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.

摘要 在医学的许多领域，对活体患者的生物标本进行病理生理学研究， 了解疾病机制。然而，由于脑活检的困难，这种策略 尚未开发用于阿尔茨海默病（AD）。因此，非常需要新颖的方法来 从活体患者中收集神经元，以捕获与AD相关的病理变化。嗅 神经上皮作为研究脑疾病的替代组织受到了极大的关注。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。作为嗅觉 神经上皮细胞包括神经干细胞/神经祖细胞，组织可以在神经干细胞/神经祖细胞分化后快速再生。 活检，这使我们能够在不损害嗅觉功能的情况下随着时间的推移重复活检。然而，经典 鼻活检平台受到其侵入性和活检组织中神经元纯度不足的限制。 为了克服这些局限性，我们最近开发了一种新的平台：一种柔软的鼻刷拭子， 神经元特异性单细胞分析。这个新的平台现在甚至更容易，甚至比一个侵入性更小。 血液抽取和神经纯度保证在单细胞分辨率。 嗅觉功能障碍是AD患者记忆力减退的早期症状; Aβ病变和tau蛋白 病理学广泛地发生在嗅觉系统中，包括嗅觉神经上皮。所以我们 假设在嗅觉神经元中检测到的磷酸化tau蛋白在苏氨酸181（pT 181-tau 通过单细胞蛋白质印迹，与轻度认知障碍患者相比， 此外，在嗅觉神经元中检测到的这种pT 181-tau水平是 与脑脊液（CSF）中检测到的pT 181-tau水平和神经认知功能相关。 事实上，我们发现从AD患者中获得的嗅觉神经元中的pT 181-tau水平显著高于 认知正常的对照组相比。在这些令人鼓舞的数据的鼓舞下，我们将研究 在30例AD患者，30例MCI受试者， 和30名认知正常的对照组（目标1）;我们将比较pT 181-tau在嗅觉神经元中的水平， 单细胞水平与来自相同个体的CSF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pT 181-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，我们希望建立一个新的，高通量的，非- 侵入性平台（鼻刷拭子，然后进行单细胞Western印迹），以研究 AD在单细胞水平上从活体患者获得的神经元中。