 9, 16, and 54, showing that Line 54 had 0.2 of the expression compared to α-synuclein E57K Line 9 and 16 and α-synuclein wild-type Line 61 transgenic mice. ( C ) Representative western blot (SDS) and ( D ) analysis of the levels of α-synuclein in the cytosolic fractions showing that Lines 9 and 16 were higher expressers of α-synuclein monomers and dimers, which was to a similar extent as Line 61. (E) Representative western blot (SDS) and ( F ) analysis of levels of monomer, dimer, and oligomer α-synuclein immunoreactivity in the membrane-bound fractions. Across all lines monomeric α-synuclein (14 kDa) was present to a greater extent in the cytosolic fraction compared to the membrane fraction. For analysis, six non-transgenic and six mThy-1 α-synuclein E57K transgenic mice (3–4 months old) from each line were used. * P

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Expressing, Mutagenesis, Construct, Western Blot

    Accumulation of proteinase K-resistant α-synuclein in the α-synuclein E57K transgenic mice. ( A ) Representative photomicrographs (frontal cortex) of brain sections from non-transgenic and α-synuclein transgenic mice immunostained with the α-synuclein polyclonal antibody (Millipore) without proteinase K (PK−, top ) and with proteinase K (PK+, bottom ) treatment. In the proteinase K-treated tissue from non-transgenic and α-synuclein transgenic mice, the antibody stained the neuropil as well as the neurons (n) in the Line 61 mice. The proteinase K+ treatment eliminated most of the neuropil immunostaining in the non-transgenic mice, while at the same time revealing α-synuclein in axons (arrowheads), as well as in dystrophic neurites in the neuropil of the α-synuclein E57K transgenic Lines 9 and 16 and the α-synuclein wild-type transgenic Line 61 mice. ( B ) Patterns of LB509 (pathological α-synuclein, top ) immunostaining in the neocortex of the non-transgenic and α-synuclein transgenic mice. The non-transgenic mice did not immunoreact with the LB509 antibody, whereas in the α-synuclein E57K transgenic mice, α-synuclein immunostaining was present in the neuropil. The α-synuclein wild-type transgenic Line 61 mice displayed α-synuclein accumulation in the neuron cell body (n) and neuropil. Patterns of p129S α-synuclein immunostaining in the neocortex of the non-transgenic and α-synuclein transgenic ( bottom ) indicated that the non-transgenic mice showed no labelling with the pSer129 antibody whereas the α-synuclein E57K transgenic mice showed α-synuclein immunostaining in the neuropil (arrowheads). The α-synuclein wild-type transgenic Line 61 mice presented α-synuclein accumulation in the neuron cell body (n) and neuropil. Scale bar = 10 µm. Four non-transgenic and four α-synuclein E57K transgenic mice (8–10 months old) from each line were used. Tg = transgenic; wt = wild-type.

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Accumulation of proteinase K-resistant α-synuclein in the α-synuclein E57K transgenic mice. ( A ) Representative photomicrographs (frontal cortex) of brain sections from non-transgenic and α-synuclein transgenic mice immunostained with the α-synuclein polyclonal antibody (Millipore) without proteinase K (PK−, top ) and with proteinase K (PK+, bottom ) treatment. In the proteinase K-treated tissue from non-transgenic and α-synuclein transgenic mice, the antibody stained the neuropil as well as the neurons (n) in the Line 61 mice. The proteinase K+ treatment eliminated most of the neuropil immunostaining in the non-transgenic mice, while at the same time revealing α-synuclein in axons (arrowheads), as well as in dystrophic neurites in the neuropil of the α-synuclein E57K transgenic Lines 9 and 16 and the α-synuclein wild-type transgenic Line 61 mice. ( B ) Patterns of LB509 (pathological α-synuclein, top ) immunostaining in the neocortex of the non-transgenic and α-synuclein transgenic mice. The non-transgenic mice did not immunoreact with the LB509 antibody, whereas in the α-synuclein E57K transgenic mice, α-synuclein immunostaining was present in the neuropil. The α-synuclein wild-type transgenic Line 61 mice displayed α-synuclein accumulation in the neuron cell body (n) and neuropil. Patterns of p129S α-synuclein immunostaining in the neocortex of the non-transgenic and α-synuclein transgenic ( bottom ) indicated that the non-transgenic mice showed no labelling with the pSer129 antibody whereas the α-synuclein E57K transgenic mice showed α-synuclein immunostaining in the neuropil (arrowheads). The α-synuclein wild-type transgenic Line 61 mice presented α-synuclein accumulation in the neuron cell body (n) and neuropil. Scale bar = 10 µm. Four non-transgenic and four α-synuclein E57K transgenic mice (8–10 months old) from each line were used. Tg = transgenic; wt = wild-type.

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Staining, Immunostaining

    Separation of oligomeric and monomeric species in extracts of wild-type and E57K transgenic mice using size exclusion chromatography-profiles ( top ) before western blot ( bottom ). ( A ) α-Synuclein extracted from α-synuclein wild-type mice with TBS+ buffer was found as monomers and small oligomers (lanes 4 and 5). ( B ) α-Synuclein E57K-transgenic mice brain extracts contained monomeric α-synuclein (lane 5 bottom ) and oligomers with a molecular weight > 200 kDa, which elute near the void volume and are retained in the stacking gel of standard SDS-PAGE (lanes 1–2). ( C ) Eluted fractions from urea-soluble extracts of α-synuclein wild-type transgenic mice reveal a monomeric α-synuclein band (lane 4) in addition to an intense absorbance peak near the void volume. ( D ) Eluted fractions from urea-soluble extracts of α-synuclein E57K transgenic mice also had an intense absorbance peak near the void volume although the oligomeric species were bigger in size than those extracted from wild-type transgenic mice (compare lanes 1–2 of C with lanes 1–4 of D ). There was no evidence of a band corresponding to monomeric α-synuclein in the α-synuclein E57K transgenic urea-soluble homogenates. EV = elution volume; HMW = high molecular weight; tg = transgenic; WB = western blot; wt = wild-type.

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Separation of oligomeric and monomeric species in extracts of wild-type and E57K transgenic mice using size exclusion chromatography-profiles ( top ) before western blot ( bottom ). ( A ) α-Synuclein extracted from α-synuclein wild-type mice with TBS+ buffer was found as monomers and small oligomers (lanes 4 and 5). ( B ) α-Synuclein E57K-transgenic mice brain extracts contained monomeric α-synuclein (lane 5 bottom ) and oligomers with a molecular weight > 200 kDa, which elute near the void volume and are retained in the stacking gel of standard SDS-PAGE (lanes 1–2). ( C ) Eluted fractions from urea-soluble extracts of α-synuclein wild-type transgenic mice reveal a monomeric α-synuclein band (lane 4) in addition to an intense absorbance peak near the void volume. ( D ) Eluted fractions from urea-soluble extracts of α-synuclein E57K transgenic mice also had an intense absorbance peak near the void volume although the oligomeric species were bigger in size than those extracted from wild-type transgenic mice (compare lanes 1–2 of C with lanes 1–4 of D ). There was no evidence of a band corresponding to monomeric α-synuclein in the α-synuclein E57K transgenic urea-soluble homogenates. EV = elution volume; HMW = high molecular weight; tg = transgenic; WB = western blot; wt = wild-type.

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Size-exclusion Chromatography, Western Blot, Molecular Weight, SDS Page

    Memory alterations in the α-synuclein E57K and α-synuclein wild-type transgenic mice. Mice were evaluated for context-dependent learning in an open field area first at 3–4 months of age and then at 8–10 months in a Kinder SmartFrame Cage Rack Station activity monitor system. ( A ) At 3–4 months of age, non-transgenic controls and α-synuclein E57K transgenic Line 54 (lower-expressing) mice had reduced levels of activity over time as they became more familiar with the environment in the cage. The α-synuclein E57K transgenic mice from Lines 9 and 16 and the α-synuclein wild-type Line 61 transgenic mice showed a similar level of activity over time. ( B ) Evaluation of the fourth day of assessment compared to non-transgenic mice suggested trends toward dishabituation in α-synuclein E57K Line 9 and α-synuclein wild-type Line 61 (one-way ANOVA with Dunnett’s multiple comparisons post hoc test). ( C ) Both the older 8–10 month non-transgenic and lower-expressing α-synuclein E57K Line 54 mice showed habituation and thus learning, whereas in the α-synuclein E57K Line 9 there was a significant difference in activity ( P

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Memory alterations in the α-synuclein E57K and α-synuclein wild-type transgenic mice. Mice were evaluated for context-dependent learning in an open field area first at 3–4 months of age and then at 8–10 months in a Kinder SmartFrame Cage Rack Station activity monitor system. ( A ) At 3–4 months of age, non-transgenic controls and α-synuclein E57K transgenic Line 54 (lower-expressing) mice had reduced levels of activity over time as they became more familiar with the environment in the cage. The α-synuclein E57K transgenic mice from Lines 9 and 16 and the α-synuclein wild-type Line 61 transgenic mice showed a similar level of activity over time. ( B ) Evaluation of the fourth day of assessment compared to non-transgenic mice suggested trends toward dishabituation in α-synuclein E57K Line 9 and α-synuclein wild-type Line 61 (one-way ANOVA with Dunnett’s multiple comparisons post hoc test). ( C ) Both the older 8–10 month non-transgenic and lower-expressing α-synuclein E57K Line 54 mice showed habituation and thus learning, whereas in the α-synuclein E57K Line 9 there was a significant difference in activity ( P

