Pathology - Theses
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ItemThe mechanism of action of CuII(atsm) for the treatment of amyotrophic lateral sclerosis( 2015)Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the progressive loss of motor neurons in the spinal cord, motor cortex and brain stem leading to complete paralysis and death, usually within 2-3 years of diagnosis. There is currently no cure for ALS and the only approved therapeutic is riluzole. However, its clinical efficacy is marginal with an average extension in survival of 3 months. A subset of ALS cases (~10%) can be attributed to genetically inherited mutations in a number of different genes (familial ALS). Mutations in the gene for Cu,Zn superoxide dismutase (SOD1) – an antioxidant enzyme – were the first to be identified. These mutations lead to a toxic gain of function but the exact nature of this toxicity remains largely unknown. There is evidence to suggest that mutations may cause incorrect metallation of SOD1 leading to aberrant catalytic chemistry and misfolding. Over-expression of the mutant forms of the human protein in mice gives rise to a phenotype that recapitulates many of the symptoms of the human condition including progressive paralysis and premature death. The PET imaging agent, diacetyl-bis(4-methylthiosemicarbazonato)Cu(II) [CuII(atsm)] has been shown to have therapeutic potential in one of these models – SOD1G93A mice. In addition, CuII(atsm) has also been shown to be protective in multiple models of Parkinson's disease. The purpose of this thesis was to further characterise the therapeutic potential of CuII(atsm) in a second model of ALS and to determine if its therapeutic mechanism involves modulation of Cu bioavailability in disease affected tissue. CuII(atsm) was shown to have similar therapeutic potential in the SOD1G37R model as in the SOD1G93A model. Survival extension and improvement in locomotor symptoms were dependent on the dose administered with the highest dose administered proving to be the most effective. No apparent therapeutic ceiling was reached. CuII(atsm) was also co-administered with riluzole with no apparent additive or detrimental effects. When administered alone, riluzole was not as effective at attenuating symptoms and survival as CuII(atsm). Additionally, CuII(atsm) was therapeutic even when given post-onset of a locomotor deficit. Even though severity of disease symptoms in these mice is dependent on mutant SOD1 expression levels, treatment with CuII(atsm) was shown to paradoxically increase the concentration of mutant SOD1 in the spinal cord of these mice. This was due to an increase in fully metallated holo SOD1 – the stable, non-toxic form of the enzyme. The holo SOD1 pool was increased by incorporation of Cu from CuII(atsm) into the Cu-deficient, Zn-containing SOD1 pool. Several other proteins also incorporated Cu from CuII(atsm) however, not all detectable cuproproteins were targets of CuII(atsm)-mediated Cu delivery. Preliminary results suggest that the cuproprotein targets of CuII(atsm) are involved in oxidative stress, metal homeostasis and Cu delivery to SOD1, potentially inhibiting the toxic action of metal-deficient SOD1 on mitochondria. The clinical and pathological similarities between familial and sporadic ALS suggest that similar pathological processes occur in both forms of the disease. There is evidence to suggest that SOD1 can cause disease in the absence of mutations and there is ample evidence implicating mitochondrial dysfunction in sporadic ALS as well as familial ALS. CuII(atsm) is therefore a promising therapeutic for the treatment of ALS and the results presented and mechanism proposed in this thesis position CuII(atsm) as an excellent candidate for translation into human clinical trials.
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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.