Journal: BMC Neuroscience

Article Title: Exogenous α-synuclein induces toll-like receptor 4 dependent inflammatory responses in astrocytes

doi: 10.1186/s12868-015-0192-0

Figure Lengend Snippet: mRNA induction of pro-inflammatory mediators by extracellular αSYN is reduced in Tlr4 −/− primary astrocytes. a Primary astrocyte-rich cultures from littermate Tlr4 +/+ and Tlr4 −/− mice were treated for 72 h with indicated concentrations of LPS (as positive control) and αSYN, or left untreated (Ø). Total RNA was isolated from the cells and semi-quantitative PCR was performed with primers specific for the indicated gene products. Expression of β-actin mRNA ( Actb ) was measured as loading control. Images representative for 5–6 independent cultures are shown. b Ethidium bromide stained RT-PCR band signals were quantified with ImageJ software and normalized to Actb . Error bars indicate standard deviation, # p < 0.05 compared to untreated samples (ANOVA Fisher’s PLSD), *p < 0.05 compared to Tlr4 −/− (Student’s t test)

Article Snippet: Primary astrocytes were treated in OptiMEM (supplemented with penicillin G and streptomycin) with 5 μM of control peptide (ctrl pep; CP7: RNTISGNIYSARRRRRRRRR) or TLR4 inhibitor peptide (viper: KYSFKLILAEYRRRRRRRRR) (both from Imgenex/Biomol) two hours prior to the addition of recombinant αSYN (0.7 μM or 3.5 μM, as indicated) or LPS (0.1 μg/ml) as positive control or left untreated (Ø).

Techniques: Positive Control, Isolation, Real-time Polymerase Chain Reaction, Expressing, Control, Staining, Reverse Transcription Polymerase Chain Reaction, Software, Standard Deviation

Journal: BMC Neuroscience

Article Title: Exogenous α-synuclein induces toll-like receptor 4 dependent inflammatory responses in astrocytes

doi: 10.1186/s12868-015-0192-0

Figure Lengend Snippet: Time course and recovery of pro-inflammatory mediator expression. a Primary astrocyte-rich cultures from littermate Tlr4 +/+ and Tlr4 −/− mice were treated for 1 h with 1.0 µg/ml LPS (as positive control) or 0.7 µM αSYN, after which the cells were allowed to recover in growth media for 1, 4 or 23 h. Parallel cultures were continuously treated with 1.0 µg/ml LPS or 0.7 µM αSYN for 2, 5 or 24 h. Control cells were left untreated (-). Total RNA was isolated and semi-quantitative PCR was performed as in Fig. a. Images representative for 4–5 independent cultures are shown. b RT-PCR band quantifications were done as described for Fig. b. Error bars indicate standard deviation, # p < 0.05 compared to untreated samples (ANOVA Fisher’s PLSD), *p < 0.05 compared to Tlr4 −/− (Student’s t test)

Article Snippet: Primary astrocytes were treated in OptiMEM (supplemented with penicillin G and streptomycin) with 5 μM of control peptide (ctrl pep; CP7: RNTISGNIYSARRRRRRRRR) or TLR4 inhibitor peptide (viper: KYSFKLILAEYRRRRRRRRR) (both from Imgenex/Biomol) two hours prior to the addition of recombinant αSYN (0.7 μM or 3.5 μM, as indicated) or LPS (0.1 μg/ml) as positive control or left untreated (Ø).

Techniques: Expressing, Positive Control, Control, Isolation, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

Journal: BMC Neuroscience

Article Title: Exogenous α-synuclein induces toll-like receptor 4 dependent inflammatory responses in astrocytes

doi: 10.1186/s12868-015-0192-0

Figure Lengend Snippet: αSYN treatment induces TLR4 dependent nuclear translocation of NF-κB in primary astrocytes. a Primary astrocyte-rich cultures from littermate Tlr4 +/+ and Tlr4 −/− mice were treated with indicated concentrations of LPS (as positive control) and recombinant human αSYN for 6 h or left untreated. The cells were fixed and immunostained using an antibody specific for p65 ( green ). Nuclei were counterstained with Hoechst 33342 ( blue ). Images were obtained with 25× objective using Axioimager microscope equipped with ApoTome Imaging system (Carl Zeiss). Scale bars correspond to 20 µm. b p65/NF-κB positive versus total cells were quantified from images as shown in ( a ). 200–400 cells per sample were analyzed. Error bars show standard deviation, *p < 0.05 (Student’s t test), n is indicated in the figure

Article Snippet: Primary astrocytes were treated in OptiMEM (supplemented with penicillin G and streptomycin) with 5 μM of control peptide (ctrl pep; CP7: RNTISGNIYSARRRRRRRRR) or TLR4 inhibitor peptide (viper: KYSFKLILAEYRRRRRRRRR) (both from Imgenex/Biomol) two hours prior to the addition of recombinant αSYN (0.7 μM or 3.5 μM, as indicated) or LPS (0.1 μg/ml) as positive control or left untreated (Ø).

Techniques: Translocation Assay, Positive Control, Recombinant, Microscopy, Imaging, Standard Deviation

Journal: BMC Neuroscience

Article Title: Exogenous α-synuclein induces toll-like receptor 4 dependent inflammatory responses in astrocytes

doi: 10.1186/s12868-015-0192-0

Figure Lengend Snippet: Extracellular αSYN is internalized by astrocytes in a TLR4 independent manner. Primary astrocytes from littermate Tlr4 +/+ and Tlr4 −/− mice ( a ) or Tlr4 +/+ mice ( b ) were continuously treated for 48 h with indicated concentrations of recombinant human synucleins or LPS or left untreated (Ø). Cells were washed twice with PBS prior to lysis. The protein lysates were immunoblotted and probed with human-specific anti-αSYN ( a , b ) and also antibody against αSYN phosphorylated at S129 ( b ). GAPDH serves as control for equal protein loading throughout. Positive control in ( b ) is brain lysate from a 18 month old Thy1[A30P]hSNCA transgenic mouse, which has high amounts of phosphorylated αSYN. N is indicated in the figure ( a ) or N = 8 ( b , c ). c Primary wild type astrocytes were treated continuously with 0.7 µM αSYN for indicated times. Protein lysates were examined by immunoblotting as above

Article Snippet: Primary astrocytes were treated in OptiMEM (supplemented with penicillin G and streptomycin) with 5 μM of control peptide (ctrl pep; CP7: RNTISGNIYSARRRRRRRRR) or TLR4 inhibitor peptide (viper: KYSFKLILAEYRRRRRRRRR) (both from Imgenex/Biomol) two hours prior to the addition of recombinant αSYN (0.7 μM or 3.5 μM, as indicated) or LPS (0.1 μg/ml) as positive control or left untreated (Ø).

Techniques: Recombinant, Lysis, Control, Positive Control, Transgenic Assay, Western Blot