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Activity Assay, Expressing

    Immunoblot analysis of α-synuclein oligomers in the α-synuclein E57K and α-synuclein wild-type transgenic mice. ( A ) Western blot evaluation indicated that all transgenic lines were positive for human α-synuclein, which was absent in the non-transgenic mice; whereas all mouse lines were positive for total α-synuclein. ( B ) After urea extraction, α-synuclein E57K Lines 9 and 16 had bands ∼70 kDa (pentamers), which were not present in the lower-expressing α-synuclein E57K Line 54, α-synuclein wild-type or non-transgenic mice, but which were present ( C ) in frontal cortex from controls and cases with dementia with Lewy bodies. ( D ) Analysis of α-synuclein in native gel reveals a faint band at ∼55 kDa for Lines 9, 16, and 54; a dense smear between 70 and 200 kDa was most prominent in Lines 9 and 16. In the α-synuclein wild-type animal line, there was a prominent band at ∼55 kDa and less smearing between 70 and 200 kDa. ( E ) Representative image of the dot blot analysis with the A11 and FILA-1 antibodies using Triton™ X-100 membrane-extracted fraction indicated that α-synuclein oligomers ( F ) were present in all α-synuclein E57K transgenic lines, and Line 61, albeit to a lesser extent than in Line 16. ( G ) Only Line 61 had significant levels of fibrils above non-transgenic background. Seeded and fibrillized dose responses of recombinant α-synuclein protein were generated for qualitative concentration analysis. For analysis, four non-transgenic and six transgenic mice (3–4 months old) from each line were used. Tg = transgenic; wt = wild-type * = p -value

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Immunoblot analysis of α-synuclein oligomers in the α-synuclein E57K and α-synuclein wild-type transgenic mice. ( A ) Western blot evaluation indicated that all transgenic lines were positive for human α-synuclein, which was absent in the non-transgenic mice; whereas all mouse lines were positive for total α-synuclein. ( B ) After urea extraction, α-synuclein E57K Lines 9 and 16 had bands ∼70 kDa (pentamers), which were not present in the lower-expressing α-synuclein E57K Line 54, α-synuclein wild-type or non-transgenic mice, but which were present ( C ) in frontal cortex from controls and cases with dementia with Lewy bodies. ( D ) Analysis of α-synuclein in native gel reveals a faint band at ∼55 kDa for Lines 9, 16, and 54; a dense smear between 70 and 200 kDa was most prominent in Lines 9 and 16. In the α-synuclein wild-type animal line, there was a prominent band at ∼55 kDa and less smearing between 70 and 200 kDa. ( E ) Representative image of the dot blot analysis with the A11 and FILA-1 antibodies using Triton™ X-100 membrane-extracted fraction indicated that α-synuclein oligomers ( F ) were present in all α-synuclein E57K transgenic lines, and Line 61, albeit to a lesser extent than in Line 16. ( G ) Only Line 61 had significant levels of fibrils above non-transgenic background. Seeded and fibrillized dose responses of recombinant α-synuclein protein were generated for qualitative concentration analysis. For analysis, four non-transgenic and six transgenic mice (3–4 months old) from each line were used. Tg = transgenic; wt = wild-type * = p -value

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Western Blot, Expressing, Dot Blot, Recombinant, Generated, Concentration Assay

    Analysis of alterations in synaptic protein markers in the α-synuclein E57K and α-synuclein wild-type transgenic mice. ( A ) Brain tissues from the fronto-temporal cortex were homogenized and separated into particulate and cytosolic fractions. The particulate fraction was used for the immunoblot analysis. Representative immunoblot analysis of antibodies against synaptophysin, synapsin 1, SNAP25 and RIM-3 comparing non-transgenic mice to the different α-synuclein transgenic lines. ( B ) Analysis of integrated pixels of the immunoblot shows a decrease in the synaptic protein synaptophysin in α-synuclein E57K Line 9, and RIM-3. In α-synuclein E57K transgenic mice from synapsin 1, levels were also significantly decreased in Lines 9, 16 and 61, but not in Line 54. ( C ) Sections were triple-labelled with antibodies against synaptophysin (SY38, red channel), synapsin 1 (green channel), and DAPI (blue channel) and imaged with the laser scanning confocal microscope. The right column represents the zoomed merged image (inset). The yellow dots represent co-localization between the two synaptic markers. ( D ) Image analysis of confocal images to estimate the percentage of synaptophysin terminals containing synapsin 1 immunostaining. Compared with non-transgenic controls, the higher-expressing α-synuclein E57K transgenic mice from Lines 9 and 16 showed a significant reduction in co-localization, as did the α-synuclein wild-type transgenic mice. No alterations were observed in mice from Line 54 (lower expressing). n = 8 mice per group, age 8–10 months. * P

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Analysis of alterations in synaptic protein markers in the α-synuclein E57K and α-synuclein wild-type transgenic mice. ( A ) Brain tissues from the fronto-temporal cortex were homogenized and separated into particulate and cytosolic fractions. The particulate fraction was used for the immunoblot analysis. Representative immunoblot analysis of antibodies against synaptophysin, synapsin 1, SNAP25 and RIM-3 comparing non-transgenic mice to the different α-synuclein transgenic lines. ( B ) Analysis of integrated pixels of the immunoblot shows a decrease in the synaptic protein synaptophysin in α-synuclein E57K Line 9, and RIM-3. In α-synuclein E57K transgenic mice from synapsin 1, levels were also significantly decreased in Lines 9, 16 and 61, but not in Line 54. ( C ) Sections were triple-labelled with antibodies against synaptophysin (SY38, red channel), synapsin 1 (green channel), and DAPI (blue channel) and imaged with the laser scanning confocal microscope. The right column represents the zoomed merged image (inset). The yellow dots represent co-localization between the two synaptic markers. ( D ) Image analysis of confocal images to estimate the percentage of synaptophysin terminals containing synapsin 1 immunostaining. Compared with non-transgenic controls, the higher-expressing α-synuclein E57K transgenic mice from Lines 9 and 16 showed a significant reduction in co-localization, as did the α-synuclein wild-type transgenic mice. No alterations were observed in mice from Line 54 (lower expressing). n = 8 mice per group, age 8–10 months. * P

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Microscopy, Immunostaining, Expressing

    Immunocytochemical analysis of the synaptic co-localization between synaptophysin and α-synuclein in the α-synuclein E57K and α-synuclein wild-type transgenic mice. Sections were double-labelled with antibodies against synaptophysin (SY38, red channel) and human α-synuclein (SY211, green channel) and imaged with the laser scanning confocal microscope. ( A ) Comparison of the patterns of co-localization between synaptophysin and α-synuclein in layer II of the neocortex across all transgenic mouse lines. The transgenic lines displayed extensive co-localization between the two synaptic markers. The split image in the column to the left (red) is synaptophysin, in the centre is human α-synuclein and to the right is the two channels merged. The box represents the magnified merged image. ( B ) Comparison of the patterns of co-localization in the CA1 region of the hippocampus across the α-synuclein E57K and α-synuclein wild-type transgenic mouse lines. All transgenic lines displayed extensive co-localization between the two synaptic markers. Six non-transgenic α-synuclein E57K transgenic mice from each line and six α-synuclein wild-type transgenic Line 61 mice (8–10 months old) were used for analysis. Scale bars in the single channel images = 25 µm; 5 µm in the enlarged merged images. Tg = transgenic; wt = wild-type.

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Immunocytochemical analysis of the synaptic co-localization between synaptophysin and α-synuclein in the α-synuclein E57K and α-synuclein wild-type transgenic mice. Sections were double-labelled with antibodies against synaptophysin (SY38, red channel) and human α-synuclein (SY211, green channel) and imaged with the laser scanning confocal microscope. ( A ) Comparison of the patterns of co-localization between synaptophysin and α-synuclein in layer II of the neocortex across all transgenic mouse lines. The transgenic lines displayed extensive co-localization between the two synaptic markers. The split image in the column to the left (red) is synaptophysin, in the centre is human α-synuclein and to the right is the two channels merged. The box represents the magnified merged image. ( B ) Comparison of the patterns of co-localization in the CA1 region of the hippocampus across the α-synuclein E57K and α-synuclein wild-type transgenic mouse lines. All transgenic lines displayed extensive co-localization between the two synaptic markers. Six non-transgenic α-synuclein E57K transgenic mice from each line and six α-synuclein wild-type transgenic Line 61 mice (8–10 months old) were used for analysis. Scale bars in the single channel images = 25 µm; 5 µm in the enlarged merged images. Tg = transgenic; wt = wild-type.

