Salivary Proteomics Identifies Transthyretin as a Biomarker of Early Dementia Conversion

Abstract

Background: Alzheimer's disease (AD) remains to date an incurable disease with a long asymptomatic phase. Early diagnosis in peripheral biofluids has emerged as key for identifying subjects at risk and developing therapeutics and preventative approaches.

Objective: We apply proteomics discovery to identify salivary diagnostic biomarkers for AD, which are suitable for self-sampling and longitudinal biomonitoring during aging.

Methods: 57 participants were recruited for the study and were categorized into Cognitively normal (CNh) (n = 19), mild cognitive impaired (MCI) (n = 21), and Alzheimer's disease (AD) (n = 17). On a subset of subjects, 3 CNh and 3 mild AD, shot-gun filter aided sample preparation (FASP) proteomics and liquid chromatography mass spectroscopy (LC-MS/MS) was employed in saliva and cerebrospinal fluid (CSF) to identify neural-derived proteins. The protein level of salivary Transthyretin (TTR) was validated using western blot analysis across groups.

Results: We found that 19.8% of the proteins in saliva are shared with CSF. When we compared the saliva and CSF proteome, 24 hits were decreased with only one protein expressed more. Among the differentially expressed proteins, TTR with reported function in amyloid misfolding, shows a significant drop in AD samples, confirmed by western blot showing a 0.5-fold reduction in MCI and AD compared to CNh.

Conclusion: A reduction in salivary TTR appears with the onset of cognitive symptoms. More in general, the proteomic profiling of saliva shows a plethora of biomarkers worth pursuing as non-invasive hallmarks of dementia in the preclinical stage.

Keywords: Alzheimer’s disease; LC-MS/MS; amyloid-β; cerebrospinal fluid; saliva; tau; transthyretin.

Fig. 1

Shotgun proteomics to compare saliva and CSF and correlation analysis of differentially expressed genes in saliva. A) GO Biological pathways for salivary proteins. B) Number of shared proteins between saliva and CSF. C) Number of shared hits between common proteins and AD related proteins. D) STRING map shows the functional association based on the string database indicating the interactome of commonly shared proteins in saliva and CSF with AD. E) Volcano plots showing differential expression in saliva and F) CSF between AD and CN. G) Abundance rank dot plots for saliva and H) CSF shows the range of expression levels for the differentially expressed proteins. I) STRING map shows the interaction of differentially expressed proteins in saliva and J) in CSF. In the STRING map, the pink line represents the known interaction that is experimentally determined, blue line represents the known interaction from curated databases, green line represents predicted interaction due to gene neighborhood, red line represents prediction due to gene fusions and dark blue line represents prediction due to gene co-occurrence.

Fig. 2

Abundance of TTR and S100A8 among the stages of AD. A) Representative images from western blot procedure to capture TTR and Tau bands, 17 kDa and 79 kDa respectively. No. of samples analyzed: CNh = 19, MCI = 21, AD = 17. Box plots with jitter across CNh, MCI, and AD groups show B) Tau expression normalized to loading control, Revert, CNh versus MCI t₃₉ = 0.291, p = 0.437; CNh versus AD t₃₅ = 0.278 p = 0.906; MCI versus AD t₃₇ = 0.185 p = 0.241, C) normalized TTR, CNh versus MCI t₃₉ = 0.007 p = 0.437; CNh versus AD t₃₅ = 0.018 p = 0.019; MCI versus AD t₃₇ = 0.845 p = 0.241. The whiskers on the boxplot represents the range of population and the horizontal line the median, ^*p < 0.05 and ^**p < 0.01.