Florey Department of Neuroscience and Mental Health - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
ItemMass Spectrometry As A Tool For Drug Development In SCN2A Developmental and Epileptic Encephalopathies( 2023-11)Mutations within the SCN2A are recognized as a prominent cause of autism spectrum disorder and a spectrum of developmental and epileptic encephalopathies (DEEs). As more patients are affected by mutations in SCN2A, it drives the need for precision medicines and to better understand the biology and pathogenesis of the disorder. The SCN2A gene encodes the voltage-gated sodium channel, Nav1.2. Antisense oligonucleotides (ASOs) are a class of drugs being developed to treat SCN2A disorders by knocking down SCN2A mRNA and therefore protein levels. In this study, targeted mass spectrometry methods are utilised to measure Nav1.2 protein levels directly and untargeted, or “discovery”, proteomic methods are used to measure the entire proteome in brain tissue collected from various SCN2A mouse models and mice treated with an experimental ASO therapy. Three SCN2A knock-in missense mutation mouse models are included in the study, each representing a phenotypic group within the SCN2A disease population. These results all support that the ASO has strong target engagement on the protein expression similar to mRNA level. The mutant mouse models are R1882Q representing the early seizure onset phenotype, R853Q representing the late seizure onset phenotype, and S1758R representing the autism with no seizure phenotype. When measuring Nav1.2 in R1882Q mouse whole-brain treated with an ED80 dose (80% knockdown Scn2a mRNA) of an Scn2a-targeting ASO, Nav1.2 was reduced 72% compared to R1882Q mice treated with a scrambled-control ASO. WT mice treated with an ED50 dose of SCN2A-targeting ASO at P30 (post-natal day 30) with brain tissue collected over a 5-week period showed consistent knockdown of Nav1.2 protein of approximately 50% 2- and 3-weeks post-injection in cerebellum, hippocampus, and cerebellum. In the mutant models of Scn2a encephalopathy, Nav1.2 expression remained unchanged in R1882Q and R853Q mutant mice compared to WT littermates while Nav1.2 expression was reduced ~50% in S1758R mutant mice compared to WT littermates, suggesting haploinsufficiency may be a major driver of the autism phenotype. Global proteomic analysis revealed several potential off-target and/or toxicity biomarkers of ASO treatment. These biomarkers were primarily associated with neuroinflammation, including neurofilament heavy (Nefh) and programmed cell death 5 (Pdcd5). Global proteomic analysis in the 3 mutant models showed unique proteomic profiles in each, with minimal overlap, suggesting the very different phenotypes also lead to differences in protein expression and dysregulation. However, dysregulated proteins across the 3 models were involved in several shared pathways, including those responsible for regulation of synaptic signalling and mitochondrial function and metabolism. The exploration of novel epileptic mouse models and mice treated with experimental antisense oligonucleotides through proteomic analysis has unveiled promising prospects for potential new biomarkers. This integrated approach has provided invaluable insights. It is anticipated that certain biomarkers identified may undergo further validation and potentially be employed in clinical trials for emerging SCN2A drugs. These biomarkers could serve to monitor disease progression and assess the effectiveness of innovative treatments, building upon prior research efforts.
-
ItemToward the structural characterisation of the relaxin receptor, RXFP1( 2021)Relaxin is a peptide hormone that is involved in several physiological processes such as pregnancy, collagen breakdown, fibrosis inhibition and vasodilation. It has been investigated for the use of several disease states such as scleroderma, fibrosis, cancer and most recently acute heart failure. Relaxin’s cognate receptor is the relaxin family peptide receptor 1 (RXFP1), an integral membrane protein belonging to the G protein-coupled receptor (GPCR) family with a complex, multistep activation mechanism which is still not well understood. Given the physiological roles of relaxin, RXFP1 is a promising target for the treatment of abovementioned conditions. However, there is currently a lack of a detailed mechanism in which relaxin mediated activation of RXFP1 occurs and this makes the design of relaxin-like compounds such as long active peptide mimetics, small molecules or biologics targeting RXFP1, or understanding and optimizing existing compounds that act at RXFP1 difficult. The lack of a detailed mechanism of RXFP1 activation can be attributed to the lack of full-length RXFP1 structures. While there are proposed models of this activation mechanism, these models were derived from studies on isolated domains of RXFP1 and thus it cannot be assumed that the findings are similar to that of a full-length RXFP1. Thus, the aim of this thesis was to work toward active and inactive state structures of full-length RXFP1 using cryo-electron microscopy (EM). By solving active and inactive state structures, we can overlay these structures to determine key conformational changes and key residues that interact with relaxin to determine a complete mode of relaxin mediated activation of RXFP1. However, these studies are hampered by the limitations of cryo-EM to study inactive state GPCRs and the low recombinant expression of WT RXFP1 which makes producing sufficient amounts of purified RXFP1 for these studies very difficult. In this thesis we optimised the expression and purification of RXFP1 for the purposes of cryo-EM studies. We also developed and optimised a novel tool, monomeric ultra-stable GFP (muGFP) as an intracellular loop 3 (ICL3) fusion partner to overcome the limitations of inactive state cryo-EM studies. We applied this to a thermostabilised variant of the alpha1A-adrenoceptor and demonstrated its utility for cryo-EM studies before applying it to RXFP1. Next, we applied an established workflow for the production of active state GPCR-G protein complexes in insect cells for cryo-EM studies to WT RXFP1 for the active state studies of the receptor. We also experimented with the expression and formation of an RXFP1-G protein complex in a mammalian expression systems. However, we were unable to proceed to cryo-EM studies of either inactive or active state RXFP1 due to inability to produce sufficient quantities of protein To overcome the limitation of poor protein yield, we developed a novel mammalian cell-based method of directed evolution. Existing methods of GPCR directed evolution are primarily E. coli based, and as RXFP1 is unable to be expressed in E. coli due to requiring post-translational modification, a mammalian system was required. We applied this novel method to RXFP1 and were able to evolve mutant #35, which demonstrated an ~9x increase in recombinant RXFP1 expression. Additionally, we also identified 2 mutants that demonstrated interesting pharmacological changes from WT. This includes a mutant that demonstrated an increase in basal signalling, and another mutant that demonstrates a decreased pEC50 for relaxin, that is a higher concentration of relaxin is required to produce an equivalent response in WT. By evolving high expressing mutant #35, we could potentially overcome the bottleneck of insufficient purified protein yield for cryo-EM studies. By applying mutant #35 to the workflows developed in this thesis, we can potentially enable downstream cryo-EM studies of RXFP1 through the ability to produce ~9x more protein than WT. Through enabling these studies, we may be able to elucidate the mechanism in which relaxin triggers RXFP1 activation in a full-length receptor. Understanding this mechanism in atomic resolution detail through cryo-EM studies could then facilitate rational drug design of novel relaxin-like mimetics for the treatment of acute heart failure or fibrosis or antagonists for the treatment of certain cancers.