s129a plasmids  (Addgene inc)

Journal: NPJ Parkinson's Disease

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

doi: 10.1038/s41531-023-00444-w

Figure Lengend Snippet: a Spontaneous excitatory (sEPSC) and inhibitory postsynaptic currents (sIPSC) measured in DIV14-18 hippocampal neurons from SNCA −/− rats transduced with αS WT or αS S129A (schematics created with BioRender.com). b Representative sEPSC traces. c Representative sIPSC traces. Scale bars, X -axis = 1 s and Y -axis = 100 pA. d sEPSC frequency in SNCA −/− neurons expressing WT αS or αS S129A. e Cumulative frequency distribution of data shown in d , expressed as percentage. f sEPSC amplitude in SNCA −/− neurons expressing αS WT or αS S129A. g Cumulative frequency distribution plot of data shown in f , expressed as percentage. Total number of individual events analyzed in panels e and g : αS WT = 1228; αS S129A = 1701. Total number of individual cells across two independent neuronal cultures ( N = 2 ) recorded in sEPSC experiments: WT αS = 21 and αS S129A n = 12. h , i sIPSC frequency between conditions as bar charts and cumulative distribution, respectively. j sIPSC amplitude in SNCA −/− neurons expressing WT or S129A αS. k Cumulative frequency distribution of data shown in j . Total number of individual events analyzed in panels i and k : αS WT = 302; αS S129A = 585. Total number of individual cells across two independent neuronal cultures recorded in sIPSC experiments: αS WT = 19 and αS S129A = 15. l , m The excitatory/inhibitory ratio of the amplitude as a bar chart and cumulative frequency distribution, respectively. E/I amplitude ratios were derived from f and j . Respective averages in f (αS WT or αS S129A) were divided by respective individual values (αS WT or αS S129A) in j to obtain E/I amplitude ratios. Each circle represents an individual cell. Recordings performed on at least three different days. Unpaired t -tests with Welch’s correction; two-tailed; mean ± SD; ns not significant; *** p < 0.001; **** p < 0.0001.

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

Techniques: Transduction, Expressing, Derivative Assay, Two Tailed Test

Journal: NPJ Parkinson's Disease

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

doi: 10.1038/s41531-023-00444-w

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

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

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

Journal: Scientific Reports

Article Title: Phosphorylated exogenous alpha-synuclein fibrils exacerbate pathology and induce neuronal dysfunction in mice

doi: 10.1038/s41598-017-15813-8

Figure Lengend Snippet: Characterization of wt-, P- and S129A- PFF. ( a ) Western blotting analysis for wt-, P- and S129A-PFF. Equal amounts of fibrils were analyzed in a 10% SDS-PAGE gel using the C20 antibody. P-PFF were detected with the α-Synuclein (phospho Ser) antibody whereas no signal was observed for the S129A- and wt-PFF. ( b ) Fibril formation monitoring by Th-S assay. Graphs show: fibril formation monitoring of the wt- and S129A-monomers incubated for 7 days (top). Comparison of the fibril content of wt-PFF and P-PFF (bottom). Monomeric α-Synuclein was used as control. The assays were performed in triplicate. ( c ) Electron microscopy images of negatively stained samples of the different types of α-Synuclein to confirm the presence of fibrils compared to the monomeric non-fibrilar α-Synuclein. Scale bar , 500 nm.

Article Snippet: pT7–7 α-Synuclein S129A was a gift from Hilal Lashuel (Addgene, plasmid # 36048) .

Techniques: Western Blot, SDS Page, Incubation, Comparison, Control, Electron Microscopy, Staining

Journal: Scientific Reports

Article Title: Phosphorylated exogenous alpha-synuclein fibrils exacerbate pathology and induce neuronal dysfunction in mice

