Cellular and Molecular Neurobiology (2019) 39:1017–1028 https://doi.org/10.1007/s10571-019-00696-2 ORIGINAL RESEARCH Enhancing the Astrocytic Clearance of Extracellular α‑Synuclein Aggregates by Ginkgolides Attenuates Neural Cell Injury Jun Hua1,2 · Nuo Yin1 · Shi Xu1 · Qiang Chen1,3 · Tingting Tao1,3 · Ji Zhang4 · Jianhua Ding1 · Yi Fan1,3 · Gang Hu1,5 Received: 9 November 2018 / Accepted: 1 June 2019 / Published online: 5 June 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract The accumulation of aggregated forms of the α-Synuclein (α-Syn) is associated with the pathogenesis of Parkinson’s dis- ease (PD), and the efcient clearance of aggregated α-Syn represents a potential approach in PD therapy. Astrocytes are the most numerous glia cells in the brain and play an essential role in supporting brain functions in PD state. In the present study, we demonstrated that cultured primary astrocytes engulfed and degraded extracellular aggregated recombinant human α-Syn. Meanwhile, we observed that the clearance of α-Syn by astrocytes was abolished by proteasome inhibitor MG132 and autophagy inhibitor 3-methyladenine (3MA). We further showed that intracellular α-Syn was reduced after ginkgolide B (GB) and bilobalide (BB) treatment, and the decrease was reversed by MG132 and 3MA. More importantly, GB and BB reduced indirect neurotoxicity to neurons induced by α-Syn-stimulated astrocytic conditioned medium. Together, we frstly fnd that astrocytes can engulf and degrade α-Syn aggregates via the proteasome and autophagy pathways, and further show that GB and BB enhance astrocytic clearance of α-Syn, which gives us an insight into the novel therapy for PD in future. Keywords α-Synuclein · Astrocytes · Autophagy · Ginkgolide B · Bilobalide Abbreviations BCA Bicinchoninic acid α-Syn α-Synuclein CM Conditioned medium ALP Autophagy–lysosome pathways DAPI 4,6-Linked amidine-2-phenylindole BB Bilobalide DLB Dementia with LBs DMSO Dimethyl sulfoxide DTT DL-Dithiothreitol Jun Hua and Nuo Yin have contributed equally to this work. GB Ginkgolide B * Yi Fan GBE Ginkgo biloba extract [email protected] GFAP Glial fbrillary acidic protein * Gang Hu LBs Lewy bodies [email protected] LC3 Microtubule-associated protein 1A/1B-light chain 3 1 Jiangsu Key Laboratory of Neurodegeneration, Department LDH Lactate dehydrogenase of Pharmacology, Nanjing Medical University, 101 Longmian Road, Nanjing 211166, Jiangsu, China LNs Lewy neuritis MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- 2 Department of Neurology & Psychology, The Fourth Clinical Medical College of Guangzhou University of Chinese zolium Medicine, 1 Fuhua Road, Shenzhen 518033, Guangdong, PBS Phosphate bufered saline China PD Parkinson’s disease 3 Neuroprotective Drug Discovery Key Laboratory of Nanjing SNpc Substantia Nigra pars compacta Medical University, 101 Longmian Road, Nanjing 211166, ThT Thiofavin T Jiangsu, China 3MA 3-Methyladenine 4 Division of Clinical Pharmacy, Department of Pharmacy, UPS Ubiquitin proteasome system The First Afliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu, China 5 Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing 210023, Jiangsu, China Vol.:(0123456789)1 3 1018 Cellular and Molecular Neurobiology (2019) 39:1017–1028 Introduction overexpression of PLK2 reduced intra-neuronal human α-Syn accumulation, and suppressed dopaminergic neu- Alpha-Synuclein (α-Syn), a 14 kDa protein (140 amino rodegeneration induced by α-Syn overexpression in a rat acids) physiologically found in presynaptic terminals of genetic model of PD (Oueslati et al. 2013). Moreover, neurons, is the major fbrillary protein in Lewy bodies clearance of α-Syn monomers and aggregates occurs via (LBs) and Lewy neuritis (LNs) (Vekrellis et al. 2011). direct proteolysis by matrix metalloprotease 9 (MMP-9) Alpha-Syn has been suggested to play important roles in (Iwata et al. 2003) or binding to molecular chaperones brain lipid metabolism, modulation of synaptic transmis- such as the proteasome (McNaught et al. 2002) and sion, and synaptic vesicle biogenesis (Kim and Lee 2008). autophagy (Oueslati et al. 2013). However, mounting evidence has indicated that α-Syn We have previously shown that aggregated α-Syn induced malfunction, especially the accumulation of misfolded cell apoptosis, and Ginkgo biloba extract (GBE), including α-Syn aggregates and genetic mutations, can also contrib- ginkgolide B (GB) and bilobalide (BB), attenuated aggre- ute to synucleinopathies such as dementia with LBs (DLB) gated α-Syn-induced cell apoptosis (Hua et al. 2017). In the and sporadic and inherited Parkinson’s disease (PD) (Kalia present study, we showed that astrocytes internalized and and Lang 2015; Spillantini et al. 1998). eliminated α-Syn aggregates in vitro, and GB and BB pro- PD is primarily characterized by selective loss of dopa- moted the degradation of intercellular α-Syn aggregates via minergic neurons in the substantia nigra pars compacta the autophagy and proteasome pathways. Moreover, GB and (SNpc) and abrogation of dopaminergic tone along the BB reduced