Influence Of Ginkgolide B And Bilobalide On The Clearance Of Extracellular α-synuclein In Astrocytes

Parkinson’s Disease(PD) is the second most prevalent neurodegenerative diseases affecting the aging population in the world, which is characterized by the degeneration of dopaminergic neurons of the SN pars compacta(SNc) in the midbrain and the reduction of dopamine(DA) in the striatum, accompanied by the pathological accumulation of Lewy body(LB) and Lewy neuritis(LN) in the residual survival neurons. As the main component of LB, the abnormal accumulation of α-syn is closely associated with the progressive pathogenesis of PD. Native α-syn is a soluble protein located mainly in the presynaptic membrane, whose physiological function involves in the regulation of DA synthesis and release, synaptic plasticity, lipid metabolism, and so on. In PD, unfolded α-syn monomers interact to form initially unstable dimers, which grow slowly to generate aggregates that accumulated in neurons leading to neurodegeneration. A53 T mutated α-syn tends to aggregate more easily than wide-type α-syn. Small amounts of α-syn can be released from neuronal cells by unconventional exocytosis, and this extracellular α-syn can be taken up by astrocytes or microglia, contributing to spreading and progression of the disease.Astrocytes account for 90% of neural cells in the CNS, and they play an indispensable role in maintaining the homeostasis of neuronal–glial networks and microenvironment in CNS. In recent years, the role of astrocytes in phagocytosis and clearance has been more highly focused. In PD, astrocytes can take up the extracellular α-syn released by neurons and degrade them in the cytoplasm. Deeper understanding of the α-syn degradation machnisms in astrocytes lays the foundation of α-syn clearance regulation, which will help to clarify the pathological process of PD and develop new prevention and treatment strategies for this disease.Ginkgolide B(GB) and Bilobalide(BB) are terpenoids extracted from ginkgo leaves, been used for years to treat cerebrovascular disease like stroke. Ginkgo leaves extracts have been report to possess a variety of biological and pharmacological activities such as DNA repairing, antioxidant and anti-inflammation. The versatility of GB and BB makes them beneficial in neurodegeneration diseases modeling like Alzheimer disease(AD) and PD. Further clarify the regulation of GB and BB on the clearance of α-syn in astrocytes will deepen the pharmacological mechanisms of GB and BB in neuroprotective effects.In the first part of the present study, recombinant WT and A53 T α-syn proteins were expressed and purified from E.coli in order to mimic extracellular α-syn released from neurons or other nerve cell types in the CNS, and we found that GB and BB protected against α-syn induced apoptosis of SH-SY5 Y. In the second part, we found that astrocytes took up and degraded the exogenous α-syn monomers and aggregates through the proteasome system and autophagy-lysosome pathway. In addition, GB and BB accelerated the clearance of α-syn aggregates in astrocytes through an enhanced autophagy. Further more, GB and BB protected against MPP+ or α-syn aggregates-induced neurotoxity. Our study provides further confirmation of the neuroprotective role of GB and BB in neurodegeneration diseases like PD and broadens the new fields in delaying the progression of PD. Part Ⅰ Purification of recombinant α-synuclein and identification of its neurotoxicity AIM: To purify recombinant α-syn monomers and aggregates, and to identify the neurotoxicity. METHODS: Recombinant plasmids p ET-28b(+)-WT and p ET-28b(+)-A53 T were constructed for the expression of WT and A53 T α-syn. DNA sequences were compared with those given by the NCBI reference sequence project. BL21(DE3) was transformed with the recombinat plasmids and then induced with IPTG for protein expression and purification. SDS-PAGE was used in determination of protein purity. Purified protein was identified by western blotting and MALDI mass spectrometric analysis. WT and A53 T monomeric α-syn(1 mg/ml) were incubated in PBS for 14 days followed by brief sonification for aggregation of α-syn. Western blotting, Th T binding assays and electron microscope were used to identify α-syn aggregates. SH-SY5 Y cells were treated with MPP+, WT /A53 T α-syn monomers and aggregates at different concentrations for 24 h followed by pre-treatment with GB or BB. MTT assay and LDH assay were used to observe cell injury of SH-SY5 Y. Flow cytometry and western blotting were used for the detection of cell apoptosis. SH-SY5 Y was observed under light field after treatment with α-syn aggregates and GB/BB. RESULTS: 1) The DNA sequences of the insert element in recombinant plasmids p ET-28b(+)-WT were matched with human SNCA given by the NCBI reference sequence project. The 157 th base of the the insert element in recombinant plasmids p ET-28b(+)-A53 T mutated from G to A compared to human SNCA. 2) SDS-PAGE showed that the purity of WT and A53 T α-syn were more than 90%; western blotting and MALDI mass spectrometric analysis confirmed the purified protein as α-syn. 3) WT and A53 T α-syn aggregates showed target bands at 55~70 k Da in the western blot analysis; the Th T binding ability of α-syn increased over time in the process of aggregation, and A53 T α-syn aggregated more quickly than WT α-syn. WT and A53 T α-syn aggregates showed fibrillar structures under electron microscope. 