doi: 10.1038/s41598-017-15813-8

Figure Lengend Snippet: Pathological α-Synuclein accumulation in the SNpc dopaminergic neurons of wt mice following stereotaxic unilateral striatal injection of three different human-PFF types (P-PFF, wt-PFF, and S129A-PFF). Animals were analyzed 60 dpi. ( a ) Confocal images showing double immunostaining for P-α-Synuclein and TH in nigral sections of PFF-injected animals. Accumulation of hyper-phosphorylated α-Synuclein (α-Synuclein phospho Ser 129) is evident in dopaminergic neurons (TH) of the ipsilateral SNpc. Pathology is absent in the ipsilateral side of PBS injected animals. The contralateral side of P-PFF injected animals shows no signs of pathologic accumulations. α-Synuclein (phospho Ser129) immunoreactivity is not detected in the ipsilateral nigra of α-Synuclein null (−/−) animals injected with P-PFF (n = 4). ( b ) Images in higher magnification are showing P-α-Synuclein accumulations induced by the different types of PFF. ( c ) Striatal tissue of injected animals (3 dpi) was extracted and immunoblotted with the 4B12 and phospho Ser 129 antibodies. Human α-Synuclein was readily detected in the striatal extracts. S129A- and wt-PFF could not be detected with the phospho Ser 129 antibody. γ-tubulin was used as a loading control (cropped gel/blot is shown). Scale bars represent 25 μm.

Article Snippet: pT7–7 α-Synuclein S129A was a gift from Hilal Lashuel (Addgene, plasmid # 36048) .

Techniques: Injection, Double Immunostaining, Control, Western Blot

Journal: Scientific Reports

Article Title: Phosphorylated exogenous alpha-synuclein fibrils exacerbate pathology and induce neuronal dysfunction in mice

doi: 10.1038/s41598-017-15813-8

Figure Lengend Snippet: Characterization of SNpc intraneuronal α-Synuclein accumulations. ( a ) Double labeling with the conformational specific α-Synuclein antibody SynO2 and TH is showing the fibrilar nature of the α-Synuclein cytoplasmic accumulations restricted to TH neurons of the ipsilateral SNpc following injections with P-, wt- and mutant S129A-PFF at 60 dpi. Contralateral side shows only background staining with the SynO2 antibody. ( b ) Representative sections of SNpc from all PFF injected animals showing the co-staining of α-Synuclein accumulations with the α-Synuclein (phospho Ser 129) and SynO2 antibodies following PK treatment for 10 min at 25 °C to expose antigenic sites. ( c ) α-Synuclein accumulations also stained positive with the C20 antibody. ( d ) Host α-Synuclein expression is essential for the formation of pathological α-Synuclein accumulations. TH-stained nigral sections (PK-treated, 10 min at 25 °C) are exhibiting α-Synuclein accumulations that are positive for the endogenous rodent α-Synuclein (D37A6) antibody. PK resistant D37A6-positive accumulations were also evident following prolonged PK treatment (1 h at 37 °C) in nigral sections until the TH signal is not detectable ( e ) α-Synuclein accumulations do not stain with the human specific anti-α-Synuclein (211) antibody. TO-PRO-3 (blue) was used as a cell nuclear marker (n = 4). Scale bars represent 25 μm.

Article Snippet: pT7–7 α-Synuclein S129A was a gift from Hilal Lashuel (Addgene, plasmid # 36048) .

Techniques: Labeling, Mutagenesis, Staining, Injection, Expressing, Marker

Journal: Scientific Reports

Article Title: Phosphorylated exogenous alpha-synuclein fibrils exacerbate pathology and induce neuronal dysfunction in mice

doi: 10.1038/s41598-017-15813-8

Figure Lengend Snippet: P-PFF exacerbate the pathology within the SNpc and significantly impair the integrity of the dopaminergic neurons. ( a ) Coronal nigral sections were immunostained for α-Synuclein (phospho Ser S129) and TH. The absolute numbers of P-accumulations that were formed within the TH positive neurons in the different fibril-injected brains were counted. As shown in representative tiled images for each of the P-, wt-, and S129A-PFF injected animals, P-PFF induced a more widespread pathology compared to the other two fibrilar types. P-PFF injected α-Synuclein null mice did not show any sign of pathology. Graph depicts the percentage of TH neurons containing P-α-Synuclein positive accumulations for each treatment group (n = 4 animals per group, 3 sections per animal). ( b ) Stereological analysis of TH-positive neurons is showing a significant loss of dopaminergic neurons in P-PFF injected animals compared to the PBS, wt-, S129A-PFF injected animals and to P-PFF α-Synuclein null (−/−) injected animals. The data are presented as a percentage of ipsilateral to contralateral side (n = 5–6 animals per group). ( c ) Decreased nigral TH positive neuron number was confirmed with VMAT2 stereological analysis following P-PFF injections (4–6 animals per group, paired Student’s t-test analysis). ( d ) Significant decrease in striatal DA levels in wt animals injected with P-PFF. The data are presented as a ratio of ipsilateral to contralateral side (n = 5–7 animals per group). ( e ) Fine motor impairment as increased errors/step in the challenging beam traversal test in P-PFF- vs. S129A- and control PBS- injected animals (n = 7–8 animals/group). Similar injections did not cause any motor impairment in null α-Synuclein (−/−) mice (n = 5 animals/group). Data represent mean values ± SEM. Differences were estimated using one-way ANOVA followed by Tukey’s post-hoc test. ( a ) p < 0,0001 ( b ) p = 0,0052 ( c ) For TH p = 0,0002, and VMAT p = 0,0062 two-tailed paired t-test ( d ) p = 0,0009 ( e ) p = 0,0163. Scale bar in ( a ) represents 250 μm.

