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Curcumin Suppresses Soluble Tau Dimers and Corrects Molecular Chaperone, Synaptic, and Behavioral Deficits in Aged Human Tau Transgenic Mice*

  • Qiu-Lan Ma
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Xiaohong Zuo
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073

    the Department of Neurobiology, Key Laboratory on Neurodegenerative Disorders of Ministry of Education, Beijing Institute of Geriatrics, Xuanwu Hospital, Capital Medical University, 10053 Beijing, China
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  • Fusheng Yang
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Oliver J. Ubeda
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Dana J. Gant
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Mher Alaverdyan
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Edmond Teng
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Shuxin Hu
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Ping-Ping Chen
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Panchanan Maiti
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Bruce Teter
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Greg M. Cole
    Correspondence
    To whom correspondence may be addressed: Veterans Greater Los Angeles Healthcare System, Geriatric Research Education and Clinical Center, 11301 Wilshire Blvd., Bldg. 113, Rm. 312, Los Angeles, CA 90073. Tel.: 310-433-0099; Fax: 310-268-4083;
    Footnotes
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Sally A. Frautschy
    Correspondence
    To whom correspondence may be addressed: Veterans Greater Los Angeles Healthcare System, Geriatric Research Education and Clinical Center, 11301 Wilshire Blvd., Bldg. 113, Rm. 312, Los Angeles, CA 90073. Tel.: 310-433-0099; Fax: 310-268-4083;
    Footnotes
    Affiliations
    From the Department of Neurology, David Geffen School of Medicine at the University of California, Los Angeles, the Geriatric, Research, and Clinical Center, Greater Los Angeles Veterans Affairs Healthcare System, Los Angeles, California 90073
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01 AG021975 (to S. A. F.), U0128583 (to S. A. F.), RC1 AG035878 (to S. A. F. and G. M. C.), AT006816 (to G. M. C.), P01 AG16570 (UCLA Alzheimer Disease Research Center; to G. M. C.), P30-AG028748 (UCLA Older Americans Independence Center; NIA), and K08 AG34628 (to E. T.). This work was also supported by Alzheimer's Association Grant NIRG-07-59659 (to Q-L. M.), the UCLA Mary S. Easton Translation Center (S. A. F., G. M. C.), and Veterans Affairs Merit grants (to G. M. C. and S. A. F.). S.A.F. and G.M.C. are coinventors on a UC Regents and Veteran's pending patent on a bioavailable curcumin formulation, which has been licensed by UC Regents to Verdure Sciences, Indianapolis, IN. These authors contributed equal funding to this project.
    This article contains supplemental Figs. S1–S3
    1 Senior coauthors.
Open AccessPublished:December 21, 2012DOI:https://doi.org/10.1074/jbc.M112.393751
      The mechanisms underlying Tau-related synaptic and cognitive deficits and the interrelationships between Tau species, their clearance pathways, and synaptic impairments remain poorly understood. To gain insight into these mechanisms, we examined these interrelationships in aged non-mutant genomic human Tau mice, with established Tau pathology and neuron loss. We also examined how these interrelationships changed with an intervention by feeding mice either a control diet or one containing the brain permeable beta-amyloid and Tau aggregate binding molecule curcumin. Transgene-dependent elevations in soluble and insoluble phospho-Tau monomer and soluble Tau dimers accompanied deficits in behavior, hippocampal excitatory synaptic markers, and molecular chaperones (heat shock proteins (HSPs)) involved in Tau degradation and microtubule stability. In human Tau mice but not control mice, HSP70, HSP70/HSP72, and HSP90 were reduced in membrane-enriched fractions but not in cytosolic fractions. The synaptic proteins PSD95 and NR2B were reduced in dendritic fields and redistributed into perikarya, corresponding to changes observed by immunoblot. Curcumin selectively suppressed levels of soluble Tau dimers, but not of insoluble and monomeric phospho-Tau, while correcting behavioral, synaptic, and HSP deficits. Treatment increased PSD95 co-immunoprecipitating with NR2B and, independent of transgene, increased HSPs implicated in Tau clearance. It elevated HSP90 and HSC70 without increasing HSP mRNAs; that is, without induction of the heat shock response. Instead curcumin differentially impacted HSP90 client kinases, reducing Fyn without reducing Akt. In summary, curcumin reduced soluble Tau and elevated HSPs involved in Tau clearance, showing that even after tangles have formed, Tau-dependent behavioral and synaptic deficits can be corrected.
      Background: Various types of Tau aggregates in AD brains may differentially impact neurodegeneration.
      Results: Curcumin selectively suppresses soluble Tau dimers and corrects molecular chaperone, synaptic, and behavioral deficits.
