Limits...
Exogenous α-synuclein induces toll-like receptor 4 dependent inflammatory responses in astrocytes.

Rannikko EH, Weber SS, Kahle PJ - BMC Neurosci (2015)

Bottom Line: The mRNA induction of pro-inflammatory cytokines, inducible nitric oxide synthase and cyclooxygenase-2 was significantly reduced in TLR4 knockout astrocytes.In contrast, the uptake of exogenous αSYN was unaffected by TLR4 knockout.Extracellular αSYN can activate pro-inflammatory TLR4 pathways in astrocytes, whereas αSYN uptake is independent of TLR4.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Faculty of Medicine, Hertie Institute for Clinical Brain Research, University of Tübingen, Otfried Müller Str. 27, 72076, Tübingen, Germany. emmy.rannikko@ki.se.

ABSTRACT

Background: The pathological hallmarks of Parkinson's disease are intracellular inclusions composed mainly of misfolded α-synuclein (αSYN). Under physiological conditions αSYN is mostly localized in synapses. In addition, a portion of αSYN is secreted to the extracellular space, where it may be sequestered by neighboring cells and could induce inflammatory responses. The mechanisms of αSYN internalization and signal transduction are not unequivocally clarified. In this work we investigated in primary mouse astrocytes the involvement of toll-like receptor 4 (TLR4) in the induction of inflammatory responses upon exposure to purified human αSYN produced in bacteria.

Results: The mRNA induction of pro-inflammatory cytokines, inducible nitric oxide synthase and cyclooxygenase-2 was significantly reduced in TLR4 knockout astrocytes. The αSYN-mediated activation of c-Jun N-terminal kinases and p38 mitogen-activated protein kinase tended to be diminished, and nuclear translocation of the p65 subunit of nuclear factor κB was abolished in TLR4 knockout astrocytes. In contrast, the uptake of exogenous αSYN was unaffected by TLR4 knockout.

Conclusions: Extracellular αSYN can activate pro-inflammatory TLR4 pathways in astrocytes, whereas αSYN uptake is independent of TLR4.

No MeSH data available.


Related in: MedlinePlus

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4562100&req=5

Fig3: 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

Mentions: Activation of TLR4 leads to phosphorylation and degradation of IκB and thus allows the transcription factor complex NF-κB to translocate to the nucleus. Primary Tlr4+/+ and Tlr4−/− astrocytes were treated with different concentrations of the positive control LPS or recombinant human αSYN for 6 h. The cells were immunostained for p65, a component of the class II NF-κB protein complex, and p65 positive nuclei were quantified. As expected, LPS treated cells showed an increased number of p65 positive nuclei in both Tlr4+/+ and Tlr4−/− cells, however to a lesser extent in the knock out astrocytes (Fig. 3). The recombinant αSYN induced NF-κB translocation in Tlr4+/+ cells in a concentration dependent manner, whereas in Tlr4−/− astrocytes very few p65 positive nuclei were detected. Thus indicating that extracellular αSYN can induce the nuclear translocation of NF-κB in a TLR4-dependent manner.Fig. 3


Exogenous α-synuclein induces toll-like receptor 4 dependent inflammatory responses in astrocytes.

Rannikko EH, Weber SS, Kahle PJ - BMC Neurosci (2015)

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4562100&req=5

Fig3: 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
Mentions: Activation of TLR4 leads to phosphorylation and degradation of IκB and thus allows the transcription factor complex NF-κB to translocate to the nucleus. Primary Tlr4+/+ and Tlr4−/− astrocytes were treated with different concentrations of the positive control LPS or recombinant human αSYN for 6 h. The cells were immunostained for p65, a component of the class II NF-κB protein complex, and p65 positive nuclei were quantified. As expected, LPS treated cells showed an increased number of p65 positive nuclei in both Tlr4+/+ and Tlr4−/− cells, however to a lesser extent in the knock out astrocytes (Fig. 3). The recombinant αSYN induced NF-κB translocation in Tlr4+/+ cells in a concentration dependent manner, whereas in Tlr4−/− astrocytes very few p65 positive nuclei were detected. Thus indicating that extracellular αSYN can induce the nuclear translocation of NF-κB in a TLR4-dependent manner.Fig. 3

Bottom Line: The mRNA induction of pro-inflammatory cytokines, inducible nitric oxide synthase and cyclooxygenase-2 was significantly reduced in TLR4 knockout astrocytes.In contrast, the uptake of exogenous αSYN was unaffected by TLR4 knockout.Extracellular αSYN can activate pro-inflammatory TLR4 pathways in astrocytes, whereas αSYN uptake is independent of TLR4.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Faculty of Medicine, Hertie Institute for Clinical Brain Research, University of Tübingen, Otfried Müller Str. 27, 72076, Tübingen, Germany. emmy.rannikko@ki.se.

ABSTRACT

Background: The pathological hallmarks of Parkinson's disease are intracellular inclusions composed mainly of misfolded α-synuclein (αSYN). Under physiological conditions αSYN is mostly localized in synapses. In addition, a portion of αSYN is secreted to the extracellular space, where it may be sequestered by neighboring cells and could induce inflammatory responses. The mechanisms of αSYN internalization and signal transduction are not unequivocally clarified. In this work we investigated in primary mouse astrocytes the involvement of toll-like receptor 4 (TLR4) in the induction of inflammatory responses upon exposure to purified human αSYN produced in bacteria.

Results: The mRNA induction of pro-inflammatory cytokines, inducible nitric oxide synthase and cyclooxygenase-2 was significantly reduced in TLR4 knockout astrocytes. The αSYN-mediated activation of c-Jun N-terminal kinases and p38 mitogen-activated protein kinase tended to be diminished, and nuclear translocation of the p65 subunit of nuclear factor κB was abolished in TLR4 knockout astrocytes. In contrast, the uptake of exogenous αSYN was unaffected by TLR4 knockout.

Conclusions: Extracellular αSYN can activate pro-inflammatory TLR4 pathways in astrocytes, whereas αSYN uptake is independent of TLR4.

No MeSH data available.


Related in: MedlinePlus