Tau accumulation induces synaptic impairment and PNAS PLUS memory deficit by calcineurin-mediated inactivation of nuclear CaMKIV/CREB signaling Yaling Yina,1, Di Gaoa,1, Yali Wanga, Zhi-Hao Wanga, Xin Wanga, Jinwang Yea, Dongqin Wua, Lin Fanga, Guilin Pia, Ying Yanga, Xiao-Chuan Wanga, Chengbiao Lub, Keqiang Yec, and Jian-Zhi Wanga,d,2 aDepartment of Pathophysiology, School of Basic Medicine and the Collaborative Innovation Center for Brain Science, Key Laboratory of Ministry of Education of China for Neurological Disorders, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China; bDepartment of Physiology and Neurobiology, Henan Province Key Laboratory of Brain Research, Xinxiang Medical University, Xinxiang 453003, China; cDepartment of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322; and dCo-Innovation Center of Neuroregeneration, Nantong University, Nantong JS 226001, China Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved May 10, 2016 (received for review March 18, 2016) Intracellular accumulation of wild-type tau is a hallmark of sporadic tangles (13, 14) and reducing endogenous tau in mouse models Alzheimer’s disease (AD), but the molecular mechanisms underlying carrying the mutated amyloid precursor protein (APP) prevents tau-induced synapse impairment and memory deficit are poorly un- the cognitive deficits and synaptic loss (15). derstood. Here we found that overexpression of human wild-type Among many structural or functional proteins involved in syn- full-length tau (termed hTau) induced memory deficits with impair- apse development and memory formation, cAMP response ele- ments of synaptic plasticity. Both in vivo and in vitro data demon- ment binding protein (CREB) is one of the most extensively strated that hTau accumulation caused remarkable dephosphorylation studied (16, 17). CREB is a transcription factor that can regulate of cAMP response element binding protein (CREB) in the nuclear the syntheses of synapse- or memory-associated proteins (18, 19). fraction. Simultaneously, the calcium-dependent protein phos- The activity of CREB is regulated by phosphorylation and phatase calcineurin (CaN) was up-regulated, whereas the calcium/ dephosphorylation, and dephosphorylation disrupts the activ- calmodulin-dependent protein kinase IV (CaMKIV) was suppressed. ity-transcription coupling of CREB in the nuclei (20). Level of Further studies revealed that CaN activation could dephosphorylate the phosphorylated CREB at Ser133 is significantly decreased CREB and CaMKIV, and the effect of CaN on CREB dephosphoryla- in the AD hippocampus (21), and knockdown of CREB in mouse tion was independent of CaMKIV inhibition. Finally, inhibition of brain causes progressive neurodegeneration in hippocampus and CaN attenuated the hTau-induced CREB dephosphorylation with im- the dorsolateral striatum (22). Several kinases can phosphorylate proved synapse and memory functions. Together, these data indi- CREB, such as protein kinase A (PKA) (23), mitogen-activated cate that the hTau accumulation impairs synapse and memory by protein kinase (MAPK) (24), protein kinase C (PKC) (25, 26), CaN-mediated suppression of nuclear CaMKIV/CREB signaling. and calcium/calmodulin-dependent protein kinases (CaMKs) (27). Our findings not only reveal new mechanisms underlying the Among various CaMKs, CaMKIV is known to phosphorylate CREB hTau-induced synaptic toxicity, but also provide potential targets in the nuclei (28, 29). On the other hand, CREB can be dephos- for rescuing tauopathies. phorylated by protein phosphatase 1 (PP1) and calcineurin (CaN, also termed PP2B) (30, 31). In Tg2576 mice, the immunoreactivity + Alzheimer’s disease | tau | calcineurin | Ca2 /calmodulin-dependent kinase IV | CREB Significance lzheimer’s disease (AD) is the most common neurodegen- Memory deterioration is a characteristic clinical symptom in Aerative disorder characterized clinically by progressive mem- patients with Alzheimer’s disease (AD); however, the mech- ory loss (1). The extracellular precipitation of β-amyloid (Aβ)(2), anisms underlying the memory loss are poorly understood. intracellular tau accumulation forming neurofibrillary tangles (3), Here, we found that intraneuronal tau accumulation, the and profound synapse degeneration are hallmark pathologies in hallmark pathology seen in AD brains, induced a remarkable AD brains (4, 5). Studies show that formation of neurofibrillary dephosphorylation/inactivation of nuclear cAMP response ele- tangles is positively correlated with the degree of dementia ment binding protein (CREB), an important memory-associated symptoms (6), and the Aβ toxicity needs the presence of tau (7). protein. Further studies