      Conclusion: A drug increasing HSPs involved in Tau clearance reduced Tau dimers and improved cognition.
      Significance: Curcumin that reduced Tau dimers and increased molecular chaperones was efficacious without altering insoluble Tau.

      Introduction

      Insoluble intracellular Tau deposits represent pathological signatures for several neurodegenerative diseases, including Alzheimer disease (AD).
      The abbreviations used are: AD
      Alzheimer disease
      hTau
      wild-type human Tau
      p-Tau
      hyperphosphorylated microtubule-associated protein Tau
      NFT
      neurofibrillary tangle
      HSP
      heat shock protein
      MWM
      Morris water maze
      CHIP
      carboxyl terminus of HSP70-interacting protein
      PSD
      postsynaptic density
      Cur
      curcumin
      NMDA
      N-methyl-d-aspartate.
      In AD, multiple factors, including the dysregulation of Tau kinases and phosphatases (
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      Tau pathology in Alzheimer disease and other tauopathies.
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      Hyperphosphorylated Tau in parahippocampal cortex impairs place learning in aged mice expressing wild-type human tau.
      ), can lead to accumulation of insoluble filaments of hyperphosphorylated microtubule-associated protein Tau (p-Tau) that form neurofibrillary tangles (NFTs). Cortical NFTs can correlate with severity of cognitive impairment in AD (
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      Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease.
      ). However, recent work bolsters the hypothesis that soluble Tau is a critical player in cognitive and synaptic dysfunction (
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      Evidence that non-fibrillar Tau causes pathology linked to neurodegeneration and behavioral impairments.
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      Preparation and characterization of neurotoxic Tau oligomers.
      ). After P301L Tau transgene repression, improvement of memory and slowing of neuronal loss occurs without reducing NFT density that can be dissociated from cognitive decline and neuronal death (
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      Tau suppression in a neurodegenerative mouse model improves memory function.
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      • Janus C.
      Accumulation of pathological Tau species and memory loss in a conditional model of tauopathy.
      ). In fact, irrespective of NFT inclusions, neurons from rTg4510 mice demonstrate similar electrophysiological deficits, dendritic atrophy, and spine loss (
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      • Luebke J.I.
      Structural and functional changes in Tau mutant mice neurons are not linked to the presence of NFTs.
      ), and in other mutant Tau models synapse loss precedes NFT deposition (
      • Mocanu M.M.
      • Nissen A.
      • Eckermann K.
      • Khlistunova I.
      • Biernat J.
      • Drexler D.
      • Petrova O.
      • Schönig K.
      • Bujard H.
      • Mandelkow E.
      • Zhou L.
      • Rune G.
      • Mandelkow E.M.
      The potential for β-structure in the repeat domain of Tau protein determines aggregation, synaptic decay, neuronal loss, and coassembly with endogenous Tau in inducible mouse models of tauopathy.
      ,
      • Eckermann K.
      • Mocanu M.M.
      • Khlistunova I.
      • Biernat J.
      • Nissen A.
      • Hofmann A.
      • Schönig K.
      • Bujard H.
      • Haemisch A.
      • Mandelkow E.
      • Zhou L.
      • Rune G.
      • Mandelkow E.M.
      The β-propensity of Tau determines aggregation and synaptic loss in inducible mouse models of tauopathy.
      ). Furthermore, aged mice, expressing native human Tau but lacking NFTs, still develop memory deficits in conjunction with increased soluble hyperphosphorylated Tau and synapse loss (
      • Kimura T.
      • Yamashita S.
      • Fukuda T.
      • Park J.M.
      • Murayama M.
      • Mizoroki T.
      • Yoshiike Y.
      • Sahara N.
      • Takashima A.
      Hyperphosphorylated Tau in parahippocampal cortex impairs place learning in aged mice expressing wild-type human tau.
      ). This suggests that strategies such as Tau epitope-specific vaccines (
      • Boutajangout A.
      • Quartermain D.
      • Sigurdsson E.M.
      Immunotherapy targeting pathological Tau prevents cognitive decline in a new tangle mouse model.
      ) or drugs that specifically alter selective Tau assemblies may target pathogenic soluble p-Tau species without impacting non-pathogenic p-Tau species (
      • Schneider A.
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      • von Bergen M.
      • Mandelkow E.
      • Mandelkow E.M.
      Phosphorylation that detaches Tau protein from microtubules (Ser-262, Ser-214) also protects it against aggregation into Alzheimer paired helical filaments.
      ).
      The heat shock protein (HSP) system is implicated in the regulation of microtubule stability and disposal of misfolded toxic Tau via multiple mechanisms (
      • Dou F.
      • Netzer W.J.
      • Tanemura K.
