Development of single molecule techniques for nanoscale imaging of toxic protein species in vitro and in cells.

Proteins are one of the fundamental building blocks for life, but in order to work correctly, proteins need to be in the right shape, or, in scientific language, they need to maintain their fold. When proteins misfold, they can take on aberrant shapes and often collapse in the form of insoluble deposits, which can be toxic to neurons in the brain. Indeed, major diseases such as Alzheimer's or Parkinson's diseases (PD), are characterized by the formation of insoluble deposits of proteins which polymerize in the form of long filaments, called amyloid fibrils. Unfortunately, the molecular interactions that lead to fibril formation and subsequent aggregation are not yet elucidated; therefore, high resolution techniques that characterize the ultrastructure of fibrils are in high demand. Electron microscopy is widely used for this purpose, but its invasive nature prevents it from following fibril formation in time and EM requires elaborate sample fixation and mounting protocols. Atomic force microscopy can also image fibrils at high resolution, but it is limited to surface topology. Although fluorescence microscopy can complement these techniques, conventional optical methods are limited by diffraction and provide a resolution far too coarse to provide information in protein fibrils / aggregates. Recently, variants of single molecule fluorescence techniques have been developed that provide 10-20 nm resolution i.e. much smaller than the wavelength of the probing light. How do they work? A sample is tagged with so called photoactivatable fluorophores which can be switched on and off with pulses of light; one can thus think of each fluorophore as a switchable light bulb. By switching on a few individual light bulbs at any one time and taking an image, the positions of the active light bulbs can be determined with very high precision (10-20 nm). By repeating this process thousands of times, activating different fluorophores each time, and taking as many images the precise location of each fluorophore can be determined and information on the underlying structure can be obtained (as an analogy one may think of a Christmas tree, 'labelled' with lots of lightbulbs: If the positions of the individual lightbulbs are accurately known, information on the underlying shape/structure of the tree can be regained). It is with such so called single molecule fluorescence techniques we would like to gain, for the first time, dynamic information on the sizes and shapes of fibrillar structures formed by alphasynclein (AS), a protein that misfolds and lies at the heart of PD. We will complement this research with other optical techniques developed by us, which also give us molecular scale resolution of protein aggregates forming in cells, and also in test solutions. If successful we will end up with tools that give us much more direct information on the aggregation process in cells than has hitherto been possible. It would be exciting for example, if we were to see differences in the AS protein deposits formed in cells treated with potential anti-aggregation drugs compared to those which have not been treated. In any case we will end up with new knowledge and tools, with which to study protein function and 'misbehaviour'. The proposed work brings together the expertise of molecular and chemical biologists, physicists, and engineers who have put in place the building blocks to tackle this important problem in neurodegenerative disease with novel optical tools.

This research will lead to the development of single molecule optical superresolution methods to a problem of fundamental imporatance in neurodegenerative disease research. Who will benefit from this research (see also academic beneficiaries section above)? The National Physics Laboratory (NPL) will benefit by applying and expanding on their expertise in single molecule biophysics area and will obtain expertise in PALM and STORM imaging The UK community as a whole will benefit from these developments through NPL's role as the national provider of metrological services to the UK R&D community. This guarantees an effective means of technology diffusion both to UK academia and industry. The UK health and pharmaceutical sectors will benefit: The possibility to quantify the presence of toxic protein species in their biological environment offers opportunities to validate the effectiveness of potential drugs and explore potential means of therapeutic intervention (e.g. anti-aggregating drugs). The research will help the UK to maintain its leading role in the applied photonics field: There is huge commercialisation potential (see letter of support from Fianium) and there are opportunities for novel array detector technologies, supercontinuum technologies, and image processing tools in all of which the UK plays leading roles. Society as a whole will benefit: Through improved tools for neurodegenerative disease research, novel therapeutic and diagnostic approaches can be developed which may ultimately help in reducing some of the most devastating, costly and humiliating age related diseases. For example, in the UK alone well over 10000 new cases of Parkinson's disease are reported every year.