demonstrated that the abnormal tau These data suggest a crucial role of tau accumulation in neuro- accumulation could activate calcineurin, a calcium/calmodu- degeneration and the cognitive impairments in patients with AD. lin-dependent protein phosphatase and cause CREB dephos- As a cytoskeleton protein, how tau accumulation causes memory phorylation. Importantly, simultaneous inhibition of calcineurin deficits is not fully understood. remarkably attenuated tau-induced CREB inactivation and Synapse is the fundamental unit for learning and memory. memory deficits. These findings not only reveal new mechanisms Dysfunction of synaptic connections is recognized as the cause of underlying AD memory deficits, but also provide a potential drug memory impairments, and significant synapse loss has been ob- target for arresting tauopathies. served in mild cognitive impairment (MCI) and in earlier stages NEUROSCIENCE of AD (8). In AD mouse models, synapse impairments appear Author contributions: Y. Yin and J.Z.W. designed research; Y. Yin, D.G., Y.W., Z.H.W., X.W., J.Y., D.W., L.F., and G.P. performed research; Y. Yin, D.G., Y.W., Y. Yang, X.C.W., before the onset of memory deficit (9), whereas amelioration of C.L., K.Y., and J.Z.W. analyzed data; and Y. Yin and J.Z.W. wrote the paper. synapse loss by administration of estradiol preserves cognitive The authors declare no conflict of interest. functions (10). Earlier investigations into AD-related synaptic This article is a PNAS Direct Submission. β damages have been mainly focused on the toxic effects of A 1Y. Yin and D.G. contributed equally to this work. (11). Recently, an emerging role of tau in synaptic impairment has 2To whom correspondence should be addressed. Email: [email protected]. been shown (12). For instance, overexpression of human mutant This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tau in mice induces synaptic degeneration even in the absence of 1073/pnas.1604519113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1604519113 PNAS | Published online June 13, 2016 | E3773–E3781 Downloaded by guest on September 25, 2021 of the phosphorylated CREB was reduced and it was restored by treatment with FK506 (32). In the nuclear fraction of AD brains, a cleaved fragment of CaN (cCaN) is increased and the cleavage activates the phosphatase (33). It is not reported whether and how the human wild-type full-length tau (hereafter, hTau) ac- cumulation may affect these synapse- or memory-related proteins. By AAV-delivered overexpression of hTau in mouse hippo- campus and in cultured hippocampal neurons, we find in the present study that overexpression of hTau in hippocampal neu- rons induces synapse impairment and memory deficits. The ac- cumulation of hTau inactivated nuclear CaMKIV-CREB signaling by activating CaN, and simultaneous inhibition of CaN effectively arrested the hTau-induced CREB inactivation and rescued the synapse in morphology and the functions with improvement of the memory capacity. Results Overexpression of hTau in Hippocampal Neurons Induces Memory Deficits with Dendrite Impairment and Synaptic Dysfunction. To ex- plore the role of hippocampal tau accumulation, an early marker of the AD brain in spatial learning and memory, we injected stereotaxically the AAV-eGFP-hTau into the dorsal hippocampal CA3 of 2-mo-old mice. After 6 wk, the expression of hTau, enriched in hippocampal CA3, was confirmed by immunofluo- rescence imaging (Fig. 1A) and Western blotting using human tau- Fig. 1. Tau accumulation in the hippocampal CA3 region impairs learning specific antibody HT7 (Fig. 1B). By Morris water maze (MWM) and memory. (A) The representative immunofluorescence imaging of hip- and fear conditioning tests, we observed that the mice with hTau pocampus and the enlarged CA3 subset after infusion of AAV-eGFP-hTau for overexpression show significantly increased latency to find the 6 wk. [Scale bars: 200 μm(Left) and 20 μm(Right).] (B) Expression of hTau in hidden platform during the 6-d learning process compared with the hippocampal CA3 was confirmed by Western blotting using human tau- C specific antibody HT7. (C) The escape latency to find the hidden platform in vector controls (Fig. 1 ), suggesting impairment of special learning – by hTau. On day 8, the spatial memory was tested by removing the MWM during 6-d learning process. (D F) The escape latency to find the hidden platform, the target platform crossings, and the time spent in the platform. A remarkably increased latency to reach the platform area target quadrant measured on day 8 by removing the platform. (G) After a with decreased target platform crossings and time in target quadrant 1-wk rest, fear conditioning was