Rotation 1: Role of DNA damage in neurodegenerative disease (ataxia telangiectasia)

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Bioscience for an integrated understanding of healthy-aminobutyric acid Type-A receptors (GABAARs) are pentameric ligand-gated ion channels that principally mediate inhibitory neurotransmission in the mammalian central nervous system (CNS). Indeed, their pivotal role in the regulation of CNS activity means that they influence many key behaviours, including overall excitation-inhibition balance, sedation, addiction, cognition, and nociception. Their importance is further revealed in conditions where GABAA receptor mutations cause defective signalling and lead to neurological disorders, such as epilepsy, anxiety, and insomnia. In humans, there are 19 GABAAR subunit subtypes and these assemble into pentamers with variations in combinations being possible, giving rise to a myriad of GABAAR subtypes. The ability to pharmacologically target specific subtype combinations is, however, severely limited, which restricts our ability to use pharmacological tools to understand their contributions to brain function and in neurological disorders.Research in the Miller lab focusses on understanding, developing, and delivering novel antibody and nanobody (a type of miniature antibody) modulators with unique pharmacological properties against ion channels, including but not limited to the GABAAR. Considering the limited efficacy and numerous side effects of many small-molecule drug treatments for CNS disorders, these immunological agents offer enhanced target specificity and therefore hold massive potential as CNS research tools and therapeutics.My project would involve generating small, targeted libraries of nanobodies of 100-500 variants using site directed mutagenesis, guided by cryogenic electron microscopy (cryo-EM) structures of GABAAR structures bound by nanobodies of interest solved by the Miller lab. The current unpublished structures reveal several nanobodies that modulate function by binding across the interface between an a- and a B-subunit. Targeting by mutating key residues involved in binding one or another subunit will alter the specificity of the nanobody to improve or switch its selectivity for a particular a- plus B-subunit subtype. This represents a completely new level of "fine grain" pharmacology for GABAARs which has not previously existed. Since GABAAR subunit subtype distribution varies across the brain, dual selective ligands against a specific A- and B-subtype will better separate out discrete involvements in neuronal and animal behaviour between subtypes. The nanobody library can subsequently be screened using in-cell bioluminescence resonance energy transfer (BRET) to detect 'hits', i.e. binders with altered selectivity that require following up.Another related but distinct part of this project would involve following up on a subset of a2B3y1-GABAAR nanobody binders characterised during my 10-week rotation project. This could also include raising new nanobodies using a novel phage display library the Miller lab is currently developing to increase the number of nanobodies targeting y1-containing GABAARs, which are believed to play a role in nociception but due to an absence of selective ligands, it has proven hard to test this hypothesis.Ultimately, this project would advance our current understanding of antibody modulator design against ion channels and bring us closer to developing ion channel protein modulators for use as pharmacological research tools for conquering CNS behavioural disorders.