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The Dynamics and Turnover of Tau Aggregates in Cultured Cells

INSIGHTS INTO THERAPIES FOR TAUOPATHIES*
  • Jing L. Guo
    Correspondence
    To whom correspondence may be addressed: Center for Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, 3rd floor Maloney Bldg., 3600 Spruce St., Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909.
    Affiliations
    Department of Pathology and Laboratory Medicine, Institute on Aging and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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  • Arjan Buist
    Affiliations
    Neuroscience Department, Janssen Research and Development, a Division of Janssen Pharmaceutica NV, 2340 Beerse, Belgium
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  • Alberto Soares
    Affiliations
    Neuroscience Department, Janssen Research and Development, a Division of Janssen Pharmaceutica NV, 2340 Beerse, Belgium
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  • Kathleen Callaerts
    Affiliations
    Neuroscience Department, Janssen Research and Development, a Division of Janssen Pharmaceutica NV, 2340 Beerse, Belgium
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  • Sara Calafate
    Affiliations
    Neuroscience Department, Janssen Research and Development, a Division of Janssen Pharmaceutica NV, 2340 Beerse, Belgium
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  • Frederik Stevenaert
    Affiliations
    Neuroscience Department, Janssen Research and Development, a Division of Janssen Pharmaceutica NV, 2340 Beerse, Belgium
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  • Joshua P. Daniels
    Affiliations
    Department of Pathology and Laboratory Medicine, Institute on Aging and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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  • Bryan E. Zoll
    Affiliations
    Department of Pathology and Laboratory Medicine, Institute on Aging and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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  • Alex Crowe
    Affiliations
    Department of Pathology and Laboratory Medicine, Institute on Aging and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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  • Kurt R. Brunden
    Affiliations
    Department of Pathology and Laboratory Medicine, Institute on Aging and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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  • Diederik Moechars
    Affiliations
    Neuroscience Department, Janssen Research and Development, a Division of Janssen Pharmaceutica NV, 2340 Beerse, Belgium
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  • Virginia M.Y. Lee
    Correspondence
    To whom correspondence may be addressed: Center for Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, 3rd floor Maloney Bldg., 3600 Spruce St., Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909.
    Affiliations
    Department of Pathology and Laboratory Medicine, Institute on Aging and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health NIA Grant AG17586, the CurePSP Foundation, and a postdoctoral fellowship award (to J. L. G.) from Sophie M. Moyer and other donors of the Alzheimer's disease research program of the BrightFocus Foundation. Some coauthors work for Janssen, Pharmaceutical Companies of Johnson & Johnson. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
    This article contains supplemental Movies S1–S4.
Open AccessPublished:April 18, 2016DOI:https://doi.org/10.1074/jbc.M115.712083
      Filamentous tau aggregates, the hallmark lesions of Alzheimer disease (AD), play key roles in neurodegeneration. Activation of protein degradation systems has been proposed to be a potential strategy for removing pathological tau, but it remains unclear how effectively tau aggregates can be degraded by these systems. By applying our previously established cellular model system of AD-like tau aggregate induction using preformed tau fibrils, we demonstrate that tau aggregates induced in cells with regulated expression of full-length mutant tau can be gradually cleared when soluble tau expression is suppressed. This clearance is at least partially mediated by the autophagy-lysosome pathway, although both the ubiquitin-proteasome system and the autophagy-lysosome pathway are deficient in handling large tau aggregates. Importantly, residual tau aggregates left after the clearance phase leads to a rapid reinstatement of robust tau pathology once soluble tau expression is turned on again. Moreover, we succeeded in generating monoclonal cells persistently carrying tau aggregates without obvious cytotoxicity. Live imaging of GFP-tagged tau aggregates showed that tau inclusions are dynamic structures constantly undergoing “fission” and “fusion,” which facilitate stable propagation of tau pathology in dividing cells. These findings provide a greater understanding of cell-to-cell transmission of tau aggregates in dividing cells and possibly neurons.

