| dc.description.abstract | Neurodegenerative disorders (ND) such as Alzheimer?s disease (AD), Parkinson?s disease (PD) and amyotrophic lateral sclerosis (ALS) are one of the leading causes of death and disabilities worldwide. While their aetiologies and clinical manifestations can vary wildly, they are all characterized by the progressive loss of specific neuronal populations. The affected area of the brain or neuronal sub-type determines the primary clinical features of the disease. A common hallmark of most NDs is the presence of misfolded protein aggregates, which are thought to be drivers of disease progression; experimental evidence exists suggesting these misfolded aggregates are capable of spreading from cell to cell, propagating the disease phenotype in a prion-like manner.
An example where this may be occurring are Lewy body disorders (LBs) , which are correlated to the aggregation of the protein α-synuclein (αSN); recent findings suggest that intervention on the early stages of aggregation may be necessary to alter disease progression. There is evidence that early-stage αSN oligomers rather than amyloid fibrils are responsible for cytotoxicity, but the detailed structural characterization of these oligomers has only occurred in recent years, and experimentally following their build-up in real-time has proven elusive.
In the first chapter of this work, the formation of pathophysiologically relevant αSN oligomers prepared at physiological conditions was monitored using a battery of physico-chemical techniques including size-exclusion high performance liquid chromatography (SE-HPLC), atomic force microscopy (AFM), and diffusion nuclear magnetic resonance spectroscopy (DOSY NMR). The biological activity of the oligomers produced was assayed by testing primary cortical neuron cytotoxicity and in vivo electrophysiological effects on rats. Using time-resolved techniques, the early stages of αSN oligomer growth were quantitatively monitored as individual components within a mixture. The early aggregation pathway of α-synuclein was then compared with that of the non-disease related protein hen egg white lysozyme (HEWL). In general, the ability to kinetically monitor these aggregates without the need for fluorescent markers and/or other modifications shows promise towards the study of proteins correlated with amyloidosis.
In the second chapter, the function of the protein Ataxin-2 (Atxn2) was investigated. The Ataxin-2 (ATXN2) gene is implicated in spinocerebellar ataxia type II (SCA2), amyotrophic lateral sclerosis (ALS) and PD. The encoded protein is a therapeutic target for ALS and other related conditions. Atxn2 (or Atx2 in insects) has been shown to play a role in most stages of the messenger RNA (mRNA) life cycle, including; translational activation, translational repression, mRNA stability and in the assembly of mRNP-granules, a process mediated by intrinsically disordered regions (IDRs). Building on previous work from the laboratory demonstrating that the LSm (Smith antigen-like) domain of Atx2, which can help stimulate mRNA translation, antagonizes mRNP-granule assembly, a series of experiments was carried out on Drosophila and human Ataxin-2 proteins. Results of Targets of RNA-Binding Proteins Identified by Editing (TRIBE), co-localization and immunoprecipitation experiments indicated that a Poly-A-binding protein (PABP) interacting, PAM2 motif of Ataxin-2 may be a major determinant of the mRNA and protein content of Ataxin-2 mRNP granules. Co-immunoprecipitation experiments demonstrated that the Atxn2 LSm domain interacts with its binding partner LSM12 through a canonical LSm:LSm interaction. Transgenic experiments in Drosophila indicated that while the Atx2-LSm domain may protect against neurodegeneration, the structured PAM2- and unstructured IDR- interactions both support Atx2-induced cytotoxicity. Point mutations targeting both the Atxn2 PAM2:PABP interaction and the Atxn2 LSm:LSM12 interactions were designed and shown to successfully inhibit these associations, a promising finding from a drug-design perspective. Finally, preliminary experiments were carried out on the sequence-specific effect of the IDRs from Atxn2, Atx2 and ataxin-2-like (Atxn2L) on granule condensation, showing that these IDRs are not functionally equivalent and have different condensation propensities. Taken together the data obtained leads to a proposal for how Ataxin-2 interactions are remodelled during translational control and how structured and non-structured interactions contribute differently to the specificity and efficiency of RNP granule condensation as well as to neurodegeneration. | en |
