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Distinct Therapeutic Mechanisms of Tau Antibodies

PROMOTING MICROGLIAL CLEARANCE VERSUS BLOCKING NEURONAL UPTAKE*
  • Kristen E. Funk
    Affiliations
    Department of Neurology, Washington University in St. Louis, St. Louis, Missouri 63110
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  • Hilda Mirbaha
    Affiliations
    Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center, Dallas, Texas 75390
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  • Hong Jiang
    Affiliations
    Department of Neurology, Washington University in St. Louis, St. Louis, Missouri 63110
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  • David M. Holtzman
    Footnotes
    Affiliations
    Department of Neurology, Washington University in St. Louis, St. Louis, Missouri 63110
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  • Marc I. Diamond
    Correspondence
    To whom correspondence should be addressed: Center for Alzheimer's and Neurodegenerative Diseases, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390. Tel.: 214-648-8857.
    Affiliations
    Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center, Dallas, Texas 75390
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  • Author Footnotes
    * This work was supported by the Alzheimer's Disease Research Program of the BrightFocus Foundation (to K. E. F.) and the Tau Consortium and National Institutes of Health Grant 1R01NS071835 (to M. I. D.). M. I. D., D. M. H., and H. J. are inventors on a submitted patent “Antibodies to Tau” that is licensed by Washington University. M. I. D. is a cofounder of ARTA Biosciences. D. M. H. cofounded and is on the scientific advisory board C2N Diagnostics. D. M. H. consults for Genentech, AstraZeneca, Neurophage, and Eli Lily. H. J., D. M. H., and M. I. D. are inventors of the antibodies described here, all of which have been licensed to C2N Diagnostics, LLC.
    1 Supported by a grant from the Tau Consortium.
Open AccessPublished:June 30, 2015DOI:https://doi.org/10.1074/jbc.M115.657924
      Tauopathies are neurodegenerative diseases characterized by accumulation of Tau amyloids, and include Alzheimer disease and certain frontotemporal dementias. Trans-neuronal propagation of amyloid mediated by extracellular Tau may underlie disease progression. Consistent with this, active and passive vaccination studies in mouse models reduce pathology, although by unknown mechanisms. We previously reported that intracerebroventricular administration of three anti-Tau monoclonal antibodies (HJ8.5, HJ9.3, and HJ9.4) reduces pathology in a model overexpressing full-length mutant (P301S) human Tau. We now study effects of these three antibodies and a negative control antibody (HJ3.4) on Tau aggregate uptake into BV2 microglial-like cells and primary neurons. Antibody-independent Tau uptake into BV2 cells was blocked by heparin, consistent with a previously described role for heparan sulfate proteoglycans. Two therapeutic antibodies (HJ8.5 and HJ9.4) promoted uptake of full-length Tau fibrils into microglia via Fc receptors. Surprisingly, HJ9.3 promoted uptake of fibrils composed of the Tau repeat domain or Alzheimer disease-derived Tau aggregates, but failed to influence full-length recombinant Tau fibrils. Size fractionation of aggregates showed that antibodies preferentially promote uptake of larger oligomers (n ≥∼20-mer) versus smaller oligomers (n ∼10-mer) or monomer. No antibody inhibited uptake of full-length recombinant fibrils into primary neurons, but HJ9.3 blocked neuronal uptake of Tau repeat domain fibrils and Alzheimer disease-derived Tau. Antibodies thus have multiple potential mechanisms, including clearance via microglia and blockade of neuronal uptake. However these effects are epitope- and aggregate size-dependent. Establishing specific mechanisms of antibody activity in vitro may help in design and optimization of agents that are more effective in vivo.

