Journal: Translational Neurodegeneration

Article Title: Cerebrospinal fluid α-synuclein predicts neurodegeneration and clinical progression in non-demented elders

doi: 10.1186/s40035-020-00222-1

Figure Lengend Snippet: Scatter plots of cerebrospinal fluid α-synuclein concentrations in the diagnostic groups. The colors of the scatterplots are grouped by different diagnostic groups. The three horizontal black lines in each boxplot indicate the median and interquartile range. The whiskers extend to the minimum and maximum CSF α-synuclein data points. a CSF α-synuclein concentration in the diagnostic groups. b CSF α-synuclein concentration in the diagnostic groups stratified by Aβ pathology. c CSF α-synuclein concentration in the AD pathophysiology (tau and amyloid-β) positive and negative subgroups. A-, Aβ negative; A+, Aβ positive; T-, tau negative; T+, tau positive

Article Snippet: The levels of CSF total α-synuclein concentrations in the ADNI cohort were measured by the Luminex MicroPlex Microspheres (Luminex Corp, Austin, TX), using the biotinylated goat anti-human α-syn antibody (R&D systems, Minneapolis, MN) as the detection antibody.

Techniques: Diagnostic Assay, Concentration Assay

Journal: Translational Neurodegeneration

Article Title: Cerebrospinal fluid α-synuclein predicts neurodegeneration and clinical progression in non-demented elders

doi: 10.1186/s40035-020-00222-1

Figure Lengend Snippet: Associations between CSF α-synuclein and longitudinal neuroimaging change in ADNI. Data from linear mixed-effects models (left) and cox proportional hazards models (right) were adjusted for age, gender, educational level and APOE ε4 genotype. Hippocampal volume on the y axis was log-transformed to ensure normality. Time (years) on the x axis indicated follow-up years, in which “0” indicating the baseline. All participants were classified into High and Low groups according to their baseline CSF α-synuclein concentration (High: greater than median; Low: less than median)

Article Snippet: The levels of CSF total α-synuclein concentrations in the ADNI cohort were measured by the Luminex MicroPlex Microspheres (Luminex Corp, Austin, TX), using the biotinylated goat anti-human α-syn antibody (R&D systems, Minneapolis, MN) as the detection antibody.

Techniques: Transformation Assay, Concentration Assay

Journal: Molecular neurodegeneration

Article Title: Oligomerization of Lrrk controls actin severing and α-synuclein neurotoxicity in vivo.

doi: 10.1186/s13024-021-00454-3

Figure Lengend Snippet: Fig. 1 Modulation of Lrrk enhances α-synuclein neurotoxicity in Drosophila. a, Increasing or decreasing Drosophila Lrrk worsens the locomotor climbing defect induced by the expression of transgenic human wild type human α-synuclein, which is further enhanced by expressing mutant forms of Lrrk engineered to mimic Parkinson’s disease mutations. b, Altering Lrrk levels further enhances α-synuclein-mediated loss of hematoxylin-stained neurons in the anterior medulla, as quantified in (c) when compared to flies expressing α-synuclein alone. Scale bar represents 10 µm in (b). d, Increasing or decreasing Lrrk levels also further decreases the α-synuclein-induced loss of tyrosine hydroxylase-positive neurons in the anterior medulla (arrows), as quantified in (e) when compared to flies expressing α-synuclein alone. Scale bar represents 5 µm in (d). f, Western blotting reveals no change in α-synuclein levels with manipulation of Lrrk. The blot is reprobed for GAPDH to illustrate equivalent protein loading. Lrrk- : Lrrke03680. Lrrk-WT: wild type Lrrk overexpression. Y1383C, I1915T and GS are Lrrk mutants homologous to Parkinson’s disease-associated human LRRK2 mutants Y1699C, I2020T, and G2019S respectively. n=6 per genotype. Data are represented as mean ± SD. *p<0.05, **p<0.01, ***p<0.005, ANOVA with Bonferroni post-test analysis. Control is nSyb-GAL4, nSybQF2/+. Flies are 10 days old in (a-d) and 1 day old in (f)

Article Snippet: Primary antibodies were used at the indicated concentrations: α-synuclein (H3C), 1:350,000, Developmental Studies Hybridoma Bank; LRRK2 (N214A/34), 1: 200, NeuroMab; GAPDH, 1:10,000, Abcam; HA-11, 1:2, 000, Covance.

Techniques: Expressing, Transgenic Assay, Mutagenesis, Staining, Western Blot, Over Expression, Control

Journal: Molecular neurodegeneration

Article Title: Oligomerization of Lrrk controls actin severing and α-synuclein neurotoxicity in vivo.

doi: 10.1186/s13024-021-00454-3

Figure Lengend Snippet: Fig. 2 Manipulation of Lrrk enhances actin cytoskeletal pathology induced by α-synuclein. a, F-actin staining with fluorescent phalloidin in whole mount brains from flies expressing human α-synuclein shows increased F-actin compared to controls. F-actin is further increased when Lrrk protein is removed in homozygous Lrrke03680 mutants, or mutant Lrrk-G1914S (Lrrk-GS), homologous to the human G2019S LRRK2 mutant, is expressed, as quantified in (b) when compared to flies expressing α-synuclein alone. Scale bar in (a) represents 75 µm. c, Immunofluorescent staining with an antibody to actin demonstrates rod-like inclusions when human α-synuclein is expressed in the fly brain. Numbers of rods are increased when Lrrk is removed or Lrrk-GS is expressed (arrows), as quantified in (d) when compared to flies expressing α-synuclein alone. Scale bar in (c) represents 15 µm. n=6 per genotype. Data are represented as mean ± SD. *p<0.05, **p<0.01,***p<0.005, ANOVA with Bonferroni post- test analysis. Control is nSyb-GAL4, nSybQF2/+. Flies are 10 days old