      • Li F.
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      • Takashima A.
      • Gouras G.K.
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      • Xu H.
      Chaperones increase association of Tau protein with microtubules.
      ,
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      • Dunmore J.
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      Akt and CHIP coregulate Tau degradation through coordinated interactions.
      ,
      • Shimura H.
      • Schwartz D.
      • Gygi S.P.
      • Kosik K.S.
      CHIP-Hsc70 complex ubiquitinates phosphorylated Tau and enhances cell survival.
      ). HSC70 and HSP70 bind directly to Tau, independent of Tau phosphorylation, facilitating microtubule polymerization and limiting Tau aggregation (
      • Sarkar M.
      • Kuret J.
      • Lee G.
      Two motifs within the Tau microtubule-binding domain mediate its association with the hsc70 molecular chaperone.
      ). However, in AD, HSP dysregulation is not fully understood, making it difficult to determine if its modulation could be used as a therapeutic intervention (
      • Jinwal U.K.
      • O'Leary 3rd, J.C.
      • Borysov S.I.
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      • Jin Y.
      • Miyata Y.
      • Gestwicki J.E.
      • Dickey C.A.
      Hsc70 rapidly engages Tau after microtubule destabilization.
      ). Other HSPs could also play a role in AD. For example, HSP90 controls protein turnover using co-chaperones that recruit to the complex client proteins, particularly Tau and short-lived kinases like Fyn and Akt, which are either refolded and released or ubiquitinated and degraded (
      • Citri A.
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      • Lavi S.
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      • Yarden Y.
      Hsp90 recognizes a common surface on client kinases.
      ,
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      • Workman P.
      HSP90 as a new therapeutic target for cancer therapy. The story unfolds.
      ).
      Because AD lacks Tau mutations, we chose wild-type human Tau (hTau) transgenic mice that exhibit NFTs, neuron loss, and cognitive deficits by 12 months of age (
      • Polydoro M.
      • Acker C.M.
      • Duff K.
      • Castillo P.E.
      • Davies P.
      Age-dependent impairment of cognitive and synaptic function in the hTau mouse model of Tau pathology.
      ,
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      • Kress Y.
      • Hof P.R.
      • Duff K.
      • Davies P.
      Cell-cycle reentry and cell death in transgenic mice expressing nonmutant human Tau isoforms.
      ). We first evaluated the association of different p-Tau species with cognitive and synaptic deficits. Next we compared this association with hTau mice maintained on diets with and without curcumin, a candidate anti-Tau treatment and blood-brain barrier-permeable amyloid binding dye that recognizes amyloidogenic segments within Tau and β-amyloid (
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      • Liu J.
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      • Eisenberg D.
      Towards a pharmacophore for amyloid.
      ). Oral curcumin is known to reduce Aβ plaque burden or size (
      • Yang F.
      • Lim G.P.
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      • Ubeda O.J.
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      Curcumin inhibits formation of amyloid β oligomers and fibrils, binds plaques, and reduces amyloid in vivo.
      ,
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      ,
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      Curcumin labels amyloid pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an Alzheimer mouse model.
      ). To best emulate potential in human clinical trials in subjects who typically have existing NFT deposition, we delayed treatment until significant Tau pathology and neuron loss had developed and begun to plateau in aged hTau mice.

      DISCUSSION

      Curcumin- correction of behavioral and NMDA receptor complex deficits in the aged hTau transgenic tauopathy model was associated with altered soluble Tau dimers but not with soluble p-Tau monomer or insoluble Tau. In fact curcumin suppressed soluble Tau dimers and corrected aberrant excitatory synaptic protein expression, behavior, and HSP defects despite multiple Tau species remaining elevated. Our data suggest that HSP up-regulation likely contributes to curcumin effects on Tau dimer reduction and neuroprotection and is consistent with emerging evidence showing a major role for HSPs in Tau removal.
      Although this report is consistent with a pathogenic role of all soluble Tau in behavior deficits (
      • Santacruz K.
      • Lewis J.
      • Spires T.
      • Paulson J.
      • Kotilinek L.
      • Ingelsson M.
      • Guimaraes A.
      • DeTure M.
      • Ramsden M.
      • McGowan E.
      • Forster C.
      • Yue M.
      • Orne J.
      • Janus C.
      • Mariash A.
      • Kuskowski M.
      • Hyman B.
      • Hutton M.
      • Ashe K.H.
      Tau suppression in a neurodegenerative mouse model improves memory function.
      ,
      • O'Leary 3rd, J.C.
      • Li Q.
      • Marinec P.
      • Blair L.J.
      • Congdon E.E.
      • Johnson A.G.
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      • Koren 3rd, J.
