Beclin1 and HMGB1 ameliorate the α-synuclein-mediated autophagy inhibition in PC12 cells

Abstract

Background: Aberrant α-synuclein aggregation due to the deficiency of ubiquitin-proteasome or of autophagy characterizes the parkinson disease (PD). High mobility group box 1 (HMGB1) is a novel stress sensor to mediate the persistent neuro-inflammation and the consequent progressive neurodegeneration, via controlling the cellular autophagy/apoptosis checkpoint during inflammation. Moreover, HMGB1 has been recently indicated to involve in the autophagic degradation of α-synuclein.

Methods: In the current study, we investigated the influence of the overexpressed α-synuclein of wild type (wt) or mutant type (A53T and A30P, mt) on the cytosolic levels of HMGB1 and Beclin1 and on the starvation-induced autophagy in pheochromocytoma PC12 cells. And then we explored the overexpression of HMGB1 or of Beclin1 on the α-synuclein degradation and on the autophagy in the α-synuclein-overexpressed PC12 cells.

Results: It was demonstrated that α-synuclein overexpression inhibited the trans-location of HMGB1 from nucleus to cytosol and reduced the cytosolic level of Beclin1 in PC12 cells, and inhibited the starvation-induced autophagy via downregulating autophagy-associated markers and via reducing the autophagic vesicles in PC12 cells under starvation. On the other side, the intracellular promotion of either HMGB1 or Beclin1 upregulated the α-synuclein degradation and ameliorated the α-synuclein-mediated autophagy reduction in PC12 cells. However, the exogenous HMGB1 treatment exerted no such regulation in PC12 cells.

Conclusion: In summary, our study confirmed the positive regulation by HMGB1 and Beclin1 on the α-synuclein degradation and on the starvation-induced autophagy in PC12 cells, implying both markers as prominent targets to promote the α-synuclein degradation.

Fig. 1

Overexpression of α-synuclein with wild type (wt) or mutant type (mt) reduces cytosolic HMGB1 and Beclin1 in PC12 cells. a and b: mRNA (a) and protein (b) levels of α-synuclein in the PC12 cell overexpressing wt α-synuclein, PC12 (Syn^wt), in the PC12 cell overexpressing mt (A53T and A30P) α-synuclein, PC12 (Syn^mt), in the PC12 cell overexpressing Enhanced Green Fluorescence Protein (EGFP), PC12 (Con), or in the blank P12 cells, the protein level of α-synuclein was examined by western blot analysis; c: Western blot analysis of HMGB1 (in cytosol and in nucleus) and Beclin1 (in cytosol) in PC12, PC12 (Con), PC12 (Syn^wt) or PC12 (Syn^mt) cells; d: Relative HMGB1 level to β-actin in cytosol or to Lamin B(LMNB1) in the nucleus of PC12, PC12 (Con), PC12 (Syn^wt) or PC12 (Syn^mt) cells; e: Relative Beclin1 level to β-actin in the cytosol of PC12, PC12 (Con), PC12 (Syn^wt) or PC12 (Syn^mt) cells; Each result was averaged for triple independent experiments. Statistical significance was presented as * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance

Fig. 2

Levels of autophagy-associated markers and autophagic vesicles induced by starvation in the PC12, PC12 (Con), PC12 (Syn^wt) or PC12 (Syn^mt) cells. a: Western blotting assay of LC3-I, LC3-II, Atg 7 and mTOR in the PC12, PC12 (Con), PC12 (Syn^wt) or PC12 (Syn^mt) cells post an incubation at 37 °C in FBS-free medium for 24 hours; b: Ratio of LC3-II to LC3-I in the starvation-treated cell lines; c: Relative level of Atg 7 and mTOR levels to β-actin in each cell line post the starvation treatment; d and e: Imaging (d) and counting (e) of EGFP-positive autophagic vesicles in the cytosol of starvation-treated PC12, PC12 (Con), PC12 (Syn^wt) or PC12 (Syn^mt) cells which were transfected with pCDNA3.1-EGFP-LC3 plasmid for another 24 hours; Data was averaged for triple independent results. * p < 0.05, ** p < 0.01, ns: no significance

