Advertisement

Proteolytic shedding of the prion protein via activation of metallopeptidase ADAM10 reduces cellular binding and toxicity of amyloid-β oligomers

  • Heledd H. Jarosz-Griffiths
    Footnotes
    Affiliations
    Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, AV Hill Building, University of Manchester, Manchester Academic Health Science Centre, Oxford Road, Manchester M13 9PT,
    Search for articles by this author
  • Nicola J. Corbett
    Affiliations
    Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, AV Hill Building, University of Manchester, Manchester Academic Health Science Centre, Oxford Road, Manchester M13 9PT,
    Search for articles by this author
  • Helen A. Rowland
    Affiliations
    Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, AV Hill Building, University of Manchester, Manchester Academic Health Science Centre, Oxford Road, Manchester M13 9PT,
    Search for articles by this author
  • Kate Fisher
    Affiliations
    Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, AV Hill Building, University of Manchester, Manchester Academic Health Science Centre, Oxford Road, Manchester M13 9PT,
    Search for articles by this author
  • Alys C. Jones
    Affiliations
    Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, AV Hill Building, University of Manchester, Manchester Academic Health Science Centre, Oxford Road, Manchester M13 9PT,
    Search for articles by this author
  • Jennifer Baron
    Affiliations
    Flow Cytometry Facility Laboratory, Faculty of Biology, Medicine, and Health, University of Manchester, CTF Building, Oxford Road, Manchester M13 9PT,
    Search for articles by this author
  • Gareth J. Howell
    Affiliations
    Flow Cytometry Facility Laboratory, Faculty of Biology, Medicine, and Health, University of Manchester, CTF Building, Oxford Road, Manchester M13 9PT,
    Search for articles by this author
  • Sally A. Cowley
    Affiliations
    Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE,

    Oxford Parkinson’s Disease Centre, University of Oxford, South Parks Road, Oxford OX1 3QX,
    Search for articles by this author
  • Satyan Chintawar
    Affiliations
    Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, and

    Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX1 3QX, United Kingdom
    Search for articles by this author
  • M. Zameel Cader
    Affiliations
    Nuffield Department of Clinical Neurosciences, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, and

    Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX1 3QX, United Kingdom
    Search for articles by this author
  • Katherine A.B. Kellett
    Affiliations
    Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, AV Hill Building, University of Manchester, Manchester Academic Health Science Centre, Oxford Road, Manchester M13 9PT,
    Search for articles by this author
  • Nigel M. Hooper
    Correspondence
    To whom correspondence should be addressed. Tel.:44-161-306-5765
    Affiliations
    Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, AV Hill Building, University of Manchester, Manchester Academic Health Science Centre, Oxford Road, Manchester M13 9PT,
    Search for articles by this author
  • Author Footnotes
    1 Present address: Clinical and Translational Rheumatology, Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds LS9 7TF, United Kingdom.
    3 The abbreviations used are: ADAlzheimer’s diseaseAβamyloid-βAβOamyloid-β oligomerAPPamyloid precursor proteinADAM10a disintegrin and metalloprotease 10BACE1β-site APP cleaving enzyme 1iPSCinduced pluripotent stem cellPrPCcellular prion proteinROSreactive oxygen speciessAPPαsoluble APP fragment produced by α-secretase cleavagesAPPβsoluble sAPPβ produced by β-secretase cleavageGPIglycosylphosphatidylinositolSFKSrc family kinase(s)DPBSDulbecco’s PBSDAPI4′,6-diamidino-2-phenylindoleqPCRquantitative PCRANOVAanalysis of varianceCHAPSO3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid.
Open AccessPublished:March 14, 2019DOI:https://doi.org/10.1074/jbc.RA118.005364
      The cellular prion protein (PrPC) is a key neuronal receptor for β-amyloid oligomers (AβO), mediating their neurotoxicity, which contributes to the neurodegeneration in Alzheimer’s disease (AD). Similarly to the amyloid precursor protein (APP), PrPC is proteolytically cleaved from the cell surface by a disintegrin and metalloprotease, ADAM10. We hypothesized that ADAM10-modulated PrPC shedding would alter the cellular binding and cytotoxicity of AβO. Here, we found that in human neuroblastoma cells, activation of ADAM10 with the muscarinic agonist carbachol promotes PrPC shedding and reduces the binding of AβO to the cell surface, which could be blocked with an ADAM10 inhibitor. Conversely, siRNA-mediated ADAM10 knockdown reduced PrPC shedding and increased AβO binding, which was blocked by the PrPC-specific antibody 6D11. The retinoic acid receptor analog acitretin, which up-regulates ADAM10, also promoted PrPC shedding and decreased AβO binding in the neuroblastoma cells and in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Pretreatment with acitretin abolished activation of Fyn kinase and prevented an increase in reactive oxygen species caused by AβO binding to PrPC. Besides blocking AβO binding and toxicity, acitretin also increased the nonamyloidogenic processing of APP. However, in the iPSC-derived neurons, Aβ and other amyloidogenic processing products did not exhibit a reciprocal decrease upon acitretin treatment. These results indicate that by promoting the shedding of PrPC in human neurons, ADAM10 activation prevents the binding and cytotoxicity of AβO, revealing a potential therapeutic benefit of ADAM10 activation in AD.