the indirect neurotoxicity of α-Syn to neurons nigrostriatal pathway (Dauer and Przedborski 2003; Obeso in vitro. Herein, we elucidate that the promotion of astro- et al. 2017). However, previous studies have indicated cytic clearance of α-Syn may be a potential approach in PD that the progressive neuronal accumulation of aggregated therapy. α-Syn and the formation of LBs might trigger neuroin- fammation, neurotoxicity, and synaptic dysfunction in PD (Gao et al. 2011; Ma et al. 2014; Volpicelli-Daley et al. Materials and Methods 2011). Notably, the release of α-Syn aggregates from neu- ronal cells was signifcantly increased under PD condition Experimental Animals and Drug Administration (Jang et al. 2010), and extracellular α-Syn aggregates may cause paracrine efects on neighboring cells. Transmission All study protocols were approved by the Institutional Ani- of α-Syn aggregates from neuron-to-neuron (Tyson et al. mal Care and Use Committee of Nanjing Medical University 2017) and neuron-to-glia has been suggested as the under- (IACUC). C57BL/6 mice were provided by Model Animal lying mechanism of neuroinfammation and neurotoxicity Resource Center (MARC, Nanjing, China). All mice were in PD (Choi et al. 2018). Hence, an efcient clearance of maintained on a 12/12 h light/dark cycle with free access to this protein might represent a potential approach in PD food and water. therapy. GB and BB (purity > 99%, supplied by the National Insti- Astrocytes are the most numerous and multifunctional tutes for Food and Drug Control, China) were dissolved in type of glia cells in the central nervous system (CNS). dimethyl sulfoxide (DMSO) as stock solutions for in vitro Astrocytes are crucial to the neuronal microenvironment studies, respectively. due to its abilities to maintain tissue structure, provide nutrition and energy to neurons, and regulate synaptic Cell Culture and Treatment development and neurotransmitter uptake. Previously studies have shown that extracellular α-Syn acted on neu- Primary astrocytes were prepared from brains of 1- to ronal Toll-like receptor 2 (TLR2) might result in neuro- 2-day-old C57BL/6 mice, as previously described (Zeng degeneration by inhibition of autophagy via AKT/mTOR et al. 2007). Briefy, neonates (P1–2) were killed by rapid signaling (Kim et al. 2013), while extracellular α-Syn decapitation. The midbrain was removed, and then the tis- activated microglia via NF-κB and p38 MAPK pathway, sue was dissociated with 0.25% tryptase (VWR Life Sci- thereby resulted in neuroinfammatory factors produc- ence, USA) at 37 °C and terminated by Dulbecco’s Modifed tion (Kim et al. 2015). While extracellular α-Syn aggre- Eagle’s Medium (DMEM, Gibco Lab., USA) supplemented gates can be taken up by neurons (Lee et al. 2008a, b) and with 10% Fetal Bovine Serum (FBS, Gibco Lab., USA), glial cells (Lee et al. 2008a, b) by endocytosis, it is not and 100 mg/mL streptomycin (Sigma-Aldrich Biotechnol- clear whether astrocytes are responsible for α-Syn clear- ogy, USA) and 100 U/mL penicillin (Gibco Lab., USA). ance in PD. A recent study reported that Polo-like kinase After centrifugation at 1500 rpm for 5 min, the cell pellets 2 (PLK2) enhanced α-Syn autophagic degradation, and were re-suspended and plated on a poly-lysine-treated fask (Sigma, St Louis, MO). The cultures were maintained at 1 3 Cellular and Molecular Neurobiology (2019) 39:1017–1028 1019 37 °C in a humidifed 5% CO2–95% air atmosphere. Culture and 5 μL α-Syn sample were added to the 96-well plate to media were changed 24 h later to complete medium and sub- a fnal volume of 200 μL. Phosphate bufered saline (PBS) sequently twice a week. The purity of astrocyte was > 95% was as a blank control. The reaction was carried out at room as determined with GFAP immunocytochemistry. Before temperature for 1 min, and the absorbance value was read experimental treatments, astrocytic cultures were passaged by a full-wavelength microplate reader. The excitation light once. is 446 nm and the absorption light is 482 nm. ThT binding Human neuroblastoma SH-SY5Y cells were purchased (unit a.u.) = Absorbance of Sample − Absorbance of PBS from American Type Culture Collection (ATCC, USA). blank control. Cells were cultured in DMEM (Gibco Lab., USA) contain- ing 10% FBS (Gibco Lab., USA), 100 mg/mL streptomycin (Sigma-Aldrich Biotechnology, USA), and 100 U/mL peni- FITC‑labeled α‑Syn Aggregates cillin (Gibco Lab., USA). The cultures were maintained at 37 °C in a humidifed atmosphere of 5% CO 2 and 95% air. The α-Syn aggregates were placed in 4-mL ultrafltration Before the experiments, cells were inoculated into 6-well tubes (30 kDa, Millipore, UFC803008) and centrifuged at plate or 96-well plate, and cultured in DMEM containing 7500 g for 20 min, following replaced PBS with a freshly 10% FBS for 24 h. Then the medium was replaced with prepared cross-linked reaction solution (7.56 g NaHCO3, serum-free medium with α-Syn (1 μM) or GB (10 μM)/ 1.06 g Na2CO3, 7.36 g NaCl, pH 9.0, plus ddH2O fxed BB (10 μM) or MG132 (20 μM)/3MA (5 mM) in diferent volume to 1 L).