New Tools to Image Tau Protein Form and Function

Project Summary The process by which tau moves from its soluble form to neurofibrillary tangles has been studied for many years, but only recently has the importance of an intermediate liquid-liquid phase separation step been shown. Indeed, the assortment of tau into these biomolecular condensates is increasingly seen as a key feature underlying both normal cell function and disease. These liquid-liquid phase separations often proceed liquid-to-solid phase transitions that in turn lead to the protein aggregates underlying tau protein misfolding diseases including Alzheimer’s disease. While the community can observe neurofibrillary tangles using thioflavin T and early-stage biomolecular condensates with FRET, there is no single imaging approach that can map and measure tau in its all three forms in living cells: soluble, liquid-liquid phase separation, and liquid-solid phase separation forms. This work will solve this major limitation with a peptide- based probe specific to tau and aggregation-induced emission (AIE). In AIE, a molecule analogous to a fluorophore produces light proportional to the degree of molecular sequestration and restricted movement. Signal is “off” when the molecule is “floppy” and “on” when locked in place. The use of AIE is significant, innovative, and critical here because the AIE signal will directly measure tau as it becomes compressed into increasingly confined environments (monomers to liquid condensates to fibrillary aggregates). Thus, our testable hypothesis is that fluorescence will increase with the type of condensate: soluble<liquid- liquid<liquid-solid. Aim 1 of the work will build and characterize the probe. We will test the probe with different concentrations of recombinant tau including different tau isoforms, different degrees of tau phosphorylation, and tau from different species. Aim 2 will use the probe in cultured cells (Aim 2A) and histology sections of rodent brains (Aim 2B). We will transfect cells to overexpress tau, induce tau biomolecular condensate and fibril formation, and then map and measure the location of tau forms in these cells using confocal microscopy. The PS19 animal model is available “off the shelf” and will used with histology to understand the distribution of tau BMCs in different brain regions; we will also stain the nuclear pore complex to understand the interactions of these different tau condensates with the nucleus. The significance of the work is apparent by the grave impact Alzheimer’s disease has on society. Innovation is underscored by the ability to image tau across multiple domains of condensation, the ability to map tau condensate interactions with other organelles, and the use of AIE to monitor tau molecular movement. Finally, the probe uses a peptide with known specificity to tau in contract to gold standard probes like thioflavin T. This work is feasible because of the PI’s expertise in imaging agents, peptide probes, and cell and animal models; the Co-I’s expertise in tau pathologies; and our peer-reviewed preliminary data.