Journal: Nature Communications

Article Title: Gut microbiota produces biofilm-associated amyloids with potential for neurodegeneration

doi: 10.1038/s41467-024-48309-x

Figure Lengend Snippet: a α-Syn aggregation in the presence of PBS ( N = 3), rBapB-PFF ( N = 4), rEspN-PFF ( N = 4) and α-Syn-PFF ( N = 3) (10:1 molar ratio) as measured by Th-T fluorescence over time. Time to reach exponential lag phase ( b) and half maximum ( c ). Data were analyzed using one-way ANOVA with Dunnett’s multiple comparison test. *** p < 0.0001. d Immunostaining of α-Syn in SH-SY5Y treated with BAP amyloids for 10 days. Higher magnifications of the highlighted regions are shown. Scale bars, 5 μm. e Quantification of α-Syn inclusions per cell. SH-SY5Y were treated with rBapB-PFF ( N = 34) and rEspN-PFF ( N = 37). As negative controls monomeric rBapB-MON ( N = 32), rEspN-MON ( N = 30) and the nonamyloid domain rBap_CM2 ( N = 20) were used. Ø, non-treated ( N = 59). f Colocalization of BapB and α-Syn using Bap-PST (left panels) or anti-Bap antibodies (right panels). Higher magnifications of the highlighted regions are shown in the lower panels. Scale bars, 5 μm. g Percentage of SH-SY5Y cells positive for pS129-α-Syn. Graph represents the percentage of pS129-α-Syn positive cells per microscopic field analyzed. rBapB-PFF (fields analyzed N = 23, cells N = 329); rEspN-PFF (fields analyzed N = 25, cells N = 310); Ø, non-treated (fields analyzed N = 21, cells N = 328). h Representative images of pS129-α-Syn (red) and total α-Syn (green) immunostaining. Higher magnifications of highlighted regions are shown in lower panels. Scale bars, 5 μm. i Detection of α-Syn in SH-SY5Y cell extracts treated with BAP amyloids. Stain-free gel portions are shown as a loading control (LC). MW; molecular weight. Data were analyzed using one-way ANOVA (*** p < 0.0001) with Bonferroni’s multiple comparison test ( e – g ). For all panels, data are shown as means, and error bars are shown as the SE of means. * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Article Snippet: Phosphorylated α-Syn was detected with a rabbit anti-pS129-α-Syn EP1536Y primary antibody (Abcam; ab51253) diluted 1:200 (v:v).

Techniques: Fluorescence, Comparison, Immunostaining, Staining, Molecular Weight

Journal: Translational Neurodegeneration

Article Title: Microstructural integrity of the locus coeruleus and its tracts reflect noradrenergic degeneration in Alzheimer’s disease and Parkinson’s disease

doi: 10.1186/s40035-024-00400-5

Figure Lengend Snippet: Flow chart of the study. After donor inclusion, post-mortem in situ 3D T1 and multi-shell diffusion MRI images were collected and (pre)processed: the LC was segmented and the LC tracts to the anterior cingulate cortex (ACC, blue tract), the dorsolateral prefrontal cortex (DLPFC, purple tract), the primary motor cortex (M1, green tract) and the hippocampus (yellow tract) were reconstructed, deriving the FA and the MD of the LC and its tracts. Autopsy and brain dissection were performed after the in situ MRI scans. Brain tissue blocks were formalin-fixated for 4 weeks, then dissected and paraffin-embedded. Next, 20-µm-thick sections were processed for immunohistochemistry for dopamine-beta hydroxylase (DBH), phosphorylated Ser129 α-synuclein (pSer129-αsyn), phosphorylated-tau (p-tau) and amyloid-β (Aβ), and imaged using a whole-slide scanner. Immunoreactivity in regions of interest were analyzed using Qupath, deriving the LC-noradrenergic cell density and fiber load, Lewy body (LB) density and Lewy neurite (LN) load. The group comparisons between AD, PD and controls were investigated with general linear models and the association between MRI and pathology markers was investigated with linear regression models, including age, sex and post-mortem delay as covariates. Abbreviations: DWI, diffusion-weighted tensor imaging; LC, locus coeruleus; NA, noradrenergic; IHC, immunohistochemistry; FA, fractional anisotropy; MD, mean diffusivity; LB, Lewy body; LN, Lewy neurite; PMD, post-mortem delay

Article Snippet: In brief, paraffin-embedded tissue blocks of the LC were cut into 4 × 20 µm-thick consecutive sections, and stained with monoclonal rabbit anti-dopamine-beta hydroxylase (DBH, dilution 1:400, Abcam, Cambridge, UK), rabbit anti-phosphorylated-Ser129 α-syn (clone EP1536Y, dilution 1:4000, Abcam), mouse anti-p-tau (clone AT8, dilution 1:800, ThermoFisher, Pittsburgh, PA) or mouse anti-Aβ (clone 4G8, dilution 1:5000, BioLegend, San Diego, CA) antibody.

