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

生物科学对健康氨基丁酸A型受体(GABAARs)的全面了解是一种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，它们在调节中枢神经系统活动中的关键作用意味着它们影响许多关键行为，包括整体兴奋-抑制平衡、镇静、成瘾、认知和伤害性感受。在GABAA受体突变导致信号缺陷并导致神经疾病，如癫痫、焦虑和失眠的情况下，它们的重要性进一步显现。在人类中，有19个GABAAR亚单位亚型，这些亚型组装成五聚体，可能会有不同的组合，从而产生无数的GABAAR亚型。然而，针对特定亚型组合的药理学靶向能力严重有限，这限制了我们使用药理学工具了解它们对大脑功能和神经疾病的贡献的能力。Miller实验室的研究重点是了解、开发和交付新型抗体和纳米体(一种微型抗体)调节剂，这些调节剂具有针对离子通道的独特药理特性，包括但不限于GABAAR。考虑到许多小分子药物治疗中枢神经系统疾病的有限疗效和众多副作用，这些免疫学试剂提供了增强的靶向特异性，因此具有作为中枢神经系统研究工具和治疗的巨大潜力。我的项目将涉及在米勒实验室解决的由感兴趣的纳米体结合的GABAAR结构的低温电子显微镜(Cryo-EM)结构的指导下，使用定点突变生成100-500个变异的纳米体的小型靶向文库。目前未发表的结构揭示了几个纳米体，它们通过结合A-和B-亚基之间的界面来调节功能。通过突变结合一个或另一个亚基所涉及的关键残基来靶向，将改变纳米体的特异性，以改善或改变其对特定A+B亚基亚型的选择性。这代表了GABAARs“细粒”药理的全新水平，这是以前从未存在过的。由于GABAAR亚单位亚型在整个大脑中的分布不同，针对特定A和B亚型的双重选择性配体将更好地区分不同亚型之间在神经元和动物行为方面的离散参与。随后可以使用细胞内生物发光共振能量转移(BRET)对纳米体库进行筛选，以检测“HITS”，即具有改变的选择性的需要跟进的结合剂。该项目的另一个相关但不同的部分将涉及在我10周的旋转项目中表征的a2B3y1-GABAAR纳米体结合剂的子集的跟进。这也可能包括利用米勒实验室目前正在开发的一种新型噬菌体展示文库来培育新的纳米体，以增加针对含有y1的GaBAARs的纳米体的数量，这些纳米体被认为在伤害性感受中发挥作用，但由于缺乏选择性配体，事实证明很难检验这一假设。最终，这个项目将促进我们目前对针对离子通道的抗体调节器设计的理解，并使我们更接近于开发离子通道蛋白质调节器，用作征服中枢神经系统行为障碍的药理学研究工具。

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.