Blots were probed with antibodies recognizing phosphorylated/total PERK, phosphorylated/total IRE1, and GAPDH. Geldanamycin treated with (+) or without (?) 800?nM thapsigargin (TG) for 5?h. Blots were probed with antibodies recognizing phosphorylated/total PERK, phosphorylated/total IRE1, and GAPDH. Molecular weight markers (kDa) are shown on the left. b Graphs show the relative amounts of phosphorylated/total PERK, and phosphorylated/total IRE1 after thapsigargin treatment. Data are displayed as percentage change compared to TG-treated CHO cells (100%). Values represent mean??S.E.M., em n /em ?=?4, two-way ANOVA, * em P /em ? ?0.05, ** em P /em ? ?0.01. (PDF 332 kb) 40478_2018_651_MOESM2_ESM.pdf (333K) GUID:?E1D771AD-4945-4414-8CE4-AEA879C604C5 Data Availability StatementAccording to Geldanamycin Wellcome Trusts Policy on data, software and Geldanamycin materials management and sharing, all data supporting this study will be available on request from the corresponding author. Abstract Human tauopathies including Alzheimers disease, progressive supranuclear palsy and related disorders, are characterized by deposition of pathological forms of tau, synaptic dysfunction and neuronal loss. We have previously identified a pathogenic C-terminal tau fragment (Tau35) that is associated with human tauopathy. However, it is not known how tau fragmentation affects critical molecular processes in cells and contributes to impaired physiological function. Chinese hamster ovary (CHO) cells and new CHO cell lines stably expressing Tau35 or full-length human tau were used to compare the effects of disease-associated tau cleavage on tau function and signaling pathways. Western blots, microtubule-binding assays and immunofluorescence labeling were used to examine the effects of Tau35 on tau function and on signaling pathways in CHO cells. We show that Tau35 undergoes aberrant phosphorylation when expressed in cells. Although Tau35 contain the entire microtubule-binding region, the lack of the amino terminal half of tau results in a marked reduction in microtubule binding and defective microtubule organization in cells. Notably, Tau35 attenuates insulin-mediated activation of Akt and a selective inhibitory phosphorylation of glycogen synthase kinase-3. Moreover, Tau35 activates ribosomal protein S6 kinase beta-1 signaling and the unfolded protein response, leading to insulin resistance in cells. Tau35 has deleterious effects on signaling pathways that mediate pathological changes and insulin resistance, suggesting a mechanism through which N-terminal cleavage of tau leads to the development and progression of tau pathology in human tauopathy. Our findings highlight the importance of the N-terminal region of tau for its normal physiological function. Furthermore, we show that pathogenic tau cleavage induces tau phosphorylation, resulting in impaired microtubule binding, disruption of insulin signaling and activation of the unfolded protein response. Since insulin resistance is a feature of several tauopathies, this work suggests new potential targets for therapeutic intervention. Electronic supplementary material The online version of this article (10.1186/s40478-018-0651-9) contains supplementary material, which is available to authorized users. strong class=”kwd-title” Keywords: Tau, Tauopathy, Microtubule binding, Akt, Glycogen synthase kinase-3, Insulin, Unfolded protein response Background Tauopathies are a heterogeneous group comprising dementias and movement disorders, neuropathologically characterized by prominent intracellular accumulations of neurofibrillary tangles formed of tau in neurons and glia. Accumulating evidence suggests that the conversion of physiological tau to pathological tau plays a central role in the development of tauopathy. In particular, abnormal phosphorylation and fragmentation of tau have been proposed as important post-translational modifications that lead to pathogenic forms of tau . In addition, a range Geldanamycin of inter-related cellular processes, including microtubule disorganization [18, 55, 56], activation of the unfolded protein response (UPR) [22, 34, 52], Geldanamycin activation of the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) [9, 49], and deficiencies in insulin signaling [41, 45], also promote cell dysfunction in tau-mediated neurodegeneration. However, the cellular events linking pathological changes in tau to cell dysfunction and the pathogenesis of tauopathies are largely unknown. We previously described a 35?kDa C-terminal tau fragment (Tau35), lacking the N-terminus of tau but containing all four Rabbit Polyclonal to ELAC2 microtubule-binding repeats (4R), that is present in 4R tauopathies . When expressed in transgenic mice, Tau35 induces several key features of tauopathy, including accumulation of abnormally phosphorylated tau, dysregulation of glycogen synthase kinase-3 (GSK3) activity, progressive cognitive and motor deficits, and loss of synaptic proteins . Here we have used a cell model to investigate the molecular mechanisms that are affected by Tau35 expression. Our.