<p>Tauopathies are neurodegenerative diseases defined by the accumulation of pathological tau protein in neurons and glia. Alzheimer’s disease (AD), the most common tauopathy, is characterized by the presence of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein aggregates in neurons. Emerging evidence suggests that the NFT burden correlates with neuron death and cognitive decline, contributing to disease progression. Besides AD, a similar deposition of tau inclusions is found to be associated with neurodegeneration in the brains of patients with other tauopathies including progressive supranuclear palsy, corticobasal degeneration, and Pick’s disease. These diseases display clinical, biochemical, and neuropathological heterogeneity. Little is known about how tau aggregation can lead to varied phenotypes in tauopathies, and there is no disease-modifying treatment. Thus, it is necessary to understand the role of diverse tau proteoforms in tauopathies for the development of new therapeutics to treat tauopathies, including AD.</p> <p>In the studies summarized in Chapter 2, we investigated how the molecular diversity of tau proteoforms could impact antibody-based assays of a phospho-tau variant serving as an AD biomarker. A tau variant phosphorylated on threonine 181 (pT181-tau) has been widely investigated as a potential AD biomarker in cerebrospinal fluid (CSF) and blood. pT181-tau is present in NFTs of AD brains, and CSF levels of pT181-tau correlate with overall NFT burden. Various immuno-based analytical methods, including Western blotting and ELISA, have been used to quantify pT181-tau in human biofluids. The reliability of these methods depends on the affinity and binding specificity of the antibodies used to measure pT181-tau levels. Although both of these properties could in principle be affected by phosphorylation within or near the antibody’s cognate antigen, such effects have not been extensively studied. Here, we developed a bio-layer interferometry (BLI)-based analytical assay to assess the degree to which the affinity of pT181-tau antibodies is altered by the phosphorylation of serine or threonine residues near the target epitope. Our results revealed that phosphorylation near T181 negatively affected the binding of pT181-tau antibodies to their cognate antigen to varying degrees. In particular, two of three antibodies tested showed a complete loss of affinity for the pT181 target when S184 or S185 was phosphorylated.</p> <p>In the studies outlined in Chapter 3, we examined the relative abilities of different tau proteoforms to induce seeded tau aggregation and to themselves undergo seeded aggregation in cultured cells. Accumulating evidence suggests that tau aggregates, including NFTs, spread in a stereotypical pattern across neuroanatomically connected brain regions. This spreading phenomenon is thought to occur via a prion-like mechanism involving the release of tau aggregates from a diseased neuron into the extracellular space, aggregate uptake by neighboring healthy neurons, and the formation of new aggregates in the cytosol of the recipient cells via a seeding process. Although research over the past decade has revealed key molecular events involved in the cell-to-cell transmission of tau aggregates, the impact of the protein’s domain structure and phosphorylation profile on the efficiency of prion-like propagation remains poorly defined. Here, we compared three tau variants – K18, 0N4R, and 2N4R – in terms of their aggregation and seeding efficiencies in recombinant protein solutions and in cell culture models. Our results revealed that K18 had the highest fibrillization rate and yield among the three tau variants. Recombinant preformed fibrils (PFFs) derived from all three variants had similar seeding efficiencies. Additionally, we investigated the relationship between tau phosphorylation and aggregation. We found that hyperphosphorylated tau did not undergo self-assembly in the absence of heparin, whereas it formed fibrils at low yield in the presence of the cofactor. Moreover, hyperphosphorylated tau PFFs produced under these conditions induced seeded tau aggregation in cell culture.</p> <p>Taken together, these results point to critical roles of tau proteoforms as both AD biomarkers and drivers of disease progression. Our results indicate that the presence of different combinations of phosphorylated residues near a target phospho-tau antigen can affect the accuracy of antibody-based biomarker assays. In addition, the domain structure and phosphorylation profiles of tau proteoforms associated with AD and other tauopathies likely have a profound influence on the evolution of tau pathology in these disorders. Our findings highlight the importance of accounting for the molecular diversity of tau proteoforms in tauopathies and provide valuable insights into molecular determinants influencing tau aggregation and propagation in the brains of patients.</p>