Fig. 3

HMGB1 upregulation inhibits α-synuclein accumulation and ameliorates the α-synuclein-inhibited autophagy in PC12 (Syn^wt) and PC12 (Syn^mt) cells. a: Western blot analysis of HMGB1 and α-synuclein in PC12 (Syn^wt) and PC12 (Syn^mt) cells, which were transfected with HMGB1-pcDNA3.1(+) or RFP-pcDNA3.1(+) plasmid for 12 or 24 hours; b and c: Ratio of HMGB1 to β-actin in PC12 (Syn^wt) and PC12 (Syn^mt) cells with or without HMGB1 promoted; d and e: Ratio of α-synuclein to β-actin in PC12 (Syn^wt) and PC12 (Syn^mt) cells, with or without HMGB1 promoted; f and g: Imaging (f) and counting (g) of EGFP-positive autophagic vesicles in the cytosol of starvation-treated PC12 (Syn^wt) or PC12 (Syn^mt), with or without HMGB1 promoted. Each result was averaged for triple independent experiments. Statistical significance was presented as * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance

Fig. 4

Western blot analysis of autophagy-associated markers in the PC12 (Syn^wt) or the PC12 (Syn^mt) cells, which were treated with HMGB1. a: Western blotting assay of LC3-I, LC3-II, Atg 7 and mTOR in the PC12 (Syn^wt) cells which were treated with 0, 0.2, 0.5 or 1 μg/mL HMGB1, under starvation for 24 hours; b: Ratio of LC3-II to LC3-I in the starvation-treated PC12 (Syn^wt) cells with HMGB1 treatment; c and d: Relative level of Atg 7 (c) and mTOR (d) levels to β-actin in the PC12 (Syn^wt) cells with HMGB1 treatment; e: Western blotting assay of LC3-I, LC3-II, Atg 7 and mTOR in the PC12 (Syn^mt) cells which were treated with 0, 0.2, 0.5 or 1 μg/mL HMGB1, under starvation for 24 hours; f: Ratio of LC3-II to LC3-I in the starvation-treated PC12 (Syn^mt) cells with HMGB1 treatment; g and h: Relative level of Atg 7 (g) and mTOR (h) levels to β-actin in the PC12 (Syn^mt) cells with HMGB1 treatment. Data was averaged for triple independent results, ns: no significance

Fig. 5

Beclin1 upregulation reduces α-synuclein accumulation and ameliorates the α-synuclein-inhibited autophagy in PC12 (Syn^wt) and PC12 (Syn^mt) cells. a: Western blot analysis of Beclin1 and α-synuclein in the PC12 (Syn^wt) and PC12 (Syn^mt) cells which were infected with 1 multiplicity of infection (MOI) pLenti-Beclin1 (LV-Beclin1) or pLenti-Con (LV-Con) under starvation for 12 or 24 hours; b and c: Ratio of Beclin1 to β-actin in PC12 (Syn^wt) (b) and PC12 (Syn^mt) (c) cells which were infected with LV-Beclin1 or with LV-Con virus; d and e: Ratio of α-synuclein to β-actin in PC12 (Syn^wt) (b) and PC12 (Syn^mt) (c) cells which were infected with LV-Beclin1 or with LV-Con virus; f and g: Imaging (f) and counting (g) of EGFP-positive autophagic vesicles in the starvation-treated PC12 (Syn^wt) or PC12 (Syn^mt), which were infected with LV-Beclin1 or with LV-Con virus. Each result was averaged for triple independent experiments. Statistical significance was presented as * p < 0.05, ** p < 0.01, *** p < 0.001, ns: no significance