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

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.

High mobility group box 1 (HMGB1) is a stress sensor that plays a critical role in various physiological and pathological processes including cell development, differentiation, inflammation, metabolism and death [19]. Increasing evidence demonstrates that HMGB1-dependent autophagy promotes chemotherapy resistance [20], sustains tumor metabolism [21], protects against endotoxemia [22] and involves in other pathological processes [23,24]. Inflammation has also been recognized to involve in PD [25][26][27]. Sustained inflammatory process and the activated microglia might drive the progressive degeneration of dopamine neurons in PD [28]. Moreover, the cytoplasmic HMGB1 has been indicated to control the cellular autophagy/apoptosis checkpoint during inflammation [29]. HMGB1 was demonstrated to mediate the persistent neuroinflammation and consequent progressive neurodegeneration, via stimulating the production of multiple inflammatory and neurotoxic factors [30]. In addition, HMGB1 has been recently indicated to involve in the autophagy inhibition caused by α-synuclein overexpression [31], implying a direct role in modulating autophagic degradation of α-synuclein.
In current study, we investigated the regulation of αsynuclein overexpression on the trans-location or the expression of HMGB1 and Beclin1, and on the starvationinduced autophagy in pheochromocytoma PC12 cells. And then we explored the overexpression of HMGB1 or Beclin1 on the α-synuclein degradation and on the autophagy in the α-synuclein-overexpressed PC12 cells. This study confirmed the positive regulation by endogenous HMGB1 on the autophagy-mediated α-synuclein degradation in PC12 cells.

Statistical evaluations
Quantitative data was presented as mean ± SE and was analyzed for a significant difference with the Student's t test or one way ANOVA test. A p value less than 0.05 was considered to be statistically significant.

α-synuclein overexpression inhibits the trans-location of HMGB1 and the expression of Beclin1 in PC12 cells
To investigate the regulation of α-synuclein overexpression on the trans-location or the level of HMGB1 and Beclin1, and on the starvation-induced autophagy in pheochromocytoma PC12 cells, we constructed the PC12 cell overexpressing WT α-synuclein, PC12 (Syn wt ), or the PC12 cell overexpressing MT (A53T and A30P) α-synuclein, PC12 (Syn mt ), with PC12 (Con), which overexpressed enhanced green fluorescent protein (EGFP), as control. As shown in Fig. 1a, the mRNA level of α-synuclein was significantly higher in both PC12 (Syn wt ) and PC12 (Syn mt ) cells, compared with the PC12 (Con) or blank PC12 cells (either p < 0.001 for PC12 (Syn wt ) or PC12 (Syn mt ) cells). And the protein level of α-synuclein was confirmed by the western blot analysis in the PC12 (Syn wt ) or PC12 (Syn mt ) cells (p < 0.001 respectively, Fig. 1b). And then we investigated the influence of α-synuclein overexpression on the Beclin1 level and the trans-location of HMGB1 from nucleus to cytosol. Western blotting (Fig. 1c) demonstrated that the cytosolic HMGB1 was markedly upregulated, whereas was significantly downregulated in nucleus in either PC12 (Syn wt ) or PC12 (Syn mt ) cells (p < 0.05 respectively, Fig. 1c). In addition the cytoplasmic Beclin1 was also examined with western blot analysis. It was indicated that the Beclin level was significantly downregulated in both PC12 (Syn wt ) and PC12 (Syn mt ) cells (p < 0.01 respectively, Fig. 1d). Thus, we confirmed the promotion to the trans-location of HMGB1 from nucleus to cytosol, and to the cytoplasmic level of Beclin1 in PC12 cells.

α-synuclein overexpression inhibits autophagy in PC12 cells
We then examined the regulation of the overexpressed α-synuclein on the autophagy in PC12 cells. The autophagy induction in PC12, PC12 (Con), PC12 (Syn wt ) or PC12 (Syn mt ) cells subject to starvation was examined with western blotting assay and the EGFP-LC3 reporter assay. Western blotting results (Fig. 2a) demonstrated that the conversion of LC3-I to LC3-II was markedly lower in either PC12 (Syn wt ) or PC12 (Syn mt ) cells, compared to the normal PC12 or the PC12 (Con) cells (p < 0.01 respectively, Fig. 2b). And such reduced autophagy was also confirmed by the reduced level of Atg7 and increased level of mTOR in either PC12 (Syn wt ) or PC12 (Syn mt ) cells (p < 0.01 respectively, Fig. 2b). Moreover, the EGFP-LC3 reporter assay also demonstrated that there were less GFP-positive autophagic puncta in either PC12 cells. Taken together, the α-synuclein overexpression with wild or mutant type inhibits the starvationstimulated autophagy in PC12 cells.
In addition, we also investigated the influence of exogenous HMGB1 treatment on the starvation-induced autophagy in PC12 cells. Western blotting assay (Fig. 4a) demonstrated that the treatment with 0.2 to 1 μg/mL HMGB1 did not significantly regulate the conversion of LC3-I to LC3-II (Fig. 4b) and the expression of Atg 7 (Fig. 4c) and mTOR (Fig. 4d). Therefore, the exogenous HMGB1 exerts no regulation on the starvation-induced autophagy in PC12 cells.