Article Snippet: pT7–7 α-Synuclein S129A was a gift from Hilal Lashuel (Addgene, plasmid # 36048) .

Techniques: Injection, Control, Two Tailed Test

Journal: Scientific Reports

Article Title: Phosphorylated exogenous alpha-synuclein fibrils exacerbate pathology and induce neuronal dysfunction in mice

doi: 10.1038/s41598-017-15813-8

Figure Lengend Snippet: Robust endogenous α-Synuclein accumulation is also evident in the cortex of injected animals. Coronal sections of P-, wt- and S129A- PFF injected animals were stained for α-Synuclein (phospho Ser129). ( a ) P-α-Synuclein positive inclusions (highlighted in magnification within the dashed line frame) were evident in the ipsilateral cortex of P- and wt- PFF-injected animals. Progression of pathology but in a more dispersed pattern is also evident in the contralateral cortex. S129A- PFF are not as efficient in inducing α-Synuclein accumulation on either sides. Arrows indicate the needle entry point. Graph shows phospho Ser129 mean fluorescence intensity of the ipsilateral cortex, normalized to the measured area (intensity/μm 2 ) (n = 4 animals/group, p = 0,0002). ( b ) The rodent specific anti-α-Synuclein (D37A6) antibody and ( c ) the human specific anti-α-Synuclein (211) were used for immunostaining of PK-treated sections (10 min at 25 °C to expose the antigenic sites). Pathological accumulations in the area of the cortex of both hemispheres were positively stained with the rodent specific antibody. α-Synuclein accumulations did not stain with the human specific anti-α-Synuclein (211) antibody in the ipsilateral cortex of P- and wt-PFF injected animals (n = 4). ( d ) Coronal striatal sections of P- and wt-PFF injected animals were also positively stained with the rodent specific α-Synuclein (D37A6) antibody. TO-PRO-3 (blue) was used as a cell nuclear marker. Scale bars represent 250 μm in ( a ) and 25 μm in ( b,c,d ).

Article Snippet: pT7–7 α-Synuclein S129A was a gift from Hilal Lashuel (Addgene, plasmid # 36048) .

Techniques: Injection, Staining, Fluorescence, Immunostaining, Marker

Journal: Scientific Reports

Article Title: Phosphorylated exogenous alpha-synuclein fibrils exacerbate pathology and induce neuronal dysfunction in mice

