Genetics - Theses
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ItemCellular characterisation of the insulin-regulated aminopeptidase (IRAP) in the brain(University of Melbourne, 2006)Central infusions of AT4 ligands, including Angiotensin IV and LW-Hemorphin 7, enhance memory acquisition, retention and retrieval in fear-conditioned memory tasks and spatial learning. A single acute dose of an AT4 ligand is also sufficient to reverse memory deficits caused by scopolamine treatment, perforant pathway lesions and other forms of experimentally induced amnesia models. The robust effects of these ligands on learning and memory processes are thought to be mediated by binding to the catalytic site of the insulin regulated aminopeptidase (IRAP). I RAP is also found in muscle and adipose cells, where the enzyme is found colocalised with GLUT4, the insulin regulated glucose transporter, in specialised vesicles. In response to insulin stimulation, IRAP traffics with GLUT4 from an intracellular location to the plasma membrane, from where GLUT4 mediates insulin regulated glucose uptake. The N-terminal domain of IRAP is believed to interact with multiple proteins to regulate the intracellular tethering, trafficking and possibly recycling of these vesicles. It is unknown how AT4 ligands, via binding to the catalytic site of IRAP, promote such robust memory enhancing effects. One hypothesis is based on the role of IRAP in regulating the trafficking of GLUT4 containing vesicles in muscle and adipose, suggesting that the enzyme may be present in the brain in an analogous system. In this way, modulation of IRAP activity by centrally administered AT4 ligands may result in enhanced glucose uptake into neurones. This hypothesis is supported by the findings that exogenous glucose administration promotes an equally wide range of cognition enhancing effects as demonstrated for AT4 ligands. Therefore to investigate whether IRAP location and function in the brain is analogous to that characterised in muscle and adipose cells, the studies described in this thesis aimed to: 1) Map and characterise the cellular expression of IRAP in the rodent brain 2) Determine the subcellular localisation of IRAP in neurones 3) Determine if IRAP is associated with the facilitative glucose transporter in neurones as it is in muscle and adipose 4) Investigate if IRAP may be involved in neuronal glucose uptake Using a highly specific in-house antibody raised against the unique intracellular tail of IRAP, the studies in this thesis utilised an immunohistochemical approach to characterise IRAP in the brain. Firstly, IRAP immunoreactivity was visualised in specific nuclei throughout the brain. In particular, IRAP was highly expressed in cognitive associated areas, such as the medial septum, cerebral cortex and hippocampus. IRAP immunoreactivity was also abundant in many motor and motor associated nuclei. Dual label immunohistochemistry demonstrated IRAP was exclusively expressed in neurones and was partially associated with cholinergic neurones and their projection, supporting a role for IRAP in cognitive processes. The pattern of IRAP immunoreactivity within neurones was punctate and vesicular, throughout the cell soma and extending into proximal dendrites, indicating a predominantly post-synaptic localisation. At a subcellular level, IRAP was localised to vesicles containing VAMP2, but not small synaptic vesicles at nerve terminals. A clear overlap between IRAP and markers of the Trans Golgi network and endosomal membranes was evident, although IRAP localisation was not limited to these membranes. These findings were confirmed by electron microscopy studies, that visualised IRAP specific electron-dense precipitate associated with neurosecretory vesicles and cisterns of the rough endoplasmic reticulum and golgi apparatus. In summary, these findings demonstrated the subcellular location of I RAP in neurones is analogous to that characterised in muscle and adipose. The association between IRAP expression and the different facilitative glucose transporters present in the brain was examined. IRAP immunoreactivity was found to coincide specifically with GLUT4, such that in some brain regions the subcellular localisation of I RAP and GLUT4 appeared to overlap completely. Three patterns of co-localisation were mapped and quantitated with a high degree of co-localisation noted in cognitive associated nuclei, moderate co-localisation in motor associated nuclei while low co-localisation was noted in the hypothalamus and cerebellum. In areas of high co-localisation I RAP and GLUT4 appear to be localised to the same intracellular vesicles. This subcellular co-localisation is suggestive of a role for I RAP in regulating GLUT4 activity in cognitive associated regions such as the hippocampus. Finally the role of IRAP in neuronal glucose uptake was investigated by testing the effect of AT4 ligands on hippocampal glucose uptake. Both Ang IV and LVV-H7 significantly enhanced stimulated glucose uptake specifically in areas where I RAP and GLUT4 are co-localized. Furthermore, glucose uptake was visualised to occur in the pyramidal neurones that contain colocalised I RAP and GLUT4. These results strongly suggest a role for I RAP in neuronal glucose uptake. Taken together, these studies have demonstrated that IRAP is present in the brain in localisation analogous to that found in muscle and adipose. The presence of IRAP in vesicles with the regulated glucose transporter, GLUT4, in neurones involved in cognitive processing suggests I RAP and GLUT4 may mediate inducible glucose uptake in neurones, possibly in response to heightened energy demand in neurones. Therefore the hypothesis investigated in this thesis is confirmed. Additionally these results suggest a potential molecular mechanism that may underlie the cognition enhancing effects of AT4 ligands.