      • Jones J.R.
      • Kraft C.
      • Peters M.
      • Abisambra J.F.
      • Duff K.E.
      • Weeber E.J.
      • Gestwicki J.E.
      • Dickey C.A.
      Phenothiazine-mediated rescue of cognition in Tau transgenic mice requires neuroprotection and reduced soluble Tau burden.
      ), it is the first report of a small molecule selectively reducing only soluble Tau dimers/oligomers, which corresponded to a correction of synaptic and behavior deficits. After tau repression in rTg4510 mice, memory deficits correlated with Tau dimers but also with sarkosyl-insoluble 64-kDa Tau (
      • Berger Z.
      • Roder H.
      • Hanna A.
      • Carlson A.
      • Rangachari V.
      • Yue M.
      • Wszolek Z.
      • Ashe K.
      • Knight J.
      • Dickson D.
      • Andorfer C.
      • Rosenberry T.L.
      • Lewis J.
      • Hutton M.
      • Janus C.
      Accumulation of pathological Tau species and memory loss in a conditional model of tauopathy.
      ).
      In AD, conformational changes in soluble Tau emerge early (
      • Weaver C.L.
      • Espinoza M.
      • Kress Y.
      • Davies P.
      Conformational change as one of the earliest alterations of Tau in Alzheimer's disease.
      ) leading to the formation of granular oligomers that precede NFT (
      • Maeda S.
      • Sahara N.
      • Saito Y.
      • Murayama S.
      • Ikai A.
      • Takashima A.
      Increased levels of granular Tau oligomers. An early sign of brain aging and Alzheimer's disease.
      ,
      • Haroutunian V.
      • Davies P.
      • Vianna C.
      • Buxbaum J.D.
      • Purohit D.P.
      Tau protein abnormalities associated with the progression of alzheimer disease type dementia.
      ). Tau dimers occur both with sporadic AD and FTDP-17 mutations as well as in cell and mouse Tau models (
      • Berger Z.
      • Roder H.
      • Hanna A.
      • Carlson A.
      • Rangachari V.
      • Yue M.
      • Wszolek Z.
      • Ashe K.
      • Knight J.
      • Dickson D.
      • Andorfer C.
      • Rosenberry T.L.
      • Lewis J.
      • Hutton M.
      • Janus C.
      Accumulation of pathological Tau species and memory loss in a conditional model of tauopathy.
      ,
      • Sahara N.
      • Maeda S.
      • Murayama M.
      • Suzuki T.
      • Dohmae N.
      • Yen S.H.
      • Takashima A.
      Assembly of two distinct dimers and higher-order oligomers from full-length tau.
      ). Tau-related pathogenesis (HSPs, NMDA receptor and behavior) paralleled the levels of SDS stable soluble Tau dimers in reducing gels, suggesting the involvement of stabilizing factors beyond disulfide bonds, such as cross-linking from lipid peroxidation (
      • Gamblin T.C.
      • King M.E.
      • Kuret J.
      • Berry R.W.
      • Binder L.I.
      Oxidative regulation of fatty acid-induced Tau polymerization.
      ).
      Curcumin reduced total and p-Tau dimers and restored dysregulated excitatory synaptic proteins, suggesting that Tau dimers represent a significant synaptotoxic species. Tau can induce excessive microtubule bundling, resulting in organelle transport deficits and dendritic spine loss (
      • Thies E.
      • Mandelkow E.M.
      Missorting of Tau in neurons causes degeneration of synapses that can be rescued by the kinase MARK2/Par-1.
      ). Tau also binds and bundles actin (
      • He H.J.
      • Wang X.S.
      • Pan R.
      • Wang D.L.
      • Liu M.N.
      • He R.Q.
      The proline-rich domain of Tau plays a role in interactions with actin.
      ), raising the possibility that Tau dimers disrupt dendritic spine actin dynamics, critical for normal learning and memory (
      • Hotulainen P.
      • Hoogenraad C.C.
      Actin in dendritic spines. Connecting dynamics to function.
      ). Soluble Tau species are implicated in synaptic deficits in the rTg4510 model. In control mice Tau is predominantly axonal, but in mutant mice it redistributes to dendritic spines where it is bound to actin (
      • Hoover B.R.
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      Tau mislocalization to dendritic spines mediates synaptic dysfunction independently of neurodegeneration.
      ).
      Depletion of NR2B and PSD95 in membrane-enriched lysis fractions and elevation in tangle and cytoskeletal-enriched SDS extracts suggests defects in shuttling between PSD95 complexes and lipid rafts. Our co-immunoprecipitation and synaptosome results argue that changes in these synaptic proteins are predominantly synaptic as opposed to extrasynaptic. Lipid rafts and postsynaptic densities (PSDs) normally exchange important signaling proteins including NMDA receptor subunits, PSD95, and downstream kinases (
      • Besshoh S.