      Introduction

      Alzheimer’s disease (AD)
      The abbreviations used are: AD
      Alzheimer’s disease
      amyloid-β
      AβO
      amyloid-β oligomer
      APP
      amyloid precursor protein
      ADAM10
      a disintegrin and metalloprotease 10
      BACE1
      β-site APP cleaving enzyme 1
      iPSC
      induced pluripotent stem cell
      PrPC
      cellular prion protein
      ROS
      reactive oxygen species
      sAPPα
      soluble APP fragment produced by α-secretase cleavage
      sAPPβ
      soluble sAPPβ produced by β-secretase cleavage
      GPI
      glycosylphosphatidylinositol
      SFK
      Src family kinase(s)
      DPBS
      Dulbecco’s PBS
      DAPI
      4′,6-diamidino-2-phenylindole
      qPCR
      quantitative PCR
      ANOVA
      analysis of variance
      CHAPSO
      3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid.
      is a progressive, age-associated disorder that is characterized by abnormal accumulation of proteinacious aggregates in the form of β-amyloid (Aβ) containing plaques and neurofibrillary tangles composed of hyperphosphorylated tau (
      • Blennow K.
      • de Leon M.J.
      • Zetterberg H.
      Alzheimer's disease.
      ,
      • De Strooper B.
      • Karran E.
      The cellular phase of Alzheimer's disease.
      ). Oligomers of Aβ (AβOs) appear to be the most neurotoxic species, binding to receptors on the surface of neurons and triggering a variety of downstream signaling pathways that negatively impact neuronal function and survival (reviewed in Refs.
      • Viola K.L.
      • Klein W.L.
      Amyloid beta oligomers in Alzheimer's disease pathogenesis, treatment, and diagnosis.
      and
      • Jarosz-Griffiths H.H.
      • Noble E.
      • Rushworth J.V.
      • Hooper N.M.
      Amyloid-β receptors: the good, the bad, and the prion protein.
      ). A substantial portion of AβO toxicity in AD is mediated following the initial interaction with the cellular prion protein (PrPC), which resides in cholesterol-rich lipid rafts at the neuronal surface (
      • Jarosz-Griffiths H.H.
      • Noble E.
      • Rushworth J.V.
      • Hooper N.M.
      Amyloid-β receptors: the good, the bad, and the prion protein.
      ,
      • Salazar S.V.
      • Strittmatter S.M.
      Cellular prion protein as a receptor for amyloid-β oligomers in Alzheimer’s disease.
      ). AβO binding to PrPC mediates inhibition of long-term potentiation in hippocampal slices (
      • Laurán J.
      • Gimbel D.A.
      • Nygaard H.B.
      • Gilbert J.W.
      • Strittmatter S.M.
      Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers.
      ) and memory and behavioral impairments in multiple AD mouse models (
      • Gimbel D.A.
      • Nygaard H.B.
      • Coffey E.E.
      • Gunther E.C.
      • Laurán J.
      • Gimbel Z.A.
      • Strittmatter S.M.
      Memory impairment in transgenic Alzheimer mice requires cellular prion protein.
      ,
      • Kostylev M.A.
      • Kaufman A.C.
      • Nygaard H.B.
      • Patel P.
      • Haas L.T.
      • Gunther E.C.
      • Vortmeyer A.
      • Strittmatter S.M.
      Prion-protein-interacting amyloid-β oligomers of high molecular weight are tightly correlated with memory impairment in multiple Alzheimer mouse models.
      ). The binding of AβO to PrPC leads to activation of Fyn kinase, a loss of surface N-methyl-d-aspartate receptors, and subsequent phosphorylation of tau (
      • Larson M.
      • Sherman M.A.
      • Amar F.
      • Nuvolone M.
      • Schneider J.A.
      • Bennett D.A.
      • Aguzzi A.
      • Lesná S.E.
      The complex PrPc-Fyn couples human oligomeric Aβ with pathological Tau changes in Alzheimer’s disease.
      ,
      • Um J.W.
      • Nygaard H.B.
      • Heiss J.K.
      • Kostylev M.A.
      • Stagi M.
      • Vortmeyer A.
      • Wisniewski T.
      • Gunther E.C.
      • Strittmatter S.M.
      Alzheimer amyloid-β oligomer bound to postsynaptic prion protein activates Fyn to impair neurons.
      • Rushworth J.V.
      • Griffiths H.H.
      • Watt N.T.
      • Hooper N.M.
      Prion protein-mediated toxicity of amyloid-β oligomers requires lipid rafts and the transmembrane LRP1.
      ). AβO also cause increases in reactive oxygen species (ROS), which contribute to the neurodegeneration (reviewed in Ref.
      • Chong Z.Z.
      • Li F.
      • Maiese K.
      Oxidative stress in the brain: novel cellular targets that govern survival during neurodegenerative disease.
      ).
      Given the central role of PrPC in mediating the toxicity of AβO, targeting PrPC has potential for AD therapy (reviewed in Ref.
      • Jarosz-Griffiths H.H.
      • Noble E.
      • Rushworth J.V.
      • Hooper N.M.
      Amyloid-β receptors: the good, the bad, and the prion protein.
      ). Immunotargeting (e.g. using the anti-PrPC mAb 6D11 to block the AβO binding site on PrPC) prevented the impairment in long-term potentiation caused by AβO derived from AD brain extracts (
      • Barry A.E.
      • Klyubin I.
      • Mc Donald J.M.
      • Mably A.J.
      • Farrell M.A.
      • Scott M.
      • Walsh D.M.
      • Rowan M.J.
      Alzheimer’s disease brain-derived amyloid-β-mediated inhibition of LTP in vivo is prevented by immunotargeting cellular prion protein.
      ,
      • Freir D.B.
      • Nicoll A.J.
      • Klyubin I.
      • Panico S.
      • Mc Donald J.M.
      • Risse E.
      • Asante E.A.
      • Farrow M.A.
      • Sessions R.B.
      • Saibil H.R.
      • Clarke A.R.
      • Rowan M.J.
      • Walsh D.M.
      • Collinge J.
      Interaction between prion protein and toxic amyloid β assemblies can be therapeutically targeted at multiple sites.
      ) and blocked Aβ synaptotoxicity following peripheral administration (
      • Klyubin I.
      • Nicoll A.J.
      • Khalili-Shirazi A.
      • Farmer M.
      • Canning S.
      • Mably A.
      • Linehan J.
      • Brown A.
      • Wakeling M.
      • Brandner S.
      • Walsh D.M.
      • Rowan M.J.
      • Collinge J.
      Peripheral administration of a humanized anti-PrP antibody blocks Alzheimer’s disease Aβ synaptotoxicity.
      ). Altering the conformation of AβO, disrupting AβO binding to PrPC, or displacing PrPC from lipid rafts blocked downstream cellular toxicity (
      • Rushworth J.V.
      • Griffiths H.H.
      • Watt N.T.
      • Hooper N.M.
      Prion protein-mediated toxicity of amyloid-β oligomers requires lipid rafts and the transmembrane LRP1.
      ,
      • Risse E.
      • Nicoll A.J.
      • Taylor W.A.
      • Wright D.
      • Badoni M.
      • Yang X.
      • Farrow M.A.
      • Collinge J.
      Identification of a compound that disrupts binding of amyloid-β to the prion protein using a novel fluorescence-based assay.
      ). Several of the actions of AβO, including activation of Fyn, dendritic spine loss, and tau phosphorylation, are mediated by PrPC coupling to mGluR5 (
      • Um J.W.
      • Kaufman A.C.
      • Kostylev M.
      • Heiss J.K.
      • Stagi M.
      • Takahashi H.
      • Kerrisk M.E.
      • Vortmeyer A.
      • Wisniewski T.
      • Koleske A.J.
      • Gunther E.C.
      • Nygaard H.B.
      • Strittmatter S.M.
      Metabotropic glutamate receptor 5 is a coreceptor for Alzheimer Aβ oligomer bound to cellular prion protein.
      ,
      • Haas L.T.
      • Salazar S.V.
      • Kostylev M.A.
      • Um J.W.
      • Kaufman A.C.
      • Strittmatter S.M.
      Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer's disease.
      • Beraldo F.H.
      • Ostapchenko V.G.
      • Caetano F.A.
      • Guimaraes A.L.
      • Ferretti G.D.
      • Daude N.
      • Bertram L.
      • Nogueira K.O.
      • Silva J.L.
      • Westaway D.
      • Cashman N.R.
      • Martins V.R.
      • Prado V.F.
      • Prado M.A.
      Regulation of amyloid β oligomer binding to neurons and neurotoxicity by the prion protein-mGluR5 complex.
      ), and pharmacological inhibition or allosteric modulation of mGluR5 reduced pathogenesis in AD mouse models (
      • Hamilton A.
      • Vasefi M.
      • Vander Tuin C.
      • McQuaid R.J.
      • Anisman H.
      • Ferguson S.S.
      Chronic pharmacological mGluR5 inhibition prevents cognitive impairment and reduces pathogenesis in an Alzheimer disease mouse model.
      ,
      • Haas L.T.
      • Salazar S.V.
      • Smith L.M.
      • Zhao H.R.
      • Cox T.O.
      • Herber C.S.
      • Degnan A.P.
      • Balakrishnan A.
      • Macor J.E.
      • Albright C.F.
      • Strittmatter S.M.
      Silent allosteric modulation of mGluR5 maintains glutamate signaling while rescuing Alzheimer's mouse phenotypes.
      ). Another approach has been to target Fyn directly with a specific inhibitor to rescue the memory deficits in an AD mouse model (
      • Kaufman A.C.
      • Salazar S.V.
      • Haas L.T.
      • Yang J.
      • Kostylev M.A.
      • Jeng A.T.
      • Robinson S.A.
      • Gunther E.C.
      • van Dyck C.H.
      • Nygaard H.B.
      • Strittmatter S.M.
      Fyn inhibition rescues established memory and synapse loss in Alzheimer mice.
      ). These approaches highlight that targeting PrPC or other components of the AβO-PrPC signaling complex may have therapeutic potential in AD.
      Aβ peptides are generated when the amyloid precursor protein (APP) is cleaved by the sequential action of the β-secretase (β-site APP-cleaving enzyme 1; BACE1) and the multisubunit γ-secretase complex in the amyloidogenic pathway (
      • Andrew R.J.
      • Kellett K.A.
      • Thinakaran G.
      • Hooper N.M.
      A Greek tragedy: the growing complexity of Alzheimer amyloid precursor protein proteolysis.
      ). β-Secretase cleavage of APP also releases the large soluble ectodomain fragment sAPPβ. Alternatively, APP can be cleaved via the nonamyloidogenic pathway through the action of the α-secretase, a disintegrin and metalloprotease ADAM10, precluding the formation of the Aβ peptide and generating an alternative soluble fragment sAPPα that has neuroprotective and neurotrophic properties (
      • Andrew R.J.
      • Kellett K.A.
      • Thinakaran G.
      • Hooper N.M.
      A Greek tragedy: the growing complexity of Alzheimer amyloid precursor protein proteolysis.
      ). It is generally assumed that there is competition between the α- and β-secretases for their substrate APP, resulting in a reciprocal relationship between the amyloidogenic and nonamyloidogenic APP-processing pathways. In support of this reciprocal relationship, neuronal overexpression of ADAM10 in APPV717I transgenic mice increased the secretion of sAPPα and reduced the formation of Aβ peptides (
      • Postina R.
      • Schroeder A.
      • Dewachter I.
      • Bohl J.
      • Schmitt U.
      • Kojro E.
      • Prinzen C.
      • Endres K.
      • Hiemke C.
      • Blessing M.
      • Flamez P.
      • Dequenne A.
      • Godaux E.
      • van Leuven F.
      • Fahrenholz F.
      A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model.
      ), whereas in human induced pluripotent stem cell (iPSC)-derived neurons, inhibition of BACE1 reduced sAPPβ and Aβ and increased sAPPα (
      • Ben Halima S.
      • Mishra S.
      • Raja K.M.P.
      • Willem M.
      • Baici A.
      • Simons K.
      • Brüstle O.
      • Koch P.
      • Haass C.
      • Caflisch A.
      • Rajendran L.
      Specific inhibition of β-secretase processing of the Alzheimer disease amyloid precursor protein.
      ).
      The ectodomain shedding of multiple cell surface proteins can be promoted by a variety of compounds. For example, activators of protein kinase C and the muscarinic agonist carbachol promote the shedding of APP (
      • Caporaso G.L.
      • Gandy S.E.
      • Buxbaum J.D.
      • Ramabhadran T.V.
      • Greengard P.
      Protein phosphorylation regulates secretion of Alzheimer β/A4 amyloid precursor protein.
      • Parvathy S.
      • Hussain I.
      • Karran E.H.
      • Turner A.J.
      • Hooper N.M.
      Alzheimer’s amyloid precursor protein α-secretase is inhibited by hydroxamic acid-based zinc metalloprotease inhibitors: similarities to the angiotensin converting enzyme secretase.
      ,
      • Parkin E.T.
      • Trew A.
      • Christie G.
      • Faller A.
      • Mayer R.
      • Turner A.J.
      • Hooper N.M.
      Structure-activity relationship of hydroxamate-based inhibitors on the secretases that cleave the amyloid precursor protein, angiotensin converting enzyme, CD23, and pro-tumor necrosis factor-α.
      • Canet-Aviles R.M.
      • Anderton M.
      • Hooper N.M.
      • Turner A.J.
      • Vaughan P.F.
      Muscarine enhances soluble amyloid precursor protein secretion in human neuroblastoma SH-SY5Y by a pathway dependent on protein kinase Cα, src-tyrosine kinase and extracellular signal-regulated kinase but not phospholipase C.
      ). The vitamin A analog, acitretin, promoted the α-secretase cleavage of APP by stimulating the transcription of ADAM10 via interaction with retinoic acid–responsive elements within the ADAM10 promoter (
      • Tippmann F.
      • Hundt J.
      • Schneider A.
      • Endres K.
      • Fahrenholz F.
      Up-regulation of the α-secretase ADAM10 by retinoic acid receptors and acitretin.
      ). As ADAM10 also cleaves and sheds the ectodomain of PrPC from the cell surface (
      • Taylor D.R.
      • Parkin E.T.
      • Cocklin S.L.
      • Ault J.R.
      • Ashcroft A.E.
      • Turner A.J.
      • Hooper N.M.
      Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein.
      ,
      • Altmeppen H.C.
      • Prox J.
      • Puig B.
      • Kluth M.A.
      • Bernreuther C.
      • Thurm D.
      • Jorissen E.
      • Petrowitz B.
      • Bartsch U.
      • De Strooper B.
      • Saftig P.
      • Glatzel M.
      Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo.
      • Altmeppen H.C.
      • Prox J.
      • Krasemann S.
      • Puig B.
      • Kruszewski K.
      • Dohler F.
      • Bernreuther C.
      • Hoxha A.
      • Linsenmeier L.
      • Sikorska B.
      • Liberski P.P.
      • Bartsch U.
      • Saftig P.
      • Glatzel M.
      The sheddase ADAM10 is a potent modulator of prion disease.
      ), we hypothesized that modulating ADAM10 activity, thereby altering the shedding and thus the amount of PrPC at the cell surface, would modulate the binding and toxicity of AβO.
      Here, we have used human neuroblastoma cells and iPSC-derived cortical neurons to show that carbachol and acitretin promote the shedding of cell surface PrPC through activation of ADAM10. The resulting reduction of cell surface PrPC leads to a concomitant reduction in the binding of AβO. Conversely, siRNA knockdown of ADAM10 resulted in increased cell surface PrPC and a corresponding increase in AβO binding that could be blocked with the PrPC antibody, 6D11. AβO binding to PrPC activated Fyn kinase and caused an increase in ROS that could be blocked by promoting the shedding of PrPC with acitretin. We also report that although acitretin reciprocally modulated the amyloidogenic and nonamyloidogenic processing of APP in neuroblastoma cells and rat hippocampal neurons, no such reciprocal relationship was observed in the human iPSC-derived neurons.