Techniques: In Situ, Diffusion-based Assay, Dissection, Immunohistochemistry, Imaging

Journal: STAR Protocols

Article Title: Optogenetic-mediated induction and monitoring of α-synuclein aggregation in cellular models of Parkinson’s disease

doi: 10.1016/j.xpro.2023.102738

Figure Lengend Snippet: LIPA-α-syn inclusions are positive to authentic Lewy body (LB) markers Confocal images of HEK293T cells overexpressing LIPA-α-syn exposed to blue light for 12 h (0.8 mW/mm 2 ) and stained with LB markers: ubiquitin, p62, Hsp70, and phosphorylated α-syn at S129 (pS129). (Scale bar = 10 μm).

Article Snippet: Rabbit, anti-alpha-synuclein (pS129) (EP1536Y); 1:2,000 (ICC) , Abcam , ab51253.

Techniques: Staining

Journal: STAR Protocols

Article Title: Optogenetic-mediated induction and monitoring of α-synuclein aggregation in cellular models of Parkinson’s disease

doi: 10.1016/j.xpro.2023.102738

Figure Lengend Snippet:

Article Snippet: Rabbit, anti-alpha-synuclein (pS129) (EP1536Y); 1:2,000 (ICC) , Abcam , ab51253.

Techniques: Recombinant, Transfection, Modification, Electron Microscopy, Protease Inhibitor, Lysis, Extraction, Software, Membrane, Chromatography, Microscopy, Confocal Laser Scanning Microscopy, Imaging, Dot Blot, Hybridization

Journal: PLoS ONE

Article Title: Improved Immunodetection of Endogenous α-Synuclein

doi: 10.1371/journal.pone.0023939

Figure Lengend Snippet: 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: As primary antibody, mouse monoclonal anti-α-syn antibodies 4D6 and LB509 (Santa Cruz Biotechnology) and rabbit monoclonal anti-phospho α-syn antibody EP1536Y (Epitomics, Burlingame, CA) were used at a dilution 1∶1,000, and rabbit polyclonal anti-actin antibody (Sigma) was also used at a dilution 1∶5,000.

Techniques: Western Blot, Membrane, Incubation

Journal: bioRxiv

Article Title: CK2 alpha prime and alpha-synuclein pathogenic functional interaction mediates inflammation and transcriptional dysregulation in Huntington’s disease

doi: 10.1101/2020.10.29.359380

Figure Lengend Snippet: A , Kruskal-Wallis test of module expressions between zQ175 (HD) mice and zQ175:CK2α’ (+/-) mice. The y-axis is the negative log transformed p-values. B , Expressions of module “Greenyellow” in each mouse sample. C , IPA canonical pathway analysis of module genes for module “Greenyellow”. D , Enrichment analysis of GO terms in CC (cellular component) for module genes of module “Greenyellow”. E , Network visualization of top 15% connected genes in “Greenyellow” Module. The size of the circles was scaled by the absolute value of the mean log2-fold change between zQ175 and zQ175:CK2α’ (+/-) mice. F , Marker genes and their mean log2 fold change between zQ175 and zQ175:CK2α’ (+/-) mice compared to WT. G , IPA network analysis of DGEs in zQ175:CK2α’ mice showing α-syn as the most significant upstream regulator.

Article Snippet: Primary antibodies were anti-CK2α’ (Rabbit, Novus NB100-379 and Proteintech 10606-1-AP, both 1:1000), anti-Iba1 (Rabbit, FUJIFILM Wako 019-19741, 1:1000), α-syn (Mouse, Biolegend 834303, 1:1000, clone 4D6), pS129-α-syn (Mouse, Millipore MABN826, 1:500, clone 81A and Rabbit, EP1536Y Abcam ab51253, 1:1000), GAPDH (Mouse, Santacruz sc-365062, 1:10000).