4) WT/A53 T α-syn aggregates caused SH-SY5 Y injury in a dose-dependent way; WT/A53 T α-syn aggregates induced the apoptosis of SH-SY5 Y. 5) GB and BB protected against MPP+ and α-syn aggregates-induced SH-SY5 Y injury, and inhibited α-syn aggregates-induced apoptosis. CONCLUSION: 1. WT and A53 T α-syn monomers and aggregates are successfully purified. 2. WT and A53 T α-syn aggregates induce neurotoxicity in SH-SY5 Y. 3. GB and BB play a protective role in α-syn aggregates-induced neurotoxicity. Part Ⅱ Influence of Ginkgolide B and Bilobalide on the clearance of extracellular α-synuclein in astrocytes AIM: To investigate the influence of GB and BB on the clearance of exogenous α-syn in astrocytes. METHODS: Immunocytofluorescence was used to analyze GFAP positive astrocytes in cultured mice primary astrocytes. Primary astrocytes were treated with 1 μM of WT/A53 T α-syn monomers or aggregates for the indicated times. Triton X-100-soluble or insoluble extracts and the supernatant proteins were analysed by western blotting. Primary astrocytes were treated with 1 μM of FITC-WT α-syn aggregates for 1h. Immunocytofluorescence was used to observe the co-location of α-syn and GFAP. Primary astrocytes were treated with 1 μM of WT α-syn monomers and aggregates for 1h and washed twice with fresh medium, followed by incubation in fresh medium for 2h(monomers) or 24h(aggregates) with MG132(20 μM) or 3MA(5 m M). α-syn was analyzed using western blotting. Fluorescence imaging of astrocytes was observed after incubating with Lyso Tracker and FITC-WT α-syn aggregates for 1h. Astrocytes were incubated with aggregated WT α-syn for 24 h after pretreatment with GB or BB for 2h. Western blotting and immunocytofluorescence were used for α-syn analysis. Astrocytes were treated with 1 μM of WT α-syn aggregates for 1h and washed twice with fresh medium, followed by incubation with GB or BB and MG132 or 3MA for 24 h. α-syn, LC3 and Cathepsin B contents were analyzed using western blotting or immunocytofluorescence. Astrocytes were treated with 1 μM of WT α-syn aggregates for 1h and washed twice with fresh medium, followed by incubation with GB or BB for 24 h in the presence or absence of MG132 and 3MA, Triton X-100-insoluble α-syn was analyzd using western blotting. Astrocytes were treated with GB and BB for the indicated times, p62 and LC3Ⅱ/Ⅰexpression were analyzed using western blotting. Astrocytes were pre-treated with GB and BB for 24 h before α-syn aggregates insult. Western blotting was used for α-syn analysis in the astrocytic conditioned medium. MTT assay and LDH assay were used to observe injury of SH-SY5 Y treated with astrocytic conditioned medium. RESULTS: 1) α-syn was detected in astrocytes after treatment with α-syn monomers 12 or aggregates for different times, while α-syn content in the supernatant decreased over time. 2) FITC-WT α-syn aggregates co-located with GFAP. 3) Internalized α-syn content decreased with time. 4) MG132 and 3MA reversed the reduction of internalized α-syn monomers and aggregates in astroyctes. 5) FITC-WT α-syn aggregates co-located with lysosome in astrocytes. 6) GB and BB further reduced α-syn content in astrocytes. 7) MG132 and 3MA inhibited the reduction of α-syn content with GB and BB treatment; expression of p62 decreased while LC3Ⅱ/Ⅰincreased in astrocytes after GB and BB treatment. 8) GB and BB reversed α-syn aggregates-stimulated astrocytic conditioned medium-induced decreased cell viability and increased LDH release of SH-SY5 Y. CONCLUSION: 1. Astrocytes take up extracelluar α-syn monomers and aggregates, and degrade the internalized α-syn through the proteasome system and autophagy-lysosome pathway. 2. GB and BB accelerate the clearance of α-syn aggregates in astrocytes through an activated autophagy pathway. In summary, the major contributions of the present study lie in: 1. GB and BB played a protective role in α-syn aggregates-induced neurotoxicity. GB and BB and inhibited α-syn aggregates-induced apoptosis and ameliorated α-syn aggregates-induced neuronal damage, which provides further confirmation of the neuroprotective role of GB and BB in neurodegeneration diseases like PD. 2. Astrocytes took up and degraded extracellular α-syn through the proteasome system and autophagy-lysosome pathway. Astrocytes took up exogenous α-syn monomers and aggregates, and degraded α-syn through the proteasome system or autophagy-lysosome pathway. The elimition of monomers was faster than aggregates. In addition, α-syn monomers could release to extracelluar space rapidly. These foundings provide promising experimental evidence for further studing the neuroprotective role of astrocytes in PD. 3. GB and BB accelerated the clearance of α-syn aggregates in astrocytes. GB and BB activated autophagy in astrocytes, thus promoting the clearance of α-syn aggregates in astrocytes and ameliorating α-syn aggregates-stimulated astrocytic conditioned medium-induced neurotoxicity. These findings elucidate new machnisms for the neuroprotective role of GB and BB.