      Results

      Promoting the shedding of PrPC decreases the cell surface binding of AβO

      As ADAM10 mediates the shedding of PrPC from the cell surface (
      • Taylor D.R.
      • Parkin E.T.
      • Cocklin S.L.
      • Ault J.R.
      • Ashcroft A.E.
      • Turner A.J.
      • Hooper N.M.
      Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein.
      ,
      • Altmeppen H.C.
      • Prox J.
      • Puig B.
      • Kluth M.A.
      • Bernreuther C.
      • Thurm D.
      • Jorissen E.
      • Petrowitz B.
      • Bartsch U.
      • De Strooper B.
      • Saftig P.
      • Glatzel M.
      Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo.
      ), we hypothesized that activation of ADAM10 would reduce AβO binding to cells due to shedding of its cell surface receptor PrPC. Initially, the muscarinic agonist carbachol, which increases the shedding of multiple cell surface proteins, including APP, was employed (
      • Parkin E.T.
      • Trew A.
      • Christie G.
      • Faller A.
      • Mayer R.
      • Turner A.J.
      • Hooper N.M.
      Structure-activity relationship of hydroxamate-based inhibitors on the secretases that cleave the amyloid precursor protein, angiotensin converting enzyme, CD23, and pro-tumor necrosis factor-α.
      ). The effect of carbachol on PrPC and APP shedding was monitored by detection of the soluble fragments, sPrPC and sAPPα, respectively, in the media fraction. Treatment of human SH-SY5Y cells expressing PrPC with carbachol promoted the shedding of full-length glycosylated PrPC by 1.4-fold and the α-secretase cleavage of APP by 1.5-fold (Fig. 1, A–C). To establish that carbachol was acting via activation of ADAM10, the cells were incubated with the ADAM10-selective inhibitor, GI254023X (
      • Hussain I.
      • Hawkins J.
      • Shikotra A.
      • Riddell D.R.
      • Faller A.
      • Dingwall C.
      Characterization of the ectodomain shedding of the β-site amyloid precursor protein-cleaving enzyme 1 (BACE1).
      ). On its own, the ADAM10 inhibitor significantly reduced the amount of sPrPC in the media by 83% (Fig. 1, A and B), consistent with ADAM10 being the major constitutive PrPC sheddase (
      • Altmeppen H.C.
      • Prox J.
      • Puig B.
      • Kluth M.A.
      • Bernreuther C.
      • Thurm D.
      • Jorissen E.
      • Petrowitz B.
      • Bartsch U.
      • De Strooper B.
      • Saftig P.
      • Glatzel M.
      Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo.
      ). In the presence of the ADAM10 inhibitor, carbachol failed to cause an increase in the shedding of PrPC (Fig. 1, A and B), indicating that carbachol was promoting the shedding of PrPC via activation of ADAM10. Similar to its effect on the shedding of PrPC, the ADAM10 inhibitor significantly reduced sAPPα in the media by 85% and blocked the increase in sAPPα caused by carbachol (Fig. 1, A and C). Treatment with carbachol and the GI254023X (GI) inhibitor did not significantly alter ADAM10 expression (Fig. 1D). Consistent with increasing the shedding of PrPC, carbachol treatment resulted in a decrease in cell surface expression of PrPC evaluated by ImageStream imaging cytometry (Fig. 1E). This analysis demonstrated a decrease in high-expressing PrPC cells in a population of cells treated with carbachol when compared with the control population. The decrease in cell surface PrPC was confirmed using immunofluorescence microscopy, where carbachol treatment of the cells caused a significant decrease (40%) in the amount of PrPC localized to the cell surface (Fig. 1, F and G). AβOs were prepared and characterized as described previously (
      • Rushworth J.V.
      • Griffiths H.H.
      • Watt N.T.
      • Hooper N.M.
      Prion protein-mediated toxicity of amyloid-β oligomers requires lipid rafts and the transmembrane LRP1.
      ), and their binding to the cells was monitored by immunofluorescence microscopy. Treatment of the cells with carbachol resulted in a significant decrease (62%) in the amount of AβO bound to the cell surface (Fig. 1, F and G).
      Figure thumbnail gr1
      Figure 1Promoting the shedding of PrPC decreases AβO binding in an ADAM10-dependent manner. A, immunoblots of sPrPC and sAPPα in concentrated conditioned media, and of ADAM10, PrPC, APP, and actin in cell lysates, from SH-SY5Y cells expressing PrPC incubated with or without carbachol (Cch; 20 μm) and with or without GI254023X (GI; 10 μm) in Opti-MEM for 24 h at 37 °C. sPrPC (B), sAPPα (C), and ADAM10 (D) immunoblots were quantified and are represented as a percentage of control (n = 4). E, ImageStream imaging cytometry analysis showing negative (N), middle (M), and high (H) levels of cell surface PrPC expression in SH-SY5Y cells expressing PrPC incubated without (blue) and with (red) carbachol (20 μm). F, immunofluorescence microscopy images showing staining of PrPC (green) and Aβ-biotin (red) in nonpermeabilized SH-SY5Y cells expressing PrPC incubated with or without carbachol (20 μm) in Opti-MEM for 3 h at 37 °C followed by incubation with AβO (500 nm) for 30 min at room temperature. Scale bar, 5 μm. G, quantification of PrPC cell surface staining (n = 3) and AβO cell surface binding (n = 3). H, immunoblot of PrPC in cell lysate samples, prepared from SH-SY5Y cells expressing PrPC incubated with or without carbachol (20 μm), and treated without or with N-glycanase for 16 h at 37 °C. I, quantification of deglycosylated full-length (FL) and C1 PrPC species, expressed as the ratio of FL/C1 (n = 4). Statistical analyses were one-way ANOVA with Tukey’s post hoc correction for multiple-comparison data or independent t test with Welch’s correction for two-sample comparison. Data are shown as mean ± S.E. (error bars); n.s., not significant; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.
      In addition to promoting the shedding of the entire ectodomain of PrPC through cleavage near the site of glycosylphosphatidylinositol (GPI) anchor attachment (
      • Taylor D.R.
      • Parkin E.T.
      • Cocklin S.L.
      • Ault J.R.
      • Ashcroft A.E.
      • Turner A.J.
      • Hooper N.M.
      Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein.
      ), ADAM10 has also been reported to cleave PrPC within the middle of the ectodomain (sometimes referred to as α-cleavage) (
      • Vincent B.
      • Paitel E.
      • Saftig P.
      • Frobert Y.
      • Hartmann D.
      • De Strooper B.
      • Grassi J.
      • Lopez-Perez E.
      • Checler F.
      The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein.
      ). The α-cleavage of PrPC releases an N-terminal fragment, termed N1, and leaves a C-terminal fragment, C1, tethered to the membrane via the GPI anchor. To establish whether carbachol also promoted the cleavage of PrPC within its ectodomain, samples were deglycosylated so that the C1 fragment could be detected and distinguished from unglycosylated full-length PrPC. There was no change in the ratio of full-length PrPC to C1 fragment when cells were incubated with carbachol (Fig. 1, H and I), indicating that carbachol reduces AβO binding by promoting the shedding of full-length PrPC and not by promoting cleavage within its ectodomain.

      Knockdown of ADAM10 increases Aβ oligomer binding in a PrP-dependent manner

      To confirm that ADAM10 was responsible for altering AβO binding through modulating the cell surface level of PrPC, ADAM10 was knocked down using siRNA in the SH-SY5Y cells expressing PrPC. For these experiments, we used both a SMARTpool siRNA containing four target siRNAs (Dharmacon) shown in Fig. 2 and a single, independent siRNA (Ambion) shown in Fig. 3. In the ADAM10 siRNA-treated cells, there was a significant reduction in the pro and mature forms of ADAM10 of 69% (Fig. 2, A and B) and 68% (Fig. 3, A and B) in Dharmacon-treated and Ambion siRNA–treated cells, respectively. Following ADAM10 siRNA knockdown, the amount of sPrPC was significantly reduced by 48% (Fig. 2, A and C) and by 30% (Fig. 3, A and C), and the amount of sAPPα was also significantly reduced, by 67% (Fig. 2, A and D) and 85% (Fig. 3, A and D). ADAM10 siRNA caused a 2.2-fold (Fig. 2, E and F) and a 1.3-fold (Fig. 3, E and F) increase in cell surface PrPC relative to nontargeting siRNA as assessed by immunofluorescence microscopy, and the binding of AβO to the cells was increased by 1.7-fold (Fig. 2, E and G) and 1.6-fold (Fig. 3, E and G) in the presence of ADAM10 siRNA relative to nontargeting siRNA. To establish whether this increased binding of AβO was due to the increased cell surface level of PrPC, cells were preincubated with the PrPC antibody, 6D11, which blocks the AβO binding site on PrPC (
      • Laurán J.
      • Gimbel D.A.
      • Nygaard H.B.
      • Gilbert J.W.
      • Strittmatter S.M.
      Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers.
      ). Incubation of the cells with the 6D11 antibody had no significant effect on the amount of PrPC at the cell surface (Figs. 2 (E and F) and 3 (E and F)). However, the 6D11 antibody significantly reduced AβΟ binding to the cells by 77% (Fig. 2, E and G) and by 60% (Fig. 3, E and G). In the presence of the 6D11 antibody, siRNA knockdown of ADAM10 failed to increase AβO binding (Figs. 2 (E and G) and 3 (E and G)), indicating that the increased binding of AβO upon ADAM10 knockdown was due to increased cell surface PrPC.
      Figure thumbnail gr2
      Figure 2Knockdown of ADAM10 reduces the shedding of PrPC and increases AβO binding. A, immunoblots of ADAM10 (pro- (p) and mature (m) forms), PrPC, APP, and actin in cell lysates and of sPrPC and sAPPα in concentrated conditioned media from SH-SY5Y cells expressing PrPC incubated with either siRNA targeted against ADAM10 (+) (Dharmacon, SMARTpool) or a nontargeting (−) siRNA control for 48 h followed by incubation with Opti-MEM for a further 24 h. ADAM10 (B), PrPC and sPrPC (B), and APP and sAPPα (C) immunoblots were quantified and are represented as a percentage of control (n = 4–6). E, immunofluorescence microscopy images showing staining of PrPC (green) and Aβ-biotin (red) in nonpermeabilized SH-SY5Y cells expressing PrPC following siRNA treatment targeted against ADAM10 and incubation in the absence or presence of the PrPC antibody, 6D11, for 20 min at 37 °C followed by AβO incubation (500 nm) for 30 min at room temperature. Scale bar, 5 μm. F, quantification of PrPC cell surface staining (n = 5); G, AβO binding to cells (n = 5). Statistical analyses were one-way ANOVA with Tukey’s post hoc correction for multiple-comparison data or independent t test with Welch’s correction for two-sample comparison. Data are shown as mean ± S.E. (error bars). n.s., not significant; *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
      Figure thumbnail gr3
      Figure 3Knockdown of ADAM10 with an independent siRNA sequence reduces the shedding of PrPC and increases AβO binding. A, immunoblots of ADAM10 (pro- (p) and mature (m) forms), PrPC, APP, and actin in cell lysates and of sPrPC and sAPPα in concentrated conditioned media from SH-SY5Y cells expressing PrPC incubated with either siRNA targeted against ADAM10 (+) (Ambion, single siRNA) or a nontargeting (−) siRNA control for 48 h, followed by incubation with Opti-MEM for a further 24 h. ADAM10 (B), PrPC and sPrPC (C), and APP and sAPPα (D) immunoblots were quantified and are represented as a percentage of control (n = 3). E, immunofluorescence microscopy images showing staining of PrPC (green) and Aβ-biotin (red) in nonpermeabilized SH-SY5Y cells expressing PrPC following siRNA treatment targeted against ADAM10 and incubation in the absence or presence of the PrPC antibody, 6D11, for 20 min at 37 °C followed by AβO incubation (500 nm) for 30 min at room temperature. Scale bar, 5 μm. Shown are quantification of PrPC cell surface staining (n = 5) (F) and AβO binding to cells (n = 5) (G). Statistical analyses were one-way ANOVA with Tukey’s post hoc correction for multiple-comparison data or independent t test with Welch’s correction for two-sample comparison. Data are shown as mean ± S.E. (error bars). n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.
      To confirm that ADAM10 modulated AβO binding in a PrPC-dependent manner, SH-SY5Y cells lacking PrPC (
      • Perera W.S.
      • Hooper N.M.
      Proteolytic fragmentation of the murine prion protein: role of Tyr-128 and His-177.
      ) were treated with ADAM10 siRNA, and AβO binding was assessed. In these cells, ADAM10 siRNA treatment significantly reduced the pro and mature forms of ADAM10 and the amount of sAPPα in the media (Fig. 4, A and B). In the absence of PrPC, AβO binding was <15% (Fig. 4C) of that observed in cells expressing PrPC (Figs. 2E and 3E), and this residual binding was unchanged following knockdown of ADAM10 (Fig. 4, C and D). Together, these data indicate that ADAM10 modulates AβO cell surface binding in a PrPC-dependent manner.
      Figure thumbnail gr4
      Figure 4ADAM10 fails to increase AβO binding in cells lacking PrPC. Immunoblots of ADAM10 and actin in cell lysates and of sAPPα in concentrated conditioned media from untransfected SH-SY5Y cells, which lack endogenous PrPC, incubated with either siRNA targeted against ADAM10 (+) or a nontargeting (−) siRNA control for 48 h followed by incubation with Opti-MEM for a further 24 h. B, ADAM10 and sAPPα immunoblots were quantified and are represented as a percentage of control (n = 3). C, immunofluorescence microscopy images showing staining of Aβ-biotin (red) in nonpermeabilized untransfected SH-SY5Y cells following siRNA treatment targeted against ADAM10 and incubation with AβO (500 nm) for 30 min at room temperature. Scale bar, 5 μm. D, quantification of AβO binding to cells (n = 5). Statistical analyses were independent t test with Welch’s correction for two-sample comparison. Data are shown as mean ± S.E. (error bars). **, p < 0.01; ****, p < 0.0001.

