Investigation of the functional effects of auto-immune ionotropic glutamate receptor antibodies in representative in vitro CNS preparations

Day, C. (2022) Investigation of the functional effects of auto-immune ionotropic glutamate receptor antibodies in representative in vitro CNS preparations. PhD thesis, University of Reading. doi: 10.48683/1926.00115651

Abstract/Summary

The NR1 subunit of N-methyl-D-aspartate receptors (NMDARs) and the GluR3 subunit of α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) have been identified as targets of autoantibodies (Aabs) in autoimmune encephalopathy, whereby seizures, cognitive impairment and memory loss are key symptoms. Recent studies have proposed mechanisms by which these Aabs act on their respective receptors, but their role in neuronal excitability, seizures and autoimmune epilepsy is yet to be established. Patient Aabs have been shown to bind to specific regions within the NR1 and GluR3 subunits. Therefore, peptide immunisation was used to generate Aabs in rabbits against these specific sequences, and ‘protein A’ purification to obtain total IgG, or peptide purification to obtain target specific Aabs. Binding and specificity of these Aabs were determined using a range of methodologies including enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (ICC) and immunohistochemistry (IHC). Functional effects were determined using a range of in vitro electrophysiology techniques, including two-electrode voltage-clamp on Xenopus oocytes, long-term potentiation (LTP) in hippocampal brain slices using multi-electrode arrays, and excitatory postsynaptic currents (EPSCs) from primary hippocampal neurons using whole-cell patch-clamp. This study has shown NMDAR and AMPAR Aabs generated from peptide immunisation demonstrated specificity for NR1 and GluR3 immunisation peptides as well as target-specific binding to their native proteins in ELISA, IHC and ICC. Upon further purification, NMDAR Aabs were shown to prevent the induction of LTP at Schaffer collateral-CA1 synapses, supporting the proposed Aab-induced internalisation of NMDARs mechanism of action. Acute and chronic application of AMPAR Aabs elicited a reduction in spontaneous and miniature EPSC frequency in hippocampal neurons. Our data is consistent with NMDAR Aabs decreasing the number of synaptic NMDARs via internalisation, and AMPAR Aabs acting via an inhibitory mechanism at the synaptic level, in both cases an effect consistent with a disruption to the excitatory/inhibitory network. This work provides a solid basis to address outstanding questions regarding the mechanism of both these Aabs; for example, future work using internalisation assays or applying Aabs to in vitro models of epileptiform activity to determine their roles on network activity. The basic science presented here can contribute to the development of novel AEDs with respect autoimmune epilepsy.

Item Type	Thesis (PhD)
URI	https://reading-clone.eprints-hosting.org/id/eprint/115651
Identification Number/DOI	10.48683/1926.00115651
Divisions	Life Sciences > School of Chemistry, Food and Pharmacy > School of Pharmacy
Date on Title Page	December 2021
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