Ganoderma lucidum extract ameliorates MPTP-induced parkinsonism and protects dopaminergic neurons from oxidative stress via regulating mitochondrial function, autophagy, and apoptosis

Abstract

Neuroprotection targeting mitochondrial dysfunction has been proposed as an important therapeutic strategy for Parkinson's disease. Ganoderma lucidum (GL) has emerged as a novel agent that protects neurons from oxidative stress. However, the detailed mechanisms underlying GL-induced neuroprotection have not been documented. In this study, we investigated the neuroprotective effects of GL extract (GLE) and the underlying mechanisms in the classic MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced mouse model of PD. Mice were injected with MPTP to induce parkinsonism. Then the mice were administered GLE (400 mg kg^-1 d^-1, ig) for 4 weeks. We observed that GLE administration significantly improved locomotor performance and increased tyrosine hydroxylase expression in the substantia nigra pars compact (SNpc) of MPTP-treated mice. In in vitro study, treatment of neuroblastoma neuro-2a cells with 1-methyl-4-phenylpyridinium (MPP⁺, 1 mmol/L) caused mitochondrial membrane potential collapse, radical oxygen species accumulation, and ATP depletion. Application of GLE (800 μg/mL) protected neuroblastoma neuro-2a cells against MPP⁺ insult. Application of GLE also improved mitochondrial movement dysfunction in cultured primary mesencephalic neurons. In addition, GLE counteracted the decline in NIX (also called BNIP3L) expression and increase in the LC3-II/LC3-I ratio evoked by MPP⁺. Moreover, GLE reactivated MPP⁺-inhibited AMPK, mTOR, and ULK1. Similarly, GLE was sufficient to counteract MPP⁺-induced inhibition of PINK1 and Parkin expression. GLE suppressed MPP⁺-induced cytochrome C release and activation of caspase-3 and caspase-9. In summary, our results provide evidence that GLE ameliorates parkinsonism pathology via regulating mitochondrial function, autophagy, and apoptosis, which may involve the activation of both the AMPK/mTOR and PINK1/Parkin signaling pathway.

Keywords: MPTP; apoptosis; autophagy; ganoderma lucidum extract; mitochondrial dysfunction; parkinsonism.

Fig. 1

GLE treatment promoted motor performance and protected against the loss of dopaminergic neuronal cells in MPTP-treated mice. a Time required to cross the beam in the beam walking test in each group. b Latency to fall in the rotarod test in each group. c Quantification of TH-positive cells in the substantia nigra pars compact (SNpc) (left). d IOD (integrated optical density) of TH-positive fibers in the striatum (right) among the different groups. e Representative immunohistochemical images of TH labeling in the SNpc (upper) and striatum (lower), respectively. Data are expressed as the mean ± SEM; n = 15 in each group, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group; ^#P < 0.05, ^###P < 0.001 vs. the MPTP group

Fig. 2

GLE treatment prevented cellular and mitochondrial damage in neuro-2a cells following MPP⁺ injury. Neuro-2a cells were exposed to MPP⁺ (1 mM, at the indicated time point) with or without GLE. a Cytotoxic effect of GLE at varying concentrations and incubation times on neuro-2a cells. b Effect of GLE on the MPP⁺-induced reductions in neuro-2a cell viability. Multiple concentrations of GLE were used to determine the concentration that was non-toxic to cells but caused the most significant increase in cell viability in the case of MPP⁺ injury; 800 μg/mL was the dose of GLE selected for subsequent trials. c Percentage of dead cell was determined by the trypan blue membrane permeability assay as described in the Materials and Methods. d Representative image of mitochondria membrane potential (MMP) staining in each group. MMP was assessed by JC-1 dye. e Representative fluorescence microscopy image of neuro-2a cells stained with ROS-detecting probes (green). f Concentrations of intracellular ATP using an ATP Determination Kit. Data are expressed as the mean ± SEM (at least three independent experiments were performed); ***P < 0.001 vs. the control group; ^#P < 0.05, ^###P < 0.001 vs. the MPP⁺ group

Fig. 3

GLE improved mitochondrial movement dysfunction in axons in MPP⁺-treated primary midbrain neuronal cells. a Representative view of mitochondrial movement along the axon; cultured neuronal cells were transfected with the Mito-EGFP plasmid and observed using the Live Cell Station. b The speed of moving mitochondria was calculated using Velocity Demo software. Bars represent the mean ± SEM of three independent experiments conducted in duplicate. *P < 0.05, **P < 0.01 vs. the control group; ^#P < 0.05 vs. the MPP⁺ group

Fig. 4

GLE administration regulated autophagy induced by MPP⁺ in neuro-2a cell lines. a Western blot was performed to test the effect of GLE on autophagic events at 6, 9, and 12 h after MPP⁺ treatment (left), and the immunoreactive band was quantified (right). The occurrence of autophagy was analyzed by examining the LC3-I-to-LC3-II conversion, expressed as the LC3-II/LC3-I ratio. b Representative confocal images of LC3B (green) and mitochondria (MitoTracker dye, red). Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, blue). Cells were treated with MPP⁺ (1 mM) in the absence or presence of GLE (800 μg/mL) for 6 h. Scale bar, 20 µm (left). Quantification of the percentage of cells with LC3 punctate staining (right). c Representative images of Western blot with antibodies against AMPKα, mTOR, and ULK1 (left). Quantification of these proteins from the Western blot analysis (right). d Western blot analysis of PINK1 and Parkin expression levels (left). Quantification analysis of PINK1 and Parkin expression from Western blot (right). The bands were quantified by densitometric analysis, and results shown represent the mean ± SEM of combined results from three independent experiments. *P < 0.05, ***P < 0.001 vs. the control group; ^#P < 0.05, ^###P < 0.001 vs. the MPP⁺ group

Fig. 5

GLE administration was resistant to MPP⁺-mediated apoptosis in neuro-2a cell lines. a Immunofluorescence analysis of the effect of GLE on cytochrome c released from mitochondria in MPP+-treated neuro-2a cells. Cells were treated with MPP⁺ (1 mM) in the absence or presence of GLE (800 μg/mL) for 6 h. Cells were co-stained with MitoTracker red and the anti-cytochrome c monoclonal antibody. Scale bar, 20 µm. b Immunoblot analysis of caspase-3 and caspase-9 using the caspase-3 and caspase-9 antibodies, which detected endogenous levels of full-length caspase (Pro-casp3, 35 kDa; Pro-casp9, 51 kDa) and the large fragment of caspase resulting from cleavage (a-casp3, 17 kDa; a-casp9, 39 kDa). c The optical density analysis of caspase-3 and caspase-9 proteins. *P < 0.05, **P < 0.01 vs. the control group, and ^#P < 0.05, ^##P < 0.01 vs. the MPP⁺ group. Data are the mean values ± SEM, n = 3