Development of a High-Sensitivity Method for the Measurement of Human Nasal Aß42, Tau, and Phosphorylated Tau.

Cost-effective and feasible methods for early diagnosis of Alzheimer's disease (AD) are needed. We present two methods to measure AD-related biomarkers simultaneously from one nasal smear for the purpose of diagnosing AD. Japanese men and women aged 63-85 years old were recruited in 2015-2016 for this case-control study. A total of 25 AD cases and 25 controls (22 men and 28 women) participated in this research. Nasal smears were collected from the common nasal meatus, inferior concha, middle nasal meatus, and olfactory cleft, and the proteins in the samples were analyzed by two methods, which we named PGD (Pre-treatment with guanidine- n-Dodecyl-beta-D-maltoside solution) method 1 (PGD-I) and 2 (PGD-II). The PGD-I method measured total tau and amyloid-ß (Aß)42, but no differences in median levels of total tau and Aß42 between AD cases and controls were found in any of the nasal locations. The PGD-II method measured Aß42, total tau, and phosphorylated tau, but levels of Aß40 in all nasal locations of both groups were near zero. Median levels of phosphorylated tau to total tau (p-tau/t-tau) ratios in the middle nasal meatus and in the olfactory cleft were significantly higher in AD cases than in controls, and could significantly predict AD. To assess diagnostic reliability, areas under the ROC curve were 0.74 (95% CL = 0.52-0.95, p = 0.030) for the middle nasal meatus and 0.72 (95% CL = 0.52-0.92, p = 0.029) for the olfactory cleft. Thus, PGD-I and PGD-II can detect AD-related biomarkers in nasal smears and PGD-II may be a useful tool for diagnosing AD.

ß-Amyloid peptide (1-40) in the brain reaches the nasal cavity via a non-blood pathway.

We examined the distribution patterns of human ß-amyloid (1-40) peptide labeled with iodine 125 ((125)I-Aß40) after injections into the cerebral ventricle or tail vein of rats. In rats receiving an intravenous injection, the radioactive concentration of (125)I-Aß40 in the nasal area was similar to other extracranial organs. In contrast, the caudal part of the nasal area in rats receiving an intracerebroventricular injection displayed a high level of (125)I-Aß40 radioactivity. These results suggest that Aß reaches the nasal cavity from the brain via a non-blood pathway.

Correlation of Aß deposition in the nasal cavity with the formation of senile plaques in the brain of a transgenic mouse model of Alzheimer's disease.

The deposition of ß-amyloid peptides (Aß) is commonly reported in the nasal cavity of Alzheimer's disease (AD) patients, although the pathological significance of this finding is unknown. This study compared Aß concentrations in the nasal area with those in the brain, blood, and cerebrospinal fluid, respectively. Immunohistochemical analysis identified Aß deposits in the nasal epithelium of Tg2576 mice. Enzyme-linked immunosorbent assay measurements revealed a correlation between the content of Aß42 in the nasal area and that in the brain, but not with that in the blood. These results suggest that the highly accessible nasal cavity could be a useful site for diagnostic analysis of AD based on Aß content.

Position-specific incorporation of a highly photodurable and blue-laser excitable fluorescent amino acid into proteins for fluorescence sensing.

A new fluorescent amino acid, L-2-acridonylalanine, was incorporated into proteins at specific positions using 4-base codon/anticodon strategy. The efficiency of the incorporation was high enough to obtain enough quantities of the mutants. The acridonyl group was highly fluorescent when it was excited at the wavelengths of blue-lasers and was highly photodurable compared with conventional fluorophores often used for biological analyses. The fluorescence intensity was sensitive to small changes in the polarity of the environment. When the nonnatural amino acid was incorporated into specific positions of streptavidin, the mutant protein worked as a fluorescent sensor to biotin. Similarly, when the amino acid was incorporated into camel single-chain antibody, the mutant protein sensitively responded to the antigen molecule. The high incorporation efficiency, the high photodurability, the excitability with blue-lasers, and high sensitivity to the environment make the acridonylalanine as the promising fluorescent amino acid for sensing small molecules when incorporated into specific positions of various antibodies, receptors, and enzymes.