Lipka, J. | bioRxiv Science

bioRxiv2026

A scalable human neuron model of Alzheimer's disease relevant tauopathy reveals mechanisms linking Tau fibrillization to synaptic dysfunction

Tauopathies, including Alzheimer's disease, are driven by pathological aggregation of hyperphosphorylated Tau, which disrupts synaptic integrity, impairs neuronal communication, and contributes to cognitive decline. To dissect tauopathy pathogenesis and enable therapeutic discovery, reliable and scalable human iPSC-neuron models are essential. Here, we developed two complementary iPSC-derived neuron models: an endogenous Tau seeding model, in which neurons are challenged with pre-formed Tau fragments that form paired helical filament (PHF)-consistent structures, and a Tau-0N3R overexpression seeding model to accelerate pathology. Both models recapitulate hallmark features of tauopathy, including the progressive formation of intracellular, hyperphosphorylated, sarkosyl-insoluble, and conformationally altered Tau aggregates (AT8, MC1 positive), along with synaptic and neuronal dysfunction. Cryogenic electron tomography (cryo-ET) further revealed the morphology of Tau fibrils within cells, as well as the ultrastructure of Tau fibrils trapping synaptic vesicles in situ. Using this platform, we performed integrated phosphoproteomics, high-content screening, and functional validation to identify key pathways driving Tau aggregation. MARK2-mediated phosphorylation within Tau's microtubule-binding domain emerged as an early trigger of aggregation, confirmed by site-specific mutagenesis. In parallel, small molecules targeting the PI3K/mTOR/GSK3 pathway reduced aggregation and restored synaptic function, with GSK3 inhibition lowering phosphorylation at critical aggregation-driving sites on Tau. Together, these findings establish a physiologically relevant, scalable platform for therapeutic screening that connects Tau seed uptake, site-specific phosphorylation, fibril formation, and synaptic disruption, ultimately identifying mechanistically separable intervention points along the aggregation cascade.

cell biology↗

bioRxiv2025

Development of a method for large-scale single-molecule analysis of tau proteoforms

Proteins exist as diverse proteoforms resulting from a combination of genetic variation, alternative splicing, and post-translational modifications. Current methods struggle to capture this complexity at the single-molecule level. Here we introduce Iterative Mapping of Proteoforms (IMaP), a method that enables the massively-parallel interrogation of millions to billions of single-protein molecules through iterative probing with fluorescently labeled antibodies. Using 12 site-specific antibodies, the method is capable of measuring 212 (4,096) potential proteoform groups. We used IMaP to measure proteoform group profiles of the tau protein, a key player in neurodegenerative diseases, using two pan anti-tau antibodies (Tau-13, Tau-216), three isoform-specific antibodies (Anti-0N, Anti-2N, Anti-4R), and seven phosphosite-specific antibodies (Anti-pT181, Anti-pS202+pT205, Anti-pT205, Anti-pS214, Anti-pT217, Anti-pT231, and Anti-pS396). The method demonstrates high sensitivity (detecting proteoforms at 0.1% abundance), high reproducibility (median CV <5.5%), and broad dynamic range (>3 orders of magnitude), outperforming conventional techniques in resolving closely related proteoform groups. We demonstrated that the method can be used on relevant biological samples by examining various neuronal models (iNeuron cells, organoids, MiBrains, and mouse brains) and human samples. This examination revealed 130 distinct tau proteoform groups with as many as six phosphorylation events. The non-random distribution of these phosphorylation events suggests ordered and site-specific modification processes rather than random, stochastic accumulation. Certain combinations of phosphorylation events were more abundant than others; for example, pT217 preferentially co-occurred with pT181. In validating the applicability of the assay to human disease samples, we noted a specific pattern of multiple phosphorylation events in an advanced Alzheimers disease patient that suggests a sequential pathway of pathological tau modification. Iterative Mapping of Proteoforms provides insights into proteoform complexity at the single-molecule level, with significant implications for understanding protein regulation in neurodegenerative diseases and beyond.

biochemistry↗