      Activation of ADAM10 with acitretin increases the shedding of PrPC and decreases AβO binding

      Acitretin, a synthetic retinoid, increases the expression of ADAM10 in cell culture and animals, leading to an increase in sAPPα (
      • Tippmann F.
      • Hundt J.
      • Schneider A.
      • Endres K.
      • Fahrenholz F.
      Up-regulation of the α-secretase ADAM10 by retinoic acid receptors and acitretin.
      ). As acitretin may have potential as a novel therapeutic drug for AD due to its ability to increase sAPPα in the CSF of AD patients (
      • Endres K.
      • Fahrenholz F.
      • Lotz J.
      • Hiemke C.
      • Teipel S.
      • Lieb K.
      • Tüscher O.
      • Fellgiebel A.
      Increased CSF APPs-α levels in patients with Alzheimer disease treated with acitretin.
      ), we investigated whether acitretin, via activation of ADAM10, would increase the shedding of PrPC and decrease AβO binding. Acitretin treatment of the SH-SY5Y cells expressing PrPC increased the expression of mature ADAM10 and the shedding of PrPC and sAPPα by 1.2-fold (Fig. 5, A–C). In the presence of the ADAM10 inhibitor GI254023X, acitretin failed to induce a significant increase in the amount of sPrPC (Fig. 5, A and B) or of sAPPα in the media (Fig. 5, A and C), indicating that it was acting via ADAM10. Treatment with acitretin caused an increase in ADAM10 protein (Fig. 5, A and D) and mRNA (Fig. 5E) expression. Treatment with acitretin resulted in a decrease in cell surface expression of PrPC as evaluated by ImageStream imaging cytometry (Fig. 5F). This analysis demonstrated a decrease in high-expressing PrPC cells in a population of cells treated with acitretin when compared with the control population. The decrease in cell surface PrPC was confirmed using immunofluorescence microscopy, where acitretin treatment of the cells caused a significant decrease (81%) in the amount of PrPC localized to the cell surface (Fig. 5, G and H). This decrease in cell surface PrPC with acitretin resulted in a reduction of the amount of AβO bound to the surface of the cells by 86% (Fig. 5, G and H). These data indicate that activation of ADAM10 with the retinoic acid analog acitretin increases the shedding of PrPC and decreases the binding of AβO to the cells.
      Figure thumbnail gr5
      Figure 5Acitretin increases PrPC shedding and decreases AβO binding and toxicity in an ADAM10-dependent manner. A, immunoblots of sPrPC and sAPPα in concentrated conditioned media and of ADAM10, PrPC, APP, and actin in cell lysates from SH-SY5Y cells expressing PrPC incubated with or without GI254023X (GI) (10 μm) and with or without acitretin (Acit) (20 μm) diluted in Opti-MEM for 48 h at 37 °C. sPrPC (B), sAPPα (C), and ADAM10 (D) immunoblots were quantified and are represented as a percentage of control (n = 4). E, relative expression of ADAM10 mRNA in SH-SY5Y cells expressing PrPC incubated with or without acitretin (Acit; 20 μm) (n = 6). F, ImageStream imaging cytometry analysis showing negative (N), middle (M), and high (H) levels of cell surface PrPC expression in SH-SY5Y cells expressing PrPC incubated without (blue) and with (red) acitretin (20 μm). G, immunofluorescence microscopy images showing staining for PrPC (green) and Aβ-biotin (red) in nonpermeabilized SH-SY5Y cells expressing PrPC incubated with or without acitretin (20 μm) in Opti-MEM for 48 h at 37 °C followed by incubation with AβO (500 nm) for 30 min at room temperature. Scale bar, 5 μm. H, quantification of PrPC cell surface staining (n = 5) and AβO binding to cells (n = 5). I, immunoblots for the phosphorylated species of Src family kinases (pSFK) and of total Fyn kinase in cell lysates from NB7 cells, which endogenously express PrPC, incubated with or without acitretin (20 μm) in Opti-MEM for 48 h at 37 °C followed by incubation with AβO (500 nm) for 20 min at 37 °C. J, the ratio of pSFK/Fyn was quantified and is expressed as a percentage of control (n = 5). K, ROS were measured using the ROS-Glo assay in SH-SY5Y cells expressing PrPC incubated with or without acitretin (20 μm) in Opti-MEM for 48 h at 37 °C and then treated with or without AβO (500 nm) for 90 min at 37 °C. Luminescence was measured and is represented as a percentage of control (n = 6). Statistical analyses were one-way ANOVA with Tukey’s post hoc correction for multiple-comparison data, or independent t test with Welch’s correction for two-sample comparison. Data are shown as mean ± S.E. (error bars). n.s., not significant; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

      Activation of ADAM10 prevents AβO-mediated activation of Fyn kinase and rescues AβO-mediated increase in ROS

      To establish whether promoting the shedding of PrPC through activation of ADAM10 could decrease AβO cytotoxicity, we monitored downstream activation of Fyn kinase and ROS production in cells. Fyn kinase is a member of the Src family kinases (SFK), and AβOs activate Fyn kinase by phosphorylation at Tyr-416 (
      • Um J.W.
      • Nygaard H.B.
      • Heiss J.K.
      • Kostylev M.A.
      • Stagi M.
      • Vortmeyer A.
      • Wisniewski T.
      • Gunther E.C.
      • Strittmatter S.M.
      Alzheimer amyloid-β oligomer bound to postsynaptic prion protein activates Fyn to impair neurons.
      ,
      • Rushworth J.V.
      • Griffiths H.H.
      • Watt N.T.
      • Hooper N.M.
      Prion protein-mediated toxicity of amyloid-β oligomers requires lipid rafts and the transmembrane LRP1.
      ). Treatment of cells with AβO increased pSFK416 levels 1.3-fold (Fig. 5, I and J). Pretreatment with acitretin abolished the AβO-mediated increase in pSFK (Fig. 5, I and J). To monitor the effect of AβO on ROS, cells were primed with menadione to reduce mitochondrial production of NAD(P)H and increase ROS (
      • Criddle D.N.
      • Gillies S.
      • Baumgartner-Wilson H.K.
      • Jaffar M.
      • Chinje E.C.
      • Passmore S.
      • Chvanov M.
      • Barrow S.
      • Gerasimenko O.V.
      • Tepikin A.V.
      • Sutton R.
      • Petersen O.H.
      Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells.
      ). The addition of AβO increased ROS by 1.2-fold in cells expressing PrPC (Fig. 5K), but pretreatment with acitretin abolished the increase in ROS mediated by AβO (Fig. 5K). Together, these data indicate that promoting the ADAM10-mediated shedding of PrPC blocks both the cellular binding and downstream cytotoxicity of AβO.

      Activation of ADAM10 increases the shedding of PrPC and rescues AβO-mediated increase in ROS in human iPSC-derived cortical neurons

      To determine whether activation of ADAM10 would lead to enhanced shedding of PrPC and reduce AβO toxicity in human neurons, we used iPSC-derived cortical neurons. The iPSC line OX1-clone 19 (OX1-19) was verified for pluripotency by the presence of Oct4, SSEA-4, and nanog and the absence of PAX6 using immunofluorescence microscopy (Fig. 6A). The iPSCs were differentiated into cortical neurons using the method of Shi et al. (
      • Shi Y.
      • Kirwan P.
      • Livesey F.J.
      Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks.
      ). The efficiency of cortical induction was calculated using PAX6 expression (
      • Shi Y.
      • Kirwan P.
      • Livesey F.J.
      Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks.
      ). This demonstrated that the differentiation efficiency of the OX1-19 and SBAD iPSCs was 92.3 ± 7.6 and 85.9 ± 1.9%, respectively (data described as mean ± S.E. for four cortical inductions of the OX1-19 cell line and for two cortical inductions of the SBAD cell line). The neuronal marker MAP2 was used along with the marker Tbr1 to confirm the presence of secondary progenitor cells and Satb2 to confirm the presence of upper layer neurons (Fig. 6B). Immunoblotting of the cortical neurons revealed the presence of APP, ADAM10, and PrPC (Fig. 6C).
      Figure thumbnail gr6
      Figure 6Activation of ADAM10 by acitretin decreases surface PrPC and decreases AβO toxicity in iPSC-derived neurons. A, immunofluorescence microscopy images showing staining for the pluripotency markers Pax6, Oct4, SSEA-4, and Nanog in permeabilized OX1-19 iPSCs. Scale bar, 200 μm. B, immunofluorescence microscopy images showing staining for the neuronal markers Satb2, Tbr1, and MAP2 in OX1-19 iPSCs differentiated to cortical neurons at day 60. Scale bar, 100 μm. C, immunoblots of APP, ADAM10, PrPC, and actin in cell lysates from OX1-19 iPSCs differentiated to neurons at day 65. D, sAPPα was quantified, using the MSD system, in conditioned media from day 65 OX1-19 cortical neurons incubated with or without GI254023X (GI; 10 μm) and with or without acitretin (20 μm) in Opti-MEM for 48 h at 37 °C (n = 6). E, immunofluorescence microscopy images showing staining for PrPC (green) in day 50 OX1-19 cortical neurons incubated with or without acitretin (20 μm) in Opti-MEM for 48 h at 37 °C. Scale bar, 5 μm. F, quantification of PrPC cell surface staining on nonpermeabilized MAP2-stained neurites, expressed as a percentage of control (n = 3). G, ROS were measured using the ROS-Glo assay in day 65 OX1-19 cortical neurons with or without acitretin (20 μm) in Opti-MEM for 48 h at 37 °C. Cells were incubated with 1% BSA for 10 min to block the nonspecific binding of AβO and then treated with or without AβO (2.5 μm) for 90 min at 37 °C. Luminescence was measured and is represented as a percentage of control (n = 4–7). Statistical analyses were one-way ANOVA with Tukey post hoc correction for multiple-comparison data or independent t test with Welch’s correction for two-sample comparison. Data are shown as mean ± S.E. (error bars). n.s., not significant; *, p < 0.05; **, p < 0.01; ****, p < 0.0001.
      Acitretin treatment of the iPSC-derived cortical neurons at day 65 increased sAPPα in the media by 1.2-fold (Fig. 6D). In the presence of the ADAM10 inhibitor, acitretin failed to induce an increase in the amount of sAPPα in the media (Fig. 6D). Treatment with acitretin decreased cell surface PrPC by 87% (Fig. 6, E and F). AβO treatment increased ROS in the iPSC-derived cortical neurons (Fig. 6G), and this increase was blocked by pretreatment of the neurons with acitretin (Fig. 6G). These data indicate that acitretin, through increasing ADAM10 activity and the shedding of PrPC, blocks the toxicity of AβO in human neurons.

