Development and validation of salivary tau as a biomarker for early Alzheimer’s Disease
Lady Davis Institute for Medical Research
Recent evidence from biomarkers (brain imaging and spinal fluid tests) indicate that clinical diagnosis of dementia may be occurring as much as 15 years after biomarkers show that Alzheimer Disease (AD) is present. A painless, inexpensive, non-invasive, and reliable biomarker of AD would be a significant contribution to clinical medicine. We have preliminary evidence that increased levels of tau protein in saliva might be just such a biomarker. Tau protein is present in brain neuron cells and becomes abnormally phosphorylated in AD early on. In this project we will attempt to perfect the lab assay for abnormal tau in the saliva. We will determine whether it is reliable, consistent, affected by age, gender, or freezing of samples. We will then assess saliva tau levels in older individuals who have AD, or who have very early forms of memory loss (ie., Mild Cognitive Impairment) which often lead to AD, compared with normal subjects or those with other brain diseases such as stroke. We will test the hypothesis that saliva tau becomes abnormal very early on in the disease process and can therefore be a useful biomarker. To further assess the validity of salivary tau, we will compare it to spinal fluid levels of tau in individuals already undergoing lumbar puncture for spinal fluid, and we will compare levels to memory tests and brain imaging tests. Finally, we will see if salivary tau becomes abnormal in frontotemporal dementia, a rarer brain disease often showing tau abnormalities in the brain.
Relevance to the acceleration of therapeutics for neurodegenerative diseases of aging
Early diagnosis of Alzheimer Disease (AD) may be a crucial element in developing new treatments to slow or prevent the disease. At present, early diagnosis through biomarkers depends on expensive and unavailable amyloid PET scans of the brain, or invasive lumbar puncture (spinal tap) carried out by specialists. If the saliva test is successful, we will have developed an ideal biomarker test which could make early diagnosis far more readily available. This would help select individuals for clinical trials of new medications, and potentially select individuals to receive future early therapy.
This is a very practical project. Our goal is to develop a saliva test for abnormal tau protein that will be useful in early diagnosis of Alzheimer Disease. We will establish its sensitivity, its specificity, its validity, and whether it can be used in other forms of dementia (such as frontotemporal dementia) as well. If successful, we will have an important and immediate impact on the whole field of dementia diagnosis, and make accurate diagnosis earlier and more accurate.
Alzheimer Disease (AD) is characterized by deposition of abnormal proteins in the brain (amyloid and tau proteins). Total tau (t-tau) and phosphorylated tau (p-tau) are abnormally elevated in the brain and cerebrospinal fluid (CSF) of individuals with Alzheimer Disease (AD). There has been recent exciting progress in using the changes in such proteins – measured either in the cerebrospinal fluid accessed by lumbar puncture (spinal tap), or measured in brain imaging with PET (positron emission tomography) – as a means of early diagnosis of AD. High cost, invasiveness, and lack of access to the machines limits the use of these diagnostic modes. Tau is also present in salivary gland tissue. We have had pilot results showing that tau protein may be elevated in the saliva of individuals with AD. Thus, salivary tau might emerge as a simple and inexpensive means of diagnosing AD early on. We therefore undertook a more detailed evaluation of salivary tau in a large set of individuals with various forms of brain disease.
First the technique of measuring tau protein in saliva was perfected. We then collected saliva and measured tau levels using western blot analysis, and we quantified the phosphor-tau to total tau ratio (p-tau/t-tau ratio) at different phosphorylation sites.
Results: We found that for one phosphorylation site, S396, p-tau/t-tau was significantly elevated in AD compared to normal elderly control subjects. There was no difference in normal elderly control compared to young normal subjects, nor compared to subjects with other neurological diseases. The elevation in saliva, however, did not correlate with CSF tau, nor did it correlate with brain measures – hippocampal volume, , and other neuropsychology tests. There was also a significant increase in p-tau/t-tau level in FTD subjects at S396 and S404.
Conclusion: We demonstrated significant elevation of p-tau/t-tau for one phosphorylation site, S396 in AD subjects compared to controls. Thus, the major hypothesis arising from our initial pilot work was confirmed. This strongly suggests that AD (Alzheimer Disease) is not simply a disease of brain, but a disease expressed in the rest of the body. There may be other ways to diagnose the disease using similar or other “peripheral” markers, and perhaps there are treatments that can be effective in targeting changes in the periphery. This is an unexplored area of dementia research, and our results reinforce the possibility that we should be looking more closely at AD outside of the brain.
Unfortunately, unless a biomarker clearly separates all AD from all normal subjects, it has little utility as a simply diagnostic test. In the case of salivary tau, seventy-five percent of AD subjects were above a p-tau/t-tau ratio level of 0.96, whereas this was approximately the median for NEC subjects (half scored above that level and half were below). A portion (approximately 1/3) of AD subjects did not have elevated p-tau/t-tau in their saliva. This means that there was a lot of overlap between results in AD and normal controls. In fact, at all the sites and in all diagnostics groups we considered, there was large variation in p-tau/t-tau levels, and considerable overlap with normal, which limits the utility of the test as a diagnostic biomarker. Several explanations for this variability are worth mentioning. One reason is that a small proportion of AD subjects may have variation in tau pathology itself. Another explanation may be that even for those AD subjects (the large majority) who showed elevated tau levels in the brain, a proportion of these individuals failed to express the tau peripherally in salivary gland tissue. This biological variability remains to be fully explored.