      Introduction

      Filamentous aggregates made up of hyperphosphorylated tau protein are the defining pathological feature of numerous neurodegenerative diseases, such as Alzheimer disease, corticobasal degeneration, progressive supranuclear palsy, and Pick disease, which are collectively termed tauopathies (reviewed by Ref.
      • Lee V.M.
      • Goedert M.
      • Trojanowski J.Q.
      Neurodegenerative tauopathies.
      ). Physiologically, tau is a highly soluble microtubule-associated protein important for the assembly and stability of microtubules (
      • Cleveland D.W.
      • Hwo S.Y.
      • Kirschner M.W.
      Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin.
      ,
      • Drechsel D.N.
      • Hyman A.A.
      • Cobb M.H.
      • Kirschner M.W.
      Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau.
      ). Insoluble, aggregated tau that is hyperphosphorylated and conformationally altered not only loses its physiological role of binding microtubules, but can also physically interfere with normal functioning of other cellular components (reviewed by Ref.
      • Ballatore C.
      • Lee V.M.
      • Trojanowski J.Q.
      tau-mediated neurodegeneration in Alzheimer's disease and related disorders.
      ). Strong correlations of the distribution and severity of tau pathology with clinical phenotypes of tauopathy patients (
      • Arriagada P.V.
      • Growdon J.H.
      • Hedley-Whyte E.T.
      • Hyman B.T.
      Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease.
      ,
      • Ling H.
      • Ling H.
      • de Silva R.
      • Massey L.A.
      • Courtney R.
      • Hondhamuni G.
      • Bajaj N.
      • Lowe J.
      • Holton J.L.
      • Lees A.
      • Revesz T.
      Characteristics of progressive supranuclear palsy presenting with corticobasal syndrome: a cortical variant.
      ,
      • Kouri N.
      • Murray M.E.
      • Hassan A.
      • Rademakers R.
      • Uitti R.J.
      • Boeve B.F.
      • Graff-Radford N.R.
      • Wszolek Z.K.
      • Litvan I.
      • Josephs K.A.
      • Dickson D.W.
      Neuropathological features of corticobasal degeneration presenting as corticobasal syndrome or Richardson syndrome.
      ) support the key contribution of aggregated tau to neuronal dysfunction and degeneration in these diseases, although some studies suggest pre-fibrillar tau species, such as oligomers, could be equally, if not more toxic than mature tau fibrils (
      • 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.
      ,
      • Wittmann C.W.
      • Wszolek M.F.
      • Shulman J.M.
      • Salvaterra P.M.
      • Lewis J.
      • Hutton M.
      • Feany M.B.
      Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles.
      ,
      • Yoshiyama Y.
      • Higuchi M.
      • Zhang B.
      • Huang S.M.
      • Iwata N.
      • Saido T.C.
      • Maeda J.
      • Suhara T.
      • Trojanowski J.Q.
      • Lee V.M.
      Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model.
      ,
      • Weingarten M.D.
      • Lockwood A.H.
      • Hwo S.Y.
      • Kirschner M.W.
      A protein factor essential for microtubule assembly.
      ).
      One obvious strategy to treat tauopathies is to remove intracellular tau aggregates either by promoting the disassembly of tau fibrils or by activating cellular degradation machineries to clear these toxic entities. A recent study demonstrated that it is possible to reverse mature tau inclusions together with associated neuronal deficits by suppressing soluble tau expression in a mouse model with inducible expression of tau, but the exact mechanism of tau pathology clearance was not explored (
      • Polydoro M.
      • de Calignon A.
      • Suárez-Calvet M.
      • Sanchez L.
      • Kay K.R.
      • Nicholls S.B.
      • Roe A.D.
      • Pitstick R.
      • Carlson G.A.
      • Gómez-Isla T.
      • Spires-Jones T.L.
      • Hyman B.T.
      Reversal of neurofibrillary tangles and tau-associated phenotype in the rTgTauEC model of early Alzheimer's disease.