Techniques: Transformation Assay, Marker

Journal: bioRxiv

Article Title: CK2 alpha prime and alpha-synuclein pathogenic functional interaction mediates inflammation and transcriptional dysregulation in Huntington’s disease

doi: 10.1101/2020.10.29.359380

Figure Lengend Snippet: A, α-syn (4D6 antibody) IB in the striatum of WT, zQ175 and SNCA KO and B in WT, zQ175 and zQ175:CK2α’ (+/-) mice at 12 months old. GAPDH used as loading control. C, α-syn protein levels analyzed by Image J from IB analyses (n= 5-6 mice/genotype). D, Nuclear/cytoplasmic fractionation of striatum samples from 12-month-old WT, zQ175 and SNCA KO mice. E , Quantification of nuclear α-syn from images in D (n=4 mice/genotype). F , Representative IF images of dorsal striatum sections from 12 month old WT, zQ175 and zQ175:CK2α’ (+/-) (n=3 mice/genotype) for α-syn and HTT (EM48 antibody). White arrows indicate α-syn/HTT colocalization. Scale bar, 10 μm. G, Magnification of images from F showing nuclear and cytoplasmic α-syn and HTT colocalization. Grey circles represent nuclei. White arrows indicate α-syn/HTT colocalization. Scale bar, 2 μm. H Number of cytoplasmic and I nuclear EM48 + puncta calculated using Image J Puncta analysis plugin. A total of three images per brain section and three brain sections per genotype were analyzed (n=27 images, n=3 mice/ genotype). J , Number of colocalized α-syn and EM48 + puncta calculated using Image J Puncta analysis plugin (n=3 mice/genotype). Error bars denote mean ± SEM, values were analyzed by Student’s t-test.

Article Snippet: Primary antibodies were anti-CK2α’ (Rabbit, Novus NB100-379 and Proteintech 10606-1-AP, both 1:1000), anti-Iba1 (Rabbit, FUJIFILM Wako 019-19741, 1:1000), α-syn (Mouse, Biolegend 834303, 1:1000, clone 4D6), pS129-α-syn (Mouse, Millipore MABN826, 1:500, clone 81A and Rabbit, EP1536Y Abcam ab51253, 1:1000), GAPDH (Mouse, Santacruz sc-365062, 1:10000).

Techniques: Fractionation

Journal: bioRxiv

Article Title: CK2 alpha prime and alpha-synuclein pathogenic functional interaction mediates inflammation and transcriptional dysregulation in Huntington’s disease

doi: 10.1101/2020.10.29.359380

Figure Lengend Snippet: A, pS129-α-syn (EP1536Y antibody) IB in the striatum of 12-month-old WT, zQ175 and SNCA KO (n=4 mice/genotype) . B pS129-α-syn (81A antibody) IB in the striatum of patients with HD (Vonsattel grade 3 and 4) compared to age and sex matched controls. GAPDH is used as loading control. C pS129-α-syn protein levels analyzed by Image J from images in B . Samples from grade 3 and grade 4 HD were all grouped for pS129-α-syn quantification. D , Representative pS129-α-syn IF images (81A antibody) in the dorsal striatum of 12-month-old WT, zQ175 and zQ175:CK2α’ (+/-) (n=3 mice/genotype), Scale bar, 20 μm. E, pS129-α-syn fluorescence signal was calculated using Image J from images in D (n=3 mice/genotype, n=27 images per mouse). F , Magnification of images in D showed pS129-α-syn and EM48 colocalization in zQ175 and zQ175:CK2α’ (+/-) (n=3 mice/group). G , Quantification of pS129-α-syn and EM48 colocalized puncta using Image J puncta plug in. All data are mean ± SEM. Statistical analyses were conducted by one-way ANOVA. H , Working model for the role of CK2α’ in the regulation of pS129-α-syn and HD-like phenotype.

Article Snippet: Primary antibodies were anti-CK2α’ (Rabbit, Novus NB100-379 and Proteintech 10606-1-AP, both 1:1000), anti-Iba1 (Rabbit, FUJIFILM Wako 019-19741, 1:1000), α-syn (Mouse, Biolegend 834303, 1:1000, clone 4D6), pS129-α-syn (Mouse, Millipore MABN826, 1:500, clone 81A and Rabbit, EP1536Y Abcam ab51253, 1:1000), GAPDH (Mouse, Santacruz sc-365062, 1:10000).

Techniques: Fluorescence