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Microscopy

    Analysis of synapto-dendritic loss in the frontal cortex and hippocampus from α-synuclein E57K and α-synuclein wild-type transgenic mice. Sections were immunolabelled with antibodies against synaptophysin (SY38) and MAP2, and imaged with a laser scanning confocal microscope. ( A ) Representative photomicrographs of the presynaptic terminals (SY38) and ( B ) dendrites (MAP2) in the hippocampus. Arrows indicate areas of dendritic loss in Line 16 in the CA2-3 region. Analysis was expressed as per cent area of the neuropil. ( C ) Compared with non-transgenic controls, α-synuclein E57K transgenic mice from Lines 9 and 16, as well as α-synuclein wild-type Line 61, displayed a significant reduction in synapotophysin in the frontal cortex, whereas line 54 (lower-expressing) was comparable non-transgenic littermates. In the hippocampus (hippo), synaptophysin was reduced in α-synuclein E57K Line 16 and the α-synuclein wild-type Line 61. ( D ) MAP2 was reduced in the frontal cortex in α-synuclein E57K Lines 9 and 16, as well as α-synuclein wild-type Line 61, but not in α-synuclein E57K Line 54. In the hippocampus, MAP2 was reduced across all transgenic lines. n = 8 mice per group, age 8–10 months. * P

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Analysis of synapto-dendritic loss in the frontal cortex and hippocampus from α-synuclein E57K and α-synuclein wild-type transgenic mice. Sections were immunolabelled with antibodies against synaptophysin (SY38) and MAP2, and imaged with a laser scanning confocal microscope. ( A ) Representative photomicrographs of the presynaptic terminals (SY38) and ( B ) dendrites (MAP2) in the hippocampus. Arrows indicate areas of dendritic loss in Line 16 in the CA2-3 region. Analysis was expressed as per cent area of the neuropil. ( C ) Compared with non-transgenic controls, α-synuclein E57K transgenic mice from Lines 9 and 16, as well as α-synuclein wild-type Line 61, displayed a significant reduction in synapotophysin in the frontal cortex, whereas line 54 (lower-expressing) was comparable non-transgenic littermates. In the hippocampus (hippo), synaptophysin was reduced in α-synuclein E57K Line 16 and the α-synuclein wild-type Line 61. ( D ) MAP2 was reduced in the frontal cortex in α-synuclein E57K Lines 9 and 16, as well as α-synuclein wild-type Line 61, but not in α-synuclein E57K Line 54. In the hippocampus, MAP2 was reduced across all transgenic lines. n = 8 mice per group, age 8–10 months. * P

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Microscopy, Expressing

    Analysis of neurons and astroglial cells in the α-synuclein E57K and α-synuclein wild-type transgenic mice. Sections were immunolabelled with antibodies against NeuN and GFAP and imaged with a digital bright field microscope. ( A ) Representative photomicrographs of the neocortex and from non-transgenic α-synuclein E57K, and α-synuclein wild-type Line 61 transgenic mice displaying neuronal populations (NeuN). ( B) Stereological assessment of the number of neurons in the frontal cortex, pyramidal cell layer of the CA3 of the hippocampus, and striatum was performed. In the frontal cortex, only α-synuclein E57K Line 16 showed a significant decrease in the number of NeuN-immunoreactive cells compared to non-transgenic mice. In the hippocampus, all transgenic mouse lines showed a significant reduction in NeuN-positive cells, whereas the striatum was unaffected. ( C ) Astrogliosis (GFAP, inset represents higher magnification) was apparent by visual inspection in α-synuclein E57K Lines 9 and 16, as well as in α-synuclein wild-type Line 61, and to a lesser extent in α-synuclein E57K Line 54. Scale bars: A and C = 100 µm. * P

    Journal: Brain

    Article Title: Accumulation of oligomer-prone α-synuclein exacerbates synaptic and neuronal degeneration in vivo

    doi: 10.1093/brain/awu057

    Figure Lengend Snippet: Analysis of neurons and astroglial cells in the α-synuclein E57K and α-synuclein wild-type transgenic mice. Sections were immunolabelled with antibodies against NeuN and GFAP and imaged with a digital bright field microscope. ( A ) Representative photomicrographs of the neocortex and from non-transgenic α-synuclein E57K, and α-synuclein wild-type Line 61 transgenic mice displaying neuronal populations (NeuN). ( B) Stereological assessment of the number of neurons in the frontal cortex, pyramidal cell layer of the CA3 of the hippocampus, and striatum was performed. In the frontal cortex, only α-synuclein E57K Line 16 showed a significant decrease in the number of NeuN-immunoreactive cells compared to non-transgenic mice. In the hippocampus, all transgenic mouse lines showed a significant reduction in NeuN-positive cells, whereas the striatum was unaffected. ( C ) Astrogliosis (GFAP, inset represents higher magnification) was apparent by visual inspection in α-synuclein E57K Lines 9 and 16, as well as in α-synuclein wild-type Line 61, and to a lesser extent in α-synuclein E57K Line 54. Scale bars: A and C = 100 µm. * P

    Article Snippet: Sections were immunolabelled with antibodies against human α-synuclein (SYN211) and synaptophysin (SY38, Millipore).

    Techniques: Transgenic Assay, Mouse Assay, Microscopy

    Detection of α-syn knockdown effect by immunoblotting using conventional method and improved method. In SK-MEL28 cells, shRNA of control or α-syn was expressed by lentivirus infection. Total cell lysates were loaded onto a 15% polyacrylamide gel, followed by immunoblotting using anti-α-syn antibody LB509 and anti-actin antibody. PFA (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Journal: PLoS ONE

    Article Title: Improved Immunodetection of Endogenous ?-Synuclein

    doi: 10.1371/journal.pone.0023939

    Figure Lengend Snippet: Detection of α-syn knockdown effect by immunoblotting using conventional method and improved method. In SK-MEL28 cells, shRNA of control or α-syn was expressed by lentivirus infection. Total cell lysates were loaded onto a 15% polyacrylamide gel, followed by immunoblotting using anti-α-syn antibody LB509 and anti-actin antibody. PFA (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Article Snippet: Recombinant α-Syn Protein To investigate α-syn monomers and its aggregates, we purchased recombinant human α-syn protein (Cat. #S7820, Sigma-Aldrich) that was expressed in E. coli and purified.

    Techniques: shRNA, Infection

    Immunoblotting of endogenous α-syn in various human cell lines. Total cell lysates were prepared from 11 human cell lines: SK-MEL28 (malignant melanoma), SH-SY5Y (neuroblastoma), K562 (erythroleukemia), HEK293 (human embryonic kidney cell), HeLa (cervical adenocarcinoma), HT1080 (lung fibrosarcoma), HL60 (promyelocytic leukemia), BJAB (Burkitt's lymphoma), SW620 (colon adenocarcinoma), U937 (myeloid leukemia), and 786-0 (renal cell carcinoma). The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked with 5% skim milk and incubated with a primary antibody such as anti-α-syn antibody 4D6, anti-α-syn antibody LB509, or anti-actin antibody. The membrane was then incubated with a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Journal: PLoS ONE

    Article Title: Improved Immunodetection of Endogenous ?-Synuclein

    doi: 10.1371/journal.pone.0023939

    Figure Lengend Snippet: Immunoblotting of endogenous α-syn in various human cell lines. Total cell lysates were prepared from 11 human cell lines: SK-MEL28 (malignant melanoma), SH-SY5Y (neuroblastoma), K562 (erythroleukemia), HEK293 (human embryonic kidney cell), HeLa (cervical adenocarcinoma), HT1080 (lung fibrosarcoma), HL60 (promyelocytic leukemia), BJAB (Burkitt's lymphoma), SW620 (colon adenocarcinoma), U937 (myeloid leukemia), and 786-0 (renal cell carcinoma). The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked with 5% skim milk and incubated with a primary antibody such as anti-α-syn antibody 4D6, anti-α-syn antibody LB509, or anti-actin antibody. The membrane was then incubated with a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Article Snippet: Recombinant α-Syn Protein To investigate α-syn monomers and its aggregates, we purchased recombinant human α-syn protein (Cat. #S7820, Sigma-Aldrich) that was expressed in E. coli and purified.

    Techniques: Western Blot, Incubation

    Comparison of nitrocellulose and PVDF membranes in immunoblotting of α-syn. Immunoblotting was performed using a PVDF membrane (left panels) and a nitrocellulose membrane (right panels). Briefly, total cell lysates were prepared from SK-MEL28 (positive control), SH-SY5Y (positive control), and BJAB (negative control). The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF or nitrocellulose membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked and incubated with a primary antibody (anti-α-syn 4D6, LB509, or anti-actin) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Journal: PLoS ONE

    Article Title: Improved Immunodetection of Endogenous ?-Synuclein

    doi: 10.1371/journal.pone.0023939

    Figure Lengend Snippet: Comparison of nitrocellulose and PVDF membranes in immunoblotting of α-syn. Immunoblotting was performed using a PVDF membrane (left panels) and a nitrocellulose membrane (right panels). Briefly, total cell lysates were prepared from SK-MEL28 (positive control), SH-SY5Y (positive control), and BJAB (negative control). The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF or nitrocellulose membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked and incubated with a primary antibody (anti-α-syn 4D6, LB509, or anti-actin) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Article Snippet: Recombinant α-Syn Protein To investigate α-syn monomers and its aggregates, we purchased recombinant human α-syn protein (Cat. #S7820, Sigma-Aldrich) that was expressed in E. coli and purified.

    Techniques: Positive Control, Negative Control, Western Blot, Incubation

    Immunoblotting of endogenous α-syn phosphorylated at Ser-129 in various human cell lines. Total cell lysates were prepared from 10 human cell lines: SH-SY5Y (neuroblastoma), K562 (erythroleukemia), HEK293 (human embryonic kidney cell), HeLa (cervical adenocarcinoma), HL60 (promyelocytic leukemia), Daoy (medulloblastoma), SK-MEL28 (malignant melanoma), A375 (malignant melanoma), MeWo (malignant melanoma), and WM266-4 (malignant melanoma). The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked and incubated with a primary antibody (anti-α-syn antibody LB509, anti-phospho-α-syn antibody EP1536Y, or anti-actin antibody) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (–) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Journal: PLoS ONE

    Article Title: Improved Immunodetection of Endogenous ?-Synuclein

    doi: 10.1371/journal.pone.0023939

    Figure Lengend Snippet: Immunoblotting of endogenous α-syn phosphorylated at Ser-129 in various human cell lines. Total cell lysates were prepared from 10 human cell lines: SH-SY5Y (neuroblastoma), K562 (erythroleukemia), HEK293 (human embryonic kidney cell), HeLa (cervical adenocarcinoma), HL60 (promyelocytic leukemia), Daoy (medulloblastoma), SK-MEL28 (malignant melanoma), A375 (malignant melanoma), MeWo (malignant melanoma), and WM266-4 (malignant melanoma). The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked and incubated with a primary antibody (anti-α-syn antibody LB509, anti-phospho-α-syn antibody EP1536Y, or anti-actin antibody) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (–) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Article Snippet: Recombinant α-Syn Protein To investigate α-syn monomers and its aggregates, we purchased recombinant human α-syn protein (Cat. #S7820, Sigma-Aldrich) that was expressed in E. coli and purified.

