- Pathology - Theses
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ItemDevelopment of a candidate for PET imaging of BACE1 in Alzheimer's diseaseDOWN, RUSSELL ( 2015)Alzheimer's disease (AD) is the most common form of dementia but there is currently no cure or disease modifying treatment. The pathological processes which lead to AD begin up to 20 years before the onset of symptoms, offering a large window for diagnosis and potential therapeutic intervention. β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) is an aspartyl protease involved in the pathogenesis of AD. BACE1 levels are increased in AD patients and this elevation is known to occur early in the course of the disease; however, there is currently no method for measuring cortical BACE1 levels in vivo. It is hypothesised that the development of a BACE1 positron emission tomography (PET) imaging agent could have clinical utility for AD diagnosis as well as offering other benefits to AD research. A series of analogues of the hit compound (E)-2-(5-(3,4-dimethoxyphenyl)thiazol-2-yl)-3-(4-hydroxy-3-methoxyphenyl)acrylonitrile, identified from a screen of 70 compounds against BACE1 activity, was synthesised in order to generate a structure activity relationship. Initial attempts to radiolabel the general scaffold of the compounds with the 18F isotope by direct labelling were unsuccessful due to the instability of the scaffold under radiofluorination conditions; however, a route to radiolabel the scaffold was achieved by utilising [18F]-fluorobenzaldehyde as a prosthetic labelling group. The ability of the compounds to bind to BACE1 was assessed by a surface plasmon resonance (SPR) inhibition in solution assay. While a small number of compounds displayed limited activity, IC50 values could not be generated and most compounds were inactive. In addition, most of the compounds were too insoluble to be assessed for membrane permeability using the parallel artificial membrane permeability assay (PAMPA), although a small number of more soluble compounds were shown to have high permeability. Due to the slow progress and problems encountered in developing a de novo BACE1 PET imaging candidate, it was decided to modify an existing BACE1 inhibitor to incorporate the 18F radiolabel. MK-8931 (Merck) has a high affinity for BACE1 and is the most advanced clinical candidate for BACE1 inhibition and a synthetic route was developed to introduce a fluorine atom to the molecule. Following the synthesis of MK-8931, achieved by modification of a literature procedure, a radiofluorinated analogue, (S)-2-amino-6-(4-(5-(3-[18F]-fluoroprop-1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)-3,6-dimethyl-5,6-dihydropyrimidin-4(3H)-one, was prepared; unfortunately, the amount of labelled material was low and purification was not attempted. Analysis of (S)-2-amino-6-(4-(5-(3-fluoroprop-1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)-3,6-dimethyl-5,6-dihydropyrimidin-4(3H)-one by SPR showed an IC50 of 2.05 nM compared to 5.87 nM for that of MK-8931, and the passive membrane permeability of both compounds as measured by PAMPA was very similar. These results suggest that should the radiosynthesis be optimised, (S)-2-amino-6-(4-(5-(3-[18F]-fluoroprop-1-yn-1-yl)pyridin-3-yl)thiophen-2-yl)-3,6-dimethyl-5,6-dihydropyrimidin-4(3H)-one would be a good candidate for PET imaging of BACE1.
ItemInvestigation of neuregulin1 processing by BACE1 and gamma-secretase in schizophreniaBARAKAT, ADEL ( 2014)Schizophrenia is a multifactorial complex psychiatric illness, which implicates many genes, among which is neuregulin1 (Nrg1). BACE1 and γ-secretase are known to be involved in the proteolytic processing of the Alzheimer’s disease-associated Amyloid Precursor Protein (APP), but they are also required for processing Nrg1 type III. Thus, perturbation of the proteolytic activity of these enzymes would disrupt Nrg1 signalling, and could be associated with schizophrenia (SCZ). Indeed, knockout of BACE1 or γ-secretase subunit APH-1b in mice causes SCZ-like phenotypes due to the miscleavage of Nrg1. Also, clinical profile analysis and genetic studies support presenilin2 (PS2), another γ-secretase subunit, as a susceptibility gene for SCZ. Therefore, we proposed to investigate further the expression of BACE1 and γ-secretase subunits, APH-1b and PS2, in SCZ subjects. Human brain samples from Brodmann area 6 (39 SCZ and 20 HC) were obtained from the Victorian Brain Bank. Our previous studies with these samples showed a 50% decrease in Nrg1-CTF in the SCZ group compared to HC. Western blotting analysis of BACE1 and APH-1b indicated no significant difference in the expression of these proteins between SCZ and HC. Notably, a correlation was found between BACE1 and APH-1b, in the HC group, but not in the SCZ group. In this thesis, RNA was analysed by qRT-PCR to identify and quantify the expression of four BACE1 splice variants (SV), APH-1b and PS2 mRNA. Data were analysed and expressed relative to two endogenous controls (UBC and RPLP0). BACE1 enzymatic activity of human brain samples was measured using a synthetic APP peptide cleavage assay. The four BACE1 SV were cloned in the pcDNA3.1+ mammalian expression vector and transfected in SH-SY5Y cells. The cells were analysed for BACE1 enzymatic activity using an in vitro assay, and by analysis of APP and Nrg1 cleavage products and measurement of Aβ secretion by ELISA. The colocalization of Nrg1, BACE1 SV and APP in SY5Y transfected cells was investigated by immunofluorescence (IF) microscopy. Subcellular fractionation was used to explore the trafficking of Nrg1, BACE1 SV and APP in transfected cell organelles. Results of qRT-PCR showed a statistically significant two-fold increase in the expression of BACE1 432 SV in the SCZ group compared to HC. No significant difference was found in the expression levels of APH-1b and PS2 mRNA, but significant correlations were discovered for APH-1b and PS2 in SCZ and HC groups. Particularly, there was a significant positive correlation between APH-1b and PS2 in the HC group that was not preserved in SCZ. Enzymatic assay and ELISA of cells transfected with BACE1 SV indicated high BACE1 enzymatic activity for BACE1 501 SV, and little or no activity for the other SV, including 432. It showed partial colocalisation between BACE1 SV, Nrg1 and APP. Also subcellular fractionation indicated disturbed BACE1 trafficking due to overexpression of BACE1 SV. We report for the first time a significant increase of BACE1 432 SV in SCZ premotor cortex, and a loss of correlation between APH-1b and PS2 expression. These results support that the proteolytic processing of Nrg1 may be altered in schizophrenia. The finding of the increased expression of a BACE1 SV with low enzymatic activity in SCZ post-mortem tissue sheds new light on alterations of the Nrg1 signalling pathway that have been observed in SCZ, and may lead to novel treatments for this disorder.