      ). There are two primary protein degradation systems in cells, the ubiquitin-proteasome system (UPS),
      The abbreviations used are: UPS
      ubiquitin-proteasome system
      AD
      Alzheimer disease
      ALP
      autophagy-lysosome pathway
      α-Syn
      α-synuclein
      BafA1
      bafilomycin A1
      c-lac
      clasto-lactacystin β-lactone
      CQ
      chloroquine diphosphate
      Dox
      doxycycline
      epox
      epoxomicin
      PFA
      paraformaldehyde
      PFF
      preformed fibril
      ANOVA
      analysis of variance.
      which is responsible for the turnover of soluble and/or short-lived proteins, and the autophagy-lysosome pathway (ALP), which is important for the degradation of insoluble and/or longer-lived proteins as well as damaged cellular organelles (reviewed by Ref.
      • Rubinsztein D.C.
      The roles of intracellular protein-degradation pathways in neurodegeneration.
      ). The majority of existing studies on tau degradation focused on soluble or monomeric tau, which was shown to be the substrate of both UPS and ALP (reviewed by Ref.
      • Chesser A.S.
      • Pritchard S.M.
      • Johnson G.V.
      Tau clearance mechanisms and their possible role in the pathogenesis of Alzheimer disease.
      ). Several papers demonstrated that stimulation of autophagy by trehalose or rapamycin can ameliorate tau pathology both in cultured cells and transgenic mice overexpressing mutant tau (
      • Krüger U.
      • Wang Y.
      • Kumar S.
      • Mandelkow E.M.
      Autophagic degradation of tau in primary neurons and its enhancement by trehalose.
      ,
      • Schaeffer V.
      • Lavenir I.
      • Ozcelik S.
      • Tolnay M.
      • Winkler D.T.
      • Goedert M.
      Stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy.
      ,
      • Caccamo A.
      • Magrì A.
      • Medina D.X.
      • Wisely E.V.
      • Lopez-Aranda M.F.
      • Silva A.J.
      • Oddo S.
      mTOR regulates tau phosphorylation and degradation: implications for Alzheimer's disease and other tauopathies.
      ). However, it is not clear whether autophagy participates in the basal degradation of insoluble tau without pharmacological activation. More importantly, tau aggregates induced purely by overexpression of mutant tau in these models may be qualitatively different from authentic tau pathology developed in diseased brains (
      • Guo J.L.
      • Lee V.M.
      Seeding of normal Tau by pathological tau conformers drives pathogenesis of Alzheimer-like tangles.
      ,
      • Iba M.
      • Guo J.L.
      • McBride J.D.
      • Zhang B.
      • Trojanowski J.Q.
      • Lee V.M.
      Synthetic Tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of Alzheimer's-like tauopathy.
      ). Furthermore, whereas inhibition of ALP has been shown to induce tau deposition (
      • Bi X.
      • Haque T.S.
      • Zhou J.
      • Skillman A.G.
      • Lin B.
      • Lee C.E.
      • Kuntz I.D.
      • Ellman J.A.
      • Lynch G.
      Novel cathepsin D inhibitors block the formation of hyperphosphorylated tau fragments in hippocampus.
      ,
      • Hamano T.
      • Gendron T.F.
      • Causevic E.
      • Yen S.H.
      • Lin W.L.
      • Isidoro C.
      • Deture M.
      • Ko L.W.
      Autophagic-lysosomal perturbation enhances tau aggregation in transfectants with induced wild-type tau expression.
      ,
      • Inoue K.
      • Rispoli J.
      • Kaphzan H.
      • Klann E.
      • Chen E.I.
      • Kim J.
      • Komatsu M.
      • Abeliovich A.
      Macroautophagy deficiency mediates age-dependent neurodegeneration through a phospho-tau pathway.
      ), the effect of tau aggregation on ALP has not been well studied.
      We previously established a cell model with robust tau aggregates closely resembling neurofibrillary tangles in the AD brains, whereby preformed tau fibrils (tau PFFs) were transduced into QBI-293 cells transiently transfected with mutant tau to “seed” the fibrillization of soluble tau (
      • Guo J.L.
