Journal: Neurobiology of disease

Article Title: Anti-α-synuclein c-terminal antibodies block PFF uptake and accumulation of phospho-synuclein in preclinical models of Parkinson's disease.

doi: 10.1016/j.nbd.2022.105969

Figure Lengend Snippet: Fig. 1. α-Syn PFF surface binding screen and identification of C-term blocking antibodies A) Rat primary cortical neurons (14 DIV) were treated with biotinylated- monomeric alpha-synuclein, biotinylated-oligomeric alpha synuclein, and biotinylated-PFF alpha synuclein for 2 h at concentrations of 1μg/ml, 2μg/ml and 5μg/ml. Cultures were fixed and stained for surface α-Syn species (Green) and Map2 (Red). Images from 1μg/ml treatments are shown. PFF α-Syn species has the most significant binding to the neuronal surface whereas control monomeric alpha synuclein species do not. Oligomeric α-Syn binds to a small subset of neurons. Scale bar, 50 um. B) Quantification of total surface α-Syn species signal normalized to total Map2 signal. Robust surface binding with PFF α-Syn species is observed and signal intensity increases with concentration. We selected 1 μg/ml biotinylated-PFF α-Syn for antibody blocking screen. N = 20 wells, Data represent mean ± SEM, ANOVA ****p < 0.0001. C) Diagram of experimental setup. Anti-α-Syn antibody clones at 30μg/ml and bio-PFF α-Syn at 1μg/ml were added to primary rat cortical neurons and incubated for 2 h and assessed for PFF surface staining. D) Examples images of negative control gp120, and antibody clone hits, 52F5, 31C2, and 56G11. Rat primary cortical neurons (14 DIV) were treated with biotinylated-PFF α-Syn for 2 h and fixed and stained for surface α-Syn species (Green) and Map2 (Red). Antibody clones 31C2 and 56G11 both bind to alpha synuclein at the c-terminal (111−130). Scale bar, 50 um. E) Quantification of total surface α-Syn species signal normalized as percent signal to negative control antibody gp120 for anti-alpha synuclein antibody clones screened. Antibody clones that bind at the C terminal region of α-Syn have strong activity at blocking PFF alpha synuclein binding to neuronal surface. N = 4 wells. Error bar, Data represent mean ± SEM. ANOVA All bar are ****p < 0.0001, except 5 bars indicted at NS. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To assess free unbound alpha synuclein as a measurement of target engagement, a free α-Syn assay was generated on Gyros platform using the drug antibody as capture antibody and rabbit polyclonal anti-alpha synuclein (ProteinTech, 10,842–1-AP) as detection antibody.

Techniques: Binding Assay, Blocking Assay, Staining, Control, Concentration Assay, Clone Assay, Incubation, Negative Control, Activity Assay

Journal: Neurobiology of disease

Article Title: Anti-α-synuclein c-terminal antibodies block PFF uptake and accumulation of phospho-synuclein in preclinical models of Parkinson's disease.

doi: 10.1016/j.nbd.2022.105969

Figure Lengend Snippet: Fig. 2. α-Syn C-terminal antibodies block PFF binding, internalization and induction of pS129 A) Diagram of experimental setup and time course of PFF phenotype development. Anti-α-Syn antibody clones and PFF (1 μg/ml) were added to primary rat cortical neurons and incubated for 10 days and then accessed for PFF surface staining (red), internalization (green), and pS129 induction (white). B) Example images of negative control gp120, and antibody clones, 52F5, 31C2, and 56G11. Rat primary cortical neurons (14 DIV) were treated with biotinylated-PFF α-Syn for 10 days and fixed and stained for surface alpha synuclein PFF (Red), internalized PFF (Green), and Map2 (blue). Antibody clones 31C2 and 56G11 both bind to α-Syn at the c-terminal (111–130), both showed strong blocking of PFF α-Syn surface binding and internalization at 1μg/ml and 10μg/ml of antibody concentration. Scale bar, 50uM. C) Examples images of negative control gp120, and antibody clones, 52F5, 31C2, and 56G11. Rat primary cortical neurons (14DIV) were treated with biotinylated-PFF alpha synuclein for 10 days and fixed and stained for pS129. Antibody clones 31C2 and 56G11 both bind to alpha synuclein at the c-terminal (111–130), and both showed strong blocking of PFF alpha synuclein induced pS129 induction. Scale bar, 50 um. D–F) IC50 curves of antibody clones (gp120, 56G11, 31C2, 52F5) activity in blocking extracellular PFF binding (D), intracellular PFF uptake (E), and PFF pS129 induction (F). N = 4. Error bar, Data represent mean ± SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: To assess free unbound alpha synuclein as a measurement of target engagement, a free α-Syn assay was generated on Gyros platform using the drug antibody as capture antibody and rabbit polyclonal anti-alpha synuclein (ProteinTech, 10,842–1-AP) as detection antibody.

