In-vivo evidence that high mobility group box 1 exerts deleterious effects in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model and Parkinson's disease which can be attenuated by glycyrrhizin

Abstract

High-mobility group box 1 (HMGB1) is a nuclear and cytosolic protein that is released during tissue damage from immune and non-immune cells - including microglia and neurons. HMGB1 can contribute to progression of numerous chronic inflammatory and autoimmune diseases which is mediated in part by interaction with the receptor for advanced glycation endproducts (RAGE). There is increasing evidence from in vitro studies that HMGB1 may link the two main pathophysiological components of Parkinson's disease (PD), i.e. progressive dopaminergic degeneration and chronic neuroinflammation which underlie the mechanistic basis of PD progression. Analysis of tissue and biofluid samples from PD patients, showed increased HMGB1 levels in human postmortem substantia nigra specimens as well as in the cerebrospinal fluid and serum of PD patients. In a mouse model of PD induced by sub-acute administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), systemic administration of neutralizing antibodies to HMGB1 partly inhibited the dopaminergic cell death, and reduced the increase of RAGE and tumour necrosis factor-alpha. The small natural molecule glycyrrhizin, a component from liquorice root which can directly bind to HMGB1, both suppressed MPTP-induced HMGB1 and RAGE upregulation while reducing MPTP-induced dopaminergic cell death in a dose dependent manner. These results provide first in vivo evidence that HMGB1 serves as a powerful bridge between progressive dopaminergic neurodegeneration and chronic neuroinflammation in a model of PD, suggesting that HMGB1 is a suitable target for neuroprotective trials in PD.

Keywords: High-mobility group box 1; MPTP; Parkinson's disease; receptor for advanced glycation endproducts.

Supplementary Fig. 1.

Double immunofluorescent staining for TH positive neurons (green) and HMGB1 (red) in human post mortem SNpc of control and PD cases. Occurrence of protein translocation and localization of HMGB1 within the cytoplasmic compartment of TH positive neurons (arrow) is observed in PD samples (J-Q) but not in control samples (A-H). Nuclei are stained with DAPI (blue), scale bar = 20 µm.

Supplementary Fig. 2.

Double immunofluorescence for microglia (green) and HMGB1 (red) (A-H), and GFAP (green) and HMGB1 (red) (J-Q) in human post mortem SNpc of control cases and PD cases. HMGB1 nuclear translocation is not observed in microglia (E-H) and GFAP positive astrocytes (N-Q) of PD samples. Nuclei are stained with DAPI (blue), scale bar = 20 µm.

Fig. 1

HMGB1 in dopaminergic neurons of human post-mortem substantia nigra (SNpc). Using western blotting, HMGB1 protein levels in SNpc are, as a mean, higher in six samples of Parkinson's disease (PD), compared with five control samples (A). Double immunofluorescence studies reveal cytoplasmic HMGB1 localization in TH-positive dopaminergic neurons of patients with PD (F–I), but not in control patients (B–E). *p < 0.05. Scale bar = 20 μm.

Fig. 2

HMGB1 levels in serum and cerebrospinal fluid in patients with Parkinson's disease. In the immunoassay, HMGB1 levels are significantly increased in both serum (A) and CSF (B) of 75 patients with Parkinson's disease (PD) compared to 47 controls (HC). HMGB1 serum levels of PD patients are negatively correlated with age at onset (C), and positively with disease duration (D).

Fig. 3

HMGB1 mRNA and protein levels are upregulated after MPTP treatment. In mice, real-time PCR and Western blot tests indicate that HMGB1 mRNA and protein levels are increased at day 1 after MPTP treatment. Within 4 days, HMGB1 protein (A) and mRNA levels (B) return back to initial values. Data are mean ± SEM, n = 4–6 mice per group. *p < 0.05; ANOVA with student Newman–Keuls post-hoc test.

Fig. 4

Double immunofluorescence reveals localization of HMGB1 in the nuclei with translocation to the cytosol after MPTP in GFAP-positive astrocytes (E–H) and Iba-1-positive microglia (M–P) in the substantia nigra pars compacta (2d after MPTP). Data are mean ± SEM, n = 4–6 mice per group. Scale bar = 20 μm.