      • Lee V.M.
      Seeding of normal Tau by pathological tau conformers drives pathogenesis of Alzheimer-like tangles.
      ). We now adapt this model to a stable cell line with inducible expression of mutant tau, which allows us to study the turnover of tau aggregates upon suppression of tau expression. A GFP tag attached to the overexpressed tau further enables us to directly visualize the dynamics of PFF-induced tau aggregates through live imaging. In addition, we successfully generated a clone stably carrying tau aggregates due to faithful propagation of misfolded tau seeds to daughter cells during cell division. With the improved system, we found that tau aggregates can be gradually cleared from cells when soluble tau expression is turned off and this clearance process is at least partially mediated by the ALP. However, the presence of even a minute amount of residual aggregates is sufficient to rapidly reinstate tau aggregation once tau expression is turned on again. These observations increase our understanding of cellular responses to tau aggregates, and have implications regarding therapeutic strategies directed toward modulation of tau expression or enhancement of the ALP pathway in tauopathies.

      Discussion

      By using a cell line with inducible expression of tau, we demonstrated that even high burdens of tau aggregates can be gradually removed with extended suppression of soluble tau expression, and the ALP is at least partially mediating the clearance process. Nevertheless, a small amount of aggregates remaining at the end of the clearance phase can lead to rapid re-emergence of robust tau pathology once soluble tau expression is restored. In addition, live imaging of these aggregate-bearing cells revealed that tau aggregates are dynamic structures that can undergo fusion and fission, and they can be stably carried in dividing cells due to ready partitioning into daughter cells during mitosis.
      In the past few years, mounting evidence suggests that protein aggregates involved in various neurodegenerative diseases, including tau aggregates, are self-amplifying entities that can propagate along neuroanatomical connections, leading to disease progression from restricted brain areas in the early phase of the disease to widespread brain regions in the later stages (reviewed by Ref.
      • Guo J.L.
      • Lee V.M.
      Cell-to-cell transmission of pathogenic proteins in neurodegenerative diseases.
      ). In vitro studies on disease-associated proteins such as prion and tau showed that fragmentation of long fibrils, usually achieved by sonication, is critical for efficient seeded amplification (
      • Meyer V.
      • Dinkel P.D.
      • Rickman Hager E.
      • Margittai M.
      Amplification of tau fibrils from minute quantities of seeds.
      ,
      • Knowles T.P.
      • Waudby C.A.
      • Devlin G.L.
      • Cohen S.I.
      • Aguzzi A.
      • Vendruscolo M.
      • Terentjev E.M.
      • Welland M.E.
      • Dobson C.M.
      An analytical solution to the kinetics of breakable filament assembly.
      ). Moreover, it has been further demonstrated that only short tau fibrils, but not long fibrils, can be internalized and transported by neurons (
      • Wu J.W.
      • Herman M.
      • Liu L.
      • Simoes S.
      • Acker C.M.
      • Figueroa H.
      • Steinberg J.I.
      • Margittai M.
      • Kayed R.
      • Zurzolo C.
      • Di Paolo G.
      • Duff K.E.
      Small misfolded tau species are internalized via bulk endocytosis and anterogradely and retrogradely transported in neurons.
      ). Therefore, it is unclear how the bulky, apparently immobile tau inclusions detected in the tauopathy brains could undergo cell-to-cell transmission. Previous studies suggested that molecules within fibrillar aggregates can freely dissociate from one filament and re-associate with another filament (
      • Carulla N.
      • Caddy G.L.
      • Hall D.R.
      • Zurdo J.
      • Gairí M.
      • Feliz M.
      • Giralt E.
      • Robinson C.V.
      • Dobson C.M.
      Molecular recycling within amyloid fibrils.
      ,
      • Belitzky A.
      • Melamed-Book N.
      • Weiss A.
      • Raviv U.