Techniques: Blocking Assay, Binding Assay, Clone Assay, Incubation, Staining, Negative Control, Concentration Assay, Activity Assay

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

Article Title: TrkB neurotrophic activities are blocked by α-synuclein, triggering dopaminergic cell death in Parkinson’s disease

doi: 10.1073/pnas.1713969114

Figure Lengend Snippet: α-Synuclein selectively interacts with TrkB receptors. (A) α-Syn specifically interacts with TrkB receptors. GST pull-down assay was conducted from HEK293 cells cotransfected with mammalian GST–α-Syn and HA-Trks. (B) α-Syn N terminus is implicated in binding TrkB. Different mGST-tagged α-Syn truncated were cotransfected with HA-TrkB into HEK293 cells. A GST pull-down assay was performed, and coprecipitated proteins were analyzed by immunoblotting with anti-HA (Top). Schematic diagram of α-Syn truncations (Bottom). (C) TrkB kinase domain is indispensable for α-Syn to interact with TrkB. (Top) Mapping assay for TrkB ICD required for binding to α-Syn. (Bottom) Schematic diagram of TrkB domains. (D) BDNF inhibits α-Syn/TrkB association. Cortical neurons were pretreated with K252a (100 nM) for 15 min, followed by BDNF treatment (50 ng/mL) for 30 min. Coimmunoprecipitation was performed with anti–α-Syn, and the coprecipitated proteins were analyzed by immunoblotting with anti-TrkB (Top). Cell lysates were probed with various antibodies (second through bottom panels). (E) α-Syn associates with TrkB in LBD human patient brains. Brain lysates from LBD patients were immunoprecipitated with control IgG or anti–α-Syn, and the coprecipitated proteins were analyzed by immunoblotting with anti-TrkB. (F) TrkB colocalizes with α-Syn in the LBs of PD patients. Immunofluorescent costainings with anti-TrkB or p-TrkB 706 (Green) and α-Syn (Red) were conducted with human PD brain sections. The nuclei were stained with DAPI. (Scale bar, 20 μm.)

Article Snippet: Primary antibodies to the following targets were used: GST-HRP, alpha-tubulin, beta-actin (Sigma-Aldrich), HA, myc, GFP, PY99, anti–alpha-synuclein, S129, TrkB, TrkC14, Ubiquitin P4D1 (Santa Cruz), anti-Y125, His (GE healthcare), pMAPK/MAPK, pAKT S473/AKT, Fyn, PARP, (Cell Signaling), anti-TH (Sigma-Aldrich), alpha-synuclein LB509 (Thermo Fisher Scientific), and anti-BDNF (Novus Biologicals).

Techniques: Pull Down Assay, Binding Assay, Western Blot, Mapping Assay, Immunoprecipitation, Control, Staining

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: A schematic presentation of the PAC (27M07) used to generate the transgenic mice lines. The organization of the human SNCA locus that is included in the PAC at the top panel. The three distinct SNCA -Rep1 genotypes are indicated below.