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      • Teves L.
      • Wallace M.C.
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      Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain.
      ). Shuttling of NMDA receptor components and PSD95 from the PSD to the rafts is reduced after ischemia and increased after spatial learning (
      • Besshoh S.
      • Bawa D.
      • Teves L.
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      Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain.
      ,
      • Delint-Ramírez I.
      • Salcedo-Tello P.
      • Bermudez-Rattoni F.
      Spatial memory formation induces recruitment of NMDA receptor and PSD95 to synaptic lipid rafts.
      ). Confocal microscopy confirms a Tau-dependent shift of NR2B from synaptic puncta along dendrites in WT mice to larger abnormal structures in untreated but not curcumin-treated hTau mice. This would be consistent with Tau dimer disruption of receptor localization to the PSD, a process involving Src/Fyn family kinases.
      Fyn kinases, transported by Tau, may mediate Tau dimer-induced dendritic toxicity. For example, NR2B translocation and retention in the PSD NMDA receptor complex (
      • Abe T.
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      • Ito S.
      Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr-1472 is essential for maintenance of neuropathic pain.
      ) is regulated by Fyn, a kinase that binds, travels with, and phosphorylates Tau at Tyr-18. Because Fyn is lost from synaptic sites and co-localizes with NFTs in clinical AD (
      • Ho G.J.
      • Hashimoto M.
      • Adame A.
      • Izu M.
      • Alford M.F.
      • Thal L.J.
      • Hansen L.A.
      • Masliah E.
      Altered p59Fyn kinase expression accompanies disease progression in Alzheimer's disease. Implications for its functional role.
      ) and AD models (
      • Vega I.E.
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      • Propst J.A.
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      • Lee G.
      • Yen S.H.
      Increase in Tau tyrosine phosphorylation correlates with the formation of Tau aggregates.
      ), this suggests Tau aggregation does not compromise Fyn binding but instead mislocalizes Fyn to aggregates (NFT). The hTau transgene resulted in elevations in Fyn in SDS fractions enriched in insoluble Tau, consistent with Fyn involvement in AD tauopathy. Dendritic Tau-Fyn interactions also mediate NR2B localization and Aβ oligomer synaptotoxicity (
      • Ittner L.M.
      • Ke Y.D.
      • Delerue F.
      • Bi M.
      • Gladbach A.
      • van Eersel J.
      • Wölfing H.
      • Chieng B.C.
      • Christie M.J.
      • Napier I.A.
      • Eckert A.
      • Staufenbiel M.
      • Hardeman E.
      • Götz J.
      Dendritic function of Tau mediates amyloid-β toxicity in Alzheimer's disease mouse models.
      ). Our co-immunoprecipitation data also confirm Tau-Fyn interaction in WT and hTau mice, but surprisingly curcumin enhanced this interaction in hTau mice compared with untreated hTau mice. This was companied by the elevation of PSD95 associated with NR2B complex and improved behavior, suggesting the increased Fyn/Tau interactions in hTau mice are not toxic. Because Fyn binding to Tau is modulated by both phosphorylation-regulated SH3 domains and the ratio of 3 Repeat (3R) to 4 Repeat (4R) Tau, curcumin may have altered either or both factors to increase Tau/Fyn binding. Although additional aged curcumin-treated mice are needed to clarify the mechanisms, preliminary experiments using pooled samples and AT180 recognizing Ser(P) 231 Tau suggest that curcumin reduced phosphorylation of Tau at this SH3 domain, which would increase Tau/Fyn binding (
      • Bhaskar K.
      • Yen S.H.
      • Lee G.
      Disease-related modifications in Tau affect the interaction between Fyn and Tau.
      ) and mitigate the curcumin large reduction in Fyn levels. Fyn is a key molecule determining the localization of BDNF receptor TrkB in lipid rafts and linking the TrkB with NMDA receptors (
      • Pereira D.B.
      • Chao M.V.
      The tyrosine kinase Fyn determines the localization of TrkB receptors in lipid rafts.
      ) that play an important role in spatial memory formation (
      • Mizuno M.
      • Yamada K.
      • He J.
      • Nakajima A.
      • Nabeshima T.
      Involvement of BDNF receptor TrkB in spatial memory formation.
      ).
      This is the first report to show HSP defects in the Tau model and that dimers correlated with HSP defects. HSPs are enriched at excitatory and inhibitory synapses and can regulate stabilization of synaptic receptor clusters (
      • Renner M.
      • Specht C.G.
      • Triller A.
      Molecular dynamics of postsynaptic receptors and scaffold proteins.