    Techniques: Western Blot, Incubation

    Detachment of α-syn monomers from Western-transferred membrane. Total cell lysates were prepared from SK-MEL28 (positive control), SH-SY5Y (positive control), and BJAB (negative control) cells. The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then processed using three different protocols. In Protocol A , the conventional immunoblotting method (without PFA fixation) was used. In Protocol B , the membrane was fixed with 0.4% PFA for 30 min just after Western transfer. In Protocol C , the membrane was incubated for 3 h 45 min (3.75 h) in TBS-T buffer after Western transfer and then fixed with 0.4% PFA for 30 min. Afterwards, these membranes were blocked and incubated with a primary antibody (anti-α-syn 4D6, LB509, or anti-actin) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membranes were detected by ECL-Plus detection system. Molecular size markers are shown in kilodaltons (kDa).

    Journal: PLoS ONE

    Article Title: Improved Immunodetection of Endogenous ?-Synuclein

    doi: 10.1371/journal.pone.0023939

    Figure Lengend Snippet: Detachment of α-syn monomers from Western-transferred membrane. Total cell lysates were prepared from SK-MEL28 (positive control), SH-SY5Y (positive control), and BJAB (negative control) cells. The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then processed using three different protocols. In Protocol A , the conventional immunoblotting method (without PFA fixation) was used. In Protocol B , the membrane was fixed with 0.4% PFA for 30 min just after Western transfer. In Protocol C , the membrane was incubated for 3 h 45 min (3.75 h) in TBS-T buffer after Western transfer and then fixed with 0.4% PFA for 30 min. Afterwards, these membranes were blocked and incubated with a primary antibody (anti-α-syn 4D6, LB509, or anti-actin) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membranes were detected by ECL-Plus detection system. Molecular size markers are shown in kilodaltons (kDa).

    Article Snippet: Recombinant α-Syn Protein To investigate α-syn monomers and its aggregates, we purchased recombinant human α-syn protein (Cat. #S7820, Sigma-Aldrich) that was expressed in E. coli and purified.

    Techniques: Western Blot, Positive Control, Negative Control, Incubation

    Immunoblotting of endogenous α-syn in tumor cell lines of the head. Total cell lysates were prepared from human retinoblastoma cell line Y79 and 3 human brain tumor cell lines: SH-SY5Y (neuroblastoma), U251MG (glioblastoma), and Daoy (medulloblastoma). The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked and incubated with a primary antibody (anti-α-syn antibody 4D6, anti-α-syn antibody LB509, or anti-actin antibody) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Journal: PLoS ONE

    Article Title: Improved Immunodetection of Endogenous ?-Synuclein

    doi: 10.1371/journal.pone.0023939

    Figure Lengend Snippet: Immunoblotting of endogenous α-syn in tumor cell lines of the head. Total cell lysates were prepared from human retinoblastoma cell line Y79 and 3 human brain tumor cell lines: SH-SY5Y (neuroblastoma), U251MG (glioblastoma), and Daoy (medulloblastoma). The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked and incubated with a primary antibody (anti-α-syn antibody 4D6, anti-α-syn antibody LB509, or anti-actin antibody) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Article Snippet: Recombinant α-Syn Protein To investigate α-syn monomers and its aggregates, we purchased recombinant human α-syn protein (Cat. #S7820, Sigma-Aldrich) that was expressed in E. coli and purified.

    Techniques: Western Blot, Incubation

    Immunoblotting of endogenous α-syn in various melanoma cell lines. Total cell lysates were prepared from 4 human melanoma cell lines (SK-MEL28, A375, MeWo, and WM266-4) and 1 mouse melanoma cell line (B16). The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked and incubated with a primary antibody (anti-α-syn antibody 4D6, anti-human α-syn antibody LB509, or anti-actin antibody) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Journal: PLoS ONE

    Article Title: Improved Immunodetection of Endogenous ?-Synuclein

    doi: 10.1371/journal.pone.0023939

    Figure Lengend Snippet: Immunoblotting of endogenous α-syn in various melanoma cell lines. Total cell lysates were prepared from 4 human melanoma cell lines (SK-MEL28, A375, MeWo, and WM266-4) and 1 mouse melanoma cell line (B16). The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked and incubated with a primary antibody (anti-α-syn antibody 4D6, anti-human α-syn antibody LB509, or anti-actin antibody) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Article Snippet: Recombinant α-Syn Protein To investigate α-syn monomers and its aggregates, we purchased recombinant human α-syn protein (Cat. #S7820, Sigma-Aldrich) that was expressed in E. coli and purified.

    Techniques: Western Blot, Incubation

    Detection of endogenous α-syn. A , Conventional immunoblotting of various human cell lines. Total cell lysates were prepared from 6 human cell lines: SK-MEL28 (malignant melanoma), SH-SY5Y (neuroblastoma), HEK293 (human embryonic kidney cell), HeLa (cervical adenocarcinoma), HT1080 (lung fibrosarcoma), and SW620 (colon adenocarcinoma). The lysates (3 µl) containing ∼10 µg proteins were loaded per lane onto a 15% SDS-polyacrylamide gel. After SDS-PAGE and Western transfer onto a PVDF membrane, immunoblotting was performed using anti-α-syn antibody 4D6 ( upper panel ). To demonstrate near equal loading amount of total cell lysates, immunoblotting using anti-actin antibody was also performed ( lower panel ). Molecular size markers are shown in kilodaltons (kDa). B , α-Syn immunostaining of various human cell lines. The indicated cell lines were fixed with PFA, stained with DAPI, permeabilized with 0.1% TritonX-100, and immunostained with mouse monoclonal anti-α-syn antibody 4D6. After washing, cells were labeled with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody. The cells were then analyzed by fluorescence microscopy. The localization of endogenous α-syn is shown by the green fluorescence of Alexa Fluor 488. Nuclear counterstaining is shown by the blue fluorescence of DAPI. Scale bar indicates 10 µm.

    Journal: PLoS ONE

    Article Title: Improved Immunodetection of Endogenous ?-Synuclein

    doi: 10.1371/journal.pone.0023939

    Figure Lengend Snippet: Detection of endogenous α-syn. A , Conventional immunoblotting of various human cell lines. Total cell lysates were prepared from 6 human cell lines: SK-MEL28 (malignant melanoma), SH-SY5Y (neuroblastoma), HEK293 (human embryonic kidney cell), HeLa (cervical adenocarcinoma), HT1080 (lung fibrosarcoma), and SW620 (colon adenocarcinoma). The lysates (3 µl) containing ∼10 µg proteins were loaded per lane onto a 15% SDS-polyacrylamide gel. After SDS-PAGE and Western transfer onto a PVDF membrane, immunoblotting was performed using anti-α-syn antibody 4D6 ( upper panel ). To demonstrate near equal loading amount of total cell lysates, immunoblotting using anti-actin antibody was also performed ( lower panel ). Molecular size markers are shown in kilodaltons (kDa). B , α-Syn immunostaining of various human cell lines. The indicated cell lines were fixed with PFA, stained with DAPI, permeabilized with 0.1% TritonX-100, and immunostained with mouse monoclonal anti-α-syn antibody 4D6. After washing, cells were labeled with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody. The cells were then analyzed by fluorescence microscopy. The localization of endogenous α-syn is shown by the green fluorescence of Alexa Fluor 488. Nuclear counterstaining is shown by the blue fluorescence of DAPI. Scale bar indicates 10 µm.

    Article Snippet: Recombinant α-Syn Protein To investigate α-syn monomers and its aggregates, we purchased recombinant human α-syn protein (Cat. #S7820, Sigma-Aldrich) that was expressed in E. coli and purified.

    Techniques: SDS Page, Western Blot, Immunostaining, Staining, Labeling, Fluorescence, Microscopy

    Immunoblotting of endogenous α-syn in various mouse tissues. Tissue lysates were prepared from C57BL/6J mice. The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked and incubated with a primary antibody (anti-α-syn antibody 4D6 or anti-actin antibody) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Journal: PLoS ONE

    Article Title: Improved Immunodetection of Endogenous ?-Synuclein

    doi: 10.1371/journal.pone.0023939

    Figure Lengend Snippet: Immunoblotting of endogenous α-syn in various mouse tissues. Tissue lysates were prepared from C57BL/6J mice. The protein samples (∼10 µg) were loaded onto a 15% SDS-polyacrylamide gel and electrophoresed, followed by Western transfer onto a PVDF membrane. The membrane was then fixed with or without 0.4% PFA for 30 min. Afterwards, the membrane was blocked and incubated with a primary antibody (anti-α-syn antibody 4D6 or anti-actin antibody) and a secondary antibody conjugated with horseradish peroxidase. Protein bands on the membrane were detected by ECL-Plus detection system. Symbols (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Article Snippet: Recombinant α-Syn Protein To investigate α-syn monomers and its aggregates, we purchased recombinant human α-syn protein (Cat. #S7820, Sigma-Aldrich) that was expressed in E. coli and purified.

