The study of the mechanisms of protein aggregation is of central importance to understanding fundamental biological processes such as protein assembly and turnover, as well as pathologies that are characterised by the presence of protein aggregates - such as Parkinson’s and Alzheimer’s diseases.
With the latter there is in fact a relatively short list of proteins or peptides the aggregation of which is linked to diseases, including the following examples.
|Disease||Proteins or peptides found in aggregates||Further reading|
|Alzheimer’s disease||Aβ peptide and hyperphosphorylated tau||Haass and Selkoe Nature Reviews Mol Cell Bio 2007|
|Parkinson’s disease||ɑ-Synuclein||Stefanis CSH Perspectives in Medicine 2012|
|Huntington’s disease||Huntingtin with polyglutamine expansion||Bates Lancet 2003|
|Other polyglutamine diseases (DRPLA, SCA1-3)||Atrophin-1, ataxins or AR||Shao & Diamond Hum Mol Genet 2007|
|Fronto-temporal dementia with Parkinsonism||Hyperphosphorylated tau protein||D’Souza et al PNAS 1999|
|Amyotophic lateral sclerosis (ALS)||TDP-43 (TAR DNA binding protein of 43kDa) and FUS (Fused in Sarcoma, an RNA binding protein)||Neumann et al Science 2006|
|Prion diseases (kuru, Creutzfeldt-Jakob disease etc)||Prion protein||Aguzzi & Heikenwalder Nat Rev Microbiol 2006|
A great deal remains to be understood concerning the mechanisms of formation and nature of these pathological protein aggregates. However, probing such aggregates can be challenging because of their supramolecular nature, their heterogeneity, and their often dynamic nature.
One common experimental approach for monitoring aggregates is the use of the Thioflavin-T (ThT) assay. ThT fluorescence is enhanced upon binding to beta-sheet rich structures such as amyloid fibrils. However, this interaction is not perfectly specific, with some evidence suggesting that ThT fluorescence can occur following binding to monomers or oligomers (Groenning, Journal of Chemical Biology 2010). There is also the possibility that the ThT label that binds the beta-sheets of the fibril throughout the experiment may affect the behaviour of the protein during measurement. Finally, some amyloid fibrils are not detected by ThT fluorescence and can provide false negative results - for example if the fibrils are packed in a way that the surface for ThT binding is not present (Nilsson, Methods 2004) .
The Fluidity One addresses these problems by measuring the size of aggregates as they form in a label independent manner.
Case Study: Assessment of α-synuclein amyloid fibril growth
α-Synuclein is an abundantly expressed neuronal protein that contributes to a host of neurological conditions characterised by Lewy body formations, including Parkinson’s disease and dementia. α-Synuclein aggregation results in insoluble, beta-sheet rich amyloid fibrils, the growth of which is of key interest in studying the development of these diseases.
Comparison of Microfluidic Diffusional Sizing (MDS) and ThT Spectroscopy found that measurement of α-Synuclein fibril growth is highly comparable between the two approaches.