Pathology - Theses
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ItemInvestigation of neuregulin1 processing by BACE1 and gamma-secretase in schizophrenia( 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.
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ItemInvestigating the functional role of the amyloid precursor protein’s copper binding domain( 2011)Alzheimer’s disease is a progressive and eventually fatal neurodegenerative disorder characterized by specific proteins deposition in the brain: amyloid plaques and tau tangles. While age represents the major risk factor for AD, the mechanisms triggering the pathology remain unclear. According to the amyloid cascade hypothesis, accumulation of the main component of the amyloid plaques, the β-amyloid (Aβ) peptide, is crucial for the onset of the disease. This phenomenon is mainly accounted for by mis-metabolism of the Amyloid Precursor Protein (APP) from which Aβ is derived and by low Aβ clearance. Copper dyshomeostasis, which has been reported in AD, contributes to Aβ accumulation by influencing APP metabolism. Conversely, APP regulates copper homeostasis, a process in which imbalances can lead to oxidative stress and inflammatory response. This study investigated the relationship between APP and copper in terms of APP-mediated copper homeostasis and toxicity, as well as copper-mediated APP metabolism. The role of the APP copper binding domain (CuBD), and in particular the copper binding site histidine residues, was examined with the results revealing that the domain mediates APP metabolism and structure stability. This study has significantly contributed to the understanding of APP CuBD in modulating APP metabolism and stability, and highlights the potential of this domain as a novel therapeutic target in AD.