Fig. 5

HMGB1 is translocated into the cytoplasm 1 day after MPTP treatment in dopaminergic neurons. Double immunofluorescence in mouse midbrain reveals localization of HMGB1 in the nuclei of TH-positive dopaminergic neurons after saline (A–D, white arrow), with a clear translocation to the cytosol after MPTP (E–H, white arrows), which is significantly reduced in mice receiving HMGB1-neutralizing antibody (I–L) or glycyrrhizin (M–P) (2d after MPTP). (Q) Stereological cell counting in epifluorescence field was performed for TH-positive neurons in SNpc. TH-positive cells presenting staining for HMGB1 in the cytoplasm portion were considered positive to HMGB1 translocation from nuclei to cytoplasm. *p < 0.05, ***p < 0.001 compare to MPTP mice group treated with saline (Newman–Keuls post-hoc test). Data are generated with 5 mice per group. Value are presented with mean ± SEM. Scale bar = 20 μm.

Fig. 6

Neutralizing HMGB1 rescues dopaminergic neurons from MPTP toxicity. Numbers of TH-positive neurons in the SNpc are comparable between saline-treated mice receiving and not receiving HMGB1-neutralizing antibody (upper panels; A–B). Three weeks after MPTP treatment, mice initially treated with HMGB1-neutralizing antibody show higher numbers of TH-positive neurons, than do mice not receiving the antibody (lower panel in A and B). Accordingly, depletion of striatal dopaminergic fibres and striatal dopamine depletion assessed three weeks after MPTP are significantly less severe in mice receiving HMGB1-neutralizing antibody (C, D). Cell counts were performed with stereology. OD was assessed by use of ScionImage Scion Corp., Frederick, Maryland, USA). Striatal monoamine levels were assessed by HPLC. *p < 0.05, compared to respective, MPTP treated, mice groups which did not receive HMGB1 antibody (Newman–Keuls post-hoc test). Data are generated with six mice per group, values are presented with mean ± SEM. AB, antibody.

Fig. 7

HMGB1 influences MPTP toxicity via RAGE and TNF-α. Two days after MPTP treatment, immunoblotting reveals higher levels of RAGE (A,B) and TNF-α protein (A,C) in mice initially also receiving HMGB1-neutralizing antibody, compared to mice not receiving the antibody. COX-2 protein levels are comparable between the differentially treated groups (A, D). Data are mean ± SEM, n = 4–6 mice per group. *p < 0.05; ANOVA with student Newman–Keuls post-hoc test.

Fig. 8

Glycyrrhizin rescues dopaminergic neurons dose-dependently from MPTP toxicity. Mice receiving initial i.p. glycyrrhizin show higher numbers of TH-positive neurons in the SNpc (A, C) as well as more striatal dopamine-positive fibres (B, D), than do mice without glycyrrhizin treatment three weeks after MPTP treatment. This effect is dose-dependent (A-D). Cell counts were performed with stereology. Optical density of striatal TH positive fibres was assessed by use of Scion Image Scion Corp., Frederick, Maryland, USA). Striatal monoamine levels were assessed by HPLC. *p < 0.05, **P < 0.01 compared to respective mice groups which did not receive glycyrrhizin. Data are generated with six mice per group. Values are presented with mean ± SEM. n = 4–5 mice per group; ANOVA with student Newman–Keuls post-hoc test.

Fig. 9

Glycyrrhizin significantly reduces MPTP-induced HMGB1 and RAGE but not S100B levels. In the immunoblot of the ventral midbrain area containing the SNpc, levels of HMGB1 and RAGE proteins one day after MPTP treatment are reduced in mice also receiving i.p. glycyrrhizin. This effect was dose-dependent and reached significance in the group receiving the high dose of glycyrrhizin. S100B, another ligand of RAGE, did not show these changes. Low levels of RAGE, TNF-α and COX-2 were detected in control mice receiving saline (data not shown). Data are mean ± SEM, n = 4–6 mice per group. *p < 0.05; ANOVA with student Newman–Keuls post-hoc test.