doi: 10.1038/s41598-017-15813-8

Figure Lengend Snippet: Biochemical profile of α-Synuclein in PFF-injected animals. ( a ) Midbrain Triton-X soluble samples of injected animals showed no difference in α-Synuclein levels between the ipsi-and contralateral side in all fibrilar types and the control PBS- injected animals (α-Synuclein monomer is shown in cropped gel/blot). GAPDH was used as a loading control (cropped gel/blot is shown) (n = 5–7 brains/group). Similar in ( b ) no differences were found for the Triton-X soluble fraction in the area of the cortex (α-Synuclein monomer is shown in cropped gel/blot) (n = 4 brains/group). ( c ) P- and wt-PFF injections resulted in a shift of the SDS-soluble α-Synuclein in higher molecular weight species ipsilaterally in the midbrain. These SDS-soluble α-Synuclein fraction was significantly enriched in the P-PFF injected side compared to the wt-PFF treatment (n = 5 brains/group). High molecular weight species in both treatments were also positive for the phospho Ser 129 α-Synuclein antibody. No difference was observed in the SDS-soluble α-Synuclein monomer levels between the ipsi- and the contralateral side for the P-, wt-, S129A-PFF and the PBS-injected animals (α-Synuclein monomer is shown in cropped gel/blot) (n-6–7 animals/group). γ-tubulin was used as a loading control (cropped gel/blot is shown). ( d ) Immunoblot for the SDS-soluble fraction extracted from the cortex of injected animals showed that α-Synuclein SDS-soluble high molecular weight species are formed readily in P-PFF-injected animals in both sides compared to the wt treatment. Densitomentry of the ipsilateral α-Synuclein levels confirmed the significant difference between the treatments (n = 5 animals/group). α-Synuclein null mice (−/−) injected with P-PFF did not show any positive signal with the Syn-1 antibody. The observed α-Synuclein species were phosphorylated in nature as seen following immunoblotting with the phospho Ser 129 antibody. No difference was observed in the SDS-soluble α-Synuclein monomer levels between the ipsi- and the contralateral side for all types of injected animals (α-Synuclein monomer is shown in cropped gel/blot) (n = 5–6 animals/group). γ-tubulin was used as a loading control (cropped gel/blot is shown). Data represent mean values ± SEM. Differences were estimated using one-way ANOVA followed by Tukey’s post-hoc test and paired two-tailed Student’s t-test. ( c ) p = 0,0037 ( d ) p = 0,0053.

Article Snippet: pT7–7 α-Synuclein S129A was a gift from Hilal Lashuel (Addgene, plasmid # 36048) .

Techniques: Injection, Control, Western Blot, Molecular Weight, High Molecular Weight, Two Tailed Test

Journal: Scientific Reports

Article Title: Phosphorylated exogenous alpha-synuclein fibrils exacerbate pathology and induce neuronal dysfunction in mice

doi: 10.1038/s41598-017-15813-8

Figure Lengend Snippet: Increased uptake and faster seeding of the endogenous α-Synuclein in primary neurons following P-PFF treatment ( a ) Mouse primary cultures (6div) were treated with PFF for 8 and 24 hours. Following Triton-X extraction, internalized fibrils were visualized with the human specific α-Synuclein antibody 4B12. Increased uptake of P-PFF was observed as early as 8 h. The uptake was quantified by densitometry and found to be significantly increased for P-PFF in both time points compared to wt- or S129A- PFF. P-PFF uptake was further verified using the phospho Ser 129 antibody. β-Actin was used as a loading control (cropped gel/blot is shown). ( b ) Sarcosyl-soluble α-Synuclein species were also increased following P-PFF treatment of primary cortical neurons. γ-tubulin was used as a loading control (cropped gel/blot is shown). ( c ) Immunocytochemistry with the rodent specific α-Synuclein antibody (D37A6) in primary mouse cortical cultures treated with the three fibrilar types. P-PFF seed the endogenous α-Synuclein more effectively compared to the other fibrilar types and PBS control treated neurons for 5 days. However ( d ) 8-day-treated neurons exhibit similar levels of endogenous α-Synuclein signal for both the wt- and P-PFF treatments, in contrast to the significant lower levels of S129A-treated neurons. Scatter plots present the mean fluorescence intensity/cell of three independent experiments (n≈90 single cells/condition per replicate). β Tubulin III (Tuj 1) was used as a neuronal marker. TO-PRO-3 (blue) was used as a cell nuclear marker. All data represent mean values ± SEM from three independent experiments. Statistical significance was obtained by ANOVA followed by Tukey’s post-hoc test for ( a ) and non parametric Kruskal-Wallis test followed by Dunn’s post-hoc test for ( c and d ). ( a ) For 8 h p < 0,0001 and for 24 h p = 0,002, ( c ) p < 0,0001 ( d ) p < 0,0001.Scale bar in ( c and d ) represent 25 μm.

Article Snippet: pT7–7 α-Synuclein S129A was a gift from Hilal Lashuel (Addgene, plasmid # 36048) .

Techniques: Extraction, Control, Western Blot, Immunocytochemistry, Fluorescence, Marker