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Transgenic Assay

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: Serial analyses to confirm the mouse lines in the study. Presented is an example analysis performed for transgenic line ‘A’, carrying the human SNCA -Rep1 261 bp allele. ( A ) FISH analysis of metaphase chromosomes obtained from the mouse's spleen was used to determine the insertion site and to estimate the range of the copy number. Red-labeled human-PAC 27M07; green-labeled mouse BAC corresponding to mouse chromosome 9, used as a reference for copy number estimation; blue-DAPI. ( B ) RT–PCR to the 27M07 PAC was used to confirm the RNA expression of the human SNCA in the brain of an F1 mouse. ( C ) Western blot using the mouse monoclonal LB509 anti-human-SNCA (Zymed), to confirm the protein expression of the human SNCA in the brain of an F1 mouse. For (B) and (C), each lane represents analysis performed using a mouse-brain sample harvested from the following mouse lines: Rep1 261-A, the 261/261 line ‘A’ transgenic; PAC, control PAC containing the original Rep1 allele transgenic ; Wt, wild type FVB; hSNCA, recombinant human SNCA protein.

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Transgenic Assay, Labeling, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Western Blot, Expressing, Control, Recombinant

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: Effect of the SNCA -Rep1 promoter genotypes on human SNCA -mRNA expression levels in transgenic mouse brains. ( A ) Validation curve of the Δ real-time assay for relative quantization of human SNCA -mRNA relative to mouse Syp-mRNA in the brain. Relative efficiency plots of human- SNCA and mouse- Syp were formed by plotting the log input amount (ng of total RNA) versus the ΔCt = [Ct( SNCA )−Ct( Syp )]. The slope is 0.045, which indicated the validation of the ΔCt calculation in the range of 0.1–100 ng RNA. ( B ) Fold levels of human SNCA -mRNA were assayed by real-time RT–PCR and calculated relative to mouse Syp -mRNA reference control using the 2 −ΔCt method. No detectable products of the human SNCA -mRNA reaction were observed when WT mouse brain-cDNA was used as template. The risk genotype 261/261 correlates with significant higher SNCA -mRNA levels than the protective genotype 259/259 ( P < 0.0001). The deleted SNCA -Rep1 genotype correlates with significant lower SNCA -mRNA levels than genotypes 261/261 and 259/259 ( P < 0.0001). In total, 72 mice were analyzed; for each genotype (261/261, 259/259 and Δ/Δ), the box plot represents the analysis performed using two transgenic lines, 12 animals from each line (6 males and 6 females), comprising 24 total brain samples per genotype, each of which was analyzed twice independently. The average values are presented by ‘X’. The box plot shows the median (horizontal line inside the box) and the 25th and 75th percentiles (horizontal borders of the box). The range between the 25th and 75th percentiles is the interquartile range. The whiskers show the minimal and maximal values inside the main data body.

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Expressing, Transgenic Assay, Biomarker Discovery, Quantitative RT-PCR, Control

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: Analyzing SNCA proteins in F2 transgenic mouse brains. Representative western blotting ( A ) and ELISA ( B ) results from 24 age- and gender-matched F2 transgenic mouse brains are shown for the three distinct genotypes in six mouse lines. Specimens were assayed with a combination of polyclonal (pAb) and monoclonal (mAb) anti-SNCA antibodies. Equal volumes of whole-brain extracts were either directly loaded onto reducing SDS–PAGE (A) or first adjusted for their total protein content, then diluted at 1: 5000 and subsequently loaded onto ELISA plates (B). Sandwich ELISA (hSA-3/211-B; red bars to the right of each pair) and ELISA (hSA-3/Syn1-B; blue bars on the left of each pair) were carried out on the same 384-well plate in triplicates. Note the near identical monitoring of the recombinant human SNCA protein standards by both assays (left); the absence of any absorbance signal in snca -null (KO) mouse brain by either assay and the absence of signal with the human SNCA-specific ELISA in WT (non-transgenic) mouse brain (center); the fact that the total SNCA signal measuring murine and human protein (blue bars, left) is always stronger than the human-specific signal alone (red bars, right).