      Activation of ADAM10 increases the neuroprotective sAPPα but does not decrease Aβ production in human neurons

      Activation of ADAM10 has been reported to increase the production of the neuroprotective sAPPα and, reciprocally, decrease Aβ production (
      • Tippmann F.
      • Hundt J.
      • Schneider A.
      • Endres K.
      • Fahrenholz F.
      Up-regulation of the α-secretase ADAM10 by retinoic acid receptors and acitretin.
      ). However, whether this reciprocal relationship occurs in human neurons upon activation of ADAM10 has not been reported. To determine this, we assessed the effect of acitretin on the relative amounts of sAPPα, sAPPβ, and Aβ peptides in both the SH-SY5Y cells and the iPSC-derived neurons. In the SH-SY5Y cells, acitretin caused a significant increase in sAPPα (Fig. 5C) and a reciprocal significant decrease in sAPPβ (Fig. 7A). The decrease in sAPPβ was paralleled by a decrease in both Aβ40 and Aβ42 following acitretin treatment (Fig. 7, B and C). However, although acitretin increased sAPPα in the iPSC-derived neurons (Fig. 6C), there was no reciprocal decrease in sAPPβ or Aβ (Fig. 7, D–F). To ascertain whether this lack of reciprocal effect of acitretin on the nonamyloidogenic and amyloidogenic pathways was a feature of neurons, the effect of acitretin on rat primary hippocampal neurons was investigated. Acitretin treatment of the hippocampal neurons increased the nonamyloidogenic cleavage of APP (Fig. 7G) and caused a reciprocal decrease in both Aβ40 and Aβ42 (Fig. 7, H and I). Thus, in both the SH-SY5Y cells and the rat hippocampal neurons, activation of the nonamyloidogenic processing of APP results in a reciprocal decrease in the amyloidogenic processing pathway, whereas in the human iPSC-derived neurons, no such reciprocal relationship was observed.
      Figure thumbnail gr7
      Figure 7The effect of acitretin on Aβ production is cell type–dependent. sAPPβ (A), Aβ40 (B), and Aβ42 (C) were quantified by MSD analysis in conditioned media from SH-SY5Y cells expressing PrPC incubated with or without acitretin (20 μm) in Opti-MEM for 48 h at 37 °C (n = 3). sAPPβ (D), Aβ40 (E), and Aβ42 (F) were quantified by MSD analysis in conditioned media from day 65 cortical neurons incubated with or without acitretin (20 μm) in Opti-MEM for 48 h at 37 °C (n = 6 differentiations of two iPSC lines, OX1-19 and SBAD). G–I, immunoblotting and quantification of sAPPα in concentrated conditioned media (G) and quantification of Aβ40 (H) and Aβ42 (I) by MSD analysis in conditioned media from rat primary hippocampal neurons incubated with or without acitretin (20 μm) in Opti-MEM for 48 h at 37 °C (n = 3). Data are expressed as a percentage of control. Statistical analyses were Mann–Whitney U test for n = 3 and independent t test with Welch’s correction for n > 3. Data are shown as mean ± S.E. (error bars). n.s., not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

