By IF we detected an overall increase in tau

By IF we detected an overall increase in tau in A152T neurons, with particular accumulation of P-tau in the cell body, consistent with somatodendritic redistribution of tau, as described for tauopathies and Alzheimer’s disease (de Calignon et al., 2012; Fong et al., 2013; Li et al., 2011; Sydow et al., 2016). We did not detect A152T-specific changes in cell morphology, as reported in another A152T model (Fong et al., 2013), which may be due to differences in methodologies and line-specific variability. Nonetheless, the overall conclusion of tau accumulation, somatodendritic redistribution, and tau-mediated neuronal toxicity in A152T cells was a common observation, unmasked either by additional stress (Figure 7) or by genetic manipulation (A152T homozygous line in Fong et al., 2013). Taken together, these two studies are complementary by demonstrating that even with technical variability, key aspects in pre-clinical disease events were corroborated. We also reveal here, for an FTD A152T proband-derived neuronal culture, a decrease in tau solubility, again most accentuated for P-tau. Although we did not detect tau puncta-like nobiletin per se, Triton X-insoluble tau and the HMW species detected are consistent with oligomeric species of reduced solubility. Based on our findings we propose a model in which accumulation of P-tau and oligomeric tau of reduced solubility are early disease-causal phenotypes, consistent with other A152T animal models demonstrating tau-mediated toxicity independent of aggregation (Maeda et al., 2016; Pir et al., 2016). To explore the mechanisms of tau-mediated toxicity, we focused on specific proteostasis pathways shown previously to be involved in tauopathy, including the HSP70/HSP90 chaperone machinery (Blair et al., 2013), UPR markers (Stutzbach et al., 2013), mitochondrial function and oxidative stress (Schulz et al., 2012), and autophagy (Wang and Mandelkow, 2012). We report on disruption of the UPS and autophagy pathways, in addition to UPR and oxidative stress induction, in A152T neurons. Disruption of protein clearance is a strong model for tau-mediated toxicity in neurodegeneration, and is consistent with the observed accumulation of total tau protein. Also consistent with our findings, a recent study identified alterations of the endosomal-lysosomal pathway associated with another tau mutation (Wren et al., 2015); yet another study identified LAMP1 and ubiquitin as cerebrospinal fluid markers of neurodegeneration, reinforcing the value of our system in the identification of tauopathy biomarkers (Heywood et al., 2015). In turn, mounting evidence points to UPR activation “in close connection with early stages of tau pathology” (Abisambra et al., 2013; Stoveken, 2013), and to mitochondrial dysfunction and vulnerability to oxidative stress as common aspects in iPSC-derived models of tauopathy (Ehrlich et al., 2015; Iovino et al., 2015). Tau-mediated disruption of proteostasis was further revealed by increased sensitivity of A152T neurons to exogenous “stressors,” unveiling toxicity phenotypes and pathways that may be affected in the early stages of tau pathology, offering a powerful method for phenotypic profiling of patient-derived neurons. Not all stressor compounds tested affected neuronal viability (not shown), revealing pathway-specific vulnerability. These phenotypes were tau dependent as shown by CRISPR/Cas9-mediated downregulation of MAPT expression in A152T neurons, which rescued neuronal stress vulnerability without affecting cell viability or integrity (Figures 7H and S6M). Most importantly, we demonstrate that stress vulnerability can be rescued pharmacologically by pre-treating neurons with the autophagy activator rapamycin, highlighting the value of this model for small-molecule screening. This is the first time, in a human neuronal cell context and with endogenous tau expression, that a compound has been shown to clear tau and consequently rescue toxicity. This result also further corroborates a model of tauopathy associated with disruption or inefficiency of protein clearance.