Alpha synuclein in Parkinson’s Disease: determining the role of helical alpha synuclein using stapled peptides

Neurodegeneration, the progressive and irrevocable loss of neuronal structure, is quickly becoming an \nimposing health concern in a globally ageing society. While specific neurodegenerative conditions \nexhibit specific clinical symptoms and progressions, a common neuropathological feature is the \nmisfolding, oligomerisation and fibrillation of certain proteins causing neuronal stress and death. \n \nParkinson’s disease, PD, has long been characterised by the death of nerve cells focused in the \nsubstantia nigra pars compacta region of the midbrain and deposition of large protein aggregates, called \nLewy Bodies, throughout the central nervous system. More recently, the protein which forms these \ninclusion bodies was identified as alpha synuclein, αSyn, a ubiquitous neuroprotein with no known \nfunction. Furthermore, persons with mutations in the SNCA gene, which codes for αSyn, exhibit PD \nprogression at a far younger age with a more severe phenotype, positively linking αSyn with PD. \n \nαSyn is an intrinsically disordered protein, IDP, and generally persists as such in solution and inside \nbacterial and mammalian cells. However, when in contact with a lipid bilayer the protein will embed \nupon the surface in an amphipathic alpha helical conformation and can also aggregate, forming toxic \noligomeric and fibrillar species containing significant β-sheet identity. Its function as a helical \napolipoprotein and subcellular localisation to both the nucleus and synapse has led researchers to \nsuggest that αSyn has a role synaptic transmission and release. However, knocking out the protein does \nnot reduce viability or produce pathological abnormalities in neuronal structure. The helical form of the \nprotein may also persist as transient, metastable helical bundles which are non-toxic and resist \naggregation. While a number of studies and tools have been reported and developed to investigate the \ntoxic oligomeric/fibrillar forms of αSyn, very little attention has been accorded to the helical \nconformation. This thesis will redress this balance by producing tools which will allow us to mimic the \nhelical form of αSyn, promote the active refolding of the full-length protein using a stable, helical \npeptide template and produce antibodies which recognise helical αSyn specifically for use in discovery \nand chaperone-like refolding. \n \nIn Chapter 2 a region of αSyn (14 amino acids) was identified with a unique primary sequence located \nwithin a mutation prone section of the protein. Peptide ‘stapling’ technologies were then employed \nusing a panel of monosubstituted ‘staple’ diastereomers, to produce a highly helical portion of αSyn. \nUsing several other protein targets particular diastereomeric ‘staple’ combinations were analysed for \nobvious trends in helical content. Using solution NMR, backbone refined three dimensional structures \nof these helical peptides were produced which showed that they were faithful structural homologues of \ntheir parent helical proteins. \n \nIn Chapter 3 the drug-like properties and therapeutic potential of stable, helical αSyn peptides were \ninvestigated. Using fluorescently labelled peptide substrates, ‘stapled’ peptides were shown to be far \nmore cell penetrant than their wild type equivalents and demonstrated that the mechanism for cellular \nuptake appears to be specific. Furthermore, under harsh proteolytic conditions the ‘stapled’, helical \npeptides were far more resistant to hydrolysis than wild type or ‘stapled’, poorly helical peptides. The \n‘stapled’ peptides were also highly soluble and did not appear to aggregate in a time-dependent manner. \nUsing ion mobility mass spectrometry, it was shown that incubation of full-length protein with the \n‘stapled’, helical peptides caused a contraction in the hydrodynamic radius of the protein. However, \nusing solution NMR no active refolding of αSyn was observed when under the same conditions. Rather \nsmall perturbations in chemical shift were apparent which did not suggest that the αSyn protein folded \ninto a discrete structural conformation, such as an alpha helix. \n \nIn Chapter 4 the stable, helical αSyn peptide was employed as a conformational model and unique \nantigen in antibody discovery. Immunisation with the ‘stapled’, helical αSyn peptide initially produced \na pool of polyclonal antibodies with a half log specificity for the helical peptide. After bespoke affinity \nchromatography this was increased to three log orders of specificity. Initial immunocytochemistry did \nnot detect any helical αSyn protein in SH-SY5Y cells. To validate the helical epitope on the full-length \nprotein in vitro an assay based around flow cytometry of synthetic vesicle structures was developed, \nwith their synthesis, characterisation and binding of the αSyn protein described.