      ,
      • Machado P.
      • Rostaing P.
      • Guigonis J.M.
      • Renner M.
      • Dumoulin A.
      • Samson M.
      • Vannier C.
      • Triller A.
      Heat shock cognate protein 70 regulates gephyrin clustering.
      ) as well as clearance of misfolded Tau (
      • Wang Y.
      • Martinez-Vicente M.
      • Krüger U.
      • Kaushik S.
      • Wong E.
      • Mandelkow E.M.
      • Cuervo A.M.
      • Mandelkow E.
      Tau fragmentation, aggregation and clearance. The dual role of lysosomal processing.
      ). Tau transgene-dependent defects in HSP70/HSP72 and HSP70 were found in the membrane fraction, and there was a trend for reduction in HSP90. HSPs stabilize microtubules and control Tau degradation (
      • Dou F.
      • Netzer W.J.
      • Tanemura K.
      • Li F.
      • Hartl F.U.
      • Takashima A.
      • Gouras G.K.
      • Greengard P.
      • Xu H.
      Chaperones increase association of Tau protein with microtubules.
      ,
      • Sarkar M.
      • Kuret J.
      • Lee G.
      Two motifs within the Tau microtubule-binding domain mediate its association with the hsc70 molecular chaperone.
      ,
      • Jinwal U.K.
      • O'Leary 3rd, J.C.
      • Borysov S.I.
      • Jones J.R.
      • Li Q.
      • Koren 3rd, J.
      • Abisambra J.F.
      • Vestal G.D.
      • Lawson L.Y.
      • Johnson A.G.
      • Blair L.J.
      • Jin Y.
      • Miyata Y.
      • Gestwicki J.E.
      • Dickey C.A.
      Hsc70 rapidly engages Tau after microtubule destabilization.
      ,
      • Dolan P.J.
      • Johnson G.V.
      A caspase cleaved form of Tau is preferentially degraded through the autophagy pathway.
      ). Thus the transgene-dependent reductions in HSPs in the membrane fraction of aged hTau mice may contribute to microtubule instability or a failure to prevent aggregation or remove toxic Tau aggregates. HSP70 and HSP90 can promote Tau solubility and its binding to microtubules as well as reduce insoluble Tau and p-Tau (
      • Dou F.
      • Netzer W.J.
      • Tanemura K.
      • Li F.
      • Hartl F.U.
      • Takashima A.
      • Gouras G.K.
      • Greengard P.
      • Xu H.
      Chaperones increase association of Tau protein with microtubules.
      ). Overexpression of inducible HSP70 reduces soluble and insoluble Tau levels in aged mice (
      • Petrucelli L.
      • Dickson D.
      • Kehoe K.
      • Taylor J.
      • Snyder H.
      • Grover A.
      • De Lucia M.
      • McGowan E.
      • Lewis J.
      • Prihar G.
      • Kim J.
      • Dillmann W.H.
      • Browne S.E.
      • Hall A.
      • Voellmy R.
      • Tsuboi Y.
      • Dawson T.M.
      • Wolozin B.
      • Hardy J.
      • Hutton M.
      CHIP and Hsp70 regulate Tau ubiquitination, degradation, and aggregation.
      ). However, with our late post-tangle intervention, we observed HSP levels inversely correlated only with soluble Tau oligomers but not insoluble Tau. Our findings may be specific to late intervention after NFT formation and in the plateau phase of insoluble Tau. In AD, HSP90 negatively correlates with granular Tau oligomers, suggesting that saturation of the HSP90 chaperone system may promote nascent Tau aggregation (
      • Sahara N.
      • Maeda S.
      • Yoshiike Y.
      • Mizoroki T.
      • Yamashita S.
      • Murayama M.
      • Park J.M.
      • Saito Y.
      • Murayama S.
      • Takashima A.
      Molecular chaperone-mediated Tau protein metabolism counteracts the formation of granular Tau oligomers in human brain.
      ), consistent with curcumin and HSP protection or clearance of still soluble misfolded Tau.
      The stimulatory effects of curcumin on levels of select HSPs may in part be explained by HSP90 binding and/or inhibitory activity (
      • Giommarelli C.
      • Zuco V.
      • Favini E.
      • Pisano C.
      • Dal Piaz F.
      • De Tommasi N.
      • Zunino F.
      The enhancement of antiproliferative and proapoptotic activity of HDAC inhibitors by curcumin is mediated by Hsp90 inhibition.
      ). HSP90 inhibition typically increases HSP90 and HSP70 levels via feedback to HSF-1 (
      • Dickey C.A.
      • Koren J.
      • Zhang Y.J.
      • Xu Y.F.
