Targeting autophagy as a therapeutic strategy for identification of liganans from Peristrophe japonica in Parkinson’s disease

Abstract

Dear Editor, PD is characterized by the loss of dopaminergic neurons in substantia nigra, and the loss of dopamine resulting in motor deficit. Its main pathological hallmarks include the genetic mutations of gene such as α-synuclein. Increasing study showed that the dopaminergic neurons in midbrain are sensitive and damaged by the PD toxins. 6-Hydroxydopamine (6-OHDA) is widely used to induce lesion of nigrostriatal dopaminergic system in the PD model systems, including nerve cells, Caenorhabditis elegans (C. elegans), and rats. Autophagy (mitophagy) is a cellular self-digestive process that engulfs the damaged organelles, including injured mitochondria and misfolded proteins such as α-synuclein. Therefore, enhanced autophagy plays an important role in neuroprotection via cellular degradation of damaged mitochondria or mutant proteins in neurodegenerative diseases such as PD. Peristrophe japonica (PJ), a traditional Chinese medicine, is reported to exhibit anti-inflammatory, anti-bacterial, and anti-oxidative effects. However, the neuroprotective components of PJ and the mechanism remain un-elucidated. In this study, we found that the total ethanol extract of PJ (PJTEE) could protect against 6-OHDA-induced damage in PC-12 cells (Supplementary Fig. 1). Meanwhile, PJ-TEE exhibited potent autophagy effect by dose-dependently increasing the number of GFP-LC3 puncta formation in stable RFP-GFP-LC3 U87 cells (Supplementary Fig. 2a). Based on the autophagy activity-guided chemical separation, PJ-TEE was extracted using petroleum ether, ethyl acetate, and n-butanol reagents, and the components in these fractions were analyzed by UHPLC-DAD-TOF/MS (Supplementary Fig. 2b). In stable RFP-GFP-LC3 U87 and PC-12 cells, ethyl acetate fraction (EF) was demonstrated to induce the strongest autophagy effect (Supplementary Fig. 2c–e). After subfractionation of EF to 34 fractions, F11 to F34 were found to induce autophagy in stable RFP-GFP-LC3 U87 and PC-12 cells (Supplementary Fig. 3). Finally, 3 liganans, including justicidin A (JA), justicidin B (JB), and justicidin C (JC) (Fig. 1a) in F11, were isolated and identified by UHPLC-DAD-TOF/MS and nuclear magnetic resonance (NMR) instruments (Supplementary Fig. 4). To further confirm the autophagy effect of F11 was attributed to JA, JB, and JC, stable RFP-GFP-LC3 U87, PC-12, and SHSY5Y cells were adopted. The results showed that JA, JB, and JC dosedependently increased the number of GFP-LC3 puncta formation in stable RFP-GFP-LC3 U87 cells (Fig. 1b), and LC3-II protein expression in both PC-12 and SHSY5Y cells (Fig. 1c and Supplementary Fig. 5 and 7). Among them, JA time-dependently increased the ratio of RFP-LC3/GFP-LC3 puncta in stable RFP-GFPLC3 U87 cells (Supplementary Fig. 6 and Video 1). Meanwhile, JA, JB, and JC increased the ratio of RFP-LC3/GFP-LC3 puncta and the number of autophagosome in PC-12 cells (Supplementary Fig. 8a, b). In addition, LY294002 (LY) and Bafilomycin A1 (Baf) inhibited the autophagic sequestration and autophagosome-lysosome fusion induced by JA, JB, and JC (Fig. 1e and Supplementary Fig. 8c, d). All these evidences suggested that the liganans induced autophagic flux in neurons. Then, the mechanistic study demonstrated that JA, JB, and JC activated autophagy via the AMPK/ ULK1, Raf/MEK/ERK, but mTOR-independent signaling pathways (Fig. 1d and Supplementary Fig. 9, 10). In addition, compound C (CC, an AMPK inhibitor) and SCH772984 (SCH, an ERK inhibitor) decreased the number of GFP-LC3 puncta formation in stable RFP-GFP-LC3 U87 cells and LC3-II protein expression in PC-12 cells (Fig. 1e and Supplementary Fig. 11). Furthermore, JA, JB, and JC increased the number of GFP-LC3 puncta formation and LC3-II protein expression in MEF Atg7 but not in MEF Atg7 cells (Fig. 1f and Supplementary Fig. 12, 13), suggesting that JA, JB, and JC induced autophagy via Atg7. Moreover, Parkin/PINK1-mediated mitophagy was also activated by JA, JB, and JC as revealed by the colocalization of MitoTracker with GFP-LC3 puncta and the decreased GFP/RFP ratio illuminated by mCherry-GFP-FIS1101-152 (mito-QC) (Supplementary Fig. 14, 15 and Video 2). Next, the neuroprotective effect of JA, JB, and JC was examined in 6-OHDA-induced PC-12 and SHSY5Y cells. Our results showed that JA, JB, and JC dose-dependently increased the cell viability (Fig. 1g), improved cell morphology, and inhibited cell apoptosis (Supplementary Fig. 16a–d). Consistently, LY and CC could attenuate the effect of JA, JB, and JC on the improvement of cell viability (Supplementary Fig. 16e–h). The flow cytometric result demonstrated that JA, JB, and JC significantly decreased ROS production in 6-OHDA-induced PC-12 and SHSY5Y cells (Fig. 1h and Supplementary Fig. 16i, j). In addition, JA, JB, and JC could protect against oxidative damage in H2O2-induced PC-12 and SHSY5Y cells (Supplementary Fig. 17). Furthermore, JA, JB, and JC inhibited the oligomerization of α-synuclein in PC-12 cells and decreased the levels of WT-, A53T-, A30P-, and E46K-α-synuclein in MEF cells via Atg7 (Supplementary Fig. 18). However, JA, JB, and JC neither inhibited MAO activity nor activated muscarinic (M) receptor (Supplementary Fig. 19). Taken together, JA, JB, and JC exerted neuroprotective effect in 6-OHDA-, H2O2-, and αsynuclein-induced cell models of PD. To validate the autophagy and neuroprotective effect of JA, JB, and JC in vivo, both C. elegans and rat models of PD were employed. BC12921 strain expressing GFP-p62 fusion protein and DA2123 strain expressing GFP-LGG-1 fusion protein were used to detect the autophagic activity in C. elegans. The results showed that JA, JB, and JC significantly decreased the expression of p62 and increased GFP-LGG-1 punctate formation (Fig. 1i). Then, the neuroprotective effect of JA, JB, and JC was evaluated in 6-OHDA-induced BZ555 strain expressing GFP in its dopaminergic neurons, and NL5901 strain expressing human α-synuclein protein tagged with YFP in the body wall muscle. The results showed that JA, JB, and JC significantly decreased the ROS levels (Supplementary Fig. 20a), inhibited the degeneration of dopaminergic neurons (Fig. 1j), and improved the food sensing ability (Supplementary Fig. 20b) in 6-OHDA-induced BZ555 strain, as