    Techniques: Mouse Assay, Western Blot, Incubation

    Detection of recombinant α-syn species by immunoblotting using conventional method and improved method. Indicated amounts of recombinant proteins were separated on a 12% SDS-polyacrylamide gel and transferred onto a PVDF membrane, followed by membrane fixation with or without 0.4% PFA. Afterwards, the membrane was blocked with skim milk and sequentially incubated with anti-α-syn antibody 4D6 and a secondary antibody. Finally, bands of recombinant α-syn species were detected by ECL-plus detection system. PFA (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Journal: PLoS ONE

    Article Title: Improved Immunodetection of Endogenous ?-Synuclein

    doi: 10.1371/journal.pone.0023939

    Figure Lengend Snippet: Detection of recombinant α-syn species by immunoblotting using conventional method and improved method. Indicated amounts of recombinant proteins were separated on a 12% SDS-polyacrylamide gel and transferred onto a PVDF membrane, followed by membrane fixation with or without 0.4% PFA. Afterwards, the membrane was blocked with skim milk and sequentially incubated with anti-α-syn antibody 4D6 and a secondary antibody. Finally, bands of recombinant α-syn species were detected by ECL-plus detection system. PFA (+) and (−) indicate presence and absence of membrane fixation with 0.4% PFA, respectively. Molecular size markers are shown in kilodaltons (kDa).

    Article Snippet: Recombinant α-Syn Protein To investigate α-syn monomers and its aggregates, we purchased recombinant human α-syn protein (Cat. #S7820, Sigma-Aldrich) that was expressed in E. coli and purified.

    Techniques: Recombinant, Incubation

    The effect of calcium and synaptic vesicles on alpha-synuclein aggregation. a Alpha-synuclein aggregation measured by ThT fluorescence using 100 µM monomeric alpha-synuclein under shaking conditions. The presence of 2.5 mM calcium increased the aggregation kinetics of alpha-synuclein compared to 1 mM EGTA, both in the presence or absence of synaptic vesicles (black and red). For the EGTA-containing groups, there was a trend towards faster aggregation of alpha-synuclein in the presence of synaptic vesicles (blue vs. gray). Three biological repeats, n = 18 for all conditions, where n represents single wells. values represent mean ± s.e.m. b TEM images of alpha-synuclein fibrils formed in the presence of 2.5 mM calcium or 1 mM EGTA either in the presence or absence of synaptic vesicles. Differences in the morphology of alpha-synuclein fibrils were observed for fibrils formed in the presence of 2.5 mM calcium in the absence and presence of synaptic vesicles. In the presence of synaptic vesicles, alpha-synuclein fibrils showed increased lateral bundling and shortening. Alpha-synuclein aggregation in the presence of 1 mM EGTA led to substantially less fibrils, however the fibrils formed, retained their morphological phenotype compared to the fibrils formed in the presence of 2.5 mM calcium. Alpha-synuclein fibrils formed in the presence of synaptic vesicles plus 1 mM EGTA showed an intermediate phenotype, with bundled, but more elongated fibril structures than found in the synaptic vesicles and 2.5 mM calcium group. Experiments were repeated twice

    Journal: Nature Communications

    Article Title: C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction

    doi: 10.1038/s41467-018-03111-4

    Figure Lengend Snippet: The effect of calcium and synaptic vesicles on alpha-synuclein aggregation. a Alpha-synuclein aggregation measured by ThT fluorescence using 100 µM monomeric alpha-synuclein under shaking conditions. The presence of 2.5 mM calcium increased the aggregation kinetics of alpha-synuclein compared to 1 mM EGTA, both in the presence or absence of synaptic vesicles (black and red). For the EGTA-containing groups, there was a trend towards faster aggregation of alpha-synuclein in the presence of synaptic vesicles (blue vs. gray). Three biological repeats, n = 18 for all conditions, where n represents single wells. values represent mean ± s.e.m. b TEM images of alpha-synuclein fibrils formed in the presence of 2.5 mM calcium or 1 mM EGTA either in the presence or absence of synaptic vesicles. Differences in the morphology of alpha-synuclein fibrils were observed for fibrils formed in the presence of 2.5 mM calcium in the absence and presence of synaptic vesicles. In the presence of synaptic vesicles, alpha-synuclein fibrils showed increased lateral bundling and shortening. Alpha-synuclein aggregation in the presence of 1 mM EGTA led to substantially less fibrils, however the fibrils formed, retained their morphological phenotype compared to the fibrils formed in the presence of 2.5 mM calcium. Alpha-synuclein fibrils formed in the presence of synaptic vesicles plus 1 mM EGTA showed an intermediate phenotype, with bundled, but more elongated fibril structures than found in the synaptic vesicles and 2.5 mM calcium group. Experiments were repeated twice

    Article Snippet: For combined confocal/STED imaging, 50 µM human WT alpha-synuclein was complemented with 10% of alpha-synuclein N122C mutant labeled with ATTO-647N dye (05316, Sigma-Aldrich).

    Techniques: Fluorescence, Transmission Electron Microscopy

    Calcium binding to the C terminus of alpha-synuclein and lipid binding. a 1 H- 15 N HSQC NMR spectrum of alpha-synuclein in the absence (red) and in the presence of calcium (green, 1.6 mM calcium). Major chemical shift perturbations in the presence of calcium are located at the C terminus of alpha-synuclein (red arrows with assigned amino acid residues), whereas peak broadening (blue arrows with assigned amino acid residues) can be seen within the NAC-region. b Fitting of alpha-synuclein calcium binding ( K D ) from 1 H- 15 N HSQC NMR spectra at increasing calcium concentrations, where L indicates the number of Ca 2+ ions interacting with one alpha-synuclein molecule. c Calcium-bound alpha-synuclein species directly observed by mass spectrometry. Electrospray ionization mass spectra were acquired under identical instrument conditions for samples incubated with or without calcium. Multiple alpha-synuclein species were observed upon charge deconvolution of the ion envelope for the 9 + –19 + charge states, inclusive. The masses correspond to alpha-synuclein: calcium complexes up to a stoichiometry of 1:6. d Lipid pull-down experiment using lipids from Folch brain extracts, recombinant alpha-synuclein and various ions. Western blot of the amount of protein pulled down shows that more alpha-synuclein was pulled down by the lipids in the presence of calcium. Neither potassium, sodium, nor magnesium increased alpha-synuclein lipid binding to the same extent. ** p = 0.0011, 0.0022, and 0.0090 for comparison of 1 mM CaCl 2 with alpha-syn control, 50 mM KCl, and 150 mM NaCl, respectively. Calculated using one-way ANOVA with Tukey’s post-hoc correction, graphs indicate mean ± s.e.m. N = 3 for all groups, corresponding to three biological repeats, d.f. 12

    Journal: Nature Communications

    Article Title: C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction

    doi: 10.1038/s41467-018-03111-4

    Figure Lengend Snippet: Calcium binding to the C terminus of alpha-synuclein and lipid binding. a 1 H- 15 N HSQC NMR spectrum of alpha-synuclein in the absence (red) and in the presence of calcium (green, 1.6 mM calcium). Major chemical shift perturbations in the presence of calcium are located at the C terminus of alpha-synuclein (red arrows with assigned amino acid residues), whereas peak broadening (blue arrows with assigned amino acid residues) can be seen within the NAC-region. b Fitting of alpha-synuclein calcium binding ( K D ) from 1 H- 15 N HSQC NMR spectra at increasing calcium concentrations, where L indicates the number of Ca 2+ ions interacting with one alpha-synuclein molecule. c Calcium-bound alpha-synuclein species directly observed by mass spectrometry. Electrospray ionization mass spectra were acquired under identical instrument conditions for samples incubated with or without calcium. Multiple alpha-synuclein species were observed upon charge deconvolution of the ion envelope for the 9 + –19 + charge states, inclusive. The masses correspond to alpha-synuclein: calcium complexes up to a stoichiometry of 1:6. d Lipid pull-down experiment using lipids from Folch brain extracts, recombinant alpha-synuclein and various ions. Western blot of the amount of protein pulled down shows that more alpha-synuclein was pulled down by the lipids in the presence of calcium. Neither potassium, sodium, nor magnesium increased alpha-synuclein lipid binding to the same extent. ** p = 0.0011, 0.0022, and 0.0090 for comparison of 1 mM CaCl 2 with alpha-syn control, 50 mM KCl, and 150 mM NaCl, respectively. Calculated using one-way ANOVA with Tukey’s post-hoc correction, graphs indicate mean ± s.e.m. N = 3 for all groups, corresponding to three biological repeats, d.f. 12

    Article Snippet: For combined confocal/STED imaging, 50 µM human WT alpha-synuclein was complemented with 10% of alpha-synuclein N122C mutant labeled with ATTO-647N dye (05316, Sigma-Aldrich).

