Groom, Anthony J.; (2002) The involvement of ionotropic AMPA receptors in the pathogenesis of neurological diseases. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The interest in glutamate in the pathogenesis of neurodegenerative disorders has rapidly expanded since the early descriptions of the excitotoxic properties of amino acids in the central nervous system. With the identification of the numerous glutamate receptors, the contribution of the various ionotropic and metabotropic receptor subclasses to excitotoxicity has been intensely studied. A wealth of data support a role for AMPA receptor in acute neurodegeneration, exemplified by neuronal loss during stroke. More recently, however, the importance of low level glutamate exposure in the evolution of chronic neurodegenerative conditions has begun to emerge. This thesis describes two aspects of AMPA receptor pathogenesis in vivo, (i.) The possible role for AMPA receptor activation and subsequent neurodegeneration within the context of acute and chronic rodent models of the inflammatory, demyelinating disorder, multiple sclerosis, and (ii.) the neurodegenerative phenotype associated with a, putatively, AMPA-mediated, spontaneous development of spasticity in the Han/Wistar spa/spa rat. Experimental autoimmune encephalomyelitis in rodents reproduces physiological and pathological features of multiple sclerosis, including CNS inflammation, myelin breakdown and axonal loss. In two acute experimental autoimmune encephalomyelitis models, both competitive and non-competitive AMPA-receptor antagonists were effective in ameliorating disease severity. The efficacy in vivo correlated with the potency of the compounds in an in vitro AMPA receptor assay. The protective effects were independent of immunosuppressive or anti-inflammatory effects, as assessed by quantification of CNS inflammation and an in vitro T-cell proliferation assay. Ultrastructural and immunohistochemical analysis revealed markers of axonal damage and a selective neurodegeneration of ventral horn motor neurons which had a direct T- cell-mediated component. The reduction in neuronal density was prevented by an AMPA receptor antagonist. Glutamate-mediated neuronal cell death within the mature CNS is reported to occur via both necrosis and apoptosis, however, classification of the latter is often based on little more than TUNEL staining. More recently, several investigators have employed ultrastructural analysis to describe an additional form of cell death in neurons of the adult CNS. The Han/Wistar spa/spa rat shows chronic neurodegeneration beginning around 3-4 weeks of age. The CNS location of damage correlates with the presentation of motor deficits. The selective neurodegeneration in these animals is due, at least in part to, AMPA receptor activation, since the neuronal loss is reversed by AMPA receptor antagonists in vivo. Using ultrastructural analysis, neurodegeneration was revealed in discrete brain regions and whilst certain elements of apoptosis were preserved, such as reduction of cell volume and chromatin condensation with maintenance of organelles and cellular and nuclear membrane integrity, others like chromatin margination, membrane blebbing, or formation of apoptotic bodies were not observed. In addition, biochemical assessment of cell death showed that DNA breakdown occurred in many regions associated with known degeneration and at time points that were consistent with the onset of degeneration in these areas. However, whilst the crucial executor caspase, caspase-3, was active in the olfactory bulb where apoptosis of glial cells is known to be ongoing, activity was not observed in the other regions of neurodegeneration. This may suggest that this neuronal death in adult neurons is caspase-3 independent which would argue against classical apoptosis. These studies open up a hitherto unexplored area of research and therapeutics within the field of multiple sclerosis and also question the role of classical neuronal apoptosis within the context of the adult central nervous system.

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