Use of super-resolution microscopy to visualize the interaction between Alzheimer therapeutic antibodies and Aβ aggregates

阿尔茨海默病（AD）是一种影响数百万人的进行性神经退行性疾病 国际吧目前AD的药物治疗是对症和无效的， 基于抗A β单克隆抗体（mAb）或分泌酶抑制剂的治疗令人失望。一 最近抗A β治疗失败的原因，即使是那些症状前开始的，是Aβ 在AD中积累的组装体在构象上是多样的，并且目前可用的mAb不靶向AD中的组装体。 主要神经毒性物种。因此，通过以下方法彻底表征分子机制至关重要： 哪些治疗性mAb与Aβ相互作用并影响其组装、结构和毒性。 推测AD的病理过程是由神经毒性Aβ寡聚体（Aβo）与受体结合而开始的 蛋白质或脂质在神经元的表面，最终导致突触功能障碍和退化。在 在以前的研究中，我们使用超分辨率显微镜直接观察β-受体相互作用， 纳米尺度。我们发现一种已知的Aβ受体，即细胞朊病毒蛋白PrPC，特异性抑制 Aβ纤维通过一种独特的机制聚合，在这种机制中，它特异性地结合到快速生长的末端， 从而阻断了该端的极化伸长。PrPC结合神经毒性寡聚体和原纤维 以类似的方式，这表明它可能识别所有这些上的共同的，末端特异性的结构基序， 组件.另外的实验表明，另外两种先前描述的Aβ受体（FcγRIIb和Fc γRIIb）也可能与Aβ受体结合。 LilrB 2）以类似的方式行事。总之，我们的结果表明，神经毒性信号通过几种不同的 受体可通过与纤维状和寡聚Aβ配体的常见分子相互作用而被激活。 在这些研究中使用的实验方法开辟了探索的机制， 影响Aβ聚合或毒性的其他药物的作用，特别是抗A β mAb，如目前 在临床试验中进行广泛的测试。在本申请中，我们提出表征 四种临床阶段抗体（aducanumab、gantenerumab、bapineuzumab和solanezumab）的作用，以及 作为一组构象依赖性抗体，识别Aβ的寡聚体、前纤维状和纤维状形式。 首先，我们将使用生化测定来测量这些mAb对Aβ聚集过程的影响。然后， 我们将利用单分子SRM来确定mAb在单个Aβ上的定位 集料.此外，我们将直接测量原纤维的伸长率，以及初级和次级纤维的伸长率。 成核过程，在存在和不存在每种mAb的情况下。最后，我们将确定这些mAb如何 影响Aβ与三种已知受体蛋白PrPC、FcγRIIb和LilrB 2之间的相互作用。这 该提案将为设计改进的单克隆抗体奠定基础，这些抗体选择性地改变Aβ中的特定步骤。 组装过程，并阻断寡聚体聚集体与细胞表面受体的相互作用， 它们的神经毒性。

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects millions of people worldwide. Current pharmacological treatments for AD are symptomatic and ineffective, and clinical trials of therapies based on anti-Aβ monoclonal antibodies (mAbs) or secretase inhibitors have been disappointing. One reason for the recent failures of anti-Aβ therapies, even those begun presymptomatically, is that the Aβ assemblies that accumulate in AD are conformationally diverse, and currently available mAbs do not target the primary neurotoxic species. Therefore, it is critical to thoroughly characterize the molecular mechanisms by which therapeutic mAbs interact with Aβ and influence its assembly, structure, and toxicity. It is presumed that AD pathology starts by the binding of neurotoxic Aβ oligomers (Aβo) to receptor proteins or lipids on the surface of neurons, resulting ultimately in synaptic dysfunction and degeneration. In previous studies, we have used super-resolution microscopy to directly visualize β-receptor interactions at the nanometer scale. We find that one documented Aβ receptor, the cellular prion protein, PrPC, specifically inhibits the polymerization of Aβ fibrils via a unique mechanism in which it binds specifically to the rapidly growing end of each fibril, thereby blocking polarized elongation at that end. PrPC binds neurotoxic oligomers and protofibrils in a similar fashion, suggesting that it may recognize a common, end-specific, structural motif on all of these assemblies. Additional experiments suggest that two other previously described Aβ receptors (FcγRIIb, and LilrB2) act in a similar fashion. Taken together, our results suggest that neurotoxic signaling by several different receptors may be activated by common molecular interactions with both fibrillar and oligomeric Aβ ligands. The experimental approach used in these studies opens up the possibility of probing the mechanism of action of other agents that affect Aβ polymerization or toxicity, in particular anti-Aβ mAbs such as those currently undergoing extensive testing in clinical trials. In this application, we propose to characterize the mechanism of action of four clinical stage antibodies (aducanumab, gantenerumab, bapineuzumab and solanezumab), as well as a panel of conformation-dependent antibodies that recognize oligomeric, pre-fibrillar, and fibrillar forms of Aβ. First, we will measure the effect of these mAbs on Aβ aggregation processes using biochemical assays. Then, we will take advantage of single molecule, SRM to determine the localization of the mAbs on the individual Aβ aggregates. In addition, we will directly measure fibril elongation rates, as well as primary and secondary nucleation processes, in the presence and absence of each mAb. Finally, we will determine how these mAbs affect the interaction between Aβ and three of its documented receptor proteins; PrPC, FcγRIIb, and LilrB2. This proposal will lay the groundwork for the design of improved mAbs that selectively alter specific steps in the Aβ assembly process, and that block the interaction of oligomeric aggregates with cell surface receptors that transduce their neurotoxic effects.