Longitudinal profiling of oligomeric Aβ in human nasal discharge reflecting cognitive decline in probable Alzheimer's disease

Abstract

Despite clinical evidence indicating a close relationship between olfactory dysfunction and Alzheimer's disease (AD), further investigations are warranted to determine the diagnostic potential of nasal surrogate biomarkers for AD. In this study, we first identified soluble amyloid-β (Aβ), the key biomarker of AD, in patient nasal discharge using proteomic analysis. Then, we profiled the significant differences in Aβ oligomers level between patient groups with mild or moderate cognitive decline (n = 39) and an age-matched normal control group (n = 21) by immunoblot analysis and comparing the levels of Aβ by a self-standard method with interdigitated microelectrode sensor systems. All subjects received the Mini-Mental State Examination (MMSE), Clinical Dementia Rating (CDR), and the Global Deterioration Scale (GDS) for grouping. We observed higher levels of Aβ oligomers in probable AD subjects with lower MMSE, higher CDR, and higher GDS compared to the normal control group. Moreover, mild and moderate subject groups could be distinguished based on the increased composition of two oligomers, 12-mer Aβ*56 and 15-mer AβO, respectively. The longitudinal cohort study confirmed that the cognitive decline of mild AD patients with high nasal discharge Aβ*56 levels advanced to the moderate stage within three years. Our clinical evidence strongly supports the view that the presence of oligomeric Aβ proteins in nasal discharge is a potential surrogate biomarker of AD and an indicator of cognitive decline progression.

Figure 1

Soluble Aβ oligomers are detected in the nasal discharges from probable AD group (pAD). Immunoblotting verification; soluble Aβ oligomers were detected in the nasal discharges of pAD group. (A) Identification of Aβ oligomer, assessed by western blot (WB; A11) with or without immunoprecipitation (IP: 6E10) using samples (1; pAD1 and 2; pAD2) from pAD group. (B) Representative data for A11-immunoreaactive (left) and D54D2-immunoreaactive (right) soluble Aβ oligomers are detected in nasal discharges of pAD group (4; pAD1, pAD2, pAD3 and pAD4) and normal group (4; normal 1, normal 2, normal 3 and normal 4). (C) The total levels of soluble Aβ species in nasal discharges were measured between the normal and pAD groups using CLASS method (self-standard ratio (a.u.)). (D) Quantification of soluble Aβ*56 protein. Expression levels of proteins were quantified using stereological analysis (ImageJ program). (E) Quantification of soluble AβO protein. Expression levels of proteins were quantified using stereological analysis (ImageJ program). Data are represented as means ± SEMs from three independent experiments. Value of samples were identified as outliers through Grubbs’ test, also called the ESD method. For statistical analysis, paired t-test was performed. Statistical significances are denoted (***P < 0.001).

Figure 2

The specific composition of soluble Aβ in nasal discharges from different stages of probable AD group (pAD). (A) The total levels of soluble Aβ species using CLASS method (self-standard ratio (a.u.)) in nasal discharges were measured in (Normal (22), mild stage (25) and moderate stage (13) of pAD groups. (B) Quantification of soluble Aβ*56 + AβO protein levels. Expression levels of proteins were quantified using stereological analysis (ImageJ program) in Normal (18), mild stage (22) and moderate stage (17) of pAD groups. (C) Quantification of soluble Aβ*56 protein. Expression levels of proteins were quantified using stereological analysis (ImageJ program) in Normal (18), mild stage (22) and moderate stage (17) of pAD groups. (D) Quantification of soluble AβO protein. Expression levels of proteins were quantified using stereological analysis (ImageJ program) in Normal (12), mild stage (9) and moderate stage (10) of pAD groups. Data are represented as means ± SEMs from three independent experiments. Value of samples were identified as outliers through Grubbs’ test, also called the ESD method. For the statistical analysis, one-way ANOVA was performed, followed by Dunnett’s post hoc test. Statistical significance is denoted (ns > 0.05, ***P < 0.001). In addition, paired t-test was performed. Statistical significances are denoted (ns > 0.05, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001).

Figure 3

The association between soluble Aβ oligomer levels and cognitive performance over time (for 3 years). (A) MMSE change pattern over 3 years (baseline is 1st year). (B) GDS change pattern over 3 years (baseline is 1st year). Marginally significance on interaction by two-way RMANOVA (P = 0.050). High group show higher change of GDS compare to Low group on 3rd year. Statistical significance is denoted (ns > 0.05 and *P < 0.05) followed by Bonferroni post hoc test. (C–D) Correlation analysis between soluble Aβ oligomer levels and slope of cognitive performance. Increased levels of soluble Aβ oligomers in nasal discharge are associated with declining cognitive status over time. This effect is constant over time; levels of soluble Aβ oligomers in nasal discharge are significantly associated with declining cognitive status after 3 years.

Figure 4

Schematic diagram which laid advantage out of AD diagnosis using nasal discharge.