Tau accumulation triggers STAT 1‐dependent memory deficits by suppressing NMDA receptor expression
2019; Springer Nature; Volume: 20; Issue: 6 Linguagem: Inglês
10.15252/embr.201847202
ISSN1469-3178
AutoresXiaoguang Li, Xiao‐Yue Hong, Yali Wang, Shu‐Juan Zhang, Jun‐Fei Zhang, Xia‐Chun Li, Yan‐Chao Liu, Dong‐Shen Sun, Qiong Feng, Jinwang Ye, Yuan Gao, Dan Ke, Qun Wang, Hong‐lian Li, Keqiang Ye, Gong‐Ping Liu, Jian‐Zhi Wang,
Tópico(s)Alzheimer's disease research and treatments
ResumoArticle13 May 2019free access Source DataTransparent process Tau accumulation triggers STAT1-dependent memory deficits by suppressing NMDA receptor expression Xiao-Guang Li Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Xiao-Yue Hong Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Ya-li Wang Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Key Laboratory for the Brain Research of Henan Province, Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang, China Search for more papers by this author Shu-Juan Zhang Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Jun-Fei Zhang Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Xia-Chun Li Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yan-Chao Liu Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Dong-Shen Sun Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Qiong Feng Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Jin-Wang Ye Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yuan Gao Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Dan Ke Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Qun Wang Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Hong-lian Li Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Keqiang Ye orcid.org/0000-0002-7657-8154 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Gong-Ping Liu Corresponding Author [email protected] Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China Search for more papers by this author Jian-Zhi Wang Corresponding Author [email protected] orcid.org/0000-0002-6216-8525 Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China Search for more papers by this author Xiao-Guang Li Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Xiao-Yue Hong Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Ya-li Wang Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Key Laboratory for the Brain Research of Henan Province, Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang, China Search for more papers by this author Shu-Juan Zhang Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Jun-Fei Zhang Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Xia-Chun Li Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yan-Chao Liu Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Dong-Shen Sun Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Qiong Feng Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Jin-Wang Ye Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yuan Gao Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Dan Ke Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Qun Wang Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Hong-lian Li Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Keqiang Ye orcid.org/0000-0002-7657-8154 Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA Search for more papers by this author Gong-Ping Liu Corresponding Author [email protected] Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China Search for more papers by this author Jian-Zhi Wang Corresponding Author [email protected] orcid.org/0000-0002-6216-8525 Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China Search for more papers by this author Author Information Xiao-Guang Li1,2,‡, Xiao-Yue Hong1,‡, Ya-li Wang1,3, Shu-Juan Zhang1, Jun-Fei Zhang1, Xia-Chun Li1, Yan-Chao Liu1, Dong-Shen Sun1, Qiong Feng1, Jin-Wang Ye1, Yuan Gao1, Dan Ke1, Qun Wang1, Hong-lian Li1, Keqiang Ye4, Gong-Ping Liu *,1,5 and Jian-Zhi Wang *,1,5 1Key Laboratory of Ministry of Education of China and Hubei Province for Neurological Disorders, Department of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2Clinic Center of Human Gene Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 3Key Laboratory for the Brain Research of Henan Province, Department of Physiology and Neurobiology, Xinxiang Medical University, Xinxiang, China 4Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA 5Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 027 83692625; E-mail: [email protected] *Corresponding author. Tel: +86 027 83693881; E-mail: [email protected] EMBO Rep (2019)20:e47202https://doi.org/10.15252/embr.201847202 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Intracellular tau accumulation forming neurofibrillary tangles is hallmark pathology of Alzheimer's disease (AD), but how tau accumulation induces synapse impairment is elusive. By overexpressing human full-length wild-type tau (termed hTau) to mimic tau abnormality as seen in the brain of sporadic AD patients, we find that hTau