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

健康氨基丁酸a型受体（GABAARs）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）中的抑制性神经传递。事实上，它们在中枢神经系统活动调节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、认知和伤害感受。在GABAA受体突变引起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，它们的重要性进一步得到揭示。在人类中，有19种GABAAR亚基亚型，这些亚型组合成五聚体，组合的变化是可能的，从而产生了无数的GABAAR亚型。然而，针对特定亚型组合的药理学能力严重有限，这限制了我们使用药理学工具来了解它们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理解、开发和提供具有独特药理特性的新型抗体和纳米体（一种微型抗体）调节剂，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，这些免疫药物提供了增强的靶点特异性，因此作为中枢神经系统研究工具和治疗方法具有巨大的潜力。我的项目将涉及使用位点定向诱变技术，在Miller实验室解决的纳米体结合GABAAR结构的低温电子显微镜（cro - em）结构的指导下，生成100-500个变体的小型靶向纳米体文库。目前未发表的结构揭示了几种通过结合a-和b -亚基之间的界面来调节功能的纳米体。通过突变参与结合一个或另一个亚基的关键残基来靶向，将改变纳米体的特异性，从而改善或改变其对特定a + b亚基亚型的选择性。这代表了以前不存在的GABAARs的“细颗粒”药理学的一个全新水平。由于GABAAR亚基亚型在整个大脑中的分布是不同的，针对特定a和b亚型的双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。纳米体文库随后可以使用细胞内生物发光共振能量转移（BRET）进行筛选，以检测“命中”，即需要跟踪的选择性改变的粘合剂。这个项目的另一个相关但不同的部分将涉及跟踪我在10周轮换项目中表征的a2B3y1-GABAAR纳米体粘合剂的子集。这也可能包括使用Miller实验室目前正在开发的一种新的噬菌体展示库来培养新的纳米体，以增加靶向含有y1的GABAARs的纳米体的数量，这种纳米体被认为在伤害感觉中起作用，但由于缺乏选择性配体，这一假设很难得到验证。最终，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，作为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.