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

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. 1a. Images representative for 4–5 independent cultures are shown. b RT-PCR band quantifications were done as described for Fig. 1b. 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)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: 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. 1a. Images representative for 4–5 independent cultures are shown. b RT-PCR band quantifications were done as described for Fig. 1b. 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)

Mentions: Time course experiments confirmed the inductions within few hours of Nos2 and Cox-2 as well as Il-6 and Il1b, which were abolished in Tlr4−/− astrocytes (Fig. 2). Tnfa was similarly induced by αSYN while again only basal expression remained in Tlr4−/− astrocytes. Washout of agonists showed the reversible nature of astrocyte stimulations (Fig. 2). We also measured NO release from stimulated astrocytes (Additional file 1: Figure S1). Similar to LPS, αSYN treatment clearly induced NO release, although overall the variance of this assay was high. Nevertheless, administration of viper peptide that inhibits TLR4 largely abolished NO release, in contrast to the control peptide (Additional file 1: Figure S1). In conclusion, the mRNA induction of pro-inflammatory mediators by extracellularly applied αSYN involves TLR4.Fig. 2


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

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

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. 1a. Images representative for 4–5 independent cultures are shown. b RT-PCR band quantifications were done as described for Fig. 1b. 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)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: 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. 1a. Images representative for 4–5 independent cultures are shown. b RT-PCR band quantifications were done as described for Fig. 1b. 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)
Mentions: Time course experiments confirmed the inductions within few hours of Nos2 and Cox-2 as well as Il-6 and Il1b, which were abolished in Tlr4−/− astrocytes (Fig. 2). Tnfa was similarly induced by αSYN while again only basal expression remained in Tlr4−/− astrocytes. Washout of agonists showed the reversible nature of astrocyte stimulations (Fig. 2). We also measured NO release from stimulated astrocytes (Additional file 1: Figure S1). Similar to LPS, αSYN treatment clearly induced NO release, although overall the variance of this assay was high. Nevertheless, administration of viper peptide that inhibits TLR4 largely abolished NO release, in contrast to the control peptide (Additional file 1: Figure S1). In conclusion, the mRNA induction of pro-inflammatory mediators by extracellularly applied αSYN involves TLR4.Fig. 2

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