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Transgenic Assay, Western Blot, Enzyme-linked Immunosorbent Assay, SDS Page, Sandwich ELISA, Recombinant

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: SNCA - Rep1 expansion increases SNCA protein concentration in mammalian brain. ( A ) Quantification of human SNCA protein in transgenic mouse brain homogenates using sandwich ELISA [hSA-3/211-B] and a 384-well plate format (triplicates; 50 µl/well); a total of 72 mice were analyzed. Concentration values (in ng/µl) were interpolated from the standard curve ( Supplementary Material, Fig. S3A ) and then corrected for the calculated gene copy number in each mouse line. Values were grouped according to the three genotypes examined, i.e. Rep1-261 bp; Rep1-259 bp; Δ-Rep1 (ratio: female:male, 50:50 in all lines). ( B ) Relative ratio of the human SNCA concentration (as shown in A) to the total SNCA protein concentration. The latter was measured using sandwich ELISA [hSA3/Syn1-B] on 384-well plates quantifying both mouse Snca and human SNCA ( Supplementary Material, Fig. S3B ; after correction for gene copy number: Supplementary Material, Fig. S3C ). Bar graph shown in (B) represents values obtained in (A) divided by those shown in Supplementary Material, Fig. S3C .

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Protein Concentration, Transgenic Assay, Sandwich ELISA, Concentration Assay

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: Effect of the SNCA -Rep1 promoter genotypes on human SNCA -mRNA expression levels in transgenic mouse whole blood. ( A ) Validation curve of the Δ real-time assay for relative quantization of human SNCA -mRNA relative to mouse Gapdh -mRNA in blood. Relative efficiency plots of human- SNCA and mouse- Gapdh were formed by plotting the log input amount (ng of total RNA) versus the ΔCt = [Ct( SNCA )−Ct( Gapdh )]. The slope is 0.1, which indicated the validation of the ΔCt calculation in the range of 0.1–100 ng RNA. ( B ) Fold levels of human SNCA -mRNA were assayed by real-time RT–PCR and calculated relative to mouse Gapdh -mRNA reference control using the 2 −ΔCt method. No detectable products of the human SNCA -mRNA reaction were observed when WT mouse blood-cDNA was used as template. The risk genotype, 261/261, correlates with higher SNCA -mRNA levels than the protective genotype, 259/259. The deleted SNCA -Rep1 genotype shows no significant correlation with SNCA -mRNA levels in comparison with genotypes 261/261 and 259/259 ( P = 0.524). In total, 72 mice were analyzed; for each genotype (261/261, 259/259 and Δ/Δ), the box plot represents the analysis performed using two transgenic lines, 12 animals from each line (6 males and 6 females); overall, 24 whole-blood samples (per genotype) were each repeated twice, independently. The average values are presented by ‘X’. The box plot shows the median (horizontal line inside the box) and the 25th and 75th percentiles (horizontal borders of the box). The range between the 25th and 75th percentiles is the interquartile range. The whiskers show the minimal and maximal values inside the main data body.

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Expressing, Transgenic Assay, Biomarker Discovery, Quantitative RT-PCR, Control, Comparison

Journal: Human Molecular Genetics

Article Title: Expansion of the Parkinson disease-associated SNCA- Rep1 allele upregulates human α-synuclein in transgenic mouse brain

doi: 10.1093/hmg/ddp265

Figure Lengend Snippet: SNCA - Rep1 expansion does not lead to detectable SNCA protein concentration differences in mouse blood. ( A ) Quantification of human SNCA protein in transgenic mouse blood homogenates using sandwich ELISA [hSA-3/211-B] and 384-well plate format (triplicates; 50 µl/well); a total of 72 mice were analyzed. Signals were specific for human SNCA protein because whole blood from WT and snca -knockout mice did not generate ELISA signals above the background ( Supplementary Material, Fig. S4A ). Values were then corrected for the calculated gene copy number in each mouse line and grouped according to the three genotypes examined, i.e. Rep1-261 bp; Rep1-259 bp; Δ-Rep1 (ratio: female:male, 50:50 in all lines). ( B ) Quantification of total SNCA protein concentration levels in transgenic mouse blood was carried out with sandwich ELISA [hSA3/Syn1-B] and 384-well plates. This ELISA measures both mouse snca and human SNCA proteins ( Supplementary Material, Fig. S4B ); values (in ng/µl) were then corrected for gene copy number.

