Subcellular translocation of molecules associated with synapse specific plasticity

McNair, Kara M. (2005) Subcellular translocation of molecules associated with synapse specific plasticity. PhD thesis, University of Glasgow.

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Activity-dependent remodelling of synaptic connections is a fundamental process by which information is acquired (learning) and stored for subsequent retrieval (memory). Remodelling of glutamatergic synapses displays distinct temporal components with an initial modification of pre-existing synaptic proteins, for example by phosphorylation, followed by transcription- and translation-dependent late phases. Using a proteomics (differential gel electrophoresis (DIGE))-based approach, we have investigated global changes in protein expression in response to pharmacological, synaptic and behavioural stimulation of glutamatergic synapses in area CA1 of the rodent hippocampus. In initial experiments, mouse hippocampal slices were subjected to pharmacological activation of glutamate receptors and protein extracts from cornu ammonis (CA)1 subfields were harvested at 4hrs following incubation. The entire hippocampal proteome was subsequently subjected to 2 dimensional (2D) gel electrophoresis to separate component proteins and enable the quantification of individual protein spot abundance levels compared with control tissues. Of the 2946 protein spots resolved, 79 showed significantly altered abundance levels following activation of glutamate receptors when compared to vehicle controls (35 increased / 44 decreased abundance (-1.91 to 1.9 fold change), all p<0.05). Inclusion of a treatment group incorporating the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovaleric acid (D-AP5) revealed that for the majority of spots (58 of 79) altered abundance was sensitive to NMDA receptor blockade consistent with previous reports of NMDA receptor activation being linked to the regulation of multiple plasticity-related genes. Following in-gel tryptic digestion and matrix assisted laser desorption/ ionisation - time of flight (Maldi-tof) / Q-star mass spectrometry, database searching revealed the identity of 39 protein spots displaying glutamate receptor activation-dependent modulation of expression. Identified proteins belonged to a diverse variety of functional classes including those associated with glutamate receptor cycling (e.g. n-ethylmaleimide sensitive fusion protein (NSF)), cytoskeleton-associated proteins, proteins involved in vesicle trafficking and metabolic and ubiquitination proteins as well as proteins with as yet no known association with either glutamate receptors or plasticity mechanisms (e.g. DJ-1). Pharmacological stimulation of glutamate receptors was also investigated in isolated dendritic fields as an attempt to identify those proteins which that are regulated locally at glutamatergic synapses of area CA1 in the rat hippocampus. Finally, analysis of hippocampus and somatosensory cortex from the brains of TASIO transgenic mice was carried out by DiGE. The transgenic mouse line TASIO over expresses the 695-amino acid isoform of the human amyloid precursor protein which itself contains the Swedish double familial AD mutation. These mice show an age dependent increase in the deposition of beta amyloid in the brain in addition to spatial learning and working memory deficits. Previous reports that the pathophysiology in these mice was greater in the cortex region than the hippocampal formation is further suggested by the fact that more (32 compared to 23) protein spots demonstrated significant changes in expression levels in the cortex region of these mice compared to the hippocampus. In summary, the novel proteomics based approach adopted for this study was successfully used to highlight the regulation of proteins involved in plasticity-related processes in the mammalian brain, including synapse specific forms of plasticity. In addition, the identification of many of these regulated protein molecules provides interesting and novel insights into the molecular events occurring in plasticity-related processes including glutamatergic afferent stimulation and long term potentiation. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Neurosciences.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Cobb, Dr. Stuart
Date of Award: 2005
Depositing User: Enlighten Team
Unique ID: glathesis:2005-71484
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 14:31
Last Modified: 12 Aug 2021 14:16

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