      Discussion

      Numerous studies have validated PrPC as a key neuronal receptor for AβO and highlighted the intrinsic role it plays in the activation of multiple downstream targets, leading to neuronal impairment in AD (reviewed in Refs.
      • Jarosz-Griffiths H.H.
      • Noble E.
      • Rushworth J.V.
      • Hooper N.M.
      Amyloid-β receptors: the good, the bad, and the prion protein.
      and
      • Salazar S.V.
      • Strittmatter S.M.
      Cellular prion protein as a receptor for amyloid-β oligomers in Alzheimer’s disease.
      ). As cleavage of PrPC by ADAM10 regulates the amount of PrPC at the cell surface (
      • Taylor D.R.
      • Parkin E.T.
      • Cocklin S.L.
      • Ault J.R.
      • Ashcroft A.E.
      • Turner A.J.
      • Hooper N.M.
      Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein.
      ,
      • Altmeppen H.C.
      • Prox J.
      • Puig B.
      • Kluth M.A.
      • Bernreuther C.
      • Thurm D.
      • Jorissen E.
      • Petrowitz B.
      • Bartsch U.
      • De Strooper B.
      • Saftig P.
      • Glatzel M.
      Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo.
      • Altmeppen H.C.
      • Prox J.
      • Krasemann S.
      • Puig B.
      • Kruszewski K.
      • Dohler F.
      • Bernreuther C.
      • Hoxha A.
      • Linsenmeier L.
      • Sikorska B.
      • Liberski P.P.
      • Bartsch U.
      • Saftig P.
      • Glatzel M.
      The sheddase ADAM10 is a potent modulator of prion disease.
      ), we hypothesized that modulation of ADAM10 activity, through altering cell surface PrPC, would impact AβO binding and toxicity. Here, we show that increasing ADAM10 activity promoted the shedding of cell surface PrPC and, as a result, blocked the binding of AβO to the surface of neurons and decreased their cytotoxicity as measured by activation of Fyn kinase and increase in ROS.
      Initially, we used the muscarinic agonist carbachol to promote the shedding of PrPC based on the observation that carbachol promotes the shedding of APP, likely through the activation of ADAM10 (
      • Parkin E.T.
      • Trew A.
      • Christie G.
      • Faller A.
      • Mayer R.
      • Turner A.J.
      • Hooper N.M.
      Structure-activity relationship of hydroxamate-based inhibitors on the secretases that cleave the amyloid precursor protein, angiotensin converting enzyme, CD23, and pro-tumor necrosis factor-α.
      ). Carbachol decreased the amount of PrPC at the cell surface, which resulted in reduced binding of AβO to the cells. Through the use of a selective ADAM10 inhibitor, we show for the first time that carbachol is acting via ADAM10 to promote the shedding of both PrPC and APP. To confirm that ADAM10 was altering AβO binding through modulating cell surface PrPC, we used siRNA knockdown to reduce ADAM10, which resulted in increased cell surface presentation of PrPC and a concomitant increase in AβO binding. Although various other proteins have been reported to act as cell surface receptors for AβO (reviewed in Ref.
      • Jarosz-Griffiths H.H.
      • Noble E.
      • Rushworth J.V.
      • Hooper N.M.
      Amyloid-β receptors: the good, the bad, and the prion protein.
      ), the effect of carbachol activation of ADAM10 on AβO binding was mediated specifically via PrPC. This was evidenced by (i) binding being blocked using the 6D11 antibody, whose epitope corresponds to the AβO-binding site on PrPC, and (ii) siRNA knockdown of ADAM10 in cells lacking PrPC failing to increase AβO binding. The AβO used here have been well-characterized using biophysical and immunological techniques and correspond to fibrillar oligomers recognized by the conformation-specific OC antibody (
      • Rushworth J.V.
      • Griffiths H.H.
      • Watt N.T.
      • Hooper N.M.
      Prion protein-mediated toxicity of amyloid-β oligomers requires lipid rafts and the transmembrane LRP1.
      ). Such OC-reactive AβO correlated with the onset and severity of AD in human brain (
      • Tomic J.L.
      • Pensalfini A.
      • Head E.
      • Glabe C.G.
      Soluble fibrillar oligomer levels are elevated in Alzheimer's disease brain and correlate with cognitive dysfunction.
      ) and with cognitive decline and tau aggregation and phosphorylation in a transgenic AD mouse model (
      • Chabrier M.A.
      • Blurton-Jones M.
      • Agazaryan A.A.
      • Nerhus J.L.
      • Martinez-Coria H.
      • LaFerla F.M.
      Soluble Aβ promotes wild-type tau pathology in vivo.
      ). Whether activation of ADAM10 will influence the cellular binding of other oligomeric species of Aβ will depend on whether their receptors are also susceptible to ADAM10-mediated shedding.
      In addition to using carbachol to activate ADAM10, we also employed the synthetic retinoid, acitretin, which releases all-trans-retinoic acid from cellular retinoic acid–binding proteins. This allows the all-trans-retinoic acid to bind to retinoid acid receptor transcription complexes, which in turn bind to retinoid binding sites on the ADAM10 promoter, leading to increased ADAM10 activity (
      • Tippmann F.
      • Hundt J.
      • Schneider A.
      • Endres K.
      • Fahrenholz F.
      Up-regulation of the α-secretase ADAM10 by retinoic acid receptors and acitretin.
      ). Acitretin has previously been reported to increase the α-secretase cleavage of APP by ADAM10 both in cellular and animal models (
      • Tippmann F.
      • Hundt J.
      • Schneider A.
      • Endres K.
      • Fahrenholz F.
      Up-regulation of the α-secretase ADAM10 by retinoic acid receptors and acitretin.
      ). Similarly, we report that acitretin promoted the shedding of APP and PrPC, both in SH-SY5Y cells and iPSC-derived neurons, which was blocked by the ADAM10-specific inhibitor. Thus, using two independently acting activators of ADAM10, we clearly show that promoting the activity of this metalloprotease reduces the cellular binding and cytotoxicity of AβO through modulating cell surface PrPC.
      ADAM10 promotes the shedding of the ectodomain of PrPC by cleaving the protein after residue 228, close to the site of attachment of the GPI anchor (residue 231) (
      • Taylor D.R.
      • Parkin E.T.
      • Cocklin S.L.
      • Ault J.R.
      • Ashcroft A.E.
      • Turner A.J.
      • Hooper N.M.
      Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein.
      ). However, it has also been proposed that ADAM10 is responsible for the α-cleavage of PrPC between amino acids 110 and 111 just C-terminal to the AβO-binding domain (residues 95–105) (
      • Laurán J.
      • Gimbel D.A.
      • Nygaard H.B.
      • Gilbert J.W.
      • Strittmatter S.M.
      Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers.
      ,
      • Chung E.
      • Ji Y.
      • Sun Y.
      • Kascsak R.J.
      • Kascsak R.B.
      • Mehta P.D.
      • Strittmatter S.M.
      • Wisniewski T.
      Anti-PrPC monoclonal antibody infusion as a novel treatment for cognitive deficits in an Alzheimer's disease model mouse.
      ), releasing the soluble N1 fragment containing the AβO-binding domain and leaving the cell-associated C1 fragment (
      • Vincent B.
      • Paitel E.
      • Saftig P.
      • Frobert Y.
      • Hartmann D.
      • De Strooper B.
      • Grassi J.
      • Lopez-Perez E.
      • Checler F.
      The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein.
      ), an action that would also be predicted to reduce AβO binding to the cell surface. The N1 fragment has been shown to bind AβOs and suppress their toxicity in cultured murine hippocampal neurons and in an in vivo mouse model of Aβ-induced memory dysfunction, leading to the suggestion that up-regulation of N1 production could act as a cellular mechanism to protect against AβO toxicity (
      • Guillot-Sestier M.V.
      • Sunyach C.
      • Ferreira S.T.
      • Marzolo M.P.
      • Bauer C.
      • Thevenet A.
      • Checler F.
      α-Secretase-derived fragment of cellular prion, N1, protects against monomeric and oligomeric amyloid β (Aβ)-associated cell death.
      ,
      • Fluharty B.R.
      • Biasini E.
      • Stravalaci M.
      • Sclip A.
      • Diomede L.
      • Balducci C.
      • La Vitola P.
      • Messa M.
      • Colombo L.
      • Forloni G.
      • Borsello T.
      • Gobbi M.
      • Harris D.A.
      An N-terminal fragment of the prion protein binds to amyloid-β oligomers and inhibits their neurotoxicity in vivo.
      ). Carbachol has previously been reported to increase the α-cleavage of PrPC in HEK293 cells through the action of ADAM17 (
      • Alfa Cissá M.
      • Sunyach C.
      • Slack B.E.
      • Fisher A.
      • Vincent B.
      • Checler F.
      M1 and M3 muscarinic receptors control physiological processing of cellular prion by modulating ADAM17 phosphorylation and activity.
      ,
      • Guillot-Sestier M.-V.
      • Sunyach C.
      • Druon C.
      • Scarzello S.
      • Checler F.
      The α-secretase-derived N-terminal product of cellular prion, N1, displays neuroprotective function in vitro and in vivo.
      ). Here, we show that in the SH-SY5Y cells, which express functional muscarinic receptors (
      • Canet-Aviles R.M.
      • Anderton M.
      • Hooper N.M.
      • Turner A.J.
      • Vaughan P.F.
      Muscarine enhances soluble amyloid precursor protein secretion in human neuroblastoma SH-SY5Y by a pathway dependent on protein kinase Cα, src-tyrosine kinase and extracellular signal-regulated kinase but not phospholipase C.
      ,
      • Vaughan P.F.
      • Walker J.H.
      • Peers C.
      The regulation of neurotransmitter secretion by protein kinase C.
      ), the carbachol-stimulated shedding of PrPC and APP is due to activation of ADAM10, not ADAM17. Furthermore, we were unable to detect the soluble N1 fragment in the conditioned medium from the SH-SY5Y cells following carbachol treatment (data not shown). The differences between our work and that previously published (
      • Alfa Cissá M.
      • Sunyach C.
      • Slack B.E.
      • Fisher A.
      • Vincent B.
      • Checler F.
      M1 and M3 muscarinic receptors control physiological processing of cellular prion by modulating ADAM17 phosphorylation and activity.
      ,
      • Guillot-Sestier M.-V.
      • Sunyach C.
      • Druon C.
      • Scarzello S.
      • Checler F.
      The α-secretase-derived N-terminal product of cellular prion, N1, displays neuroprotective function in vitro and in vivo.
      ) may reflect the use of different cell lines in which the relative levels of ADAM10 and ADAM17 differ and/or that in the SH-SY5Y cells, the N1 fragment is rapidly metabolized. However, as activation of ADAM10 in the SH-SY5Y cells did not influence the ratio of full-length PrPC to the C1 fragment, this indicates that the α-cleavage of PrPC is unaltered in our experimental model, ruling out a contribution of α-cleavage of PrPC to the mechanism by which ADAM10 activation blocks AβO binding.
      AβOs mediate a range of cellular actions that contribute to their neurotoxicity in AD. Binding of AβO to PrPC leads to activation of Fyn kinase (
      • Um J.W.
      • Nygaard H.B.
      • Heiss J.K.
      • Kostylev M.A.
      • Stagi M.
      • Vortmeyer A.
      • Wisniewski T.
      • Gunther E.C.
      • Strittmatter S.M.
      Alzheimer amyloid-β oligomer bound to postsynaptic prion protein activates Fyn to impair neurons.
      ,
      • Rushworth J.V.
      • Griffiths H.H.
      • Watt N.T.
      • Hooper N.M.
      Prion protein-mediated toxicity of amyloid-β oligomers requires lipid rafts and the transmembrane LRP1.
      ), which in turn phosphorylates N-methyl-d-aspartate receptors, altering their cell surface distribution (
      • Um J.W.
      • Nygaard H.B.
      • Heiss J.K.
      • Kostylev M.A.
      • Stagi M.
      • Vortmeyer A.
      • Wisniewski T.
      • Gunther E.C.
      • Strittmatter S.M.
      Alzheimer amyloid-β oligomer bound to postsynaptic prion protein activates Fyn to impair neurons.
      ), and directly phosphorylates tau on Tyr-18 (
      • Larson M.
      • Sherman M.A.
      • Amar F.
      • Nuvolone M.
      • Schneider J.A.
      • Bennett D.A.
      • Aguzzi A.
      • Lesná S.E.
      The complex PrPc-Fyn couples human oligomeric Aβ with pathological Tau changes in Alzheimer’s disease.
      ). AβOs also induce cytotoxicity through increasing ROS (
      • Crews L.
      • Masliah E.
      Molecular mechanisms of neurodegeneration in Alzheimer's disease.
      ), although to date whether this effect is mediated via their binding to PrPC has not been reported. Here, we show that activation of ADAM10 blocked the AβO-dependent activation of Fyn phosphorylation mediated by PrPC. Furthermore, and for the first time, we report that in both SH-SY5Y cells and human iPSC-derived neurons, AβO binding to PrPC increases cellular ROS and that this increase in ROS can be blocked by removing cell surface PrPC upon ADAM10 activation. Thus, ADAM10 activation can ameliorate the downstream cytotoxicity induced by AβO binding to PrPC and represents another potential therapeutic approach to disrupt the AβO-PrPC signaling complex in AD.
      Genetic analyses of families with late-onset AD revealed two rare mutations (Q170H and R181G) in the pro-domain of ADAM10 that attenuated its α-secretase activity and shifted APP processing toward β-secretase–mediated cleavage with a 2–3-fold increase in Aβ levels, enhanced Aβ plaque load, and reactive gliosis (
      • Kim M.
      • Suh J.
      • Romano D.
      • Truong M.H.
      • Mullin K.
      • Hooli B.
      • Norton D.
      • Tesco G.
      • Elliott K.
      • Wagner S.L.
      • Moir R.D.
      • Becker K.D.
      • Tanzi R.E.
      Potential late-onset Alzheimer’s disease-associated mutations in the ADAM10 gene attenuate α-secretase activity.
      ,
      • Suh J.
      • Choi S.H.
      • Romano D.M.
      • Gannon M.A.
      • Lesinski A.N.
      • Kim D.Y.
      • Tanzi R.E.
      ADAM10 missense mutations potentiate β-amyloid accumulation by impairing prodomain chaperone function.
      ). Based on our work with human iPSC-derived neurons, it is likely that these mutations in ADAM10 will also lead to increased cell surface PrPC and enhanced AβO binding and cytotoxicity, which may contribute to the AD phenotype in individuals with such ADAM10 mutations.
      The general consensus is that there is competition between the α- and β-secretases for their substrate APP. For example, in murine primary cortical neurons, ADAM10 knockdown increased sAPPβ and Aβ (
      • Kuhn P.-H.
      • Wang H.
      • Dislich B.
      • Colombo A.
      • Zeitschel U.
      • Ellwart J.W.
      • Kremmer E.
      • Rossner S.
      • Lichtenthaler S.F.
      ADAM10 is the physiologically relevant, constitutive α-secretase of the amyloid precursor protein in primary neurons.
      ), and in human iPSC-derived neurons, inhibition of BACE1 reduced sAPPβ and Aβ and increased sAPPα (
      • Ben Halima S.
      • Mishra S.
      • Raja K.M.P.
      • Willem M.
      • Baici A.
      • Simons K.
      • Brüstle O.
      • Koch P.
      • Haass C.
      • Caflisch A.
      • Rajendran L.
      Specific inhibition of β-secretase processing of the Alzheimer disease amyloid precursor protein.
      ). However, other studies have failed to observe such a reciprocal relationship (
      • Colombo A.
      • Wang H.
      • Kuhn P.H.
      • Page R.
      • Kremmer E.
      • Dempsey P.J.
      • Crawford H.C.
      • Lichtenthaler S.F.
      Constitutive α- and β-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines.
      ). On the whole, however, activation of ADAM10 has been reported to result in an increase in sAPPα with a reciprocal decrease in sAPPβ and Aβ peptides in various cell and transgenic mouse models (
      • Postina R.
      • Schroeder A.
      • Dewachter I.
      • Bohl J.
      • Schmitt U.
      • Kojro E.
      • Prinzen C.
      • Endres K.
      • Hiemke C.
      • Blessing M.
      • Flamez P.
      • Dequenne A.
      • Godaux E.
      • van Leuven F.
      • Fahrenholz F.
      A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model.
      ,
      • Tippmann F.
      • Hundt J.
      • Schneider A.
      • Endres K.
      • Fahrenholz F.
      Up-regulation of the α-secretase ADAM10 by retinoic acid receptors and acitretin.
      ,
      • Hoey S.E.
      • Williams R.J.
      • Perkinton M.S.
      Synaptic NMDA receptor activation stimulates α-secretase amyloid precursor protein processing and inhibits amyloid-β production.
      ,
      • Moss M.L.
      • Powell G.
      • Miller M.A.
      • Edwards L.
      • Qi B.
      • Sang Q.-X.A.
      • De Strooper B.
      • Tesseur I.
      • Lichtenthaler S.F.
      • Taverna M.
      • Zhong J.L.
      • Dingwall C.
      • Ferdous T.
      • Schlomann U.
      • Zhou P.
      • et al.
      ADAM9 inhibition increases membrane activity of ADAM10 and controls α-secretase processing of amyloid precursor protein.
      ). For example, in APPV717I transgenic mice, moderate neuronal overexpression of ADAM10 increased the secretion of sAPPα, reduced the formation of Aβ peptides, and prevented their deposition in plaques, whereas expression of mutant catalytically inactive ADAM10, which acts in a dominant negative manner, led to an increase in the number and size of plaques in the double transgenic mice (
      • Postina R.
      • Schroeder A.
      • Dewachter I.
      • Bohl J.
      • Schmitt U.
      • Kojro E.
      • Prinzen C.
      • Endres K.
      • Hiemke C.
      • Blessing M.
      • Flamez P.
      • Dequenne A.
      • Godaux E.
      • van Leuven F.
      • Fahrenholz F.
      A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model.
      ). Stimulation of ADAM10 promoter activity with the vitamin A analogue acitretin led to an increase of mature ADAM10 protein that increased the ratio between α- and β-secretase activity in SH-SY5Y cells, whereas intracerebral injection of acitretin in APP/PS-1 transgenic mice led to a reduction of Aβ40 and Aβ42 (
      • Tippmann F.
      • Hundt J.
      • Schneider A.
      • Endres K.
      • Fahrenholz F.
      Up-regulation of the α-secretase ADAM10 by retinoic acid receptors and acitretin.
      ). Thus, we were somewhat surprised that although activating ADAM10 with acitretin led to a reduction in Aβ levels in the SH-SY5Y cells and in the murine hippocampal neurons, no such decrease in Aβ40, Aβ42, or sAPPβ was observed in iPSC-derived neurons from two different individuals, despite there being a similar increase in sAPPα in all three cell models. This result with human neurons is consistent with the result from a pilot clinical trial in which 21 mild to moderate AD patients were treated with acitretin for 4 weeks. In the AD patients, acitretin caused a significant increase in CSF sAPPα when compared with a placebo control group, but there was not a reciprocal significant decrease in sAPPβ, and Aβ42 was unchanged between the two groups (
      • Endres K.
      • Fahrenholz F.
      • Lotz J.
      • Hiemke C.
      • Teipel S.
      • Lieb K.
      • Tüscher O.
      • Fellgiebel A.
      Increased CSF APPs-α levels in patients with Alzheimer disease treated with acitretin.
      ). Together, these observations suggest that the reciprocal relationship between the nonamyloidogenic and amyloidogenic APP-processing pathways may not hold true in human neurons when α-secretase cleavage is stimulated.
      Regardless of whether activation of ADAM10 leads to a decrease in Aβ by shifting the balance of APP processing between the nonamyloidogenic and amyloidogenic pathways, activation of ADAM10 could be beneficial in AD by acting through other mechanisms. Activation of ADAM10 will directly increase sAPPα that has neuroprotective, neurotrophic, and neurogenic properties (
      • Caillá I.
      • Allinquant B.
      • Dupont E.
      • Bouillot C.
      • Langer A.
      • Müller U.
      • Prochiantz A.
      Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone.
      ,
      • Mattson M.P.
      • Cheng B.
      • Culwell A.R.
      • Esch F.S.
      • Lieberburg I.
      • Rydel R.E.
      Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the β-amyloid precursor protein.
      • Ring S.
      • Weyer S.W.
      • Kilian S.B.
      • Waldron E.
      • Pietrzik C.U.
      • Filippov M.A.
      • Herms J.
      • Buchholz C.
      • Eckman C.B.
      • Korte M.
      • Wolfer D.P.
      • Müller U.C.
      The secreted β-amyloid precursor protein ectodomain APPsα is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice.
      ), elevates adult neurogenesis in the hippocampus (
      • Suh J.
      • Choi S.H.
      • Romano D.M.
      • Gannon M.A.
      • Lesinski A.N.
      • Kim D.Y.
      • Tanzi R.E.
      ADAM10 missense mutations potentiate β-amyloid accumulation by impairing prodomain chaperone function.
      ), and has been reported to decrease Aβ generation by directly associating with BACE1 and modulating its activity (
      • Obregon D.
      • Hou H.
      • Deng J.
      • Giunta B.
      • Tian J.
      • Darlington D.
      • Shahaduzzaman M.
      • Zhu Y.
      • Mori T.
      • Mattson M.P.
      • Tan J.
      Soluble amyloid precursor protein-α modulates β-secretase activity and amyloid-β generation.
      ). In addition, as we have demonstrated here, activation of ADAM10 will increase the shedding of PrPC to reduce the cellular binding and downstream toxicity of AβO. However, as well as shedding APP and PrPC, ADAM10 also proteolytically cleaves more than 90 membrane proteins in the central nervous system, many of which are essential for brain development and normal physiological functions (
      • Saftig P.
      • Lichtenthaler S.F.
      The α secretase ADAM10: a metalloprotease with multiple functions in the brain.
      ,
      • Kuhn P.H.
      • Colombo A.V.
      • Schusser B.
      • Dreymueller D.
      • Wetzel S.
      • Schepers U.
      • Herber J.
      • Ludwig A.
      • Kremmer E.
      • Montag D.
      • Müller U.
      • Schweizer M.
      • Saftig P.
      • Bräse S.
      • Lichtenthaler S.F.
      Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function.
      ). Thus, whether activation of ADAM10 will only have beneficial effects is unclear, a situation that may be exacerbated if activation were to occur over a prolonged period of time as required to treat a chronic condition such as AD.
      We have shown that activation of ADAM10 promotes the proteolytic shedding of the AβO receptor PrPC from the surface of human neuroblastoma cells and iPSC-derived neurons. This shedding of PrPC reduces the binding of AβO to cells and blocks their cytotoxicity as monitored by activation of Fyn kinase and increase in ROS. These data provide the first indication that modulating cell surface PrPC may contribute to the therapeutic potential of ADAM10 activation in AD and contribute to the neurodegeneration observed in individuals with mutations in ADAM10. In addition, we report that activation of ADAM10 does not result in a decrease in Aβ levels in the human iPSC-derived neurons, a result consistent with the results from a study following acitretin administration in humans (
      • Endres K.
      • Fahrenholz F.
      • Lotz J.
      • Hiemke C.
      • Teipel S.
      • Lieb K.
      • Tüscher O.
      • Fellgiebel A.
      Increased CSF APPs-α levels in patients with Alzheimer disease treated with acitretin.
      ) and highlighting that human iPSC-derived neurons are a valuable model system to explore the mechanisms underlying AD.

