Florey Department of Neuroscience and Mental Health - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
ItemSpatial and temporal surveillance of the mechanisms controlling proteome foldedness via a FRET-based biosensor( 2018)Proteostasis (protein homeostasis) is essential for keeping the proteome functional. This process controls protein synthesis, folding and degradation and involves hundreds of genes, including those encoding chaperones, to form extensive quality control (QC) networks (Kim et al., 2013). Imbalances in proteostasis are implicated in a range of aggregation-based neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS), Huntington’s and Alzheimer’s diseases (Morimoto et al., 2014; Vilchez et al., 2014). Currently there is a lack of capacity to quantitatively measure proteostasis imbalance and therefore we are limited in understanding how proteostasis imbalance manifests during disease. A new biosensor system has been developed by our lab to address this shortfall. The biosensor is a genetically encoded unfolded “bait” flanked by two fluorescent proteins to assay foldedness by fluorescence resonance energy transfer (FRET). Proteostasis efficiency is reported by measurement of the efficiency to which the bait interacts with the QC network. In this master’s project, the biosensor was further targeted to organelles to allow for a higher degree of spatiotemporal control. Signalling peptides were used to target the biosensor to specialised microenvironments, and successful targeting was achieved in the Golgi apparatus and nucleus. Investigations into nuclear proteostasis revealed the biosensor behaved predictably to chaperone overexpression (Hsp40 and Hsp70 co-expression) or inhibition (Hsp70 or Hsp90 inhibition). Polyglutamine (PolyQ) expansions of non-pathogenic (Q25) to pathogenic (Q72) lengths reduced the biosensor foldedness and decreased aggregation, which is consistent with an increase in chaperone supply. The biosensor was also adapted to express in the body wall muscles of Caenorhabditis elegans to examine change in proteostasis across age and in an organismal context. The biosensor was successfully expressed in the model organism, with potential sub-microscopic and variant biosensor expression level confounding data analysis. The C. elegans reporter lines were successfully crossed with lines expressing Aβ (1-42) demonstrating the ability of the biosensor to report on disease states. Moving forward, the generation of low-expression, single-copy C. elegans biosensor lines would allow for steady, matched expression and enhanced capacity for comparison between worm lines.
-
ItemApplying matrix assisted laser desorption-ionization mass spectrometry to identify and quantify isoforms of amyloid beta in human brain( 2017)Amyloid beta (Aβ) plays a central pathogenic role in Alzheimer's Disease (AD), however its great heterogeneity remains largely unexplored as most research focuses on only two specific species: Aβ1-40 and Aβ1-42. More than fifty truncated species of amyloid beta have been identified, as well as a multitude of additional post translational modifications including pyroglutamation, oxidation and glycosylation. This spectrum of isoforms is either cumbersome or impossible to resolve with prevailing methods such as ELISA and western blots. Matrix Assisted Laser Desorption-Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS) allows specific and simultaneous identification of these species. My thesis employed this method, with prior enrichment of donated brain samples with amyloid beta immunoprecipitation, to characterise these isoforms in different fractions of human brain: the cytosolic (TBS) fraction, the peripheral membrane/vesicular (Na2CO3) fraction, the integral lipid/membrane (urea/detergent) fraction, and the polymerised fibrillary (formic acid) fraction. At least fourteen different Aβ isoforms were resolved, most in the formic acid fraction and membrane fraction, with relatively less in the sodium carbonate fraction. Extending the methodology, isotopically labelled ‘heavy’ internal standards were utilised to allow for absolute quantification of some prominent truncations, including Aβ4-42 and Aβ1-42. This showed a mean of 704 pmol/g wet brain of Aβ4-42 in formic acid fractions of brains with Alzheimer’s pathology, and 438 pmol/g in the membrane fractions of brains with Alzheimer’s pathology. Aβ1-42 was present in the formic acid fractions and membrane fractions of those with Alzheimer’s Disease in mean values of 1.3 nmol/g and 232 pmol/g respectively. These values were greater than those in the control brains. It was also seen that Aβ4-42 ionised approximately 1.3 fold the extent of AβH1-42, and approximately 3 fold that of AβH1-40. Noting the difficulties inherent in ionising entire species of amyloid beta, enzymatic cleavage with Lys-N was undertaken to improve reliability and throughput of MALDI-TOF MS analysis. Lys-N is a metalloendopeptidase that preferentially cleaves N-terminals of lysine groups. These cleaved products of Aβ are more amenable to MALDI-TOF MS than products of C-terminal cleavage. Lys-N was first isolated from the fruiting bodies of pleurotus ostreatus, achieving a 3.94 fold purification. This was then applied to synthetic amyloid beta and MALDI-TOF MS analysis, preferentially resolving N terminal truncated isoforms of Aβ. With further refinement, this method would allow a specific and robust approach to characterising and quantifying multiple species of Aβ to picomole amounts.