What defines the seeding and cross-seeding potential of amyloid particles?

A number of devastating human brain disorders, for example Alzheimer's disease (AD), Hungtington's diseases, diabetes type 2 and transmissible spongiform emcephalopathies (TSEs), are associated with the abnormal folding of proteins. The net result of this misfolding is the formation of large insoluble protein deposits and small toxic protein particles in a state called amyloid. The deposition of aggregated protein material in various tissues (e.g. brain, liver etc) is one of the many common characteristics shared by these diseases. Importantly, these disorders also share a similar method by which the misfolded and aggregated proteins are propagated in the disease conditions. In this process, different proteins in the cell are triggered to undergo a major change in their structure to form the highly robust amyloid state. One crucial step in amyloid formation is that the addition of preformed amyloid particles, the seeds, can greatly accelerate amyloid growth, and this phenomenon is called seeding. In some cases these seed particles are considered as infectious entities, capable of transmitting the disease to neighbouring cells, tissues, or another individual of the same or a different species, as in the case of the TSEs and possibly in other amyloid diseases such as Alzheimer's disease. The current and projected impact of these diseases on human health and welfare cannot be understated yet the fundamental question of how is the amyloid state propagated through seeding remains to be fully resolved.

In some of the diseases associated with protein misfolding, more than one type of amyloid aggregate may exist where each type of aggregate is made of a protein with a different amino acid sequence. For example, in Alzheimer's disease the co-existence of various amyloid forms of the diagnostic amyloid-beta protein and at least one other different protein has been reported in patients including the presence of transmissible prion protein aggregates. Furthermore, recent reports have suggested that the onset of prion disease can be influenced and possibly enhanced by the presence of amyloid-beta deposits. This co-existence of two different amyloids in the same patient can be potentially explained by the interaction between misfolded proteins with each other, accelerating their respective conversions to the amyloid state. Therefore, the amyloid seeding and cross-seeding process is also potentially involved in the devastating synergetic effects in amyloid diseases.

Our aim is to study the fundamental process of amyloid seeding by a combination of test tube-based in vitro approaches as well as cell-based in vivo approaches using the baker's yeast Saccharomyces cerevisiae as a safe and experimentally tractable model. In our project we will map the seeding potency of well-characterised amyloid seed samples, monitoring the growth of the amyloid fibrils using natural seeds or seeds formed from other amyloid proteins, so called "cross-seeding". We will then investigate how "cross-seeding" occurs in the yeast cell using a novel yeast prion-based assay. Since yeast prions are infectious but non-toxic, this system allows us to follow amyloid formation and propagation without causing cell death and therefore we can investigate the fundamental principles of cross-seeding in a living cell. Overall, our project will allow us to establish the nature and spectrum of the potential interactions between misfolded proteins and the dependence, if any, on cellular components in generating this important disease-associated amyloid forms.

Amyloid formation proceeds through a nucleated assembly mechanism. Addition of preformed amyloid particles to the reaction, so called 'seeds', accelerates the formation of amyloid aggregates. We will investigate the molecular mechanism of amyloid seeding and cross-seeding in vitro and using yeast cell-based models of amyloid assemblies.

The seeding potential of four different amyloidogenic protein sequences will be investigated in vitro and in vivo: the yeast prion protein Sup35, including mutants that inhibit the propagation of the prion to different extents, and three human proteins, amyloid-beta, alpha-synuclein and amylin. Amyloid seeds will be formed both in vitro and in a cellular environment by overexpression of the monomeric precursors in S. cerevisiae. The amyloid fibrils formed from each protein will be used as seeds to accelerate the subsequent formation of amyloid aggregates in vitro from either the same monomeric precursors (seeding) or other protein monomers (cross-seeding). Formation of fibrils will be monitored using ThT fluorescence kinetics assays and EM/AFM imaging. Next, the cell extracts containing in vivo-generated amyloid particles will be used in in vitro seeding and cross-seeding reactions to investigate how cellular components may affect the efficacy and specificity of seeds. Finally, to study seeding mechanism in a living cell, seeds of the three human proteins or Sup35 mutants will be used to transfect yeast sphaeroplasts and assayed for their ability to generate the yeast prion [PSI+]. The efficiency of the yeast transfection experiments will be monitored and compared with the in vitro results.

The data generated by the experiments will be globally analysed to construct a new mechanistic model in order to resolve generic properties of amyloid seeding and cross-seeding. The model will provide with insight for the mechanism by which formation of amyloid aggregates by different proteins can affect the aggregation of other proteins.

The results that emerge from this project will be of significance to researchers working in the academic and industrial biomedical sectors with an interest in protein misfolding, amyloid and prion disorders such as Alzheimer disease and diabetes type 2. Each of these diseases has the potential to become a worldwide health time bomb with the aging of the population. This will result in an ever-increasing financial burden for European and worldwide economies. For example, in UK alone, dementia is thought to affect about 800,000 people, with the cost to society estimated at £23bn. The number of people with Type 2 diabetes in the UK is rising rapidly and is set to reach five million by 2025, and it is a condition that costs the NHS over £10 billion a year. With the average lifespan of humans in the UK already 82 years for women and 78 years for men, and with an estimated 1 in 4 chance of contracting Alzheimer Disease by that age of 85, the emerging crisis we face is evident. There is, therefore, an urgent need for effective strategies for early diagnosing, preventing and treating amyloid-associated diseases. Amyloid-associated disorders share common mechanisms of protein misfolding and aggregation. Moreover, one amyloid disease could increase the risk of other misfolding events in the same organism. Our findings will, therefore, also make a significant contribution to our fundamental understanding of the molecular mechanism of amyloid seeding for both, homologous and non-homologous, proteins and therefore potentially provides key tools for diagnostic and drug developments targeting amyloid associated diseases. In the longer term this has the potential of having profound benefits to human health and the UK economy since the costs of caring for individuals suffering from dementias is already in the £billions annually and will only continue to rise as does the numbers of cases of dementia associated with amyloid-associated disorders. The amyloid diseases also have adverse socioeconomic impact beyond the affected individuals themselves including carers and dependent family members. Thus, early stages diagnosis, improvements in the post-symptomatic treatment and improvements in the pre-symptomatic prevention of such disorders will have high potential for a broad and positive impact felt in the wider society.