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

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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.