      The dynamic nature of amyloid β(1–40) aggregation.
      ). This dynamic equilibrium between aggregates and their subunits was also supported by an in vivo study on tau (
      • Sydow A.
      • Mandelkow E.M.
      ‘Prion-like’ propagation of mouse and human tau aggregates in an inducible mouse model of tauopathy.
      ). By visualizing GFP-tagged tau aggregates in living cells, our current study provides direct evidence of the dynamics of intracellular tau aggregates at the macroscopic level. Not surprisingly, small tau aggregates appear to be mobile entities that are highly diffusible in cells and have a great propensity to coalesce into larger aggregates when soluble tau expression is high. More interestingly, even visually cumbersome aggregates can readily undergo morphological restructuring and fracture into smaller pieces during cell division, which get passed along into daughter cells. In some instances, small aggregates also seemed to dissociate from the edge of large aggregates in the absence of cell division. Assuming that this dynamic nature of tau aggregates also occurs in neurons, these results suggest that seemingly inert tau inclusions can release small seeds for transport along axons, with subsequent release and spreading to other neurons. Furthermore, fragmentation of tau aggregates into daughter cells offers a potential mechanistic explanation for pathological tau transmission in glial cells, which are shown to proliferate in neurodegenerative diseases (reviewed by Ref.
      • Glass C.K.
      • Saijo K.
      • Winner B.
      • Marchetto M.C.
      • Gage F.H.
      Mechanisms underlying inflammation in neurodegeneration.
      ).
      We showed here that the ALP was involved in the clearance of tau aggregates when soluble tau expression was turned off, as supported by frequent colocalization of focal tau inclusions with autophagy markers and slower reduction of insoluble tau upon lysosomal inhibition. However, it is possible that spontaneous disassembly of tau aggregates into smaller species, such as oligomers and even monomers, may also be taking place. In fact, it has been demonstrated in vitro that amyloid fibrils made up of tau, Aβ, or α-Syn readily undergo disaggregation when diluted in monomer-free solution (
      • Shammas S.L.
      • Garcia G.A.
      • Kumar S.
      • Kjaergaard M.
      • Horrocks M.H.
      • Shivji N.
      • Mandelkow E.
      • Knowles T.P.
      • Mandelkow E.
      • Klenerman D.
      A mechanistic model of tau amyloid aggregation based on direct observation of oligomers.
      ,
      • Harper J.D.
      • Wong S.S.
      • Lieber C.M.
      • Lansbury Jr., P.T.
      Assembly of A beta amyloid protofibrils: an in vitro model for a possible early event in Alzheimer's disease.
      ,
      • Cremades N.
      • Cohen S.I.
      • Deas E.
      • Abramov A.Y.
      • Chen A.Y.
      • Orte A.
      • Sandal M.
      • Clarke R.W.
      • Dunne P.
      • Aprile F.A.
      • Bertoncini C.W.
      • Wood N.W.
      • Knowles T.P.
      • Dobson C.M.
      • Klenerman D.
      Direct observation of the interconversion of normal and toxic forms of α-synuclein.
      ,
      • Tang L.
      • Li H.T.
      • Du H.N.
      • Zhang F.
      • Hu X.F.
      • Hu H.Y.
      Study of the disassembly-assembly process of α-synuclein fibrils by in situ atomic force microscopy.
      ), which is somewhat analogous to the cellular condition when soluble tau expression is suppressed. Reduced concentration of soluble tau in the cytoplasm may shift the dynamic equilibrium of tau aggregation, resulting in a net disassembly of aggregates. This hypothesis of concentration-dependent equilibrium is supported by live imaging of clone 4.1 cells, which showed that dissociation of small aggregates from large inclusions was accompanied by a gradual decrease in the overall sizes of aggregates when tau expression was turned off, but not when cells were highly expressing tau. Moreover, ineffective lysosomal degradation of insoluble tau when tau expression is high in our cell model suggests the ALP may be incapable of handling relatively bulky tau aggregates, although the latter appear to be actively recruiting autophagosomes. Therefore, spontaneous disintegration of large tau aggregates is likely occurring upon suppression of tau expression to facilitate degradation by the ALP. The lack of obvious colocalization of lysosomes with tau inclusions may imply that the conspicuous aggregates are rarely delivered into the lysosomes, and that the actual tau species being degraded are probably very short filaments or oligomers that dissociate from the large aggregates during disassembly.