accumulation activates JAK2 to phosphorylate STAT1 (signal transducer and activator of transcription 1) at Tyr701 leading to STAT1 dimerization, nuclear translocation, and its activation. STAT1 activation suppresses expression of N-methyl-D-aspartate receptors (NMDARs) through direct binding to the specific GAS element of GluN1, GluN2A, and GluN2B promoters, while knockdown of STAT1 by AAV-Cre in STAT1flox/flox mice or expressing dominant negative Y701F-STAT1 efficiently rescues hTau-induced suppression of NMDAR expression with amelioration of synaptic functions and memory performance. These findings indicate that hTau accumulation impairs synaptic plasticity through JAK2/STAT1-induced suppression of NMDAR expression, revealing a novel mechanism for hTau-associated synapse and memory deficits. Synopsis Tau accumulation, one hallmark of Alzheimer's disease, induces synaptic impairment by activating JAK2/STAT1 signaling, which transcriptionally suppresses N-methyl-D-aspartate receptors. Downregulation of STAT1 ameliorates synaptic function and memory performance in mice. Accumulation of hTau triggers JAK2-dependent STAT1 dimerization, activation and nuclear translocation. STAT1 activation directly suppresses N-methyl-D-aspartate receptor expression. Downregulation of STAT1 rescues hTau-induced N-methyl-D-aspartate receptor suppression. Introduction Intracellular accumulation of tau forming neurofibrillary tangles is one of the two hallmarks in Alzheimer's disease (AD), the most common neurodegenerative disorder in the elderly 1, 2. Abnormal tau accumulation is positively correlated with neurodegeneration and memory deterioration 3, 4, and the total tau level in cerebrospinal fluids has an inverse correlation with memory score in AD patients 5, 6. The axonal tau pathology in hippocampus is critical for the clinical presentation of dementia and may constitute an anatomical substrate of clinically verifiable memory dysfunctions 3. The human tau transgenic mice recapitulate features of human tauopathies and cognitive deficits 7, 8. Tau is essential for β-amyloid-induced synaptic toxicity 9, while tau knockout attenuates neuronal dysfunction and prevents behavioral deficits in transgenic mice expressing human amyloid precursor protein (APP) without altering high Aβ level in the brain 10, 11. These clinical evidence and laboratory evidence strongly suggest that tau abnormality plays a pivotal role in AD-like synapse and memory impairments. As a cytoskeleton protein, the originally characterized function of tau is to promote microtubule assembly and maintain the stability of microtubules, which is essential for axonal transport 12, 13. Tau hyperphosphorylation dissociates microtubules and thus disrupts axonal transport 14-18. Recent studies suggest that tau phosphorylation is actively involved in regulating cell viability 19-21. Tau proteins are largely located in the neuronal axons in physiological conditions 22; it is also reported that the postsynaptic location of Fyn is tau-dependent, suggesting the dendritic distribution of tau 23. Upon hyperphosphorylation or cleavage, tau accumulated in dendritic spines where it interacts with the postsynaptic proteins and thus induces synaptic dysfunction 24, 25. Intracellular accumulation of tau causes mitochondrial dysfunction and mitophagy deficits by increasing mitochondrial membrane potential 26, 27. Tau accumulation also disrupts intracellular calcium signaling leading to activation of calcineurin and CREB dephosphorylation in primary neuron cultures 4. These hypothesis-driven studies partially disclose the mechanisms underlying the toxic effects of tau. However, the molecular mechanism underlying hTau-induced synapse impairment is not fully understood. In the present study, we employed a large-scale screening approach to explore novel molecular mechanisms underlying tau toxicities. By using whole-genome mRNA chip and the transcription factor activation profiling array, we found that overexpressing hTau upregulated JAK2/STAT1 signaling, and simultaneous downregulating STAT1 by hippocampal infusion of AAV-Cre in STAT1flox/flox mice or by overexpressing dominant negative STAT1 mutant mitigates the hTau-induced synaptic and memory deficits. We also found that STAT1 can directly bind to the specific GAS elements in GluN1, GluN2A, or GluN2B promoter and thus suppress expression of the NMDARs, which reveals a novel mechanism underlying hTau-induced synapse impairment and memory deficit. Results Intracellular hTau accumulation induces activation of STAT1 During our studies on tau, we often observe that overexpressing hTau proteins result in changes in other proteins. We thus speculate that hTau accumulation may influence gene expression. To test this, we first conducted a whole-genome mRNA chip screening in hTau-overexpressed HEK293 cells. Indeed, we detected significant alterations in the level of 520 mRNA molecules (235 increased and 285 decreased) in