      Introduction

      Tauopathies are neurodegenerative diseases characterized by the accumulation within neurons of the microtubule associated protein Tau (
      • Mandelkow E.-M.
      • Mandelkow E.
      Biochemistry and cell biology of tau protein in neurofibrillary degeneration.
      ). These include Alzheimer disease (AD),
      The abbreviations used are: AD
      Alzheimer disease
      amyloid-β
      RD
      repeat domain
      ODS
      octadecyl sulfate
      SEC
      size exclusion chromatography
      IP
      immunoprecipitation
      FcγR
      Fc γ receptor
      HSPG
      heparan sulfate proteoglycan
      PD
      polymeric dextran
      Bis-Tris
      2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol.
      which also features amyloid-β (Aβ) accumulation, and myriad dementia syndromes associated only with Tau accumulation. Tau pathology correlates with other measures of disease progression including neuronal dysfunction, synaptic loss, and functional decline (
      • 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.
      ,
      • Bancher C.
      • Braak H.
      • Fischer P.
      • Jellinger K.A.
      Neuropathological staging of Alzheimer lesions and intellectual status in Alzheimer's and Parkinson's disease patients.
      ,
      • 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.
      ,
      • Small S.A.
      • Duff K.
      Linking Aβ and tau in late-onset Alzheimer's disease: a dual pathway hypothesis.
      ), and progresses along neuroanatomical pathways in disease-specific patterns (
      • Braak H.
      • Braak E.
      Neuropathological stageing of Alzheimer-related changes.
      ,
      • Braak H.
      • Braak E.
      Staging of Alzheimer's disease-related neurofibrillary changes.
      ,
      • Braak H.
      • Thal D.R.
      • Ghebremedhin E.
      • Del Tredici K.
      Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years.
      ). The mechanism of progression is incompletely understood, but we and others have posited that Tau aggregates escape from diseased neurons into the extracellular space prior to entering adjacent, or synaptically connected cells. Aggregates then convert monomeric Tau to an amyloid by acting as a conformational template. This may mediate the relentless spread of Tau pathology in humans (
      • Holmes B.B.
      • Diamond M.I.
      Prion-like properties of Tau protein: the importance of extracellular Tau as a therapeutic target.
      ). Importantly, therapeutic interventions that prevent the uptake of aggregate seeds into naive neurons or promote microglial clearance might inhibit disease progression.
      Microglia, the brain's resident immune cells, might clear antibody-bound Tau from the extracellular space. A recent study reported that microglia display modest levels of phagocytic capacity for Tau oligomers that are increased by LPS activation (
      • Majerova P.
      • Zilkova M.
      • Kazmerova Z.
      • Kovac A.
      • Paholikova K.
      • Kovacech B.
      • Zilka N.
      • Novak M.
      Microglia display modest phagocytic capacity for extracellular tau oligomers.
      ). It is not clear how microglia might clear antibody-bound Tau. Considering that pro-inflammatory cytokines are chronically heightened in the aging brain (
      • Godbout J.P.
      • Chen J.
      • Abraham J.
      • Richwine A.F.
      • Berg B.M.
      • Kelley K.W.
      • Johnson R.W.
      Exaggerated neuroinflammation and sickness behavior in aged mice following activation of the peripheral innate immune system.
      ) as well as neurodegenerative diseases (
      • Griffin W.S.T.
      Inflammation and neurodegenerative diseases.
      ,
      • Mrak R.E.
      • Griffin W.S.T.
      Potential inflammatory biomarkers in Alzheimer's disease.
      ,
      • Streit W.J.
      • Conde J.R.
      • Fendrick S.E.
      • Flanary B.E.
      • Mariani C.L.
      Role of microglia in the central nervous system's immune response.
      ), the typical disease state may be sufficient to induce phagocytic activity in microglia.
      The model of transcellular propagation suggests that extracellular Tau might be vulnerable to antibody-mediated therapies. Importantly, we and others have observed that passive immunization with anti-Tau antibodies reduces pathology in several mouse models of tauopathy (
      • Asuni A.A.
      • Boutajangout A.
      • Quartermain D.
      • Sigurdsson E.M.
      Immunotherapy targeting pathological tau conformers in a tangle mouse model reduces brain pathology with associated functional improvements.
      ,
      • Boutajangout A.
      • Ingadottir J.
      • Davies P.
      • Sigurdsson E.M.
      Passive immunization targeting pathological phospho-tau protein in a mouse model reduces functional decline and clears tau aggregates from the brain.
      ,
      • Chai X.
      • Wu S.
      • Murray T.K.
      • Kinley R.
      • Cella C.V.
      • Sims H.
      • Buckner N.
      • Hanmer J.
      • Davies P.
      • O'Neill M.J.
      • Hutton M.L.
      • Citron M.
      Passive immunization with anti-Tau antibodies in two transgenic models: reduction of Tau pathology and delay of disease progression.
      ,
      • Yanamandra K.
      • Kfoury N.
      • Jiang H.
      • Mahan T.E.
      • Ma S.
      • Maloney S.E.
      • Wozniak D.F.
      • Diamond M.I.
      • Holtzman D.M.
      Anti-tau antibodies that block tau aggregate seeding in vitro markedly decrease pathology and improve cognition in vivo.
      ,
      • Yanamandra K.
      • Jiang H.
      • Mahan T.E.
      • Maloney S.E.
      • Wozniak D.F.
      • Diamond M.I.
      • Holtzman D.M.
      Anti-tau antibody reduces insoluble tau and decreases brain atrophy.
      ), but the mechanisms are unknown. Several theories have been proposed, including inhibition or reversal of fibril formation (
      • Bacskai B.J.
      • Kajdasz S.T.
      • McLellan M.E.
      • Games D.
      • Seubert P.
      • Schenk D.
      • Hyman B.T.
      Non-Fc-mediated mechanisms are involved in clearance of amyloid-β in vivo by immunotherapy.
      ,
      • Solomon B.
      • Koppel R.
      • Frankel D.
      • Hanan-Aharon E.
      Disaggregation of Alzheimer β-amyloid by site-directed mAb.
      ), clearance of intraneuronal fibrils via the lysosome (
      • Congdon E.E.
      • Gu J.
      • Sait H.B.R.
      • Sigurdsson E.M.
      Antibody uptake into neurons occurs primarily via clathrin-dependent Fcγ receptor endocytosis and is a prerequisite for acute tau protein clearance.
      ), and blocking neuronal uptake of Tau seeds (
      • Yanamandra K.
      • Kfoury N.
      • Jiang H.
      • Mahan T.E.
      • Ma S.
      • Maloney S.E.
      • Wozniak D.F.
      • Diamond M.I.
      • Holtzman D.M.
      Anti-tau antibodies that block tau aggregate seeding in vitro markedly decrease pathology and improve cognition in vivo.
      ). We hypothesized that therapeutic antibodies might promote uptake of Tau aggregates into microglia. We have tested this idea using two forms of recombinant Tau protein fibrillized in vitro, as well as AD-derived Tau assemblies, evaluating the effects of antibodies in cell culture systems.

      Author Contributions

      K. E. F. and M. I. D. designed the study and wrote the paper. H. M. prepared the SEC-separated Tau aggregates. H. J. and D. M. H. supplied the experimental antibodies. All authors analyzed the results and approved the final version of the manuscript.

      Acknowledgments

      We are grateful to Nigel Cairns, Erin Householder, the technical support of the Betty Martz Laboratory for Neurodegenerative Research, and the Knight Alzheimer's Disease Research Center at Washington University in St. Louis for brain tissue, supported by the following National Institutes of Health grants: P50AG005681 (Knight Alzheimer's Disease Research Center) and P01AG003991 (Healthy Aging and Senile Dementia).

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