      Although prior work (
      • Polydoro M.
      • de Calignon A.
      • Suárez-Calvet M.
      • Sanchez L.
      • Kay K.R.
      • Nicholls S.B.
      • Roe A.D.
      • Pitstick R.
      • Carlson G.A.
      • Gómez-Isla T.
      • Spires-Jones T.L.
      • Hyman B.T.
      Reversal of neurofibrillary tangles and tau-associated phenotype in the rTgTauEC model of early Alzheimer's disease.
      ) and our study suggest it is possible to remove intracellular tau aggregates by suppressing soluble tau expression, there are several factors that should be considered in adopting this strategy as a therapeutic approach for tauopathies. First, one of the detrimental consequences of tau aggregation is suggested to be reduced microtubule stability due to sequestration of soluble tau (
      • Brunden K.R.
      • Zhang B.
      • Carroll J.
      • Yao Y.
      • Potuzak J.S.
      • Hogan A.M.
      • Iba M.
      • James M.J.
      • Xie S.X.
      • Ballatore C.
      • Smith 3rd, A.B.
      • Lee V.M.
      • Trojanowski J.Q.
      Epothilone D improves microtubule density, axonal integrity, and cognition in a transgenic mouse model of tauopathy.
      ). This deficit will be exacerbated by reducing tau expression. Second, our study reveals that even miniscule amounts of tau aggregates remaining after clearance are sufficient to promote robust pathology if soluble tau expression is restored, and irreversible clearance only occurred with a more extended period of soluble tau removal. Moreover, in an earlier study using transgenic mice with inducible expression of tau, neurofibrillary tangles continued to accumulate in mice when the suppression of soluble tau expression was incomplete (
      • 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.
      ). These findings pose challenges for a potential therapeutic approach of reducing normal tau, as it is unclear how long it would take for well established tau inclusions to be thoroughly cleared from human brains without residual seeding-competent tau species, and it is also technically challenging and potentially unsafe to maintain appreciable suppression of tau expression on a long-term basis. Third, our live imaging experiment showed that small tau aggregates generated during the clearance phase are exceptionally mobile, rendering them possibly even more harmful to cells than the bulkier and more consolidated aggregates, as has been proposed for oligomers and protofibrils (reviewed in Refs.
      • Caughey B.
      • Lansbury P.T.
      Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders.
      and
      • Haass C.
      • Selkoe D.J.
      Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.
      ). Moreover, the highly diffusible misfolded tau seeds, if not thoroughly cleared, may spread more readily along neuroanatomically connected pathways, leading to even wider distribution of pathology once tau expression is resumed.
      Our observation that QBI-293 cells are able to stably propagate tau inclusions for months without obvious cellular toxicity suggests aggregated tau could be well tolerated by cells. Although we cannot exclude intrinsic differences in resilience between immortalized dividing cells in culture and post-mitotic neurons in human brain, our study reveals that the toxicity of tau aggregates could be partly determined by their effects on protein degradation systems. In our clone 4.1 cells, despite local recruitment of autophagosomes by tau aggregates, there is no appreciable accumulation of ALP components overall and the autophagy flux remains normal. We also found few interactions between the UPS and tau inclusions. In short, both the UPS and ALP are not strongly responding to tau aggregation and remain largely unperturbed in our cell model. On the other hand, significant accumulation of autophagic vacuoles (pre-lysosomal autophagy vesicles) is observed in AD brains, especially in the dystrophic neurites and perikarya of neurons containing tau filaments (
      • Nixon R.A.
      • Wegiel J.
      • Kumar A.