hTau-expressing cells compared with those expressing the empty vector (Appendix Fig S1), suggesting that intracellular hTau accumulation indeed influences gene transcription. To confirm this point, we measured activity of the transcription factors in nuclear fraction by transcription factor activation profiling array (Appendix Tables S1 and S2), in which the activity of 96 transcription factors was monitored using a collection of biotin-labeled DNA probes based on the consensus sequences of individual transcription factor DNA-binding sites (Signosis). The results showed that the activity of STAT1 and CBF was significantly increased, while the activity of HNF1, HOX4C, PLAG1, SMUC, VDR, SF-1, and PIT1 decreased remarkably in cells overexpressing hTau (Fig 1A and B). In protein level measured by Western blotting, only elevation of STAT1 but not CBF was shown in total extracts and the nuclear fraction (Appendix Fig S2). Figure 1. Overexpression of hTau activates STAT1 with an increased nuclear translocation in vitro A, B. Overexpression of wild-type full-length human tau (hTau, also termed tau441 or tau40 or tau2N4R) induced significant alterations of nine transcription factors screened by using Transcription Factor Activation Profiling Plate Array II, in which 96 transcription factors (Appendix Tables S1 and S2) were monitored. The empty vector was transfected as a control (Ctrl). C–F. Expression of hTau (probed by HT7) increased total and the phosphorylated STAT1 at Tyr701 (pY-STAT1) in whole-cell extracts (C, D) and the nuclear fraction (E, F) measured by Western blotting (n = 4). G. The representative immunofluorescent images and quantitative analysis show significantly increased STAT1 signal in the nuclear fraction of HEK293 cells with overexpression of hTau compared with the empty vector control (eGFP) (n = 5). Scale bar, 10 μm. H. Overexpression of hTau most significantly increased STAT1 monomer and dimer formation in nuclear fraction (Nu) measured by Western blotting. I. Overexpression of hTau increased STAT1 activity in HEK293 cells detected by luciferase assay (n = 4). J. Overexpression of hTau increased STAT1-DNA-binding activity in HEK293 cells measured by electrophoretic mobility shift assay (EMSA). * indicates STAT1/DNA complex. Data information: Data were presented as mean ± SD (Mann–Whitney test). *P < 0.05; **P < 0.01; ***P < 0.001 vs Ctrl. Source data are available online for this figure. Source Data for Figure 1 [embr201847202-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Herein, we focused on STAT1 which has been implicated in cognitive functions 28, 29. We demonstrated that overexpressing hTau remarkably increased the activation-dependent phosphorylation of STAT1 at Tyr701 (pY-STAT1) in both cell lysates (Fig 1C and D) and the nuclear fraction (Fig 1E and F) with an enhanced nuclear translocation (Fig 1G) and dimerization (Fig 1H) of STAT1 measured by Western blotting and immunofluorescence imaging. Activation of STAT1 by overexpressing hTau was also detected by TF luciferase assay (Fig 1I). By EMSA using an oligonucleotide probe containing STAT1 binding site, we also found that hTau accumulation increased binding of STAT1 to DNA and this association was disrupted by using cold probe (Fig 1J). To verify the specificity of STAT1 activation, we studied the effects of TDP-43 and α-synuclein on STAT1 in HEK293 cells. The results showed that overexpression of TDP-43 or α-synuclein did not significantly alter the levels of STAT1 and p-STAT1 (Fig EV1A and B), indicating that STAT1 activation may be specific to tau. To further identify the minimal fragments of hTau on STAT1 activation, we constructed truncated tau plasmids covering different length of N-terminal and C-terminal tau fragments. After expressed these tau fragments in HEK293 cells, we observed that the N-terminal (tau1-368, tau1-255, tau1-197) but not C-terminal (tau256-441) tau could upregulate STAT1; and the currently identified minimal fragment able to induce STAT1 activation was tau1-197 while the shorter tau fragments including tau1-44, tau1-150, tau121-150, and tau121-197 had no stimulating effect on STAT1 (Fig EV1A, B, D and E). These data suggest that both full-length and the N-terminal tau fragments can active STAT1. Click here to expand this figure. Figure EV1. Overexpressing N-terminal tau fragments not TDP-43 or α-synuclein activate STAT1 A, B. Plasmids of TDP-43, α-synuclein, or hTau fragments or the vector control were transfected into HEK293 cells for 48 h, and then, the protein levels of STAT1 and pY-STAT1 were detected by Western blotting in the total extracts (A) and the nuclear fraction (B). C. Adeno-associated virus (AAV)-eGFP-TDP43 or AAV-eGFP-α-synuclein or the vector control was transfected into the primary hippocampal neurons (6 div) and the neurons were cultured for another 48 h, and then, the protein levels of NMDARs were detected by Western blotting. D, E. The subdivided hTau fragments