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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

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)
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Fig1: 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)

Mentions: To investigate if TLR4 plays a role in inflammatory reactions to exogenous αSYN, primary littermate Tlr4+/+ and Tlr4−/− astrocytes were treated with recombinant αSYN, and LPS as a positive control. The expression of Nos2, Il-6, Il1b, Cox-2, Tnfa and Ngf mRNA was investigated by semi-quantitative PCR. LPS dramatically induced the expression of Nos2, Il-6, Il1b, and Cox-2 in Tlr4+/+ astrocytes (Fig. 1). Tnfa expression was also enhanced by LPS treatment while Ngf expression was less consistently altered. Though significantly reduced (Fig. 1b), some induction by LPS could be detected also in Tlr4−/− astrocytes. This might reflect the ability of LPS to activate additional receptors, such as TLR2 [31]. Treatment with 0.7 µM αSYN induced the expression of Nos2, Il-6, Il1b, and Cox-2 in Tlr4+/+ but no significant induction was detected in Tlr4−/− astrocytes (Fig. 1b). Tnfa expression was elevated by αSYN in Tlr4+/+ astrocytes, but remained at basal levels in Tlr4−/− astrocytes.Fig. 1


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

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

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)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4562100&req=5

Fig1: 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)
Mentions: To investigate if TLR4 plays a role in inflammatory reactions to exogenous αSYN, primary littermate Tlr4+/+ and Tlr4−/− astrocytes were treated with recombinant αSYN, and LPS as a positive control. The expression of Nos2, Il-6, Il1b, Cox-2, Tnfa and Ngf mRNA was investigated by semi-quantitative PCR. LPS dramatically induced the expression of Nos2, Il-6, Il1b, and Cox-2 in Tlr4+/+ astrocytes (Fig. 1). Tnfa expression was also enhanced by LPS treatment while Ngf expression was less consistently altered. Though significantly reduced (Fig. 1b), some induction by LPS could be detected also in Tlr4−/− astrocytes. This might reflect the ability of LPS to activate additional receptors, such as TLR2 [31]. Treatment with 0.7 µM αSYN induced the expression of Nos2, Il-6, Il1b, and Cox-2 in Tlr4+/+ but no significant induction was detected in Tlr4−/− astrocytes (Fig. 1b). Tnfa expression was elevated by αSYN in Tlr4+/+ astrocytes, but remained at basal levels in Tlr4−/− astrocytes.Fig. 1

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