Abstract
Early drug discovery for new psychiatric medicines relies heavily on the use of in vitro assays. At present, this work is typically conducted through ectopic expression of human proteins of interest in cells that are often neither human nor neural in origin. As our appreciation of the complexity inherent to receptor pharmacology has increased, these heterologous expression systems have come under increasing scrutiny as a means to evaluate the mode of action of novel neuropsychiatric drug candidates. These limitations likely contribute to the high attrition rate observed as these compounds progress to the clinic. The use of native human neural cells during initial drug development has the potential to overcome some of these problems.This thesis describes work towards the development of an in vitro drug discovery platform using a!human, conditionally immortalised, cortically derived, neural stem cell line (CTX0E16/02). Protocols were developed to enable robust and reproducible differentiation of these human neural stem cells into cell-types normally found in the adult cerebral cortex –different GABAergic neuronal subtypes, glutamatergic neurons, astrocytes and potentially oligodendrocytes. Neurons! grown under these conditions were also shown to be electrically active. By investigating ligand-induced Ca2+ and Erk1/2 signalling, cells within differentiated cortical cultures were shown to express functional receptors, for a range of neurotransmitters, including dopamine, serotonin, glutamate, GABA, histamine, norepinephrine and acetylcholine. Many of these receptors are known targets of currently available neuropsychiatric compounds, making this platform ideal for studying drugs that modulate the activity of the human brain.
Data presented here highlighted the importance of using native neural cells to interrogate the signalling consequences of ligand-receptor interactions. For instance, the reported full 5MHT2A-specific agonist –TCBM2 –was shown to demonstrate inverse agonism with respect! to! intracellular Ca2+ accumulation, while cholinergic stimulation was shown to provoke muscarinic receptor-mediated Ca2+ influx rather!than an expected mobilisation from intracellular stores. Most importantly, however, effects of a therapeutically relevant concentration of the antipsychotic, haloperidol, could be detected using these differentiated CTX0E16/02 cultures. However, this effect was only apparent in the presence of simulated neurophysiological tone. This finding has important implications regarding the way in which the mechanism of action of complex drug-receptor interactions is experimentally investigated in vitro.
| Date of Award | 2012 |
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| Original language | English |
| Awarding Institution |
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