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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

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
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Fig4: 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

Mentions: Different studies have indicated that a variety of cell types, for example neuronal cells, microglia and astrocytes, are able to internalize extracellular αSYN [26, 32]. The mechanism of the uptake of the monomeric form has been suggested to differ from the uptake of oligomeric and fibrillar αSYN [33]. We investigated if primary Tlr4+/+ and Tlr4−/− astrocytes cultures were able to internalize the recombinant αSYN. Indeed, after 48 h of continuous incubation an immunoblot signal for human αSYN could be detected in samples that had been treated with 0.07 or 0.7 µM wild-type αSYNwt, mutant αSYNA30P or phosphorylation-deficient αSYNS129A (Fig. 4a). No considerable difference in the internalization of different αSYN variants or between the genotypes of astrocytes could be detected. Pathological αSYN is extensively phosphorylated at S129 and at this site phosphorylated αSYN is found in Lewy bodies [34]. To see if the internalized αSYN is phosphorylated in the astrocytes we treated primary astrocyte cultures with recombinant αSYN for 48 h. No signal could be detected with a phospho-S129 αSYN specific antibody (Fig. 4b). As a positive control for the antibody we used brain lysate from an aged SNCA transgenic mouse, which harbors excessive amounts of S129 phosphorylated αSYN [35]. Also the αSYNS129A mutant was internalized (Fig. 4b), suggesting that phosphorylation at S129 is not a predominant factor in astrocytic uptake of αSYN.Fig. 4


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

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

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
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4562100&req=5

Fig4: 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
Mentions: Different studies have indicated that a variety of cell types, for example neuronal cells, microglia and astrocytes, are able to internalize extracellular αSYN [26, 32]. The mechanism of the uptake of the monomeric form has been suggested to differ from the uptake of oligomeric and fibrillar αSYN [33]. We investigated if primary Tlr4+/+ and Tlr4−/− astrocytes cultures were able to internalize the recombinant αSYN. Indeed, after 48 h of continuous incubation an immunoblot signal for human αSYN could be detected in samples that had been treated with 0.07 or 0.7 µM wild-type αSYNwt, mutant αSYNA30P or phosphorylation-deficient αSYNS129A (Fig. 4a). No considerable difference in the internalization of different αSYN variants or between the genotypes of astrocytes could be detected. Pathological αSYN is extensively phosphorylated at S129 and at this site phosphorylated αSYN is found in Lewy bodies [34]. To see if the internalized αSYN is phosphorylated in the astrocytes we treated primary astrocyte cultures with recombinant αSYN for 48 h. No signal could be detected with a phospho-S129 αSYN specific antibody (Fig. 4b). As a positive control for the antibody we used brain lysate from an aged SNCA transgenic mouse, which harbors excessive amounts of S129 phosphorylated αSYN [35]. Also the αSYNS129A mutant was internalized (Fig. 4b), suggesting that phosphorylation at S129 is not a predominant factor in astrocytic uptake of αSYN.Fig. 4

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