      Experimental procedures

      Cell culture

      SH-SY5Y human neuroblastoma cells were stably transfected with the cDNA encoding murine PrPC containing the 3F4 epitope tag (human M108/M111) as described previously (
      • Perera W.S.
      • Hooper N.M.
      Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.
      ). Both untransfected and PrPC-expressing SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum. NB7 human neuroblastoma cells were cultured in RPMI 1640 medium (Sigma) supplemented with 10% (v/v) fetal bovine serum. Cells were maintained in a humidified incubator at 37 °C in a 5% CO2, 95% air atmosphere.

      Culture and differentiation of induced pluripotent stem cells

      The iPSC lines, OX1-19 (obtained from S. Cowley, University of Oxford) (
      • van Wilgenburg B.
      • Browne C.
      • Vowles J.
      • Cowley S.A.
      Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions.
      ) and SBAD03-05 (obtained from StemBANCC) (
      • Baud A.
      • Wessely F.
      • Mazzacuva F.
      • McCormick J.
      • Camuzeaux S.
      • Heywood W.E.
      • Little D.
      • Vowles J.
      • Tuefferd M.
      • Mosaku O.
      • Lako M.
      • Armstrong L.
      • Webber C.
      • Cader M.Z.
      • Peeters P.
      • et al.
      Multiplex high-throughput targeted proteomic assay to identify induced pluripotent stem cells.
      ) were maintained on Matrigel (BD Biosciences) in mTeSR1 medium (StemCell Technologies) containing 50 units/ml penicillin and 50 μg/ml streptomycin (Life Technologies, Inc.) in a humidified incubator at 37 °C in a 5% CO2, 95% air atmosphere. iPSCs were differentiated to cortical neurons as described previously (
      • Shi Y.
      • Kirwan P.
      • Livesey F.J.
      Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks.
      ), using dual-SMAD inhibition by 1 μm dorsomorphin and 10 μm SB431452 (Tocris). Following successful differentiation, neural progenitor cells were replated on day 35 postinduction at 250,000 cells/well onto polyornithine and laminin-coated (Sigma) 6-well tissue culture plates and cultured until day 65 postinduction with medium changes every 2–3 days. iPSC pluripotency and successful cortical neuron differentiation were confirmed using immunofluorescence microscopy with appropriate markers.

      Preparation of rat primary hippocampal neurons

      Primary neurons were prepared from the hippocampi of 1–2-day-old Wistar rats and cultured as described previously (
      • Watt N.T.
      • Taylor D.R.
      • Kerrigan T.L.
      • Griffiths H.H.
      • Rushworth J.V.
      • Whitehouse I.J.
      • Hooper N.M.
      Prion protein facilitates uptake of zinc into neuronal cells.
      ). Neurons were cultured for 14 days with medium changes every 3–4 days.

      Activation and inhibition of ADAM10

      To activate ADAM10, cells were incubated in Opti-MEM containing GlutaMAX (Life Technologies) containing either 20 μm carbachol (Sigma) for 24 h or 20 μm acitretin (Sigma) for 48 h. To inhibit ADAM10, cells were incubated with 10 μm GI254023X (Tocris), a selective ADAM10 inhibitor, for either 24 or 48 h in Opti-MEM. DMSO only–treated cells were used for comparison with treated cells.

      RNAi studies

      siRNA specific for human ADAM10 and a nontargeting sequence were obtained as SMARTpools from Dharmacon (Thermo Fisher Scientific). An additional, single siRNA sequence for human ADAM10 was also obtained for verification experiments (Ambion). SH-SY5Y (untransfected or PrPC-expressing) cells were seeded into T75 flasks or 24-well plates in routine culture medium and allowed to adhere overnight. The cell monolayers were washed twice with Dulbecco’s PBS (DPBS), and a 25 nm (final concentration) SMARTpool siRNA solution was delivered as a complex with DharmaFECT-1 transfection reagent (Dharmacon) in serum-containing DMEM for 48 h. Cells were then washed twice in DPBS prior to incubation in Opti-MEM for 24 h.

      Conditioned medium, cell lysate, and cell membrane preparation

      Conditioned medium was harvested, and cell debris was pelleted by centrifugation at 500 × g for 5 min. A 1-ml sample of conditioned medium was removed for immunoassay and stored at −20 °C. Remaining conditioned media were concentrated to 100 μl in a Vivapsin 20-ml concentrator (10,000 molecular weight membrane) by centrifugation at 1,900 × g for ∼1 h in a bench top centrifuge at 4 °C. Cells were washed in PBS (Lonza), harvested, and pelleted at 1,400 × g for 3 min. Cells were lysed on ice for 30 min in radioimmune precipitation assay buffer (50 mm Tris/HCl, 150 mm NaCl, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, pH 8.0) containing protease and phosphatase inhibitor mixtures (Roche Diagnostics Ltd.). Cell lysates were clarified by centrifugation at 12,460 × g for 10 min at 4 °C, and the clarified lysate was stored at −20 °C before use. For the preparation of membranes, cells were resuspended in 3 ml of 50 mm HEPES, pH 7.5, and sonicated at amplitude 7 μm for 30 s using a Soniprep150 disintegrator. The cell suspension was then centrifuged at 2,500 × g for 10 min at 4 °C to pellet cell membranes and nuclei. The supernatant was then centrifuged in a Beckman Coulter Optima at 100,000 × g for 1 h at 4 °C. Membranes were resuspended in 50 mm Tris/HCl, pH 7.5, 2 mm EDTA, 150 mm NaCl, and 1% (w/v) CHAPSO. Protein concentration of all samples was determined by a bicinchoninic acid assay.

      Deglycosylation of cell lysate samples

      All deglycosylation solutions were purchased from Prozyme. Cell lysates were made up to 100 μg of protein in a 150-μl volume and boiled for 5 min in 20% (v/v) 5× N-glycanase reaction buffer and 4% denaturation solution. After cooling, 4% detergent solution was added to each tube. To one of the tubes, 0.5% (v/v) N-glycanase (200 milliunits) was added and incubated at 37 °C for 16 h.

      Immunoblotting

      Samples were made up in dissociation buffer (1× dissociation buffer (100 mm Tris-HCl, 2% (w/v) SDS, 10% (v/v) glycerol, 100 mm DTT, 0.02% (w/v) bromphenol blue, pH 6.8) and heated at 95 °C for 5 min. Proteins were resolved by SDS-PAGE on 7–17% acrylamide Tris-glycine gels and then transferred to Hybond polyvinylidene difluoride membranes (GE Healthcare). Following electrotransfer, the membranes were blocked for 1 h in PBS with 0.1% Tween 20 (PBST) and 5% (w/v) nonfat milk and then incubated with primary antibody overnight at 4 °C. Antigens were probed using the following primary antibodies: anti-APP (22C11, Millipore), SAF32 (anti-PrP N terminus, Cayman Chemical), 8H4 (anti-PrP residues 175–185), anti-ADAM10 antibody (Abcam), 6E10 (anti-Aβ(1–17), Merck Biosciences), AC15 (anti-β-actin) and synapsin 1 (Sigma), 2B3 (anti-human sAPPα, Immuno-Biological Laboratories), and anti-phospho-Src family kinase (Tyr-416; Cell Signaling Technology). Primary antibodies were detected by incubation with horseradish peroxidase–conjugated secondary antibody, both in PBST containing 2% BSA. Bound horseradish peroxidase conjugates were visualized using the ECL® detection system with a Syngene Gbox XT4 (Syngene). Densitometric analysis was performed using Genetools analysis software (Syngene).

