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.

We will examine inhibitory transmission and neural circuit activity, focussing on GABA-ARs and their specific modulation by neurosteroids in the amygdala, in the context of mental health disorders. Our aim is to understand how dysfunctional mechanisms involving GABA inhibition and GABA-AR modulation can precipitate mental illness by studying an area of the brain that is critical for emotional processing. We will use genetic, cellular, molecular and behavioural methods to examine the interplay between neurosteroids and GABA-ARs in the amygdala.

Genetic engineering will be used to selectively remove neurosteroid modulation from alpha3- and alpha5-GABA-ARs in specific amygdala cell types. Whole-cell patch clamp recording will assess synaptic and tonic GABA inhibition and their effects on excitability using native receptors in acute brain slice preparations before proceeding to whole animals to assess neural circuitry and EEG activity. Key aspects of GABA channel function, including spontaneous gating and the effects of phosphorylation will also be assessed. Application of molecular biological, biochemical and electrophysiological methods will allow us to scrutinise receptor post-translational modifications, trafficking and function. Behavioural experiments will ascertain the importance of neurosteroid regulation of amygdala GABA-ARs for anxiety phenotypes, fear processing, and cognition, for which operators will be blinded for genotype.

In parallel, we will use our high-resolution structural approaches (x-ray crystallography, cryo-EM), in collaboration with our pharmaceutical partners, to examine the binding of endogenous and synthetic neurosteroids to GABA-ARs, with the aim of developing new GABAAR-specific neurosteroids as novel therapeutics. Overall, our programme aims to improve the understanding of how dysfunctional inhibition within the brain impacts on mental health and to provide new approaches for the treatment of specific disorders.

Our programme is projected to have broad ranging effects on diverse groups in terms of academic, economic and societal impact. Initial impact will be centred on academics and industrial collaborators with longer term impact projected to benefit clinically-trained healthcare professionals and individuals suffering from mental health disorders.

The benefit to academics in neuroscience will derive from advancing our understanding of the role of inhibition within a key part of the brain that is involved in emotional processing - the amygdala. This important brain region is complex and as such has largely resisted detailed scrutiny of molecular mechanisms which will be highly significant for mental health. Through the application of our novel approaches to altering gene expression in specific amygdala cell-types, our work will provide a clearer picture as to the importance of inhibitory circuits and their associated GABA-ARs for amygdala function. Furthermore, it is equally important to understand how neurosteroids, a group of powerful, naturally-produced, chemical modulators in the brain control neuronal and network excitability in the amygdala via their actions on GABAARs. This work will therefore provide fundamental insight into inhibitory mechanisms underlying normal physiological brain activity. However, our programme will also have wider applicability since dysfunctional inhibitory transmission and aberrant amygdala function strongly associate with a range of neuropsychiatric conditions, in particular, anxiety, depression, and stress disorders. Interrogating the function and manipulating the effectiveness of neurosteroids will bring additional long-term impact that will have outreach and benefit to all the groups above and also to psychiatrists and endocrinologists who associate with mental health studies and their treatment.

Our ongoing collaborations with the pharmaceutical industry incorporates further impact into our programme by providing excellent opportunities for translation into more effective future mental health therapies. We will explore new neurosteroid molecules docked into their binding sites which will form the rational basis for designing novel, receptor-selective neurosteroids. This will have significant impact not only for industrial colleagues but also for clinical practitioners. Indeed, in the longer term, we would predict that the outcomes from our programme have the potential to lead to improved quality of life for mental health patients, family members, and carers. These outcomes will be formed from an improved understanding of: the role of GABA inhibition and modulation of synaptic and extrasynaptic GABA-AR isoforms in the amygdala; the function of the amygdala during healthy and disease conditions; and especially in how best to utilise the potency and binding selectivity of neurosteroids in modulating inhibition for improved therapeutic effect.

Our outcomes may become patentable, and as before with other biologic-based treatment modalities that we are investigating, UCL-Business (ULC-B) will be our guide towards protecting IP. They have excellent support structures to enable us to proceed and exploit our discoveries in discussions with healthcare investors and pharma.

Greater understanding as to how neurosteroids and GABA-ARs are involved in mental health disorders should facilitate the identification of new drug targets in disease pathology with fewer side effects. This is of vital importance for all sectors of society given the increasing prevalence of mental health disorders for which there is an urgent clinical need for new therapeutic initiatives.