      • Yu W.H.
      • Peterhoff C.
      • Cataldo A.
      • Cuervo A.M.
      Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study.
      ). Also unlike our cell model, a subset of neurofibrillary tangles are ubiquitinated in AD brains, although ubiquitination was suggested to be a late event (
      • Cole G.M.
      • Timiras P.S.
      Ubiquitin-protein conjugates in Alzheimer's lesions.
      ,
      • Brion J.P.
      • Power D.
      • Hue D.
      • Couck A.M.
      • Anderton B.H.
      • Flament-Durand J.
      Heterogeneity of ubiquitin immunoreactivity in neurofibrillary tangles of Alzheimer's disease.
      ,
      • He Y.
      • Duyckaerts C.
      • Delaère P.
      • Piette F.
      • Hauw J.J.
      Alzheimer's lesions labelled by anti-ubiquitin antibodies: comparison with other staining techniques: a study of 15 cases with graded intellectual status in ageing and Alzheimer's disease.
      ). Because degradation by UPS requires unfolding and translocation of substrates into the catalytic core of proteasome, this pathway is ill-suited at degrading physically cumbersome protein aggregates. Our current study suggests that, despite a modulatory role of the ALP in clearing tau aggregates when soluble tau expression is suppressed, the ALP is also inefficient at handling large tau aggregates, and similar findings were made in our earlier study on α-Syn aggregates (
      • Tanik S.A.
      • Schultheiss C.E.
      • Volpicelli-Daley L.A.
      • Brunden K.R.
      • Lee V.M.
      Lewy body-like α-synuclein aggregates resist degradation and impair macroautophagy.
      ). Furthermore, when cells fail to clear large tau or α-Syn aggregates by activating both UPS and ALP, the excessive engagement of degradation machineries with aggregates may actually compromise the degradation of other proteins, resulting in cellular dysfunction in the long run. In fact, reduced proteasome activity found in AD brains was demonstrated to be caused by neurofibrillary tangles interacting with proteasomes (
      • Keck S.
      • Nitsch R.
      • Grune T.
      • Ullrich O.
      Proteasome inhibition by paired helical filament-tau in brains of patients with Alzheimer's disease.
      ,
      • Keller J.N.
      • Hanni K.B.
      • Markesbery W.R.
      Impaired proteasome function in Alzheimer's disease.
      ). Therefore, whereas it is generally believed that activating cellular degradation systems represents a promising therapeutic strategy for neurodegenerative diseases characterized by protein aggregation, our studies lead us to propose that a more beneficial approach may be to identify means to inhibit unproductive interactions between protein aggregates and components of the protein degradation systems, so as to free the latter for their normal functions.
      In conclusion, our data provide new understandings of the dynamics of tau aggregates in mediating cell-to-cell transmission in dividing cells and perhaps in neurons. Moreover, our elucidation of the turnover of tau aggregates, as well as their interactions with protein degradation systems, offers new insights into potential therapeutic strategies for neurodegenerative tauopathies. In particular, our data raise concerns about a therapeutic approach of lowering tau expression, and call for a reconsideration of therapeutic attempts to enhance protein degradation systems. Indeed, our results suggest that small molecules or biologics that can prevent tau aggregates from engaging cellular degradation machineries may allow for the continued function of these critical cellular components, thereby reducing cellular stress and toxicity.

      Author Contributions

      J. L. G. conceived and designed the study, performed and analyzed the experiments, and wrote the paper. V. M. L., D. M., and K. R. B. conceived, designed and coordinated the study, and edited the paper. A. B., A. S., K. C., S. C., F. S., J. P. D., B. E. Z., and A. C. performed and analyzed the experiments. All authors reviewed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Dawn Riddle, Lakshmi Changolkar, and Chris Chung for technical assistance, Dr. Selcuk Tanik for technical advice, and all the other members of the Center for Neurodegenerative Disease Research for their help and support. Monoclonal antibody PHF-1 was a generous gift from Dr. Peter Davies.

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