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

摘要 在许多医学领域，对活体患者的生物标本的病理生理学研究促进了我们的 对疾病机制的理解。然而，由于脑活组织检查的困难，这种策略已经 不是为阿尔茨海默病(AD)开发的。因此，非常需要新的方法来 收集活体患者的神经元，以捕捉与AD相关的病理变化。嗅觉 神经上皮细胞作为研究脑部疾病的替代组织受到了极大的关注。多项研究 已经在分子水平上证明了嗅觉神经元/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/神经前体细胞，组织可以在 活组织检查，它允许我们随着时间的推移重复活组织检查，而不会损害嗅觉功能。然而，经典的 鼻活检平台的侵袭性和活检组织中神经元的纯度不够高是其局限性。 为了克服这些限制，我们最近开发了一个新平台：一个柔软的鼻刷拭子，然后是一个 针对神经元的单细胞分析。这一新平台现在比 抽血，神经纯度在单细胞分辨率下得到保证。 嗅觉障碍是AD患者强健记忆缺陷的早期症状；Aβ损害和tau 病理广泛发生在嗅觉系统，包括嗅神经上皮。因此，我们 假设在嗅神经元中检测到苏氨酸181位的磷酸化tau蛋白(pT181-tau) 与轻度认知受试者相比，AD患者的单细胞Western blotting增加 损害(MCI)和对照，此外，在嗅神经元中检测到的这种pT181-tau水平是 与脑脊液中检测到的pT181-tau水平和神经认知功能有关。 事实上，我们发现阿尔茨海默病患者嗅觉神经元中pT181-tau的水平明显更高 在我们的初步研究中，来自认知正常对照组的人。在这些充满希望的数据的鼓舞下，我们将研究 单细胞分辨pT181-tau在30例AD患者，30例MCI患者， 和30名认知正常的对照组(目标1)；我们将比较在以下时间检测到的嗅神经元pT181-tau的水平 单细胞水平与从相同个体的脑脊液中检测到的水平(目标2)；我们将确定 在单细胞水平检测到的嗅觉神经元中pT181-tau水平与神经心理学有关 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、高通量和非 侵袭性平台(鼻刷拭子后单细胞Western blotting)研究鼻咽癌的病理生理 在单细胞水平上，从活着的患者中获得的神经元中的AD。

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.