Progressive accumulation and misfolding of tau are critical drivers in the pathogenesis of Alzheimer’s disease (AD), frontotemporal dementia (FTD) and other related diseases. Increasing evidence from tauopathy studies suggests that dysfunction in the autophagy-lysosomal pathway (ALP) plays a major role in these diseases. Under normal conditions, the ALP is responsible for maintaining proteostasis by degrading and recycling damaged organelles and misfolded proteins. A full understanding of ALP dysregulation in tauopathies is increasingly within reach, but first requires new tools that can monitor temporal changes in the pathway in human neurons. The currently available cellular biosensors that are commonly used to track ALP dysfunction only offer static snapshots of individual components of the ALP, (e.g., autophagosome number, lysosomal acidification) limiting their capacity to elucidate how tau perturbs the complex and delicately balanced dynamics of this system. To remedy this, we have developed two complementary fluorescence-based biosensors that report on real- time nuclear translocation and dimerization of Transcription Factor EB (TFEB), the master regulator of autophagy and lysosomal biogenesis. The first biosensor monitors subcellular TFEB localization using high-content imaging; the second biosensor uses high-resolution time-resolved FRET measurements that sensitively report on TFEB dimerization. Using these biosensors, which have not previously been reported in the tau field, we have already shown that: 1) tau induces pathological translocation of TFEB into the nucleus; 2) mutant tau alters nuclear import kinetics of TFEB; and 3) TFEB activation involves its homo-dimerization in the nucleus. To prove the feasibility of these tools, we did the initial optimizations in highly scalable model cell systems (N2a and HEK293 cell lines), but we recognize that those are not sufficient models for tauopathy or for future applications of the technology. Thus, this proposal takes the critical next steps in the engineering of these TFEB biosensors by translating them into human iPSC (hiPSC) glutamatergic neurons from healthy and AD/FTD patient lines. We will exploit our patented rapid differentiation protocol to generate hiPSC glutamatergic neurons expressing our TFEB biosensors. We will then establish the baseline for native TFEB dynamics under non-stressed conditions. Having done so, we will investigate perturbations induced by known small molecule effectors of tau and ALP (including torin1 and forskolin). Lastly, we will study a limited set tau of mutations using AD/FTD patient derived hiPSC lines (Tau Consortium) to classify which most strongly compromise TFEB function in neurons. Establishing this new technology is significant because it will: 1) enable future studies elucidating biological connections between ALP dysfunction and tauopathy; and 2) will provide a platform for the first high-throughput screening campaigns that target tau-induced de...