Understanding the Molecular Origins of the Toxicity of Alpha-synuclein in Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder afflicting 2% of the global population over 65 years of age. It is generally recognised that the hallmark of PD is the deposition of insoluble fibrils of human alpha-synuclein, a pre-synaptic 140-residue protein, in Lewy bodies. There is also significant genetic evidence implicating alpha-synuclein in the pathogenesis of PD, with point mutations (A30P, E46K, H50Q, G51D and A53T) and gene triplication known to cause dominantly inherited early onset PD.

The proposal will achieve a conclusive understanding of the structural bases of the toxicity of prefibrilar alpha-synuclein oligomers, which are generally acknowledged to be the most toxic species in the aetiology of PD. This is a top goal that is associated with significant experimental and theoretical challenges owing to the transient nature of these protein states. Indeed, there is currently a poor understanding of the nature of alpha-synuclein oligomers, including the structural and molecular bases of their toxicity.

We believe that our research proposal comes at a most opportune time, since we now have all the tools to perform such a study. Our goal is to unveil the molecular bases of the cellular toxicity of alpha-synuclein oligomers by characterising and comparing the structural properties of two types of pre-fibrilar oligomers having similar morphologies but significantly different levels of toxicity (Cremades et al Cell, 2012, 149:1048-59). By using solution and solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with computational biology, we will determine the structural properties of these oligomers and investigate the origins of their selective interaction with biological lipid membranes. Our analysis will be extended to pathological mutants of alpha-synuclein and will be complemented by biophysical and cellular experiments.

The aimed interdisciplinary characterisation of the molecular bases of alpha-synuclein oligomers/membrane interactions will have significant impact on the wider academic community studying the molecular bases of amyloid diseases (including Parkinson's, Alzheimer's and Diabetes type II) and wider disciplines such as protein science, biochemistry, NMR spectroscopy, molecular simulations, cellular and molecular biophysics. It is anticipated that the outcomes of this research may directly translate into knowledge leading to new therapeutic approaches in Parkinson's disease and other amyloid-associated disorders.

In this project we assembled a cohort of complementary skills and combined our longterm research programmes to carry out an interdisciplinary study of the structural bases of the toxicity of prefibrilar alpha-synuclein oligomers in view of the crucial interaction with the membrane, which represents the key step in the aetiology of Parkinson's disease. Our research proposal comes at a most opportune time, since we now have all the tools, background research and materials to perform such an ambitious study. The proposal is articulated on three independent and complementary streams of increasing scale of complexity (i.e. from oligomers structure to membrane interaction and to the effects of pathological mutations).

To fulfil this ambitious goal, we will carry out an investigation of solid-state NMR (ssNMR), solution NMR and computational biology that will provide an accurate structural characterisation of toxic and non-toxic oligomers, previously individuated by a part of the team (Cremades et al, Cell, 2012 149:1048-59). Moreover, to probe the selective interaction of the two types of oligomers with a variety of lipid membranes, we will adapt our recent approach that successfully unveiled the nature of the interaction between monomeric alpha-synuclein and lipid membranes (Fusco et al, Nature Communications, 2014 5:3827). The structural investigation will be complemented biophysical and cellular toxicity measurements to define the underlying structural bases of cellular toxicity of alpha-synuclein.

The outcomes of this research impact both the academic sector and the industry as well as both basic and applied sciences. The project will provide a key knowledge toward the identification of new therapies to combat Parkinson's and other amyloid neurodegenerative diseases. Moreover, the general understanding of amyloid toxicity will have an impact on the general research in biomaterials and new biotechnologies. This will enhance dramatically our ability to study key molecular processes that are relevant to both academia and industry and extend the applicability of current spectroscopic methods to more complex processes.

INDUSTRIAL BENEFICIARIES: The project is likely to provide a significant impact to pharma industry by advancing the use of NMR spectroscopy to understand complex molecular processes. Direct beneficiaries include those pharmaceutical companies working at the definition of therapeutic approaches to combat amyloid diseases such as Alzheimer's and Parkinson's. An additional area for applied sciences is the exploitation of new biomaterials based on amyloid fibrils. In order to develop such biotechnologies, it is necessary understanding and controlling the processes by which protein oligomers elicit cellular toxicity, which is the top goal of this project. More broadly, the interdisciplinary approaches here proposed will enhance our ability to characterise the mechanisms of heterogeneous biological processes, with key impact on many bio-industrial areas.

MEDICAL CHALLENGES IN NEURODEGENERATIVE DISORDERS: Understanding the molecular bases of alpha-synuclein toxicity is a key target for Parkinson's research. By producing structural data on toxic and non-toxic oligomers, the project will set the scenario for drug discovery toward the identification of effective therapeutic molecules to target toxic species at the onset and development of Parkinson's disease.

DISSEMINATION: We will make every effort to ensure that research is disseminated widely to the research community by the Open Access publication in high-impact journals, presentation at international research meetings and the development of new collaborations. Furthermore, protocols and algorithms developed for the analysis of complex NMR data will be made available through our websites at Imperial College and University of Cambridge and the Collaborative Computing project for NMR (CCPN: http://www.ccpn.ac.uk/ccpn).
We are highly committed to make our findings available to the wider public. Presentations explaining how NMR spectroscopy assists in the drug development, Parkinson's research and scientific discovery will be made School-oriented presentations and Schools Open Days. The press offices at Imperial will assist in disseminating discoveries via the popular science press and other media formats.

TRAINING: In the proposed research we will employ and develop state-of-the-art methods of NMR spectroscopy to contribute in the wide area of structural biology. This provides an excellent platform to enhance training of PDRAs, PhDs as well as undergraduate students at the interface of biology, spectroscopy, medicine and chemical engineering.