Beclin1 overexpression promotes autophagy and α-synuclein degradation in the α-synuclein-overexpressed PC12 cells
Given the high importance of Beclin1-dependent autophagy in the α-synuclein degradation [10,12], we then investigated the influence of Beclin1 overexpression on the α-synuclein degradation and the starvation-induced autophagy in both PC12 (Syn wt ) and PC12 (Syn mt ) cells. As shown in Fig. 5a, the lentivirus-mediated Beclin1 overexpression in protein level was significant in both cell lines (Fig. 5b and c) Fig. 5e). In addition, 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 we examined the starvation-induced autophagic vesicles in each cell line with the EGFP-LC3 reporter assay. It was indicated in Fig. 5f and g that the infection with 1 MOI LV-Beclin1 promoted more autophagic vesicles than the LV-Con infection in either PC12 (Syn wt ) or PC12 (Syn mt ) cells (p < 0.05). Thus, we also confirmed the positive regulation by Beclin1 on the starvationinduced autophagy in PC12 cells.

Discussion
HMGB1, as a cytokine-like factor [33], is increasingly recognized as a novel autophagy regulator via interfering with the PIK3C3 complex [34]. Nucleus-to-cytosol translocated HMGB1 competitively binds to Beclin1 and subsequently induces autophagy [35]. However, α-synuclein has been shown to impair the autophagy [36,37]. In particular, αsynuclein could bind to both cytoplasmic and nuclear 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 HMGB1, block HMGB1-Beclin1 binding, whereas to strengthen the Beclin1-BcL2 binding [31], and thus to inhibit autophagy. And such inhibition could be restored by the Beclin 1 overexpression. In the present study, we found that the overexpression of either WT or MT αsynuclein markedly reduced the cytoplasmic levels of both 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 HMGB1 and Beclin1 in PC12 cells, implying the reduced HMGB1 and Beclin1 might contribute to the α-synucleinmediated autophagy inhibition in PC12 cells. Impaired autophagy has been indicated to correlate with the α-synuclein aggregation and neurodegeneration in PD [38][39][40][41]; and the stimulated autophagy could reduce the accumulation of α-synuclein in cells and in mouse brain [12,42], and could even rescue midbrain dopamine neurons from α-synuclein toxicity [43]. On the other side, α-synuclein has been shown to impair autophagy [36,37]. The overexpression of either WT or MT α-synuclein inhibits autophagy in pheochromocytoma PC12 cells [31], impairs neurite outgrowth of primary midbrain neurons, affects neurite branching [44]. In this study, we reconfirmed such autophagy inhibition by α-synuclein overexpression in PC12 cells, the overexpression of wild-type or mutant-type α-synuclein significantly downregulated the starvation-induced autophagy via inhibiting the mTOR/Atg 7 signaling. Therefore, we also confirmed the autophagy inhibition by the overexpressed α-synuclein in PC12 cells. And it implies that the reduced HMGB1 and Beclin1 might contribute to the reduced autopahgy in the α-synuclein-overexpressed PC12 cells.
HMGB1 is also known as the high-mobility group protein 1 (HMG-1). As a chromatin-associated nuclear protein, it is a critical regulator of autophagy. And the pharmacological inhibition of HMGB1 cytoplasmic translocation limits starvation-induced autophagy. Moreover, only endogenous HMGB1 has been indicated to regulate the Bcl-2-Beclin1 binding, via the direct interact with Beclin 1 [34,35], and thus regulating the convergence of autophagy and apoptosis, via interacting with antiapoptotic Bcl-2-like proteins [45]. Our study confirmed that either endogenous HMGB1 or Beclin 1 overexpression promoted the α-synuclein degradation in PC12 cells. Moreover, the overexpressed HMGB1 or Beclin 1 markedly ameliorated the α-synuclein-mediated autophagy reduction in the α-synuclein-overexpressed (either WT or MT) PC12 cells, implying the promotion by HMGB1 to the α-synuclein degradation might be autophagy-dependent. And such amelioration might be dependent on the HMGB1-Beclin1 interaction.
Interesting, our results indicated that the extracellular HMGB1 exerted no regulatory role on the starvationinduced autophagy, posing no influence on the levels of mTOR and Atg 7 in both PC12 (Syn wt ) and PC12 (Syn mt ) cells. HMGB1 is a secretary cytokine from activated macrophages and monocytes [46] to mediate inflammation [33], via binding to receptor for advanced glycation endproducts (RAGE) [47] or to toll-like receptor (TLR) [48]. However, the present study confirmed that the promotion by HMGB1 was not RAGE-or TLRdependent. In addition, the gain-of-function strategy also confirmed the promotion to the α-synuclein degradation and the autophagy induction by Beclin1 overexpression in PC12 cells. Taken together, we speculated that endogenous HMGB1 and Beclin1 might promote the autophagic degradation of α-synuclein. Therefore, the endogenous HMGB1 and Beclin1 exerts protective role in the cells against the α-synuclein accumulation.

Conclusion
In summary, α-synuclein with wild-type or mutant-type inhibited autophagy in PC12 cells via inhibiting the HMGB1 and Beclin1. On the other side, the cytosolic promotion to HMGB1 or to Beclin1 up-regulates the autophagic degradation of α-synuclein via increasing the autophagy in PC12 cells. Therefore, the endogenous HMGB1 and Beclin1 present protective role in the cells against the α-synuclein accumulation.