Article Snippet: Of note is that three internal controls were compared: mouse glyceraldehyde-3-phosphate dehydrogenase ( Gapdh Mm99999915_g1), mouse alpha-synuclein ( Snca Mm00447331_m1), mouse enolase 2 ( Eno2 Mm00469062_m1) and synaptophysin ( Syp Mm00436850_m1) using different total RNA amounts (0.1–100 ng) from whole-brain tissue and whole-blood samples of a transgenic mouse.

Techniques: Protein Concentration, Transgenic Assay, Sandwich ELISA, Knock-Out, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Prognostic stratification of sepsis through DNA damage response based RiskScore system: insights from single-cell RNA-sequencing and transcriptomic profiling

doi: 10.3389/fimmu.2024.1345321

Figure Lengend Snippet: Validation of characteristic genes in vivo (A) Relative expression levels of DDR-related genes in control and sepsis groups (n=4 in each group). (B) Relative expression levels of ARL4C in control, sepsis, sepsis+Ad-shNC, and sepsis+Ad-shARL4C group (n=8 in each group). (C) Expression levels of inflammatory factors (IL-1β, TNF-α, IL-10, and IL-18) in the peripheral blood of rat with sepsis+Ad-shNC and sepsis+Ad-shARL4C (n=8 in each group). (D) Survival status of rats in each group (n=10 in each group). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. NS, no significant difference.

Article Snippet: In summary, adenoviral vectors containing shRNA sequences specifically targeting the ARL4C gene in rats (Ad-shARL4C) alongside a non-targeting control shRNA sequence (Ad-shNC), both sourced from RiboBio in Guangzhou, China, were utilized.

Techniques: Biomarker Discovery, In Vivo, Expressing, Control

Journal: Frontiers in Immunology

Article Title: Prognostic stratification of sepsis through DNA damage response based RiskScore system: insights from single-cell RNA-sequencing and transcriptomic profiling

doi: 10.3389/fimmu.2024.1345321

Figure Lengend Snippet: Validation of ARL4C in vitro . (A) Flow cytometry detected the apoptosis rate in the Control, Sepsis, Sepsis+Lv-shNC, and Sepsis+Lv-shARL4C groups. (n=4 in each group) (B) Flow cytometry detected the ROS production in the Control, Sepsis, Sepsis+Lv-shNC, and Sepsis+Lv-shARL4C groups. (n=4 in each group), *** p < 0.001, **** p < 0.001. NS, no significant difference.

Article Snippet: In summary, adenoviral vectors containing shRNA sequences specifically targeting the ARL4C gene in rats (Ad-shARL4C) alongside a non-targeting control shRNA sequence (Ad-shNC), both sourced from RiboBio in Guangzhou, China, were utilized.

Techniques: Biomarker Discovery, In Vitro, Flow Cytometry, Control

Journal: IBRO Neuroscience Reports

Article Title: Phosphorylated alpha-synuclein distribution in the colonic enteric nervous system of patients with diverticular disease

doi: 10.1016/j.ibneur.2025.02.009

Figure Lengend Snippet: Comparison of Age, Gender Distribution, and P-aSyn Immunoreactivity Across Control, Diverticulosis (Dose), and Diverticulitis (Ditis) Cohorts. P-aSyn IR: phosphorylated alpha-synuclein immunoreactivity.

Article Snippet: Briefly, after deparaffinization in xylol and rehydration in alcohol with descending concentrations, tissue sections were pretreated with citrate buffer (pH 6.0, 95°C, water bath) for 25 minutes, followed by overnight incubation with rabbit-anti-phosphorylated alpha-synuclein antibody (1/1000, D1R1R, Cell signaling Technology) in antibody diluent (Thermo Fisher Scientific, Carlsbad, USA).

Techniques: Comparison, Control

Journal: NPJ Parkinson's Disease

Article Title: Antiviral innate immunity induces alpha synuclein phosphorylation at serine129 in neurons independent of aggregation

doi: 10.1038/s41531-026-01297-9

Figure Lengend Snippet: a Immunofluorescence analysis (IFA) of brain tissue from patients with West Nile virus (WNV) encephalitis (n = 5 patients) compared to uninfected control brain tissue ( n = 4, bar=50 μm) from the midbrain. IFA analysis of individual sections for b mean fluorescence intensity of pS129 asyn, c Pearson co-localization coefficient measurement of pS129 asyn and MAP2 expression, and d Pearson co-localization coefficient measurement of Stat2 with pS129 asyn. * p = <0.05, Welch’s t -test, 2-tailed.

