MICA: Multi-parametric and super-resolution imaging of amyloidogenic proteins

Alzheimer's, like other neurodegenerative diseases, is caused by aberrant forms of proteins which tend to accumulate and aggregate into shapes, which are believed to be toxic to the brain cells of affected patients. The two proteins thought to cause Alzheimer's are called amyloid-beta and tau. The precise mechanisms by which these proteins aggregate into toxic forms are unknown, but there is accumulating evidence that the shape and size of forming aggregates is intricately linked to the onset and progression of disease. Yet there are no tools to study the growth of toxic aggregates in living cells, they are simply too small to be observed with traditional techniques. For the development of potential therapies against Alzheimer's, a microscopic understanding of the events leading to aggregation, and their consequence on affected cells, is crucial. Being able to observe protein aggregation in brain cells would permit us to relate the appearance of protein "clumps" with their toxic effect. Do affected cells malfunction? In what way? What aggregate species cause the cells to malfunction? Can we identify the toxic species leading cells to 'misbehave'? Can we inhibit the formation of "toxic shapes" in living cells through the addition of potential therapeutic agents? Can we stop the spreading of toxic species from cell-to-cell? Answers to these questions require radically new approaches to look into cells, at unprecedented resolution. In our group we have recently developed a new kind of microscope, a so called optical super-resolution microscope, that is so powerful that it is capable of visualising the shape of protein aggregates directly in cultured brain cells, which we use as model systems of disease. We were the first researchers to demonstrate that it is possible to obtain 'images' of toxic protein shapes in neurones, and here we propose to use this newly developed tool to shed new light on the mechanisms that lead to the onset and propagation of disease.
The aims of our proposed research project are: 1) to identify specific species of amyloid-beta and tau in neurones and other cells present in the brain and to relate the shape, and size of these species to the appearance of disease symptoms. 2) to study amyloid-beta and tau trafficking in and out of cells and to infer on the implications of this on the possible mechanisms of disease progression. 3) to see whether potential inhibitors of protein aggregation and propagation have an effect on what we observe within the cells we study. We will test several promising anti-aggregation anti-propagation compounds that our collaborators provide.
Through the development and application of advanced microscopic imaging techniques developed in our group we aim to gain insight into the molecular level processes that lie at the heart of Alzheimer's disease and other forms of dementia. Our techniques enable us to investigate the mechanisms that lead to the misfolding and spreading of proteins and their aggregation into toxic structures in live cell model systems at very high resolution, giving insight into the mechanisms of disease and its propagation. This understanding may help in the future search for potential therapeutic agents. The knowledge and tools generated here will provide a rational basis for the development of biomarkers/diagnostics that will enable physicians to: 1) detect the disease in its earliest stages when only biochemical changes are apparent; 2) identify the stage of the disease in order to tailor therapies for individual patients; and 3) monitor response to that treatment. Knowledge of these pathways will also provide targets for therapies to repair these pathways, thereby preventing or even reversing the disease.

Amyloid proteins such as Abeta and tau are known to misfold and, eventually, to aggregate into insoluble deposits.

Questions that remain unsolved include: What are the neurotoxic amyloid species? Can the misfolded state propagate from one cell to another and has this an impact on the neuropathology? Do extracellular chaperones impact on degradation or aggregation pathways of amyloid species? The applicant's group specialises in the development and application of advanced microscopy techniques for the functional study of protein self-assembly reactions in neurodegenerative disease. We will use these tools to address the following questions:

- Abeta and tau aggregates are likely to be composed of ensembles of oligomers with different sizes and conformations and potentially differing neurotoxicity. We will use novel fluorescence-based sensors to: a) characterise the biophysical properties of these ensembles in live cells with a spatial resolution on the molecular scale; and b) correlate their biophysical properties with effects on neuropathology.

- There is increasing evidence in the literature that intracellular Abeta plays an important role in disease. In addition, the propagation of tau pathology appears to be crucial in spreading the disease to non-affected brain areas. We aim to characterise the trafficking mechanisms responsible for amyloid proteins being taken up by cells and/or released in the extracellular space.

- We will screen for different inhibitors of Abeta or tau mediated neuropathology. Firstly, we will investigate different inhibitors of amyloid proteins propagation. Secondly, we will test the potential of extracellular chaperones to interfere with amyloid protein degradation or aggregation.

Over 820,000 people were affected by dementia in the UK in 2010, 24 million worldwide in 2005. This number is forecast to increase rapidly, making it one of the most pressing health issues in developed countries. The annual cost of dementia was over £23 billion in 2009. Our research is part of a global effort to assist the discovery of a cure for these devastating and costly diseases. Through the development and application of advanced microscopic imaging techniques developed in our group we aim to gain a mechanistic insight into the molecular level processes that lie at the heart of Alzheimer's disease and other forms of dementia. Our techniques enable us to investigate the mechanisms that lead to the misfolding of proteins and their aggregation into toxic structures in live cell model systems at molecular scale resolution, giving insight into the mechanisms of disease and its propagation. This understanding may help in the future search for potential therapeutic agents. The potential beneficiaries from this research are therefore:
- Neurobiologists studying neurodegenerative diseases (see previous section on academic beneficiaries)
- Pharmaceutical companies designing drugs will benefit from a better understanding of protein misfolding, aggregation, and dysfunction. They will also benefit from the technology we develop which could be optimised into methods for screening novel drugs and the investigation of their mode of action.
- Patients suffering from Alzheimer's disease and other forms of dementia, their carers and the community at large will benefit from a better understanding of the molecular and cellular events, which are involved in the onset and propagation of dementia. Drugs that may develop from such an understanding would greatly increase their quality and dignity of life as well as that of their relatives and carers.
- The project provides career opportunities for the named researchers through involvement and training in a cutting edge research environment on a topic of great socioeconomic importance. They will be trained in the development and application of state of the art molecular imaging methods, which finds use both in academic as well as in industrial research environments (e.g. in pharmaceutical companies).
- The National Physics laboratory will benefit through our collaboration in the development of optical super-resolution methods: the application of these techniques to address question of biomedical importance will provide incentives to further develop these methods (see letter of support). As the UK's national provider of measurement services, NPL will serve industry and academia on the whole with the provision of these technologies.
- The Neuroscience Consortium led by Prof. P. St. George-Hyslop, comprising research groups from across the UK and abroad will also benefit. Our technologies are critical to support the activities of these researchers, and our infrastructure and technical expertise will be made available to them free of charge.