      • Jinwal U.K.
      • Birnbaum M.J.
      • Monks B.
      • Sun M.
      • Cheng J.Q.
      • Patterson C.
      • Bailey R.M.
      • Dunmore J.
      • Soresh S.
      • Leon C.
      • Morgan D.
      • Petrucelli L.
      Akt and CHIP coregulate Tau degradation through coordinated interactions.
      ). Curcumin elevated HSP90 protein in both cytosolic- and membrane-enriched fractions, which could enhance misfolded Tau clearance by CHIP/HSP90. HSP90 inhibition is also consistent with curcumin correction of transgene-dependent HSP70 and HSP70/HSP72 defects. Furthermore, both Tau dimer and the HSP90 client kinase Fyn (
      • Citri A.
      • Harari D.
      • Shohat G.
      • Ramakrishnan P.
      • Gan J.
      • Lavi S.
      • Eisenstein M.
      • Kimchi A.
      • Wallach D.
      • Pietrokovski S.
      • Yarden Y.
      Hsp90 recognizes a common surface on client kinases.
      ,
      • Maloney A.
      • Workman P.
      HSP90 as a new therapeutic target for cancer therapy. The story unfolds.
      ) were reduced by curcumin (Fig. 2C) without impacting Ser(P)-262 Tau (not shown), a Tau species unrecognized by the CHIP/HSP90 complex (
      • Dickey C.A.
      • Koren J.
      • Zhang Y.J.
      • Xu Y.F.
      • Jinwal U.K.
      • Birnbaum M.J.
      • Monks B.
      • Sun M.
      • Cheng J.Q.
      • Patterson C.
      • Bailey R.M.
      • Dunmore J.
      • Soresh S.
      • Leon C.
      • Morgan D.
      • Petrucelli L.
      Akt and CHIP coregulate Tau degradation through coordinated interactions.
      ).
      However, brain levels of curcumin were only ∼200 nm, well below the ∼6 μm IC50 for HSP90 inhibition by curcumin and in our hTau mice HSP levels were altered in the absence of HSP70 or HSP90 mRNA increases (not shown) induced by HSP90 inhibitors. Unlike classical HSP induction by toxins and HSP90 inhibitors, curcumin up-regulated HSP70 only in the membrane fraction but did not reduce other AD-relevant HSP90 client proteins such as Akt (not shown). Furthermore, similar levels of brain curcumin did not increase HSP levels in wild-type mice (not shown). Thus, curcumin may act via an alternative mechanism selectively altering HSP90 co-chaperone interactions, for example through a high affinity (6 nm) HSP90 binding site of unknown function (
      • Giommarelli C.
      • Zuco V.
      • Favini E.
      • Pisano C.
      • Dal Piaz F.
      • De Tommasi N.
      • Zunino F.
      The enhancement of antiproliferative and proapoptotic activity of HDAC inhibitors by curcumin is mediated by Hsp90 inhibition.
      ).
      In addition to correcting transgene-dependent defects, curcumin also increased membrane-bound HSC70, a neuroprotective synaptic and lipid raft component (
      • Chen J.
      • Wanming D.
      • Zhang D.
      • Liu Q.
      • Kang J.
      Water-soluble antioxidants improve the antioxidant and anticancer activity of low concentrations of curcumin in human leukemia cells.
      ) required for Tau chaperone-mediated autophagy (
      • Wang Y.
      • Martinez-Vicente M.
      • Krüger U.
      • Kaushik S.
      • Wong E.
      • Mandelkow E.M.
      • Cuervo A.M.
      • Mandelkow E.
      Tau fragmentation, aggregation and clearance. The dual role of lysosomal processing.
      ). Therefore, curcumin actions on Tau may be mediated by HSP90- and HSP70/HSC70-related mechanisms.
      We cannot fully exclude HSP-independent mechanisms such as curcumin binding to NFTs (
      • Mohorko N.
      • Repovs G.
      • Popovi M.
      • Kovacs G.G.
      • Bresjanac M.
      Curcumin labeling of neuronal fibrillar Tau inclusions in human brain samples.
      ) and PHF β-pleated sheet cores (
      • Landau M.
      • Sawaya M.R.
      • Faull K.F.
      • Laganowsky A.
      • Jiang L.
      • Sievers S.A.
      • Liu J.
      • Barrio J.R.
      • Eisenberg D.
      Towards a pharmacophore for amyloid.
      ), which could directly inhibit Tau formation. Alternatively, because amyloid probes can induce HSPs (
      • Alavez S.
      • Vantipalli M.C.
      • Zucker D.J.
      • Klang I.M.
      • Lithgow G.J.
      Amyloid-binding compounds maintain protein homeostasis during ageing and extend lifespan.