were transfected into the HEK293 cells for 48 h, and then, the protein levels of STAT1 and pY-STAT1 were detected by Western blotting in total extracts (D) and the nuclear fraction (E). Asterisks indicated the bands which matched with fragments’ predicted molecular weight. Source data are available online for this figure. Download figure Download PowerPoint These in vitro data indicate that intracellular hTau accumulation induces STAT1 activation. To test the in vivo effects of hTau accumulation on STAT1, we first injected stereotaxically AAV-hTau into the mouse hippocampi and measured the alterations of STAT1 and pY-STAT1 after 1 month. Expression of hTau was confirmed by Western blotting (Fig 2A), and fluorescent imaging and immunohistochemistry (Fig EV2A). Accumulation of misfolded tau was shown by Thioflavin-S and Bielschowsky silver staining (Fig EV2B). Overexpression of hTau significantly increased total STAT1 and pY-STAT1 in hippocampal extracts and the nuclear fraction (Fig 2A and B) without changing VDR, PLAG1, and SMUC (Fig EV2C), suggesting a relatively specific effect of hTau on STAT1. Infection of control AAV-eGFP did not activate STAT1 (Fig EV2D). By co-staining of nuclear translocation of STAT1 with NeuN, IBA1, and GFAP, we found that the neuronal staining of STAT1 was most significant (Fig EV3). Elevation of STAT1 and pY-STAT1 was also detected in the hippocampi of 9-month- and 12-month-old hTau transgenic mice (Fig 2C and D and Appendix Fig S3A). By transfecting Syn-hTau-AAV into the hippocampus, we found that the neuron-specific overexpression of hTau also significantly increased total STAT1 and pY-STAT1 in hippocampal extracts and the nuclear fraction (Appendix Fig S4A and B). In the cortex of AD patients, both total and pY-STAT1 were also significantly increased (Fig 2E–G). Further, STAT1 mRNA also increased significantly in hTau-expressed tissues (Fig 2H). These data provide the in vivo and human evidence for the role of tau accumulation in activating STAT1. Figure 2. Overexpression of hTau upregulates phosphorylated STAT1 in vivo A, B. AAV-hTau-eGFP (AAV-hTau) or the empty vector AAV-eGFP (1.13 × 1013 v.g./ml) was stereotaxically injected into hippocampal CA3 of 3-month-old C57 mice. After 1 month, the increased levels of STAT1 and pY-STAT1 in hippocampal total extracts and the nuclear fraction were detected in hTau group by Western blotting (n = 6, Mann–Whitney test). C, D. The increased STAT1 and pY-STAT1 in hippocampal total extracts and the nuclear fraction of 12-month-old hTau transgenic mice were measured by Western blotting (n = 4, Mann–Whitney test). E, F. The representative images of STAT1 and pY-STAT1 in the brain of AD patients probed by co-immunohistochemical staining and quantitative analysis (hematoxylin stains nuclei, purple; DAB stains the target proteins, brown; n = 5–6 slices). Arrowheads indicated typical nuclear staining of STAT1/pY-STAT1. G. The increased AT8 (pS202/pT205), STAT1, and pY-STAT1 in cortex total extracts of AD patients were measured by Western blotting (n = 3, Student's t-test). H. STAT1 mRNA was analyzed by qRT-PCR in AAV-hTau- or AAV-eGFP infected hippocampal tissues (n = 6, Mann–Whitney test). Data information: Data were presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 vs eGFP, wt, or Ctrl. Source data are available online for this figure. Source Data for Figure 2 [embr201847202-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Overexpressing hTau induces tau aggregation and does not affect protein level of VDR, PLAG1, and SMUC Adeno-associated virus (AAV)-eGFP expressing wild-type full-length human tau (hTau) or the empty AAV vector (eGFP) (1.13 × 1013 v.g./ml) was stereotaxically injected into the hippocampal CA3 of 3-month-old C57 mice. One month later, the infected virus was shown by direct fluorescence (left panels) and immunohistochemical staining of HT7 (specifically reacts with human tau, right panel), respectively. Scale bar, 200 μm; 100 μm (enlarged picture). The representative images of Thioflavin S and Bielschowsky silver staining showed tau aggregation in the hippocampus of the virus-injected mice. AAV-hTau or the empty vector (eGFP) (1.13 × 1013 v.g./ml) was stereotaxically injected into the hippocampal CA3 of 3-month-old C57 mice. One month later, the protein levels of VDR (vitamin D receptor), PLAG1 (pleiomorphic adenoma gene 1), and SMUC (snail-related transcription factor) were detected by Western blotting (n = 4). AAV-eGFP (1.13 × 1013 v.g./ml) or PBS was stereotaxically injected into the hippocampal CA3 of 3-month-old C57 mice. PBS-injected tissue was used as uninfected control. One month later, the protein levels of STAT1 and pY-STAT1 were detected by Western blotting (n = 4). Data information: Data were presented as mean ± SD (Mann–Whitney test). Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. STAT1 is predominantly expressed in neurons
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