To our knowledge, this is the first study that comprehensively investigated tear levels of amyloid-beta peptides and tau protein in patients with cognitive impairment compared to healthy controls. While AB40, AB42 and total-tau were well detectable, AB38 and p-tau were detectable in only half of the collected tear fluid samples. This result may be explained by the fact that AB38 levels are also (much) lower than AB40 levels in CSF (18) and than AB40 and AB42 levels in plasma (19). Similarly, p-tau levels in CSF are five to ten times lower than total-tau levels (18). Levels of p-tau were undetectable in tear fluid samples from healthy controls. This is an interesting finding since the determination of p-tau in CSF may increase the specificity and sensitivity in the detection of AD as opposed to total-tau (20). However, tests with higher sensitivity for p-tau are needed to confirm this observation in tear fluid. In all cases, highly sensitive assays are essential for tear fluid analysis due to limited sample volume.
The tear fluid level of t-tau in dementia, MCI and SCD patients was significantly higher than in HC participants. However, tear t-tau levels did not differ significantly between the dementia, MCI and SCD groups. One individual with dementia displayed a remarkable high tear t-tau level (4.14 pg/mg), while his CSF t-tau level was also higher than the threshold (350 pg/mL) as biomarker for neurodegeneration (21).
To the best of our knowledge, there have been no studies that used immunoassays of amyloid-beta and tau for tear fluid. Previous studies on tear fluid samples of AD patients (10, 11) used mass spectrometry and were therefore not able to detect amyloid-beta and tau, since mass spectrometry is less sensitive for low abundant proteins than the immunoassays we used in our study. Additionally, detection of amyloid and tau by mass spectrometry usually employs targeted methodologies using antibody immunoaffinity enrichment. Kallo et al. (10) used mass spectrometry to compare ten priorly selected proteins in tear fluid samples from AD patients versus controls and observed differences for lipocalin-1, lactotransferrin, extracellular glycoprotein lacritin, lysozyme-C, prolactin inducible protein and dermcidin. Kenny et al. (11) used mass spectrometry on tear fluid from AD patients versus controls. They identified a panel of 12 proteins that were differentially expressed in tear fluid of AD patients compared to controls. Among these, elongation initiation factor 4E (eIF4E) was only detectable in AD samples (n = 5) and undetectable in samples from controls. EIF4E is involved in the initiation of protein synthesis by recognizing and binding the 7-methylguanosine-containing mRNA cap. The authors mention that they were not able to identify amyloid-beta or tau in their study and claim that this was probably due to their untargeted experimental set-up.
This investigation is also the first to evaluate both tear fluid and CSF. CSF was available from a subset of patients for clinical diagnostic purposes. We observed that AB42 and t-total were negatively correlated in tear fluid in the same way as in CSF. No significant correlations were observed between CSF p-tau and tear p-tau, however it must be noted that p-tau was analyzed in CSF using the p-tau-181 antibody and in tear fluid using the p-tau-231 antibody. Furthermore, p-tau levels in CSF are expressed in pg/mL while tear p-tau levels are expressed in U/mg.
We identified differences in tear volume between participants. Tear volume was lower in SCD, MCI and dementia patients compared to the HC group. This is in good agreement with previously published reports that demonstrate that neurodegenerative disease may be associated with abnormal tear function (22). For example, approximately 20% of patients with Sjögren’s syndrome (an autoimmune disorder involving the exocrine gland) have CNS involvement including dementia (23). Another factor that might explain this difference in tear volume between groups is age. The frequency of dry eye syndrome is known to be gradually increased in middle and advanced ages (24). Nevertheless, since we correct our biomarker concentrations for the tear total protein content (that was comparable across groups), differences in tear volume do not influence the biomarker results.
We observed an average tear total protein content of approximately 3 mg/mL, which is in good agreement with previously published data (25). While Kallo et al. (10) identified a significant increase in the total protein content of tear samples from AD patients versus controls, in our study, and in line with previous reports (11), we did not observe such difference. The presence of ocular comorbidities (such as ocular inflammations or infections) may underline an increased total protein content. Tear protein concentrations (3–7 mg/mL) are generally lower than blood protein concentrations (60–80 mg/mL) but higher than CSF protein concentrations (0.15 to 0.45 mg/mL). While it is common for tear biomarkers to express their concentrations per unit protein (pg/mg protein), this is usually not done for CSF biomarkers. However, CSF protein concentrations vary greatly amongst individuals (26) and can be elevated after for example ischemic stroke (27).
This study is in line with others studies that identified tear biomarkers for different neurological conditions, such as TNF-alpha (28) and oligomeric alpha-synuclein (29) for Parkinson’s disease and alpha-1 anti-chymotrypsin for multiple sclerosis (30). Together, these studies support the hypothesis that tear fluid may predispose to mirror pathophysiological changes in the central nervous system. Furthermore, the fact that amyloid-beta and tau are detectable in tear fluid might reveal new fundamental insights in the disease molecular pathology. Retinal ganglion cells and retinal pigment epithelium have been identified as major sources of amyloid-beta synthesis (31). After secretion into the vitreous humor, amyloid-beta is subsequently transported to the aqueous humor. This follows a similar pattern observed in the central nervous system, where amyloid-beta is primarily synthesized in neurons but accumulate in CSF.
Finally, in this study we investigated tear fluid from patients that visit the memory clinic with the first complaint of cognitive impairment. We believe that this approach is clinically more relevant than comparing severely affected patients to healthy controls. In the end, it are patients with early symptoms that would benefit from future tear-based biomarker tests.