Development of single-molecules techniques to study native α-Synuclein aggregates

Abstract

It is now widely recognised that α-Synuclein (αSyn) oligomers are the main pathogenic species in Parkinson’s Disease (PD). In addition to being cytotoxic they are also thought to play a central role in the interneuronal spread of αSyn pathology. There is compelling evidence to suggest that these proteoforms are released into cerebrospinal fluid, where differences in their abundance and ability to seed the formation of larger species have been used to differentiate PD patients from aged matched controls. However, αSyn oligomers are a highly heterogeneous population and the characteristics of those present in biofluids have not been clearly established, nor which species are most neurotoxic and contribute to disease onset and progression. This is largely due to the fact that these small clusters of αSyn are notoriously difficult to study using classical biochemical ensemble methods, owing to their heterogeneity, transient nature and low abundance relative to the monomeric protein (<5% of a solution of protein). For this reason, single-molecule fluorescence microscopy has been used to study them; by probing molecules one-by-one, the rare oligomeric species can be identified amongst the monomeric protein, counted and characterised. Typically, to study species in single-molecule experiments, the biomolecule of interest is covalently tagged with a fluorophore. This may affect the behaviour of the molecule, and also prevents the techniques from being applied to human samples. Attempts to study native oligomers at the single-molecule level, have relied upon amyloid binding dyes, which are non-specific, labelling other βsheet rich structures present in cerebrospinal fluid as well as αSyn.

The αSyn content of aggregates secreted by cells was investigated using an iPS cell line derived from a Parkinson’s diseased patient harbouring a triplication of the SNCA gene, which encodes for αSyn, on one allele and a derivative of this cell line with the gene deleted, SNCA knock-out. The iPS cells were differentiated into midbrain dopaminergic neurons, the primary neuronal subtype associated with the pathogensis of PD and the conditioned media was collected and used as an analogue of cerebrospinal fluid. Initial, investigations focused on utilising amyloid binding dye, thioflavin-T, to probe media samples for the presence of secreted αSyn aggregates.

This involved studying aggregates using single-molecule confocal microscopy coupled with microfluidics to count and characterise thioflavin-T active species, as well as total internal refection microscopy to directly visualise and measure their structure.

In order to facilitate specific detection of αSyn species in complex biological fluids using single-molecule confocal microscopy, the amyloid binding dye was replaced with an αSyn antibody. The need for constitutivly active dyes to be used at low picomolar concentrations in order to discretise burst of fluorescence intensity has prevented this from being achieved before now. With access to an antibody with low picomolar affinity for fibrillar αSyn, I was able to study the oligomersation of unlabelled αSyn. Despite this technique being considerably more sensitive than previously reported methods, I was unable to detect αSyn oligomers in conditioned media, even in conditions where intracellular pathology was induced via treatment with pre-formed αSyn fibrils, suggesting aggregates are rapidly internalised by cells but are then trapped and unable to escape. Observing unlabelled αSyn oligomers directly at the single-molecule level is challenging, due to the fact that specific imaging probes such antibodies are adsorb onto glass surfaces, generating a large degree of non-specific signal. Single-molecule pull-down (SiMPull) is a recently developed technique that circumvents this issue, by immobilising the protein of interest on a passivated surface that is capable of blocking antibody binding. By combining SiMPull with the principles of two-colour coincidence detection, I was able to develop a highly sensitive method for directly visualising αSyn aggregates. Applying this new methodology to conditioned media samples, I was able infer the presence of αSyn oligomers.

The predicted size of αSyn oligomers is in the range of 12-16 nm, which is substantially below the resolving power of any conventional fluorescence microscopy technique due to the diffraction limit of light. Therefore, in order to view αSyn oligomers at an appropriate scale, I combined SiMPull with a super-resolution technique, DNA-PAINT, which enabled αSyn structures to be viewed and characterised with nanometer resolution (∼ 20 nm).

Overall, the work presented here describes methodologies developed to detect and characterise native αSyn oligomers with a high degree of specificity and improved sensitivity in comparison with established techniques. Although these developments were performed on α-synuclein, they have now been adopted by others to investigate other pathogenic proteins implicated in neurodegeneration. The advance ments made will enable further investigations into the molecular mechanisms involved in neurodegerative diseases, and also for potential biomarkers to be detected and studied. This should lead to the development of diagnostic tools for neurode-generative conditions, which will enable clinicians to follow the disease course in patients, and test new therapeutic interventions.