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      Identification of novel autophagic Radix Polygalae fraction by cell membrane chromatography and UHPLC-(Q)TOF-MS for degradation of neurodegenerative disease proteins

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          Abstract

          With its traditional use in relieving insomnia and anxiety, our previous study has identified onjisaponin B from Radix Polygalae ( RP), as a novel autophagic enhancer with potential neuroprotective effects. In current study, we have further identified a novel active fraction from RP, contains 17 major triterpenoid saponins including the onjisaponin B, by the combinational use of cell membrane chromatography (CMC) and ultra-performance liquid chromatography coupled to (quadrupole) time-of-flight mass spectrometry {UHPLC-(Q)TOF-MS}. By exhibiting more potent autophagic effect in cells, the active fraction enhances the clearance of mutant huntingtin, and reduces protein level and aggregation of α-synuclein in a higher extent when compared with onjisaponin B. Here, we have reported for the first time the new application of cell-based CMC and UHPLC-(Q)TOF-MS analysis in identifying new autophagy inducers with neuroprotective effects from Chinese medicinal herb. This result has provided novel insights into the possible pharmacological actions of the active components present in the newly identified active fraction of RP, which may help to improve the efficacy of the traditional way of prescribing RP, and also provide new standard for the quality control of decoction of RP or its medicinal products in the future.

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          Most cited references41

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          Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and alpha-synuclein.

          Trehalose, a disaccharide present in many non-mammalian species, protects cells against various environmental stresses. Whereas some of the protective effects may be explained by its chemical chaperone properties, its actions are largely unknown. Here we report a novel function of trehalose as an mTOR-independent autophagy activator. Trehalose-induced autophagy enhanced the clearance of autophagy substrates like mutant huntingtin and the A30P and A53T mutants of alpha-synuclein, associated with Huntington disease (HD) and Parkinson disease (PD), respectively. Furthermore, trehalose and mTOR inhibition by rapamycin together exerted an additive effect on the clearance of these aggregate-prone proteins because of increased autophagic activity. By inducing autophagy, we showed that trehalose also protects cells against subsequent pro-apoptotic insults via the mitochondrial pathway. The dual protective properties of trehalose (as an inducer of autophagy and chemical chaperone) and the combinatorial strategy with rapamycin may be relevant to the treatment of HD and related diseases, where the mutant proteins are autophagy substrates.
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            Atg7-dependent autophagy promotes neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in Drosophila.

            Autophagy, a cellular process of cytoplasmic degradation and recycling, is induced in Drosophila larval tissues during metamorphosis, potentially contributing to their destruction or reorganization. Unexpectedly, we find that flies lacking the core autophagy regulator Atg7 are viable, despite severe defects in autophagy. Although metamorphic cell death is perturbed in Atg7 mutants, the larval-adult midgut transition proceeds normally, with extended pupal development compensating for reduced autophagy. Atg7-/- adults are short-lived, hypersensitive to nutrient and oxidative stress, and accumulate ubiquitin-positive aggregates in degenerating neurons. Thus, normal levels of autophagy are crucial for stress survival and continuous cellular renewal, but not metamorphosis.
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              Formation of Toxic Oligomeric α-Synuclein Species in Living Cells

              Background Misfolding, oligomerization, and fibrillization of α-synuclein are thought to be central events in the onset and progression of Parkinson's disease (PD) and related disorders. Although fibrillar α-synuclein is a major component of Lewy bodies (LBs), recent data implicate prefibrillar, oligomeric intermediates as the toxic species. However, to date, oligomeric species have not been identified in living cells. Methodology/Principal Findings Here we used bimolecular fluorescence complementation (BiFC) to directly visualize α-synuclein oligomerization in living cells, allowing us to study the initial events leading to α-synuclein oligomerization, the precursor to aggregate formation. This novel assay provides us with a tool with which to investigate how manipulations affecting α-synuclein aggregation affect the process over time. Stabilization of α-synuclein oligomers via BiFC results in increased cytotoxicity, which can be rescued by Hsp70 in a process that reduces the formation of α-synuclein oligomers. Introduction of PD-associated mutations in α-synuclein did not affect oligomer formation but the biochemical properties of the mutant α-synuclein oligomers differ from those of wild type α-synuclein. Conclusions/Significance This novel application of the BiFC assay to the study of the molecular basis of neurodegenerative disorders enabled the direct visualization of α-synuclein oligomeric species in living cells and its modulation by Hsp70, constituting a novel important tool in the search for therapeutics for synucleinopathies.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                24 November 2015
                2015
                : 5
                : 17199
                Affiliations
                [1 ]State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology , Macau, China
                Author notes
                Article
                srep17199
                10.1038/srep17199
                4657008
                26598009
                e0e70ff0-a55b-4303-b195-2268f2529818
                Copyright © 2015, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 03 February 2015
                : 26 October 2015
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