    Techniques: Binding Assay, Nuclear Magnetic Resonance, Mass Spectrometry, Incubation, Recombinant, Western Blot

    The C terminus of alpha-synuclein binds to synaptic vesicles upon calcium binding. a CEST-NMR experiments were performed on alpha-synuclein and synaptic vesicles in the absence (black) or presence of calcium (red, 6 mM). In the absence of calcium, the N terminus shows the strongest interaction with synaptic vesicles. Upon addition of calcium, the interaction of the C terminus and also of some residues of the NAC-region increases, which is seen as a reduction of the signal. Experiments were repeated twice. b Lipid pull-down experiment showing the transient nature of alpha-synuclein lipid binding. Western blot of the amount of alpha-synuclein pulled down by the lipids showing that calcium-induced lipid binding of alpha-synuclein is reversible upon addition of the calcium chelator EGTA. * p = 0.0263, calculated using one-way ANOVA with Tukey’s post-hoc correction, graphs indicate mean ± s.e.m. N = 6 for control and CaCl 2 , n = 4 for EGTA and CaCl 2 + EGTA, data from three biological repeats, d.f. 16. c d STORM super-resolution imaging of alpha-synuclein and VAMP2 on isolated synaptosomes displaying alpha-synuclein clustering under normal physiological conditions with 2.5 mM calcium in the extracellular buffer (upper panel). Upon calcium depletion in the extracellular buffer, using 1 mM EGTA, alpha-synuclein localization was dispersed (lower panel). d Cluster analysis of alpha-synuclein and VAMP2 immunostaining showing increased cluster size of alpha-synuclein upon calcium depletion, whereas VAMP2 cluster size is the same either in the presence of calcium or upon calcium depletion in the extracellular buffer. **** p

    Journal: Nature Communications

    Article Title: C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction

    doi: 10.1038/s41467-018-03111-4

    Figure Lengend Snippet: The C terminus of alpha-synuclein binds to synaptic vesicles upon calcium binding. a CEST-NMR experiments were performed on alpha-synuclein and synaptic vesicles in the absence (black) or presence of calcium (red, 6 mM). In the absence of calcium, the N terminus shows the strongest interaction with synaptic vesicles. Upon addition of calcium, the interaction of the C terminus and also of some residues of the NAC-region increases, which is seen as a reduction of the signal. Experiments were repeated twice. b Lipid pull-down experiment showing the transient nature of alpha-synuclein lipid binding. Western blot of the amount of alpha-synuclein pulled down by the lipids showing that calcium-induced lipid binding of alpha-synuclein is reversible upon addition of the calcium chelator EGTA. * p = 0.0263, calculated using one-way ANOVA with Tukey’s post-hoc correction, graphs indicate mean ± s.e.m. N = 6 for control and CaCl 2 , n = 4 for EGTA and CaCl 2 + EGTA, data from three biological repeats, d.f. 16. c d STORM super-resolution imaging of alpha-synuclein and VAMP2 on isolated synaptosomes displaying alpha-synuclein clustering under normal physiological conditions with 2.5 mM calcium in the extracellular buffer (upper panel). Upon calcium depletion in the extracellular buffer, using 1 mM EGTA, alpha-synuclein localization was dispersed (lower panel). d Cluster analysis of alpha-synuclein and VAMP2 immunostaining showing increased cluster size of alpha-synuclein upon calcium depletion, whereas VAMP2 cluster size is the same either in the presence of calcium or upon calcium depletion in the extracellular buffer. **** p

    Article Snippet: For combined confocal/STED imaging, 50 µM human WT alpha-synuclein was complemented with 10% of alpha-synuclein N122C mutant labeled with ATTO-647N dye (05316, Sigma-Aldrich).

    Techniques: Binding Assay, Nuclear Magnetic Resonance, Western Blot, Imaging, Isolation, Immunostaining

    Calcium and alpha-synuclein levels mediate dopamine toxicity. a - c Ventral midbrain neurons were incubated with 100 µM dopamine in the presence or absence of 5 µM isradipine. d STORM super-resolution microscopy of alpha-synuclein and synaptotagmin-1 after 72 h revealed an increase in the area of alpha-synuclein puncta, an increase in the size of synaptotagmin-1 puncta and an increased co-localization of alpha-synuclein with synaptotagmin-1 upon dopamine treatment. These effects were reversed by the Ca v 1.3 calcium channel antagonist isradipine, showing decreased size of alpha-synuclein and synaptotagmin-1 puncta and decreased co-localization. **** p

    Journal: Nature Communications

    Article Title: C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction

    doi: 10.1038/s41467-018-03111-4

    Figure Lengend Snippet: Calcium and alpha-synuclein levels mediate dopamine toxicity. a - c Ventral midbrain neurons were incubated with 100 µM dopamine in the presence or absence of 5 µM isradipine. d STORM super-resolution microscopy of alpha-synuclein and synaptotagmin-1 after 72 h revealed an increase in the area of alpha-synuclein puncta, an increase in the size of synaptotagmin-1 puncta and an increased co-localization of alpha-synuclein with synaptotagmin-1 upon dopamine treatment. These effects were reversed by the Ca v 1.3 calcium channel antagonist isradipine, showing decreased size of alpha-synuclein and synaptotagmin-1 puncta and decreased co-localization. **** p

    Article Snippet: For combined confocal/STED imaging, 50 µM human WT alpha-synuclein was complemented with 10% of alpha-synuclein N122C mutant labeled with ATTO-647N dye (05316, Sigma-Aldrich).

    Techniques: Incubation, Microscopy

    Alpha-synuclein and calcium balance the interaction of synaptic vesicles. a STED super-resolution imaging of isolated synaptic vesicles incubated with 1 mM EGTA, 200 µM calcium, 50 µM alpha-synuclein + 200 µM calcium, or 50 µM alpha-synuclein + 1 mM EGTA. Images show synaptic vesicles circled in red as detected for analysis of synaptic vesicle clustering. Scales represent 200 nm. Synaptic vesicle clustering is shown as a decrease of synaptic vesicles found as single vesicles and as an increase of synaptic vesicles found in clusters was seen upon incubation of synaptic vesicles with either increased calcium or alpha-synuclein. Note, EGTA was not able to reduce synaptic vesicle clustering in the presence of increased alpha-synuclein concentrations. ** p = 0.0023, **** p

    Journal: Nature Communications

    Article Title: C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction

    doi: 10.1038/s41467-018-03111-4

    Figure Lengend Snippet: Alpha-synuclein and calcium balance the interaction of synaptic vesicles. a STED super-resolution imaging of isolated synaptic vesicles incubated with 1 mM EGTA, 200 µM calcium, 50 µM alpha-synuclein + 200 µM calcium, or 50 µM alpha-synuclein + 1 mM EGTA. Images show synaptic vesicles circled in red as detected for analysis of synaptic vesicle clustering. Scales represent 200 nm. Synaptic vesicle clustering is shown as a decrease of synaptic vesicles found as single vesicles and as an increase of synaptic vesicles found in clusters was seen upon incubation of synaptic vesicles with either increased calcium or alpha-synuclein. Note, EGTA was not able to reduce synaptic vesicle clustering in the presence of increased alpha-synuclein concentrations. ** p = 0.0023, **** p

    Article Snippet: For combined confocal/STED imaging, 50 µM human WT alpha-synuclein was complemented with 10% of alpha-synuclein N122C mutant labeled with ATTO-647N dye (05316, Sigma-Aldrich).

    Techniques: Imaging, Isolation, Incubation

    Cross-binding competition ELISA assays. a , b , and c : The two site inhibition curves show distinct high and low binding components in plasma from Parkinson’s Disease ( PD-blue, squares ), Multiple System Atrophy ( MSA-green, triangles ) patients and normal control ( NC-black, circles ) subjects. Ten plasma samples from each group were pooled and incubated in 1:400 dilution with increasing concentration of α-, β-, and γ-synuclein monomers in combinations as indicated, with subsequent measurement of free NAbs by ELISA on plates coated with 10 μg/ml of α-synuclein. d - i : Percentage of inhibition (PI) of individual plasma samples with free d , e : α−/β-synuclein monomers, F , G : α−/γ-synuclein monomers; h , i : β−/γ-synuclein monomer on plates coated with 10 μg/mL of α-synuclein monomer. Plasma samples from Parkinson’s Disease (PD), Multiple System Atrophy (MSA) patients and normal controls (NC) were tested at 1:400 dilution in the presence of ( d , f , h ) 100 nM or ( e , g , i ) 10 nM α-, β-, and γ-synuclein monomers in combinations. Horizontal bars represent the mean values +/− SEM. Significance was tested using Man–Whitney’s U test ( P

    Journal: Molecular Neurodegeneration

    Article Title: Autoimmune antibody decline in Parkinson’s disease and Multiple System Atrophy; a step towards immunotherapeutic strategies

    doi: 10.1186/s13024-017-0187-7

    Figure Lengend Snippet: Cross-binding competition ELISA assays. a , b , and c : The two site inhibition curves show distinct high and low binding components in plasma from Parkinson’s Disease ( PD-blue, squares ), Multiple System Atrophy ( MSA-green, triangles ) patients and normal control ( NC-black, circles ) subjects. Ten plasma samples from each group were pooled and incubated in 1:400 dilution with increasing concentration of α-, β-, and γ-synuclein monomers in combinations as indicated, with subsequent measurement of free NAbs by ELISA on plates coated with 10 μg/ml of α-synuclein. d - i : Percentage of inhibition (PI) of individual plasma samples with free d , e : α−/β-synuclein monomers, F , G : α−/γ-synuclein monomers; h , i : β−/γ-synuclein monomer on plates coated with 10 μg/mL of α-synuclein monomer. Plasma samples from Parkinson’s Disease (PD), Multiple System Atrophy (MSA) patients and normal controls (NC) were tested at 1:400 dilution in the presence of ( d , f , h ) 100 nM or ( e , g , i ) 10 nM α-, β-, and γ-synuclein monomers in combinations. Horizontal bars represent the mean values +/− SEM. Significance was tested using Man–Whitney’s U test ( P

    Article Snippet: 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL or 0.5 ng/mL of recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight ( > 12 h) at 4 °C.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Incubation, Concentration Assay

    Plasma samples from MSA patients have significantly reduced binding capability to α-synuclein monomer. Average percentage (± − SEM) of inhibition (PI) of individual plasma samples with free α-synuclein monomer on plates coated with 5 μg/mL of α-synuclein monomer. Plasma samples from Parkinson’s Disease ( PD-blue squares ), Multiple System Atrophy ( MSA-green, triangles ) patients and normal controls ( NC-black, circles ) were tested at two different dilutions a 1:100 and b 1:200. The line represents the fitting of a two-site model to the data (* p

    Journal: Molecular Neurodegeneration

    Article Title: Autoimmune antibody decline in Parkinson’s disease and Multiple System Atrophy; a step towards immunotherapeutic strategies

    doi: 10.1186/s13024-017-0187-7

    Figure Lengend Snippet: Plasma samples from MSA patients have significantly reduced binding capability to α-synuclein monomer. Average percentage (± − SEM) of inhibition (PI) of individual plasma samples with free α-synuclein monomer on plates coated with 5 μg/mL of α-synuclein monomer. Plasma samples from Parkinson’s Disease ( PD-blue squares ), Multiple System Atrophy ( MSA-green, triangles ) patients and normal controls ( NC-black, circles ) were tested at two different dilutions a 1:100 and b 1:200. The line represents the fitting of a two-site model to the data (* p

    Article Snippet: 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL or 0.5 ng/mL of recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight ( > 12 h) at 4 °C.