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ItemCellular characterisation of the insulin-regulated aminopeptidase (IRAP) in the brain(University of Melbourne, 2006)Central infusions of AT4 ligands, including Angiotensin IV and LW-Hemorphin 7, enhance memory acquisition, retention and retrieval in fear-conditioned memory tasks and spatial learning. A single acute dose of an AT4 ligand is also sufficient to reverse memory deficits caused by scopolamine treatment, perforant pathway lesions and other forms of experimentally induced amnesia models. The robust effects of these ligands on learning and memory processes are thought to be mediated by binding to the catalytic site of the insulin regulated aminopeptidase (IRAP). I RAP is also found in muscle and adipose cells, where the enzyme is found colocalised with GLUT4, the insulin regulated glucose transporter, in specialised vesicles. In response to insulin stimulation, IRAP traffics with GLUT4 from an intracellular location to the plasma membrane, from where GLUT4 mediates insulin regulated glucose uptake. The N-terminal domain of IRAP is believed to interact with multiple proteins to regulate the intracellular tethering, trafficking and possibly recycling of these vesicles. It is unknown how AT4 ligands, via binding to the catalytic site of IRAP, promote such robust memory enhancing effects. One hypothesis is based on the role of IRAP in regulating the trafficking of GLUT4 containing vesicles in muscle and adipose, suggesting that the enzyme may be present in the brain in an analogous system. In this way, modulation of IRAP activity by centrally administered AT4 ligands may result in enhanced glucose uptake into neurones. This hypothesis is supported by the findings that exogenous glucose administration promotes an equally wide range of cognition enhancing effects as demonstrated for AT4 ligands. Therefore to investigate whether IRAP location and function in the brain is analogous to that characterised in muscle and adipose cells, the studies described in this thesis aimed to: 1) Map and characterise the cellular expression of IRAP in the rodent brain 2) Determine the subcellular localisation of IRAP in neurones 3) Determine if IRAP is associated with the facilitative glucose transporter in neurones as it is in muscle and adipose 4) Investigate if IRAP may be involved in neuronal glucose uptake Using a highly specific in-house antibody raised against the unique intracellular tail of IRAP, the studies in this thesis utilised an immunohistochemical approach to characterise IRAP in the brain. Firstly, IRAP immunoreactivity was visualised in specific nuclei throughout the brain. In particular, IRAP was highly expressed in cognitive associated areas, such as the medial septum, cerebral cortex and hippocampus. IRAP immunoreactivity was also abundant in many motor and motor associated nuclei. Dual label immunohistochemistry demonstrated IRAP was exclusively expressed in neurones and was partially associated with cholinergic neurones and their projection, supporting a role for IRAP in cognitive processes. The pattern of IRAP immunoreactivity within neurones was punctate and vesicular, throughout the cell soma and extending into proximal dendrites, indicating a predominantly post-synaptic localisation. At a subcellular level, IRAP was localised to vesicles containing VAMP2, but not small synaptic vesicles at nerve terminals. A clear overlap between IRAP and markers of the Trans Golgi network and endosomal membranes was evident, although IRAP localisation was not limited to these membranes. These findings were confirmed by electron microscopy studies, that visualised IRAP specific electron-dense precipitate associated with neurosecretory vesicles and cisterns of the rough endoplasmic reticulum and golgi apparatus. In summary, these findings demonstrated the subcellular location of I RAP in neurones is analogous to that characterised in muscle and adipose. The association between IRAP expression and the different facilitative glucose transporters present in the brain was examined. IRAP immunoreactivity was found to coincide specifically with GLUT4, such that in some brain regions the subcellular localisation of I RAP and GLUT4 appeared to overlap completely. Three patterns of co-localisation were mapped and quantitated with a high degree of co-localisation noted in cognitive associated nuclei, moderate co-localisation in motor associated nuclei while low co-localisation was noted in the hypothalamus and cerebellum. In areas of high co-localisation I RAP and GLUT4 appear to be localised to the same intracellular vesicles. This subcellular co-localisation is suggestive of a role for I RAP in regulating GLUT4 activity in cognitive associated regions such as the hippocampus. Finally the role of IRAP in neuronal glucose uptake was investigated by testing the effect of AT4 ligands on hippocampal glucose uptake. Both Ang IV and LVV-H7 significantly enhanced stimulated glucose uptake specifically in areas where I RAP and GLUT4 are co-localized. Furthermore, glucose uptake was visualised to occur in the pyramidal neurones that contain colocalised I RAP and GLUT4. These results strongly suggest a role for I RAP in neuronal glucose uptake. Taken together, these studies have demonstrated that IRAP is present in the brain in localisation analogous to that found in muscle and adipose. The presence of IRAP in vesicles with the regulated glucose transporter, GLUT4, in neurones involved in cognitive processing suggests I RAP and GLUT4 may mediate inducible glucose uptake in neurones, possibly in response to heightened energy demand in neurones. Therefore the hypothesis investigated in this thesis is confirmed. Additionally these results suggest a potential molecular mechanism that may underlie the cognition enhancing effects of AT4 ligands.