Probing the molecular origins of Parkinsons disease using camel single domain antibodies

Parkinsons disease is an incurable neurodegenerative condition that afflicts about 1-2% of the global population over 65 years, and whose prevalence is expected to increase even further as our population ages. The symptoms of this devastating and fatal disease include tremor, rigidity, and slowness of voluntary movements. Currently, although there are treatments to reduce these highly debilitating symptoms, no cure is available that can alter significantly the course of the disease itself. The development of a cure is hampered by our lack of understanding of the molecular processes occurring in the diseased brain. The regions of the brain that are most damaged in the disease contain thread-like assemblies of a protein called alpha-synuclein, which form deposits known as Lewy bodies. Although a clear link exists between the presence of these deposits and the disease, it is not clear whether these or other smaller aggregates are actually responsible for the loss of brain cells. A detailed understanding of the structural properties of these aggregates is still missing, but it would be vital for enabling the development of therapeutic strategies. The introduction of novel tools to control the populations of these aggregates is of great importance for their structural investigation and for identifying the species that are most toxic to brain cells. We have designed a type of small proteins (called antibody domains) that can bind to alpha-synuclein in different ways and that allow us to control the formation of these different types of aggregates. By employing these small antibody domains to control the aggregation of alpha-synuclein in cells, worms and in the brain of fruit flies, we will measure whether any of the small antibody domains are able to prevent the neurodegeneration caused by alpha-synuclein. The comparison of the observations in these organisms with those on the effects of the antibody domains in the test tube will allow us to identify which types of aggregates are most important in the development of Parkinsons disease and hence represent the best targets for developing therapies to treat or prevent this disease and other diseases related to the aggregation of alpha-synuclein, generally termed synucleinopathies. The knowledge gained from the proposed research will also contribute considerably to the advances in the elucidation of other protein aggregation disorders, such as Alzheimers disease, and the transmissible spongiform encephalopathies, including scrapie and the mad cow disease, which equally have a high impact on public health and economy.

The aggregation of alpha-synuclein and its deposition into Lewy Bodies are key pathological hallmarks of Parkinson s disease. Despite the importance of these processes, however much remains to be understood about the specific mechanism of aggregation of alpha-synuclein and its relationship to the pathogenesis of Parkinson s disease. Our objective is to address these questions by modulating alpha-synuclein aggregation, both in vitro and in vivo, with conformation-specific antibody fragments. These molecules target different epitopes of the monomeric form of alpha-synuclein and alter the in vitro and in vivo aggregation properties of alpha-synuclein. The binding of each antibody fragment to alpha-synuclein will be characterised at atomic level detail by using a wide range of methods, including NMR spectroscopy, X-ray crystallography and advanced computational techniques. These structural studies will then be combined with biophysical investigations of the effects of each antibody fragment on the kinetics of the alpha-synuclein aggregation pathways, using single molecule fluorescence correlation spectroscopy and transmission electron microscopy. Together, these data will enable an accurate molecular level dissection of the mechanisms underlying the formation of different types of alpha-synuclein aggregates, ranging from oligomers to fibrils. In order to understand the relationship between alpha-synuclein aggregation and neurodegeneration, the effects of each antibody fragment on the in vivo neurotoxicity of alpha-synuclein aggregation will be investigated with in situ and in vivo models of Parkinson s disease with different levels of complexity, from neuronal cell-line models, to C. elegans and D. melanogaster models. Various state-of the art techniques will be used to image alpha-synuclein aggregation and to obtain structural information on the species populated in the cell, such as florescence lifetime imaging (FLIM) and fluorescence recovery after photobleaching (FRAP), which are established in the host laboratory. Analysis of the effecst of each antibody fragment on neurotoxicity combined with the knowledge of how each fragment affects alpha-synuclein in vitro will enable the determination of the key pathogenic steps in the alpha-synuclein aggregation process that underlie the development of Parkinson s disease. We believe that this project has a very high potential to succeed, as it builds on the exceptionally strong expertise of the host laboratory in the study protein aggregation, in particular for alpha-synuclein, and it is most timely, as all key technologies have now been established, either directly or through collaborations.