Dynamic conformational ensembles of soluble Tau encode neuronal toxicity prior to aggregation

Tau pathology is a hallmark of Alzheimer's disease (AD) and related dementias (ADRDs). Although Tau is often described as intrinsically disordered, it is a dynamic protein with distinct but poorly defined conformations. Here we conduct a systematic time-resolved structure-function analysis of normal and pathologic Tau, including hyperphosphorylated, mutant Tau, and posttranslational-modification-mimetic Tau. To characterize dynamic conformational changes of Tau, we combined state-of-the-art hydrogen deuterium exchange mass spectrometry with structured illumination microscopy, demonstrating a novel Tau-MT binding mode: "dynamic oscillation". To correlate Tau structure with neuronal function, we evaluated axonal transport as a sensitive readout of neuronal health. Many toxic Tau forms share a common signature of increased exposure of the N-terminal phosphate activating domain (PAD) <i>in vitro</i> and <i>in vivo</i> . Aberrant exposure of PAD correlates with Tau pathology and axonal transport defects. Tau phosphorylation at S262 alone is sufficient to alter Tau-microtubule interactions beyond R1-R4 motifs, globally changing Tau conformation, disrupting "dynamic oscillation" on MTs, and inhibiting axonal transport. Frontotemporal dementia-associated P301L-Tau remains associated with microtubules but also inhibits axonal transport. Our results reveal a well-defined conformation of soluble WT Tau in neurons and its highly dynamic interaction with microtubules, altered by AD/ADRD-Tau forms. Our multidisciplinary approach comprising biochemical manipulations, innovative MS tools, advanced microscopy, cellular assays, and mouse and human data pair Tau conformations with distinct neuronal functions and pathologies in health and disease.