Article Snippet: Primary antibodies were used at the indicated concentrations: α-synuclein (H3C), 1:350,000, Developmental Studies Hybridoma Bank; LRRK2 (N214A/34), 1: 200, NeuroMab; GAPDH, 1:10,000, Abcam; HA-11, 1:2, 000, Covance.

Techniques: Staining, Expressing, Mutagenesis, Control

Journal: Molecular neurodegeneration

Article Title: Oligomerization of Lrrk controls actin severing and α-synuclein neurotoxicity in vivo.

doi: 10.1186/s13024-021-00454-3

Figure Lengend Snippet: Fig. 5 Expression of a clinically protective Lrrk mutant reduces α-synuclein neurotoxicity and decreases Lrrk oligomerization. a, Expression of Lrrk- Q1003H, analogous to the protective human LRRK2-R1398H variant, rescues the locomotor dysfunction produced by expression of α-synuclein. b- c, Lrrk-Q1003H suppresses the loss of medullary hematoxylin-stained neurons (b), as quantified in (c), and tyrosine hydroxylase immunostained neurons (d arrows), as quantified in (e) when compared to flies expressing α-synuclein alone. Scale bars represent 10 µm in (b) and 5 µm in (d). n=6 per genotype. f, Western blotting reveals no change in transgenic human α-synuclein levels with expression of Lrrk-Q1003H. The blot is reprobed for GAPDH to illustrate equivalent protein loading. g, Increased levels of F-actin as assayed by phalloidin staining on whole mount brains (top) and quantified (bottom) are reversed by expressing Lrrk-Q1003H. Scale bar in (g) represents 75 µm. h, Similarly, the formation of actin rods (top arrows) and quantified (bottom) is also rescued by expression of Lrrk-Q1003H when compared to flies expressing α-synuclein alone. Scale bar in (h) represents 15 µm. i,j, The α-synuclein induced decline in OCR is partially rescued by expression of Lrrk-Q1003H when compared to flies expressing α-synuclein alone. k, Native gel electrophoresis followed by western blotting (k) and quantitative analysis (l) demonstrates a significant reduction in the ratio of Lrrk multimer to monomer in flies expressing Lrrk-Q1003H compared to Lrrk-HA control flies. Data are represented as mean ± SD. n=3-6 per genotype. *p<0.05, ** p<0.01, ***p<0.005, ns, not significant, ANOVA with Bonferroni post-test analysis. Control is nSyb-GAL4, nSybQF2/+ in (a-j). Control is nSyb-GAL4/+ in (k). Lrrk-HA is LrrkHA, nSyb-GAL4/+ in (k,l). Flies are 10 days old in (a-e, g-l) and 1 day old in (f)

Article Snippet: Primary antibodies were used at the indicated concentrations: α-synuclein (H3C), 1:350,000, Developmental Studies Hybridoma Bank; LRRK2 (N214A/34), 1: 200, NeuroMab; GAPDH, 1:10,000, Abcam; HA-11, 1:2, 000, Covance.

Techniques: Expressing, Mutagenesis, Variant Assay, Produced, Staining, Western Blot, Transgenic Assay, Nucleic Acid Electrophoresis, Control

Journal: Journal of Biological Chemistry

Article Title: Nature-inspired design and evolution of anti-amyloid antibodies

doi: 10.1074/jbc.ra118.004731

Figure Lengend Snippet: Figure 5. Western blot analysis of the conformational and sequence specificity of the AF1 antibody. Aβ, tau, and α-synuclein [disaggregated (D) and fibril (F); 0.85 µg] were separated via SDS-PAGE and transferred to nitrocellulose membranes. The AF1-Fc antibody was used at 10 nM (1% milk). Sequence-specific antibodies against Aβ (NAB228, 1:1000 dilution, 1% milk), tau (1E1/A6, 1:5000 dilution, 1% milk) and α-synuclein (5C2, 1:10000 dilution, 1% milk) were also used as controls. Blots were imaged at short (15 s; AF1-Fc, NAB228) and long (5 min; 1E1/A6, 5C2) exposure times. The differences in the intensities of the molecular weight markers between different blots are due to the use of different secondary antibodies.

Article Snippet: Antibody binding was performed by incubating the blocked membranes with 10 mL of 100 nM scFv, 10 nM scFv-Fc fusion, 1:1000 dilution of NAB228 (2 mg/mL stock concentration; A8354, Sigma-Aldrich), 1E1/A6 (stock concentration unknown; 05-804, EMD Millipore) or 1:10000 of 5C2 (1 mg/mL stock concentration; NBP1-04321, Novus Biologicals).

Techniques: Western Blot, Sequencing, SDS Page, Molecular Weight