Identification of human biomarkers in saliva for early detection of Alzheimer's disease and other dementia related illnesses

Abstract

Alzheimer’s disease (AD) is a leading cause of death in Australia. Diagnosis is often too late to halt the widespread and irreversible brain damage caused by this disease. Early detection of AD raises the hope of treating its early pathology and thereby preventing events triggering downstream degeneration. Many studies show that altered levels of circulating proteins signal early AD changes, even before symptoms present. To date, the search for fluid biomarkers has focussed mainly on cerebral spinal fluid (CSF) and blood. Saliva is a relatively new sample type in neurodegenerative studies, and it is not clear yet whether well-studied hallmark biomarkers, such as tau and amyloid beta, reflect the same promising patterns in saliva as has been reported in CSF and blood. Neurofilament light chain (NfL), a recognised protein biomarker of neuronal damage in many diseases including AD and motor neurone disease, has successfully been detected in saliva by a handful of studies. Following a recent report on a newly found NfL fragment in the CSF of AD individuals, a novel assay was developed which could successfully measure this fragment in blood and saliva. In contrast to existing NfL assays, the assay in this thesis may also be the first to target oligomeric NfL. Future work is planned to measure this species in cohorts with AD and other neurodegenerative diseases. Building on promising results from a mass spectrometry study by our group, assays were developed to measure levels of five proteins shown previously to be significantly different in saliva from individuals with AD and mild cognitive impairment (MCI). Assay results from this work were consistent with the earlier study and all five targets had altered abundance in saliva of AD and MCI individuals. Combinations of these proteins had excellent diagnostic accuracy for distinguishing both MCI and AD from age and sex matched controls with areas under curve of 0.97 by receiver operating characteristic analysis. A key finding from this work was that persons with MCI or AD had significantly higher levels of total salivary protein. To explore further, saliva was collected by different methods and total protein measured using two commonly used methods of protein estimation. Despite their differing chemistry, the results showed that salivary protein measurements from both Bicinchoninic acid (BCA) and Bradford assays were tightly correlated. Both methods confirmed the finding of significantly higher protein levels in saliva from individuals with MCI or AD. All cohort saliva was collected with a commercial collection device not used before in AD studies. For the first time, proteins in filtrate from the device along with saliva from other methods of collection were analysed and compared using shotgun mass spectrometry. Over 3,000 proteins were identified in whole unprocessed saliva and the results clearly showed that the protein profile of the device filtrate closely aligned with saliva supernatant after centrifugation. These results will aid researchers decide which saliva collection method best satisfies their requirements as well as assisting in the reconciliation of results between saliva studies and inform design of future saliva studies.