Special LMNN Seminar - An all optical approach to study amyloid aggregation

Understanding the formation and structural characteristics of amyloid fibrils is vital for the development of therapeutics for many neurodegenerative disorders. We have developed a fluorescence-lifetime based aggregation sensor which can readily be applied in vitro and in vivo, permitting the screening of small molecule drugs against protein aggregation. In order to characterise amyloid fibril growth, we apply two colour total internal reflection microscopy and super-resolution imaging and demonstrate that there is heterogeneity in the growth rates of individual amyloid fibrils which can be attributed to structural polymorphism. We also show that Tau and heparin form composite fibrils and thus structurally prevent seeding.
In order to understand the normal physiological and pathological function of alpha-synuclein, a protein linked to Parkinson’s disease, we have applied a combination of optical and NMR spectroscopy techniques. We have discovered that alpha-synuclein acts as a calcium sensor at the pre-synapse. In particular, we show that a sub-group of alpha-synuclein-positive vesicles at the pre-synapse is specifically responsive to changes in calcium concentrations. This is in contrast to VAMP-2 positive vesicles which do not form localised clusters at the pre-synapse upon calcium exposure. NMR data indicate that this calcium sensing capacity of alpha-synuclein is mediated via the negatively charged C-terminus, which, upon calcium binding, directly interacts with synaptic vesicles. We further demonstrate that the alpha-synuclein calcium sensor needs to be finely tuned as increased levels of calcium over a prolonged time in the presence of alpha-synuclein can directly result in pathology. Similarly, using a cell model of Parkinson’s disease, toxicity can either be prevented by a decrease in the level of alpha-synuclein or by isradipine, a voltage-gated calcium channel inhibitor.
In summary, by applying primarily optical techniques we have shed light on amyloid protein function, aggregation kinetics and structural characteristics which together may help to develop therapeutic strategies against various forms of neurodegenerative diseases.