Phosphorylated tau protein detection in olfactory neurons at a single-cell resolution: needle-free biopsy for Alzheimer's disease pathophysiology study

以单细胞分辨率检测嗅觉神经元中的磷酸化 tau 蛋白：用于阿尔茨海默病病理生理学研究的无针活检

• 批准号: 10117832
• 负责人: Koko Ishizuka
• 金额: $ 45.03万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10117832
• 关键词: Alzheimer' s Disease; Alzheimer' s disease diagnosis; Alzheimer' s disease patient; Amyloid beta-42; Amyloid beta-Protein; Area; Biopsy; Bipolar Disorder; Blood; Blood Cells; Brain; Brain Diseases; Cells; Cerebrospinal Fluid; Cognitive; Collecting Cell; Collection; Comb animal structure; Consumption; Cross-Sectional Studies; Data; Deposition; Detection; Disease; Early Diagnosis; Endoscopes; Functional disorder; Individual; Lesion; Longitudinal Studies; Medicine; Memory impairment; Mental disorders; Methodology; Methods; Molecular; Molecular Analysis; Molecular Profiling; Natural regeneration; Nature; Needles; Neurocognitive; Neurodegenerative Disorders; Neurons; Neuropsychology; Nose; Olfactory Pathways; Olfactory dysfunction; Parkinson Disease; Pathologic; Pathology; Patients; Pilot Projects; Prognosis; Punch Biopsy; Reporting; Resolution; Schizophrenia; Spinal Puncture; Swab; Symptoms; Techniques; Threonine; Time; Tissues; Western Blotting; alpha synuclein; base; biomarker performance; brain cell; cognitive control; disorder control; early detection biomarkers; induced pluripotent stem cell technology; interest; mild cognitive impairment; nerve stem cell; neuroepithelium; novel; relating to nervous system; single cell analysis; tau Proteins; tau-1; tool

Abstract In many areas of medicine, pathophysiological studies of biospecimens from living patients have facilitated our understanding of disease mechanisms. However, due to the difficulty of brain biopsies, such strategies have not been developed for Alzheimer’s disease (AD). Thus, there is a great need for novel methodologies to collect neurons from living patients in order to capture pathological changes relevant to AD. The olfactory neuroepithelium has received great interest as a surrogate tissue to study brain disorders. Multiple studies have shown the neuronal validity of olfactory neurons/neuronal cells at the molecular level. As the olfactory neuroepithelium includes neural stem cells/neuroprogenitor cells, the tissue can quickly regenerate following a biopsy, which allows us to repeat biopsies over time without impairing olfactory function. However, the classic nasal biopsy platform is limited by its invasiveness and the insufficient purity of neurons in the biopsied tissue. To overcome these limitations, we recently developed a new platform: a soft nasal brush swab followed by a single-cell analysis specific for neurons. This new platform is now even easier and even less invasive than a blood draw, and neural purity is guaranteed at a single-cell resolution. Olfactory dysfunction is an early symptom preceding robust memory deficits in AD; Aβ lesions and tau pathology occur extensively in the olfactory system, including the olfactory neuroepithelium. Therefore, we hypothesized that phosphorylated tau protein at threonine 181(pT181-tau) detected in olfactory neurons through single-cell Western blotting is increased in AD patients, compared to subjects with mild cognitive impairment (MCI) and controls, and additionally that such pT181-tau levels detected in olfactory neurons are associated with the pT181-tau levels detected in cerebrospinal fluid (CSF) and with neurocognitive function. Indeed, we found that pT181-tau levels in olfactory neurons obtained from AD patients were significantly higher than those from cognitively normal controls in our pilot study. Encouraged by this promising data, we will study pT181-tau levels in olfactory neurons at the single-cell resolution in 30 patients with AD, 30 subjects with MCI, and 30 cognitively normal controls (Aim 1); We will compare pT181-tau levels in olfactory neurons detected at the single-cell level with those detected in CSF from the same individuals (Aim 2); We will determine whether pT181-tau levels in olfactory neurons detected at the single-cell level are associated with neuropsychological function (Aim 3). Through this proof-of-concept study, we hope to establish a novel, high-throughput, and non- invasive platform (nasal brush swab followed by single-cell Western blotting) to study the pathophysiology of AD in neurons obtained from living patients at the single-cell level.

抽象的 在许多医学领域，对活体患者生物样本的病理生理学研究促进了我们的研究 了解疾病机制。然而，由于脑活检的困难，此类策略已经 尚未开发用于治疗阿尔茨海默病（AD）。因此，非常需要新的方法来 从活体患者身上收集神经元，以捕获与 AD 相关的病理变化。嗅觉 神经上皮作为研究大脑疾病的替代组织引起了极大的兴趣。多项研究 已经在分子水平上显示了嗅觉神经元/神经元细胞的神经元有效性。正如嗅觉 神经上皮包括神经干细胞/神经祖细胞，该组织可以在经过一段时间后快速再生。 活检，这使我们能够随着时间的推移重复活检而不损害嗅觉功能。然而，经典 鼻活检平台因其侵入性和活检组织中神经元纯度不足而受到限制。 为了克服这些限制，我们最近开发了一个新平台：柔软的鼻刷拭子，然后是 针对神经元的单细胞分析。这个新平台现在比传统平台更简单、侵入性更小。 抽血，并在单细胞分辨率下保证神经纯度。 嗅觉功能障碍是 AD 中严重记忆缺陷之前的早期症状。 Aβ 损伤和 tau 病理广泛发生在嗅觉系统，包括嗅神经上皮。因此，我们 假设在嗅觉神经元中检测到苏氨酸 181 (pT181-tau) 磷酸化 tau 蛋白 与轻度认知障碍患者相比，AD 患者通过单细胞蛋白质印迹检测的结果增加 损伤（MCI）和对照，此外，在嗅觉神经元中检测到的 pT181-tau 水平是 与脑脊液 (CSF) 中检测到的 pT181-tau 水平以及神经认知功能相关。 事实上，我们发现从 AD 患者身上获得的嗅觉神经元中的 pT181-tau 水平明显更高 与我们的试点研究中认知正常对照的结果相比。受到这些有希望的数据的鼓舞，我们将研究 30 名 AD 患者、30 名 MCI 受试者的单细胞分辨率下嗅觉神经元中的 pT181-tau 水平 和 30 个认知正常的对照组（目标 1）；我们将比较在以下位置检测到的嗅觉神经元中的 pT181-tau 水平： 单细胞水平与同一个体脑脊液中检测到的单细胞水平（目标 2）；我们将确定是否 在单细胞水平上检测到的嗅觉神经元中的 pT181-tau 水平与神经心理学相关 功能（目标 3）。通过这项概念验证研究，我们希望建立一种新颖的、高通量的、非 侵入性平台（鼻刷拭子，然后进行单细胞蛋白质印迹）研究病理生理学 AD 在单细胞水平上从活体患者身上获得的神经元。