Article Snippet: Cells were permeabilized with 0.3% Triton X-100 in DPBS and blocked with blocking buffer (5% goat serum DPBS solution) for 1 h. Primary neurons were stained with primary antibodies against pS129 alpha synuclein (Cell Signaling, 23706) and West Nile Virus Envelope protein (Thermo Fisher, MA5-35917) in blocking buffer overnight at 4 °C.

Techniques: Immunofluorescence, Virus, Control, Fluorescence, Expressing

Journal: NPJ Parkinson's Disease

Article Title: Antiviral innate immunity induces alpha synuclein phosphorylation at serine129 in neurons independent of aggregation

doi: 10.1038/s41531-026-01297-9

Figure Lengend Snippet: Wild-type mice were inoculated with WNV 10 4 pfu, i.n., and olfactory bulb tissue was isolated for a Western blot analysis and b ELISA analysis for alpha synuclein aggregate formation. Each band of the western blot is an individual mouse. Values below each Western blot row indicate densitometry values. SVV-infected macaques underwent necropsy during acute SVV infection, and dorsal root ganglia were isolated. Dorsal root ganglia tissue from c mock-infected (top panel) and SVV-infected macaques (bottom panel) was analyzed for pS129 asyn expression (brown) using immunohistochemistry. d Image analysis of mean pixel intensity for pS129 asyn expression in SVV antigen-negative vs SVV antigen-positive samples. Each data point represents an independent section from 3 individual macaques per group, * p = 0.0002, t -test, and bar = 100 μm. e , f Wild-type C57BL/6 mice were inoculated with 10 5 PFU HSV-1 by corneal inoculation, and the trigeminal ganglia were isolated for histological analysis at 7 days for acute infection and 28 days for latent infection analysis. e Tissue sections were analyzed for pS129 asyn expression (brown) in trigeminal ganglia during acute HSV infection, latent infection, mock inoculation, and in infected tissue labeled with an isotype control antibody. f Image analysis of mean pixel intensity for pS129 asyn expression in SVV-negative vs SVV-positive samples. Each data point represents an independent section from 3 individual macaques per group, * p < 0.05, t -test, and bar = 100 μm.

Article Snippet: Cells were permeabilized with 0.3% Triton X-100 in DPBS and blocked with blocking buffer (5% goat serum DPBS solution) for 1 h. Primary neurons were stained with primary antibodies against pS129 alpha synuclein (Cell Signaling, 23706) and West Nile Virus Envelope protein (Thermo Fisher, MA5-35917) in blocking buffer overnight at 4 °C.

Techniques: Olfactory, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Immunohistochemistry, Labeling, Control

Journal: NPJ Parkinson's Disease

Article Title: Antiviral innate immunity induces alpha synuclein phosphorylation at serine129 in neurons independent of aggregation

doi: 10.1038/s41531-026-01297-9

Figure Lengend Snippet: a Experimental flow of WNV (MOI 10) inoculation and analysis of pS129 asyn and asyn aggregates. b Representative ISG expression over acute time points from 4 to 48 h post-infection (hpi) with WNV. n = 3 experimental replicates per group, per time point. Primary neurons were treated with mock inoculation or WNV (MOI 10) and analyzed at 4–48 h post-infection for expression of c pS129 asyn ( n = 57), and d total asyn aggregates ( n = 3 per time point). The dotted line indicates the limit of detection. Kruskal–Wallis test with Dunn’s correction for multiple comparisons. ** p = 0.001, **** p < 0.0001. e Primary n e urons were treated with mock or WNV (MOI 10) inoculum and then analyzed using IFA for DAPI (blue), pS129 asyn (red), ThioflavinS (green), or WNV Eprotein (white). n = 3 replicates (4 images per condition were analyzed). 40× original image magnification. f Corrected total cell fluorescence values (CTCF) for pS129 Asyn expression in WNV-inoculated neurons increased at 24 h compared to mock controls. n = 8 experimental replicates, * p = 0.02, One-way ANOVA with Tukey’s multiple comparisons test. F = 3.95.

