Optical Structural Analysis of Individual alpha-Synuclein Oligomers

Small aggregates of misfolded proteins play a key role in neurodegenerative disorders. Such species have proved difficult to study due to the lack of suitable methods capable of resolving these heterogeneous aggregates, which are smaller than the optical diffraction limit. We demonstrate here an all‐optical fluorescence microscopy method to characterise the structure of individual protein aggregates based on the fluorescence anisotropy of dyes such as thioflavin‐T, and show that this technology is capable of studying oligomers in human biofluids such as cerebrospinal fluid. We first investigated in vitro the structural changes in individual oligomers formed during the aggregation of recombinant α‐synuclein. By studying the diffraction‐limited aggregates we directly evaluated their structural conversion and correlated this with the potential of aggregates to disrupt lipid bilayers. We finally characterised the structural features of aggregates present in cerebrospinal fluid of Parkinson's disease patients and age‐matched healthy controls

Transplanting the leafy liverwort Herbertus hutchinsiae : A suitable conservation tool to maintain oceanic-montane liverwort-rich heath?

Thanks to the relevant landowners and managers for permission to carry out the experiments, Chris Preston for helping to obtain the liverwort distribution records and the distribution map, Gordon Rothero and Dave Horsfield for advice on choosing experimental sites and Alex Douglas for statistical advice. Juliane Geyer’s help with fieldwork was greatly appreciated. This study was made possible by a NERC PhD studentship and financial support from the Royal Botanic Garden Edinburgh and Scottish Natural Heritage.Peer reviewedPostprin

A natural product inhibits the initiation of a-synuclein aggregation & suppresses its toxicity

The self-Assembly of a-synuclein is closely associated with Parkinson''s disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects a-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces a-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of a-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing a-synuclein, observing a dramatic reduction of a-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson''s disease and related conditions

Multistep Inhibition of α-Synuclein Aggregation and Toxicity in Vitro and in Vivo by Trodusquemine

12 pags, 3 figs. -- The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.8b00466.The aggregation of α-synuclein, an intrinsically disordered protein that is highly abundant in neurons, is closely associated with the onset and progression of Parkinson's disease. We have shown previously that the aminosterol squalamine can inhibit the lipid induced initiation process in the aggregation of α-synuclein, and we report here that the related compound trodusquemine is capable of inhibiting not only this process but also the fibril-dependent secondary pathways in the aggregation reaction. We further demonstrate that trodusquemine can effectively suppress the toxicity of α-synuclein oligomers in neuronal cells, and that its administration, even after the initial growth phase, leads to a dramatic reduction in the number of α-synuclein inclusions in a Caenorhabditis elegans model of Parkinson's disease, eliminates the related muscle paralysis, and increases lifespan. On the basis of these findings, we show that trodusquemine is able to inhibit multiple events in the aggregation process of α-synuclein and hence to provide important information about the link between such events and neurodegeneration, as it is initiated and progresses. Particularly in the light of the previously reported ability of trodusquemine to cross the blood-brain barrier and to promote tissue regeneration, the present results suggest that this compound has the potential to be an important therapeutic candidate for Parkinson's disease and related disorders.This work was supported by the Boehringer Ingelheim Fonds (P.F.), the Studienstiftung des Deutschen Volkes (P.F.), Gates Cambridge Scholarships (R.L. and G.T.H) and a St. John’s College Benefactors’ Scholarship (R.L.), the UK Biotechnology and Biochemical Sciences Research Council (M.V. and C.M.D.), a Senior Research Fellowship award from the Alzheimer’s Society, UK, grant number (317, AS-SF-16-003) (F.A.A.), the Wellcome Trust (C.M.D., M.V., and T.P.J.K.), the Frances and Augustus Newman Foundation (T.P.J.K.), the Regione Toscana—FAS Salute—Supremal project (R.C., C.C., and F.C.), a Marie Skłodowska-Curie Actions—Individual Fellowship (C.G.), Sidney Sussex College Cambridge (G.M.), the Spanish Government—MINECO (N.C.), and by the Cambridge Centre for Misfolding Diseases (M.P., P.F., R.L., F.A.A., C.G., G.T.H., S.W.C., J.R.K., T.P.J.K., M.V., and C.M.D)

A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity.

The self-assembly of α-synuclein is closely associated with Parkinson's disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson's disease and related conditions.This work was supported by the Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), US National Institutes of Health (A.M. and A.B.); by the Boehringer Ingelheim Fonds (P.F.); by a European Research Council starting grant (to M.B.D.M. and E.A.A.N.); and by The Cambridge Centre for Misfolding Diseases. N.C. thanks the Spanish Ministry of Economy and Competitiveness (RYC-2012-12068). S.W.C. thanks the Agency for Science, Technology, and Research, Singapore for support

Screening of small molecules using the inhibition of oligomer formation in α-synuclein aggregation as a selection parameter

Abstract: The aggregation of α-synuclein is a central event in Parkinsons’s disease and related synucleinopathies. Since pharmacologically targeting this process, however, has not yet resulted in approved disease-modifying treatments, there is an unmet need of developing novel methods of drug discovery. In this context, the use of chemical kinetics has recently enabled accurate quantifications of the microscopic steps leading to the proliferation of protein misfolded oligomers. As these species are highly neurotoxic, effective therapeutic strategies may be aimed at reducing their numbers. Here, we exploit this quantitative approach to develop a screening strategy that uses the reactive flux toward α-synuclein oligomers as a selection parameter. Using this approach, we evaluate the efficacy of a library of flavone derivatives, identifying apigenin as a compound that simultaneously delays and reduces the formation of α-synuclein oligomers. These results demonstrate a compound selection strategy based on the inhibition of the formation of α-synuclein oligomers, which may be key in identifying small molecules in drug discovery pipelines for diseases associated with α-synuclein aggregation

Screening of small molecules using the inhibition of oligomer formation in α-synuclein aggregation as a selection parameter

Abstract: The aggregation of α-synuclein is a central event in Parkinsons’s disease and related synucleinopathies. Since pharmacologically targeting this process, however, has not yet resulted in approved disease-modifying treatments, there is an unmet need of developing novel methods of drug discovery. In this context, the use of chemical kinetics has recently enabled accurate quantifications of the microscopic steps leading to the proliferation of protein misfolded oligomers. As these species are highly neurotoxic, effective therapeutic strategies may be aimed at reducing their numbers. Here, we exploit this quantitative approach to develop a screening strategy that uses the reactive flux toward α-synuclein oligomers as a selection parameter. Using this approach, we evaluate the efficacy of a library of flavone derivatives, identifying apigenin as a compound that simultaneously delays and reduces the formation of α-synuclein oligomers. These results demonstrate a compound selection strategy based on the inhibition of the formation of α-synuclein oligomers, which may be key in identifying small molecules in drug discovery pipelines for diseases associated with α-synuclein aggregation