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

健康的生物科学-氨基丁酸A型受体（GABAARs）是五聚体配体门控离子通道，主要介导哺乳动物中枢神经系统（CNS）的抑制性神经传递。事实上，它们在调节CNS活动中的关键作用意味着它们影响许多关键行为，包括总体兴奋-抑制平衡、镇静、成瘾、认知和伤害感受。它们的重要性在GABAA受体突变导致信号缺陷并导致神经系统疾病（如癫痫、焦虑和失眠）的情况下进一步揭示。在人类中，存在19种GABAAR亚基亚型，并且这些亚基亚型组装成五聚体，其中组合的变化是可能的，从而产生无数的GABAAR亚型。然而，靶向特定亚型组合的能力受到严重限制，这限制了我们使用药理学工具了解其对脑功能和神经系统疾病的贡献的能力。米勒实验室的研究重点是了解，开发和提供新型抗体和纳米抗体（一种微型抗体）调节剂，其具有针对离子通道的独特药理学性质，包括但不限于GABAAR。考虑到许多小分子药物治疗CNS疾病的有限功效和众多副作用，这些免疫剂提供增强的靶向特异性，因此具有作为CNS研究工具和治疗剂的巨大潜力。我的项目将涉及使用定点诱变产生100-500个变体的纳米抗体的小型靶向文库，由低温电子显微镜（cryo-EM）结构引导的由米勒实验室解决的感兴趣的纳米抗体结合的GABAAR结构。目前未发表的结构揭示了几种纳米抗体，它们通过在a亚基和b亚基之间的界面上结合来调节功能。通过突变参与结合一个或另一个亚基的关键残基的靶向将改变纳米抗体的特异性，以改善或转换其对特定a-加B-亚基亚型的选择性。这代表了GABAAR的“细粒”药理学的全新水平，这是以前不存在的。由于GABAAR亚基亚型分布在整个大脑中各不相同，针对特定A-和B-亚型的双重选择性配体将更好地分离出亚型之间神经元和动物行为的离散参与。随后可以使用细胞内生物发光共振能量转移（BRET）筛选纳米抗体库以检测“命中”，即需要跟进的具有改变的选择性的结合剂。该项目的另一个相关但不同的部分将涉及跟进在我的10周轮换项目期间表征的a2 B3 y1-GABAAR纳米抗体结合剂的子集。这也可能包括使用米勒实验室目前正在开发的新型噬菌体展示文库来提高新的纳米抗体，以增加靶向含γ 1的GABAAR的纳米抗体的数量，这些纳米抗体被认为在伤害感受中发挥作用，但由于缺乏选择性配体，已经证明很难验证这一假设。该项目将推进我们目前对离子通道抗体调节剂设计的理解，并使我们更接近于开发离子通道蛋白调节剂，用作征服CNS的药理学研究工具行为障碍

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.