Article Snippet: Cells were permeabilized with 0.3% Triton X-100 in DPBS and blocked with blocking buffer (5% goat serum DPBS solution) for 1 h. Primary neurons were stained with primary antibodies against pS129 alpha synuclein (Cell Signaling, 23706) and West Nile Virus Envelope protein (Thermo Fisher, MA5-35917) in blocking buffer overnight at 4 °C.

Techniques: Expressing, Infection, Fluorescence

Journal: NPJ Parkinson's Disease

Article Title: Antiviral innate immunity induces alpha synuclein phosphorylation at serine129 in neurons independent of aggregation

doi: 10.1038/s41531-026-01297-9

Figure Lengend Snippet: Primary cortical neurons were treated with Poly I:C (25 μg/mL) and analyzed for a ISG expression ( n = 6) and b pS129 asyn protein expression over the indicated time points post-treatment. n = 18 replicates per time point per group. **** p < 0.0001, *** p < 0.0008. Kruskal–Wallis with Dunn’s multiple comparison test. c , d Corrected total cell fluorescence analysis of Poly I:C treated neurons at 4, 8, & 24 h post-administration (hpa) for pS129 asyn (red), ThioflavinS (green), and DAPI (blue). n = 4 experimental replicates (4 images per condition were analyzed). * p = 0.03, One-way ANOVA with Tukey’s multiple comparison test. F = 4.88.

Article Snippet: Cells were permeabilized with 0.3% Triton X-100 in DPBS and blocked with blocking buffer (5% goat serum DPBS solution) for 1 h. Primary neurons were stained with primary antibodies against pS129 alpha synuclein (Cell Signaling, 23706) and West Nile Virus Envelope protein (Thermo Fisher, MA5-35917) in blocking buffer overnight at 4 °C.

Techniques: Expressing, Comparison, Fluorescence

Journal: NPJ Parkinson's Disease

Article Title: Antiviral innate immunity induces alpha synuclein phosphorylation at serine129 in neurons independent of aggregation

doi: 10.1038/s41531-026-01297-9

Figure Lengend Snippet: Primary cortical neurons were treated with interferon-alpha (100 ng/mL) and were harvested at indicated time points for ELISA to measure a ISG expression ( n = 3–9), b pS129 asyn expression ( n = 29), and c asyn aggregate formation ( n = 9). The dotted line indicates the limit of detection. * p = 0.02, ** p = 0.001. One-way ANOVA with Tukey’s multiple comparisons test. d Primary cortical neurons were treated with interferon-alpha-a2 (100 ng/mL) and analyzed at the indicated time points by IFA for expression of pS129 asyn (red), and thioflavinS (green). n = 3 independent experimental replicates per experimental condition. Images are 40× original magnification, bar = 100 μM. e Corrected total cell fluorescence analysis of pS129 asyn expression at the indicated time points. * p = 0.005, One-Way ANOVA with Tukey’s multiple comparison test, F = 6.45. Primary cortical neurons from f WT mice and g interferon receptor-deficient mice (Ag129 mice) were treated with interferon-alpha-a2 and analyzed at 30 min for expression of pS129 asyn. n = 27–36 replicates. **** p < 0.0001. ns = not significant ( p = 0.32). Two-tailed, Welch’s t -test. Bar = 50 μM. h Primary cortical neurons (CN) were isolated from mice containing type 1 and type II interferon receptor knockout (Ag129). Neurons were treated with mock inoculum, WNV (MOI 10), and Poly I:C (PIC, 25 μg/mL) and analyzed for pS129 asyn expression at 4 h post-treatment. n = 27 = 32, ns = not significant ( p = 0.63), One-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Cells were permeabilized with 0.3% Triton X-100 in DPBS and blocked with blocking buffer (5% goat serum DPBS solution) for 1 h. Primary neurons were stained with primary antibodies against pS129 alpha synuclein (Cell Signaling, 23706) and West Nile Virus Envelope protein (Thermo Fisher, MA5-35917) in blocking buffer overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence, Comparison, Two Tailed Test, Isolation, Knock-Out