Journal: NPJ Parkinson's disease

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

doi: 10.1038/s41531-023-00444-w

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

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

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

Journal: Molecular Biology Reports

Article Title: S -Adenosylmethionine affects ERK1/2 and STAT3 pathway in androgen-independent prostate cancer cells

doi: 10.1007/s11033-022-07331-2

Figure Lengend Snippet: Overview of antibodies used in this study

Article Snippet: Cyclin A , Rabbit , ThermoFisher (PA5-36048) , 1:500.

Techniques:

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