Quantitative assessment of Tau proteoform changes in iPSC-derived neurons during differentiation

Alzheimer's disease (AD) and related neurodegenerative diseases, such as primary Tauopathies are a significant burden in healthcare due to growing prevalence. One central element in these diseases, particularly in tauopathies, is the microtubule-associated protein, Tau. There are six major isoforms generated by alternative mRNA splicing of exons 2 and 3 (either 0N, 1N, or 2N isoforms) and exon 10 (3R or 4R isoforms) resulting in the mature human brain. Specific post-translational modifications of tau have been associated with Alzheimer"s disease and have been suggested as clinical biomarkers and/or therapeutic targets. However, it remains unclear which tau proteoform(s) contribute(s) most to neural dysfunction and neurodegeneration, and how. Current methods do not adequately address the molecular diversity of Tau proteoforms in neuronal tissue. We have utilized a single-molecule detection technology, which allows for highly sensitive and precise identification of Tau proteoforms and their ratios in iPSC-derived neurons.

Here we apply this single-molecule approach with single-molecule sensitivity and a wide dynamic range to the discovery of Tau proteoforms from iPSC- derived neurons. We utilize the single-molecule detection capability to detect and assess the specific proteoforms and their relative ratios present in the tissue.

We demonstrate the detection of the proteoforms using a multi-cycle method adopting commercially available protein-isoform and PTM-specific antibodies and validate that the single-molecule platform exhibits a linear analytic response. First, to determine single-molecule binding, single protein nanoparticle-conjugates are deposited on the flow cell. In a second step, an antibody specific for a Tau epitope of interest is added to analyze all single proteins on the flow cell massively in parallel. In the third step, bound antibody is imaged by fluorescence, localizing each antibody binding event to an individual protein at a specific location. The cycle of binding, imaging, and removal is repeated multiple times to probe for Tau epitopes of interest.

Applying a panel of 8 epitope probes (256 potential proteoforms) covering several Tau isoforms and phosphoproteoforms we quantify the relative amounts of Tau proteoform in differentiated iPSC cells confirming the relative amounts of Tau isoforms indicated by Western blot. In addition, the single molecule nature of the platform also allows us to determine relative quantities of phosphorylated proteoforms present in those cells. These results clearly demonstrate the unique capability of the platform to quantify biologically relevant Tau proteoforms accelerating our understanding of the role of Tau proteoforms in Tau aggregation, a hallmark of Alzheimer's disease.