      ), it is possible that curcumin binds to Tau (
      • Landau M.
      • Sawaya M.R.
      • Faull K.F.
      • Laganowsky A.
      • Jiang L.
      • Sievers S.A.
      • Liu J.
      • Barrio J.R.
      • Eisenberg D.
      Towards a pharmacophore for amyloid.
      ) and stimulates HSPs. Nor can we exclude lipid peroxidation and nitration, which are also inhibited by curcumin (
      • Begum A.N.
      • Jones M.R.
      • Lim G.P.
      • Morihara T.
      • Kim P.
      • Heath D.D.
      • Rock C.L.
      • Pruitt M.A.
      • Yang F.
      • Hudspeth B.
      • Hu S.
      • Faull K.F.
      • Teter B.
      • Cole G.M.
      • Frautschy S.A.
      Curcumin structure-function, bioavailability, and efficacy in models of neuroinflammation and Alzheimer's disease.
      ) and influence Tau polymerization and aggregate stability (
      • Gamblin T.C.
      • King M.E.
      • Kuret J.
      • Berry R.W.
      • Binder L.I.
      Oxidative regulation of fatty acid-induced Tau polymerization.
      ,
      • Horiguchi T.
      • Uryu K.
      • Giasson B.I.
      • Ischiropoulos H.
      • LightFoot R.
      • Bellmann C.
      • Richter-Landsberg C.
      • Lee V.M.
      • Trojanowski J.Q.
      Nitration of Tau protein is linked to neurodegeneration in tauopathies.
      ,
      • Vana L.
      • Kanaan N.M.
      • Hakala K.
      • Weintraub S.T.
      • Binder L.I.
      Peroxynitrite-induced nitrative and oxidative modifications alter Tau filament formation.
      ,
      • Reynolds M.R.
      • Berry R.W.
      • Binder L.I.
      Site-specific nitration and oxidative dityrosine bridging of the Tau protein by peroxynitrite. Implications for Alzheimer's disease.
      ). However, in this paradigm, curcumin did not reduce inducible nitric-oxide synthase expression (not shown), and these alternatives do not explain the persistence of Ser(P)262 Tau after curcumin treatment. In contrast, Ser(P)262 persistence and Fyn loss are consistent with selectively enhanced HSP90 clearance.
      Prior work with 12-month-old hTau mice demonstrated spatial and non-spatial memory deficits in the presence of intact sensorimotor function (
      • Polydoro M.
      • Acker C.M.
      • Duff K.
      • Castillo P.E.
      • Davies P.
      Age-dependent impairment of cognitive and synaptic function in the hTau mouse model of Tau pathology.
      ). Our behavioral experiments with 19–20 month old hTau mice illustrate more profound deficits in hidden-platform MWM that likely reflect both the previously described memory deficits and additional motivational deficits associated with further aging in this model given the slower swim speeds in the MWM and decreased active exploration on novel object recognition. Although additional contributions of visual or motor dysfunction cannot be entirely ruled out, the hypothesized presence of apathy in untreated hTau mice is consistent with previous work demonstrating apathy in other models of AD (
      • Filali M.
      • Lalonde R.
      • Rivest S.
      Cognitive and non-cognitive behaviors in an APPswe/PS1 bigenic model of Alzheimer's disease.
      ) and correlations between apathy and cortical Tau pathology in human AD (
      • Marshall G.A.
      • Fairbanks L.A.
      • Tekin S.
      • Vinters H.V.
      • Cummings J.L.
      Neuropathologic correlates of apathy in Alzheimer's disease.
      ). Regardless of whether the behavioral deficits seen in aged hTau mice are primarily attributable to cognitive, motivational, visual, and/or motor impairments, these deficits were ameliorated by chronic curcumin treatment. We hypothesize that curcumin-related lowering of select Tau species and correction of excitatory synaptic markers associated with normal neuronal signaling underlies the improvement in MWM indices seen in treated hTau animals.
      In summary, intervention with curcumin at late stage in tangle-bearing hTau mice reduces soluble Tau oligomers and Fyn, perhaps through increasing HSP70, HSP90, and HSC70, which have the potential to clear misfolded Tau. These changes by curcumin improve the excitatory synaptic profile that may contribute to the improved behavioral performance. These data provide rationale for this bioavailable curcumin formulation approved for clinical use (
      • Gota V.S.
      • Maru G.B.
      • Soni T.G.
      • Gandhi T.R.
      • Kochar N.
      • Agarwal M.G.
      Safety and pharmacokinetics of a solid lipid curcumin particle formulation in osteosarcoma patients and healthy volunteers.
      ) to be a candidate for the treatment of tauopathies, including AD.

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