Pharmaco-circuitry of neurosteroids - regulators of mood and excitability disorders

Mental health disorders, especially anxiety, depression and stress will affect many individuals at least once during their lifetimes with debilitating consequences not just for individuals, but also for society. Operationally, synchronised neuronal activity is vital for the normal functioning of the nervous system and the key orchestrators for this are inhibitory neurotransmitter receptors that are activated by GABA. These receptors (GABA-ARs) are expressed throughout the central nervous system. They are located at specialised junctions between neurons (inhibitory synapses) where they mediate rapid neuronal inhibition. They also reside outside synapses (extrasynaptic) providing a persistent lower level tonic inhibition. Together, these forms of GABA inhibition control neuronal excitability. The importance of these receptors to the operation of the brain is exemplified by the consequences that follow their dysfunction, for example, by precipitating mental health disorders such as anxiety and depression. As a consequence, GABA-ARs are widely recognised as key therapeutic targets. There are several brain centres that are considered to be important for the initiation of anxiety and stress. Prominent amongst these is the amygdala - a centre involved in the processing of emotional reactions, but about which we know very little of the roles that GABA-ARs and their associated inhibitory circuitry play, and less still about the importance of modulation of these receptors by the neurosteroids (an important class of regulatory molecule in the brain). This is significant because our previous studies revealed that neurosteroids perform an innate anxiety-relieving (anxiolytic) role, partly mediated by alpha2-subunit-containing GABA-ARs. These receptors are present in the amygdala at inhibitory synapses. However, specific amygdala neurons involved in controlling anxiety and fear, also contain extrasynaptic alpha3-GABA-ARs (usually expressed at synapses in other brain regions) and alpha5-GABA-ARs (which exhibit a variable sensitivity to neurosteroids) implicating them both in the control of emotional responses. Thus, to further understand amygdala function in mental health disorders we need to investigate how these other types of GABA-ARs are modulated by neurosteroids to control inhibition. To achieve our aims, we will genetically engineer alpha3- and alpha5-GABA-AR isoforms in the amygdala to determine how neurosteroids regulate their function. This will significantly contribute towards our understanding of mental health disorders which can develop when neurosteroid regulation becomes dysfunctional. We will use our evolving GABA-AR protein structures to explore how neurosteroids modulate these receptors, facilitating the development of new neurosteroid molecules. Our expectation is to improve the understanding of mechanisms that may cause mental illness and to provide new approaches to its treatment. We have four main aims - (i) to characterise neurosteroid modulation of GABA-ARs in the amygdala; (ii) to genetically engineer mice ('knock-ins') with alpha3- and alpha5-GABA-ARs that are insensitive to neurosteroids; (iii) to analyse neural network and behavioural characteristics of the GABA-AR knock-in mice focusing on the consequences for anxiety and fear; and (iv) to explore GABA-AR neurosteroid binding sites at an atomic level to develop new selective steroid-based therapeutics for mental health. These knock-in lines will provide unique tools for understanding how neurosteroids modulate alpha3- and alpha5-GABA-ARs receptors to control specific neurons in the amygdala and how dysfunction in this system causes mental disorders. Using structural studies of GABA-ARs to develop new receptor subtype-selective neurosteroids in collaboration with our pharmaceutical partners, will offer the prospect of new treatments for specific mental health disorders for which there is an extensive unmet clinical need.