Post-synaptic scaffolding protein changes in Alzheimer’s disease

Mr Dustin Proctor (2010). Post-synaptic scaffolding protein changes in Alzheimer’s disease PhD Thesis, School of Chemistry & Molecular Bioscience, The University of Queensland.

       
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Author Mr Dustin Proctor
Thesis Title Post-synaptic scaffolding protein changes in Alzheimer’s disease
School, Centre or Institute School of Chemistry & Molecular Bioscience
Institution The University of Queensland
Publication date 2010-10
Thesis type PhD Thesis
Supervisor A/Prof Peter Dodd
Dr Elizabeth Coulson
Total pages 380
Total colour pages 54
Total black and white pages 326
Subjects 03 Chemical Sciences
Abstract/Summary Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and most common form of dementia. AD is clinically characterised by the gradual deterioration in a subject’s cognitive function affecting memory, behaviour and reasoning. The severity of cognitive decline correlates strongly to selective neuronal and synaptic loss in affected regions of the AD brain. The precise cause(s) of AD are unknown; but is likely to emanate from the disruption of multiple differing cellular pathways and processes, some of which are evoked by glutamate-mediated excitotoxicity. Specifically, changes in the regulation, activity and/or concentrations of the ionotropic N-methyl-D-aspartate (NMDA) and -amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) glutamate receptor which favour the influx of toxic levels of Ca2+ into nerve terminals. Glutamate receptors play important roles in long-term potentiation (LTP), synaptogenesis, memory formation, and plasticity; disruption of these processes could underlie AD symptoms particularly in early stages of the disease. Glutamate-mediated excitatory transmission is particularly disrupted in AD and synaptic loss is especially severe for glutamatergic neurons. NMDA receptors are organised into multi-protein signalling complexes localised to the dendritic spines, chiefly by interactions with the post-synaptic membrane associated guanylate kinase proteins, (PSD-MAGUK). Four PSD-MAGUK proteins are characterised, PSD-93, PSD-95, SAP-97 and SAP-102. PSD-MAGUKs act as scaffolding proteins between membrane receptors and intracellular proteins and can facilitate signal transduction cascades initiated by NMDA receptor activation. PSD-MAGUKs directly regulate synaptic expression of NMDA receptors upon binding and can indirectly modulate receptor activity through phosphorylation. The concept that a reduction in PSD-MAGUK expression in AD affected brain regions could initiate a simultaneous decline in NMDA receptors present at synapses in these areas formed the major hypothesis tested in this study. The primary objective of this project was to investigate alterations in PSD-MAGUK expression with respect to brain region and progression of the disease. Specifically, the project would endeavour to determine whether associated changes occurred at the level of transcription, translation or protein trafficking. To indentify differences at the level of transcription, a quantitative real-time PCR assay was established to measure and compare mRNA transcripts of the PSD-MAGUK proteins PSD-95 and SAP-102 between AD and control groups. Two pathologically affected regions of the AD brain, the hippocampus and the inferior temporal cortex and one relatively spared region, the occipital cortex, were chosen for the study. Quantification revealed a reduction trend in both PSD-95 and SAP-102 in the AD inferior temporal cortex but not in the occipital cortex. A decline in the pre-synaptic marker synaptophysin, was observed in both the hippocampus and inferior temporal cortex but not in the occipital cortex which was consistent with previous studies. Expression of scaffolding protein and synaptophysin transcripts was found to correlate with disease progression based on the severity of AD pathological markers, A plaques, neurofibrillary tangles and neuron loss. Transcript data was correlated to PSD-95 and SAP-102 protein expression in the same cases and areas. A sensitive immuno-detection-assay utilising recombinant protein standards was developed to measure concentrations of these two proteins. A significant reduction in both PSD-95 and SAP-102 was documented in the AD inferior temporal cortex with no differences between AD and control occipital cortex samples. No changes were observed for either of the scaffolding proteins in the hippocampus which supported mRNA findings. Protein levels of both PSD-95 and SAP-102 declined with disease progression. AD subjects containing a copy of the APOE4 allele had considerably less SAP-102 protein than subjects without a copy of the gene. However, APOE genotype had no effect on mRNA transcript levels. Immunohistochemistry was carried out on inferior temporal and occipital cortex samples for PSD-93, PSD-95, SAP-97 and SAP-102 on 5 AD and 3 control subjects. To evaluate the effect of AD pathology on scaffolding protein expression and distribution, samples were also stained for A plaques, NFTs and synapse loss using the marker synaptophysin. Expression of scaffolding proteins was concentrated to the soma and axons of pyramidal neurons the superficial cortical layers. A small reduction in PSD-93, PSD-95 and SAP-102 was observed in the AD inferior temporal cortex which was not present in the occipital cortex. There were no differences in immunoreactivity for SAP-97. Markers of Alzheimer’s disease showed no significant relationship to PSD-MAGUK immunoreactivity. PSD-MAGUKs are localised dendritic spines; unfortunately these puncta were not detected. Further investigation using more sensitive detection methods such as confocal and electron microscopy to quantify dendrite localised PSD-MAGUK changes with respect to disease is required. The results presented in this project offer further understanding of basic pathogenic processes of AD and provide a possible mechanism which could contribute to glutamate receptor and glutamatergic transmission changes occurring in AD.
Keyword Alzheimer disease
Neurodegeneration
MAGUK
Glutamate
Excitoxicity
Scaffolding protein
Post-synaptic density
PSD-95
SAP-102
NMDA receptor
Additional Notes Colour pages: 27, 32-33, 35, 44-45, 51-52, 60, 63, 73, 82, 86-87, 99-102, 105, 107, 110, 141-142, 147-148, 160-163, 165, 168, 202-203, 207-210, 212-214, 216, 218, 223, 226, 229, 232, 234, 249-250, 254-256, 379-380 Landscape pages: 94-95, 156-157, 197, 220, 234

 
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Created: Tue, 08 Feb 2011, 10:46:02 EST by Mr Dustin Proctor on behalf of Library - Information Access Service