    Techniques: Binding Assay, Inhibition

    Absent correlation of age and binding properties of anti-α-synuclein NAbs in plasma from negative controls. Percentage of maximal plasma NAbs binding to immobilized α-synuclein monomer (5 μg/mL) in younger controls (

    Journal: Molecular Neurodegeneration

    Article Title: Autoimmune antibody decline in Parkinson’s disease and Multiple System Atrophy; a step towards immunotherapeutic strategies

    doi: 10.1186/s13024-017-0187-7

    Figure Lengend Snippet: Absent correlation of age and binding properties of anti-α-synuclein NAbs in plasma from negative controls. Percentage of maximal plasma NAbs binding to immobilized α-synuclein monomer (5 μg/mL) in younger controls (

    Article Snippet: 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL or 0.5 ng/mL of recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight ( > 12 h) at 4 °C.

    Techniques: Binding Assay

    Reduced levels of high affinity anti-phosphorylated (P) α-synuclein NAbs in plasma from MSA and PD patients. Binding of anti-P-α-synuclein NAbs in plasma to immobilized a , b α-synuclein monomer (10 ng/mL) or c , d P-α-synuclein (10 ng/mL) in competitive MSD assay in the presence of a , c 1 nM or b , d 0.1 nM free P-α-synuclein. Non-specific binding is defined as the binding to the plate in the presence of 100 nM free P-α-synuclein monomer. Maximal binding is defined as the binding observed in the absence of added free α-synuclein monomer. Horizontal bars represent the mean values +/− SEM. Plasma samples from Parkinson’s Disease (PD), Multiple System Atrophy (MSA) patients and normal controls (NC) were tested at 1:400. Significance was tested using Man–Whitney’s U test ( P

    Journal: Molecular Neurodegeneration

    Article Title: Autoimmune antibody decline in Parkinson’s disease and Multiple System Atrophy; a step towards immunotherapeutic strategies

    doi: 10.1186/s13024-017-0187-7

    Figure Lengend Snippet: Reduced levels of high affinity anti-phosphorylated (P) α-synuclein NAbs in plasma from MSA and PD patients. Binding of anti-P-α-synuclein NAbs in plasma to immobilized a , b α-synuclein monomer (10 ng/mL) or c , d P-α-synuclein (10 ng/mL) in competitive MSD assay in the presence of a , c 1 nM or b , d 0.1 nM free P-α-synuclein. Non-specific binding is defined as the binding to the plate in the presence of 100 nM free P-α-synuclein monomer. Maximal binding is defined as the binding observed in the absence of added free α-synuclein monomer. Horizontal bars represent the mean values +/− SEM. Plasma samples from Parkinson’s Disease (PD), Multiple System Atrophy (MSA) patients and normal controls (NC) were tested at 1:400. Significance was tested using Man–Whitney’s U test ( P

    Article Snippet: 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL or 0.5 ng/mL of recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight ( > 12 h) at 4 °C.

    Techniques: Binding Assay

    Concentrations of α-synuclein a and α-synuclein-NAbs complexes b in plasma from Parkinson’s Disease (PD), Multiple System Atrophy (MSA) patients and normal controls (NC). Horizontal bars represent the mean values ±SEM. α-Synuclein-NAbs complex concentrations were quantified using α-synuclein standard curve. Significance was tested using Man–Whitney’s U test ( P

    Journal: Molecular Neurodegeneration

    Article Title: Autoimmune antibody decline in Parkinson’s disease and Multiple System Atrophy; a step towards immunotherapeutic strategies

    doi: 10.1186/s13024-017-0187-7

    Figure Lengend Snippet: Concentrations of α-synuclein a and α-synuclein-NAbs complexes b in plasma from Parkinson’s Disease (PD), Multiple System Atrophy (MSA) patients and normal controls (NC). Horizontal bars represent the mean values ±SEM. α-Synuclein-NAbs complex concentrations were quantified using α-synuclein standard curve. Significance was tested using Man–Whitney’s U test ( P

    Article Snippet: 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL or 0.5 ng/mL of recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight ( > 12 h) at 4 °C.

    Techniques:

    Schematic overview of the competitive ELISA technique. Before transfer onto plates coated with α-synuclein monomer, plasma samples are incubated for 1 h at RT with α-synuclein monomer. Following washing steps, the competition reaction is created by moving plasma/α-synuclein monomer samples onto the coated plate. Here, high affinity antibodies form more stable bonds with free antigen than do lower affinity antibodies. Consequently, lower affinity antibodies remain bound to the excess immobilized antigen on the plates, but high-affinity antibodies bound to the fluid-phase antigen are washed away. After several washes, the amounts of low-affinity antibodies remaining on the plates are detected by peroxidase-labelled polyclonal goat anti-human IgG (Fc fragment specific). The plate is developed by adding an enzymatic substrate to produce a visible signal

    Journal: Molecular Neurodegeneration

    Article Title: Autoimmune antibody decline in Parkinson’s disease and Multiple System Atrophy; a step towards immunotherapeutic strategies

    doi: 10.1186/s13024-017-0187-7

    Figure Lengend Snippet: Schematic overview of the competitive ELISA technique. Before transfer onto plates coated with α-synuclein monomer, plasma samples are incubated for 1 h at RT with α-synuclein monomer. Following washing steps, the competition reaction is created by moving plasma/α-synuclein monomer samples onto the coated plate. Here, high affinity antibodies form more stable bonds with free antigen than do lower affinity antibodies. Consequently, lower affinity antibodies remain bound to the excess immobilized antigen on the plates, but high-affinity antibodies bound to the fluid-phase antigen are washed away. After several washes, the amounts of low-affinity antibodies remaining on the plates are detected by peroxidase-labelled polyclonal goat anti-human IgG (Fc fragment specific). The plate is developed by adding an enzymatic substrate to produce a visible signal

    Article Snippet: 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL or 0.5 ng/mL of recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight ( > 12 h) at 4 °C.

    Techniques: Competitive ELISA, Incubation

    The two site inhibition curves show distinct high and low binding components in plasma from Parkinson’s Disease ( PD-blue, squares ), Multiple System Atrophy ( MSA-green, triangles ) patients and normal control ( NC-black, circles ) subjects. Ten plasma samples from each group were pooled and incubated with increasing concentration of α-synuclein monomer with subsequent measurement of free NAbs by ELISA or Meso Scale Discovery (MSD) assays. a ELISA: 10 μg/mL α-synuclein coating, plasma dilution 1:400; b ELISA: 5 μg/mL α-synuclein coating, plasma dilution 1:200; c MSD assay: 5 ng/mL α-synuclein coating, plasma dilution 1:500. The relative binding of anti-α-synuclein NAbs is expressed as a percentage of maximal binding attained in the assay for each sample, where competition reactions with 1000 nM α-synuclein monomer are defined as constituting non-specific (background) binding and reactions without competition reflect 100% (maximum) binding. The line represents the fitting of a two site model to the data

    Journal: Molecular Neurodegeneration

    Article Title: Autoimmune antibody decline in Parkinson’s disease and Multiple System Atrophy; a step towards immunotherapeutic strategies

    doi: 10.1186/s13024-017-0187-7

    Figure Lengend Snippet: The two site inhibition curves show distinct high and low binding components in plasma from Parkinson’s Disease ( PD-blue, squares ), Multiple System Atrophy ( MSA-green, triangles ) patients and normal control ( NC-black, circles ) subjects. Ten plasma samples from each group were pooled and incubated with increasing concentration of α-synuclein monomer with subsequent measurement of free NAbs by ELISA or Meso Scale Discovery (MSD) assays. a ELISA: 10 μg/mL α-synuclein coating, plasma dilution 1:400; b ELISA: 5 μg/mL α-synuclein coating, plasma dilution 1:200; c MSD assay: 5 ng/mL α-synuclein coating, plasma dilution 1:500. The relative binding of anti-α-synuclein NAbs is expressed as a percentage of maximal binding attained in the assay for each sample, where competition reactions with 1000 nM α-synuclein monomer are defined as constituting non-specific (background) binding and reactions without competition reflect 100% (maximum) binding. The line represents the fitting of a two site model to the data

    Article Snippet: 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL or 0.5 ng/mL of recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight ( > 12 h) at 4 °C.

