Alterations in retinal tau phosphorylation in Alzheimer's disease patients identified by mass spectrometry

Background: Most neurodegenerative diseases, including Alzheimer's disease (AD) and multiple sclerosis (MS), are associated with abnormal post-translational phosphorylation of tau (p-tau) in the brain. Studies using immunostaining techniques have revealed an accumulation of p-tau also in the AD retina and suggested this p-tau accumulation may reflect tau pathology in the brain. However, immunostainings are dependent on antibody specificity and tissue processing. Hence, further validation using additional methods is needed to identify the p-tau species in the retina and verify their relationship to AD pathology. Methods: We used mass spectrometry to measure p-tau peptides in retinal and hippocampal samples from non demented controls (NC, n=8), AD ( n=12), and MS (n=4). Peptides were first extracted with Lysis buffer to capture extracellular components and cytoplasm, and then with Ripa buffer to isolate nuclear and organelle proteins. We then analysed the differences in p-tau levels between diagnoses and explored how retinal p-tau variants correlate with hippocampal p-tau and neuropathological changes. Results: The mass spectrometry analysis of the retina revealed peaks corresponding to tau peptides phosphorylated at T181, S199/S202, T231, S396 + T403/S404, and T403/S404. These p-tau peptides were also detected in the hippocampal samples, along with additional p-tau peptides such as T217 and S262. Total tau phosphorylation and phosphorylation at S199/S202 and T231 were significantly higher in the retina of AD cases compared to NC. These two peptides, along with peptides phosphorylated at S396+T403/S404 and T403/S404, were also higher in cases with high amyloid-beta (Aβ) Braak stages compared to those with low Aβ Braak stages. Further analysis showed that higher Aβ Braak stages were associated with higher mass spectrometry peak intensities of peptides phosphorylated at S199/S202 and S396+T403/S404. Additionally, retinal p-tau peptides at S396+T403/S404 and T403/S404 2 correlated with neurofibrillary tangle (NFT) Braak stages, and p-tau peptides S396+T403/S404 in the retina were linked to corresponding phosphorylaion in the CA1 region. Conclusions: These findings underscore the connection between retinal and brain tau pathology and highlight the potential of retinal tau as a biomarker for AD diagnosis and monitoring while also deepening our understanding of tauopathies in both the retina and brain.