    Techniques: Inhibition, Binding Assay, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Reduced levels of high affinity anti-α-synuclein NAbs in plasma from MSA and PD patients. Binding of anti-α-synuclein NAbs in plasma to immobilized α-synuclein monomer (5 μg/mL) in competitive ELISA assay in the presence of a , b 50 nM or c , d 2 nM free α-synuclein. Non-specific binding is defined as the binding to the plate in the presence of 1000 nM free α-synuclein monomer. Maximal binding is defined as the binding observed in the absence of added free α-synuclein monomer. Horizontal bars represent the mean values +/− SEM. Plasma samples from Parkinson’s Disease (PD), Multiple System Atrophy (MSA) patients and normal controls (NC) were tested at two dilutions, i.e. a , c 1:100 and b , d 1:200. Significance was tested using Man–Whitney’s U test ( P

    Journal: Molecular Neurodegeneration

    Article Title: Autoimmune antibody decline in Parkinson’s disease and Multiple System Atrophy; a step towards immunotherapeutic strategies

    doi: 10.1186/s13024-017-0187-7

    Figure Lengend Snippet: Reduced levels of high affinity anti-α-synuclein NAbs in plasma from MSA and PD patients. Binding of anti-α-synuclein NAbs in plasma to immobilized α-synuclein monomer (5 μg/mL) in competitive ELISA assay in the presence of a , b 50 nM or c , d 2 nM free α-synuclein. Non-specific binding is defined as the binding to the plate in the presence of 1000 nM free α-synuclein monomer. Maximal binding is defined as the binding observed in the absence of added free α-synuclein monomer. Horizontal bars represent the mean values +/− SEM. Plasma samples from Parkinson’s Disease (PD), Multiple System Atrophy (MSA) patients and normal controls (NC) were tested at two dilutions, i.e. a , c 1:100 and b , d 1:200. Significance was tested using Man–Whitney’s U test ( P

    Article Snippet: 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL or 0.5 ng/mL of recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight ( > 12 h) at 4 °C.

    Techniques: Binding Assay, Competitive ELISA

    Specificity of anti-alpha-synuclein NAb binding. The ability of increasing concentrations of a α-synuclein monomer or b amyloidβ1–40 (Aβ1–40) to inhibit the binding of α-synuclein NAbs to α-synuclein coated ELISA plates. Data are presented as % inhibition (PI) relative to the unblocked condition. Pooled plasma samples are from 10 Parkinson’s Disease ( PD-blue, squares ), 10 Multiple System Atrophy ( MSA-green, triangles ) patients and 10 normal controls ( NC-black, circles ) were diluted 1:400. The lines in a represents the fitting of a one site model to the data

    Journal: Molecular Neurodegeneration

    Article Title: Autoimmune antibody decline in Parkinson’s disease and Multiple System Atrophy; a step towards immunotherapeutic strategies

    doi: 10.1186/s13024-017-0187-7

    Figure Lengend Snippet: Specificity of anti-alpha-synuclein NAb binding. The ability of increasing concentrations of a α-synuclein monomer or b amyloidβ1–40 (Aβ1–40) to inhibit the binding of α-synuclein NAbs to α-synuclein coated ELISA plates. Data are presented as % inhibition (PI) relative to the unblocked condition. Pooled plasma samples are from 10 Parkinson’s Disease ( PD-blue, squares ), 10 Multiple System Atrophy ( MSA-green, triangles ) patients and 10 normal controls ( NC-black, circles ) were diluted 1:400. The lines in a represents the fitting of a one site model to the data

    Article Snippet: 96-well microtiter plates (Standard MSD bind plate #L15XA-1) were coated with either 5 ng/mL or 0.5 ng/mL of recombinant α-synuclein monomer (rPeptide, #S-10001-2) in ice-cold 0.1 M carbonate buffer (pH 8.5) overnight ( > 12 h) at 4 °C.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Inhibition

    The E46K mutation in α-synuclein ablates replication of MSA prions. α-Synuclein prions were isolated from three MSA patient samples and one control sample by phosphotungstic acid precipitation and were incubated with HEK cells expressing mutated and truncated α-synuclein–YFP fusion proteins. ( A ) MSA prions were unable to replicate in cells expressing the E46K mutation. Coexpression with the A53T mutation marginally improved infection. However, coexpression of the A30P and A53T mutations resulted in robust replication of MSA prions. ( B ) Truncation of α-syn*A53T at residue 95, but not at residue 97, hindered MSA prion infection in the HEK cells. Data are shown as mean ± SD. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Familial Parkinson’s point mutation abolishes multiple system atrophy prion replication

    doi: 10.1073/pnas.1719369115

    Figure Lengend Snippet: The E46K mutation in α-synuclein ablates replication of MSA prions. α-Synuclein prions were isolated from three MSA patient samples and one control sample by phosphotungstic acid precipitation and were incubated with HEK cells expressing mutated and truncated α-synuclein–YFP fusion proteins. ( A ) MSA prions were unable to replicate in cells expressing the E46K mutation. Coexpression with the A53T mutation marginally improved infection. However, coexpression of the A30P and A53T mutations resulted in robust replication of MSA prions. ( B ) Truncation of α-syn*A53T at residue 95, but not at residue 97, hindered MSA prion infection in the HEK cells. Data are shown as mean ± SD. * P

    Article Snippet: Recombinant α-synuclein monomers (WT, A30P, E46K, and A53T; Sigma) were prepared as previously reported ( ).

    Techniques: Mutagenesis, Isolation, Incubation, Expressing, Infection

    Cell-passaged MSA prions transmit disease. ( A ) α-Synuclein prions were isolated from three MSA patient samples and one control sample by phosphotungstic acid precipitation. The MSA prions were able to significantly infect HEK cells expressing WT and mutated (A30P and A53T) α-synuclein–YFP fusion proteins. Data are shown as mean ± SD. ( B and C ) Patient sample MSA14 was used to generate stable clones infected with MSA prions (MSA14-11, MSA14-12, and MSA14-M1). Lysates from infected and uninfected cells were inoculated into TgM83 +/− mice (30 μg protein). ( B ) The Kaplan–Meier plot shows the onset of neurological signs. Dpi, days post inoculation. ( C ) α-Synuclein prions were measured from frozen half-brains in the α-syn140*A53T–YFP cell assay (× 10 3 a.u.). Circles indicate infected mice that died from disease (above dotted line); triangles indicate asymptomatic animals terminated 400 d post inoculation. Mice inoculated with cell-passaged MSA contained significantly more α-synuclein prions than controls. *** P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Familial Parkinson’s point mutation abolishes multiple system atrophy prion replication

    doi: 10.1073/pnas.1719369115

    Figure Lengend Snippet: Cell-passaged MSA prions transmit disease. ( A ) α-Synuclein prions were isolated from three MSA patient samples and one control sample by phosphotungstic acid precipitation. The MSA prions were able to significantly infect HEK cells expressing WT and mutated (A30P and A53T) α-synuclein–YFP fusion proteins. Data are shown as mean ± SD. ( B and C ) Patient sample MSA14 was used to generate stable clones infected with MSA prions (MSA14-11, MSA14-12, and MSA14-M1). Lysates from infected and uninfected cells were inoculated into TgM83 +/− mice (30 μg protein). ( B ) The Kaplan–Meier plot shows the onset of neurological signs. Dpi, days post inoculation. ( C ) α-Synuclein prions were measured from frozen half-brains in the α-syn140*A53T–YFP cell assay (× 10 3 a.u.). Circles indicate infected mice that died from disease (above dotted line); triangles indicate asymptomatic animals terminated 400 d post inoculation. Mice inoculated with cell-passaged MSA contained significantly more α-synuclein prions than controls. *** P

    Article Snippet: Recombinant α-synuclein monomers (WT, A30P, E46K, and A53T; Sigma) were prepared as previously reported ( ).

    Techniques: Isolation, Expressing, Clone Assay, Infection, Mouse Assay

    . ( A ) MSA prions from patients MSA12, MSA13, and MSA14 were unable to infect cells containing the E46K mutation alone (K) or in combination with the A53T mutation (KT), nor could they infect cells expressing α-synuclein*A53T truncated at residue 95 (1-95). ( B ) MSA prions passaged through TgM83 +/− mice showed similar selectivity in the α-synuclein−YFP cell assays. ( C and D ) Patient sample MSA14 was used to generate stable clones infected with MSA prions (MSA14-11, MSA14-12, and MSA14-M1). Lysates from these cells showed a similar infectivity profile in the α-synuclein cell lines ( C ), as did TgM83 +/− mice inoculated with the lysates ( D ). Control samples did not infect the cells. Data are shown as the mean.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Familial Parkinson’s point mutation abolishes multiple system atrophy prion replication

    doi: 10.1073/pnas.1719369115

    Figure Lengend Snippet: . ( A ) MSA prions from patients MSA12, MSA13, and MSA14 were unable to infect cells containing the E46K mutation alone (K) or in combination with the A53T mutation (KT), nor could they infect cells expressing α-synuclein*A53T truncated at residue 95 (1-95). ( B ) MSA prions passaged through TgM83 +/− mice showed similar selectivity in the α-synuclein−YFP cell assays. ( C and D ) Patient sample MSA14 was used to generate stable clones infected with MSA prions (MSA14-11, MSA14-12, and MSA14-M1). Lysates from these cells showed a similar infectivity profile in the α-synuclein cell lines ( C ), as did TgM83 +/− mice inoculated with the lysates ( D ). Control samples did not infect the cells. Data are shown as the mean.

    Article Snippet: Recombinant α-synuclein monomers (WT, A30P, E46K, and A53T; Sigma) were prepared as previously reported ( ).

    Techniques: Mutagenesis, Expressing, Mouse Assay, Clone Assay, Infection