      ImageStream imaging cytometry

      SH-SY5Y cells expressing PrPC were incubated in Opti-MEM containing GlutaMAX and either 20 μm carbachol for 24 h or 20 μm acitretin for 48 h. DMSO only–treated cells were used for comparison with treated cells. Cells were collected in PBS without metals and recovered by centrifugation (300 × g for 5 min). All subsequent procedures were carried out at 4 °C. Cells were resuspended in blocking buffer (10% donkey serum in PBS containing metals) at 20 × 106 cells/ml and incubated in primary antibody, SAF32, for 1 h before washing twice with PBS and recovering cells by centrifugation. Cells were then resuspended in blocking buffer and incubated with donkey anti-mouse Alexa Fluor® 488 (Invitrogen) for 30 min in the dark. Cells were washed twice in PBS, fixed in 3% (v/v) paraformaldehyde, and then finally resuspended in PBS before analysis using the ImageStreamX MkII imaging cytometer (Amnis). Brightfield, fluorescence, and dark field scatter images were collected at ×40 magnification for 3000 cells over six biological repeats. Cells were identified by the area and aspect ratio parameters. In focus cells were identified as having a gradient root mean square measure of >40. AlexaFluor® 488 emission was generated by a 488-nm laser set to 100 milliwatts in the INSPIRE software (Amnis). Data were analyzed in IDEAS software (Amnis) and exported to Flowjo version 10 (Tree Star, Inc.) to generate overlay histograms.

      Aβ oligomer preparation

      Synthetic biotin-Aβ(1–42) containing a 6-carbon linker between the biotin moiety and the N terminus of Aβ was purchased from AnaSpec (San Jose, CA). AβOs were prepared as described previously (
      • Rushworth J.V.
      • Griffiths H.H.
      • Watt N.T.
      • Hooper N.M.
      Prion protein-mediated toxicity of amyloid-β oligomers requires lipid rafts and the transmembrane LRP1.
      ). Briefly, Aβ peptide was dissolved in 1,1,1,3,3,3-hexafluoropropan-2-ol to break down any aggregated material, dried under a stream of N2 gas, and stored at −80 °C. Peptide films of biotin-Aβ(1–42) were dissolved in DMSO to 1 mm and then resuspended in Ham’s F-12 medium (Lonza) to a final monomer concentration of 100 μm and incubated for 18–24 h at room temperature. The preparation was then centrifuged at 14,000 × g for 20 min to pellet out any fibrillar material, and the supernatant was retained as the oligomer preparation.

      Fluorescence microscopy

      SH-SY5Y cells were cultured in growth medium to ∼60% confluence on glass coverslips before required incubations (addition of carbachol, acitretin, or GI254023X, as described) or ADAM10 siRNA treatments were carried out. For AβO-binding experiments, cells were incubated with 500 nm AβO (total peptide concentration) diluted in Opti-MEM for 30 min at 37 °C. Where indicated, a 20-min preincubation with or without 10 μg/ml 6D11 antibody (Covance, BioLegend) was carried out before incubation with AβOs. Postincubation, cells were fixed with 4% (v/v) paraformaldehyde and blocked overnight at 4 °C in DPBS with 5% (v/v) fish skin gelatin. Where required, cells were permeabilized in 0.1% Triton X-100 for 10 min at room temperature before blocking. Coverslips were subsequently incubated for 2 h in the same buffer containing primary antibody, washed, and then incubated with the corresponding fluorescently labeled secondary antibody. PrPC was detected using SAF32 antibody followed by donkey anti-mouse AlexaFluor® 488, and MAP2 was detected with anti-MAP2 (Millipore) followed by goat anti-chicken Alexa Fluor 647 Cy5-IgG (Invitrogen) and biotin-Aβ(1–42) (for detection of AβO) using Texas Red–conjugated streptavidin (Invitrogen). Nuclei were counterstained by washing briefly in DAPI stain, and coverslips were mounted onto glass slides using Fluoromount-G (Southern Biotech). Cells were visualized using a DeltaVision optical restoration microscopy system (Applied Precision). Data were collected from 12 0.5-μm-thick optical sections, and three-dimensional data sets were deconvolved using the softWoRx program (Applied Precision). Images analyzed were individual z-sections taken from the middle of the data stack, representing a section through the center of the cell. The number of cells analyzed is indicated in individual figure legends. Fluorescence around the cell membrane was quantified using ImageJ software as described previously (
      • Taylor D.R.
      • Watt N.T.
      • Perera W.S.
      • Hooper N.M.
      Assigning functions to distinct regions of the N-terminus of the prion protein that are involved in its copper-stimulated, clathrin-dependent endocytosis.
      ). This was plotted as pixel intensity versus distance around the cell using Microsoft Excel, and then the percentage of cell surface with detectable staining was calculated from multiple images.
      For the fluorescence microscopy of iPSCs and iPSC-derived cortical neurons, cells were grown on laminin-coated coverslips, fixed with 4% (v/v) paraformaldehyde, and blocked for 4 h at room temperature in DPBS with 10% (v/v) donkey serum. Where required, cells were permeabilized in 0.2% Triton X-100 for 4 min at room temperature before blocking. Coverslips were then incubated with iPSC-specific markers (PAX6, Oct4, SSEA-4, and nanog; Abcam) to check pluripotency and with neuronal markers (Satb2, Tbr1, and MAP2; Abcam) to confirm differentiation. Each primary antibody was detected with the corresponding secondary antibody, Alexa Fluor 488, 594, or 647. DAPI stain was applied, and coverslips were mounted using ProLong Gold containing DAPI (Southern Biotech). Cells were visualized using an EVOS FL cell imaging system (Thermo Fisher Scientific).

      qPCR

      SH-SY5Y cells expressing PrPC were incubated in Opti-MEM containing GlutaMAX and 20 μm acitretin for 48 h. DMSO only-treated cells were used for comparison with treated cells. Cells were harvested, and RNA was extracted using the RNeasy plus kit (QIAgen) according to the manufacturer’s instructions. cDNA was synthesized using 1 μg of prepared RNA using the iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions. The mRNA expression level of ADAM10 was analyzed by real-time qPCR using the SYBR Green method (Applied Biosystems) with the sense and antisense primers reported previously (
      • Cui L.
      • Gao Y.
      • Xie Y.
      • Wang Y.
      • Cai Y.
      • Shao X.
      • Ma X.
      • Li Y.
      • Ma G.
      • Liu G.
      • Cheng W.
      • Liu Y.
      • Liu T.
      • Pan Q.
      • Tao H.
      • et al.
      An ADAM10 promoter polymorphism is a functional variant in severe sepsis patients and confers susceptibility to the development of sepsis.
      ). Samples were analyzed in triplicate on a Quantstudio 3 (Life Technologies), and relative expression was calculated using ribosomal qPCR as the control.

      Multiplex immunoassay

      Aβ40 and Aβ42 were measured using the V-PLEX Aβ peptide panel 1 (6E10) assay (MSD (Meso Scale Discovery), catalog no. K15200E). sAPPα and sAPPβ were measured using the sAPPα/sAPPβ multiplex assay kit (MSD, catalog no. K15120E) according to the manufacturer’s instructions. Assay plates were blocked, and conditioned cell medium samples and standards buffered with 500 mm HEPES, pH 7.4, to a final concentration of 50 mm were loaded in duplicate. Following washing and secondary antibody incubation, assays were read using the MESO QUICKPLEX SQ 120 imager and analyzed using MSD Workbench 4.0 software. The protein concentration of the conditioned medium was determined by a bicinchoninic acid assay, and sAPPα, sAPPβ, and Aβ levels were corrected for total protein concentration.

      ROS assay

      For the detection of ROS in cells, H2O2 production was measured with the ROS-GloTM H2O2 assay system (Promega). SH-SY5Y cells expressing PrPC were seeded onto black-walled, clear bottom 96-well plates and allowed to adhere overnight. Cells were then incubated with or without 20 μm acitretin diluted in Opti-MEM for 48 h before incubation with or without AβO (500 nm) in the presence of 10 μm menadione (Sigma) and the H2O2 substrate at 37 °C for 90 min. ROS-Glo detection solution and signal enhancer were then added, and, after a 20-min incubation at room temperature, luminescence was measured with a Synergy HT Bio-Tek fluorimeter using Gen5 software. For experiments with cortical neurons, the above method was used with the following modifications. Cells were plated at day 35 and cultured until day 65. Following acitretin incubation, 1% BSA was added to cell cultures for 10 min to block the nonspecific binding of AβO to the laminin coating on the plates prior to incubation with 2.5 μm AβOs.

      Statistical analysis

      Data were analyzed as stated in the figure legends, and n numbers are specified. For statistical analysis, data were analyzed using GraphPad Prism version 7.00. A normal distribution was assumed for all cell data as mean values were recorded from a population of cells (based on the assumption that cells from a clonal population will respond in a similar manner), and therefore parametric analyses were performed. For comparison between two data sets, an independent t test was applied with Welch’s correction (equal S.D. values not assumed). For the rat primary hippocampal neuron data, a Mann–Whitney U test was used to compare between groups, as a normal distribution could not be assumed or determined from the sample size. For multiple comparisons, a one-way ANOVA with Tukey’s post hoc correction for pairwise comparisons was used. Data are shown as mean ± S.E., and p < 0.05 was considered significant. Levels of significance are defined in the figure legends.

      Author contributions

      H. H. J.-G., K. A. B. K., and N. M. H. designed the study. H. H. J.-G., N. J. C., H. A. R., K. F., A. C. J., G. J. H., J. B., and K. A. B. K. performed the research. S. A. C., S. C., and M. Z. C. provided reagents. H. H. J. G., N. J. C., K. F., G. J. H., K. A. B. K., and N. M. H. analyzed the data. H. H. J.-G., K. A. B. K., and N. M. H. wrote the paper. All authors read and approved the final manuscript.

      Acknowledgments

      The Bioimaging Facility microscopes used in this study were purchased with grants from the BBSRC, Wellcome Trust, and the University of Manchester Strategic Fund. The ImagestreamX MkII used in this study was purchased with funding from the Medical Research Council and accessed through the University of Manchester Flow Cytometry Core Facility.

      References

        • Blennow K.
        • de Leon M.J.
        • Zetterberg H.
        Alzheimer's disease.
        Lancet. 2006; 368 (16876668): 387-403
        • De Strooper B.
        • Karran E.
        The cellular phase of Alzheimer's disease.
        Cell. 2016; 164 (26871627): 603-615
        • Viola K.L.
        • Klein W.L.
        Amyloid beta oligomers in Alzheimer's disease pathogenesis, treatment, and diagnosis.
        Acta Neuropathol. 2015; 129 (25604547): 183-206
        • Jarosz-Griffiths H.H.
        • Noble E.
        • Rushworth J.V.
        • Hooper N.M.
        Amyloid-β receptors: the good, the bad, and the prion protein.
        J. Biol. Chem. 2016; 291 (26719327): 3174-3183
        • Salazar S.V.
        • Strittmatter S.M.
        Cellular prion protein as a receptor for amyloid-β oligomers in Alzheimer’s disease.
        Biochem. Biophys. Res. Commun. 2017; 483 (27639648): 1143-1147
        • Laurán J.
        • Gimbel D.A.
        • Nygaard H.B.
        • Gilbert J.W.
        • Strittmatter S.M.
        Cellular prion protein mediates impairment of synaptic plasticity by amyloid-β oligomers.
        Nature. 2009; 457 (19242475): 1128-1132
        • Gimbel D.A.
        • Nygaard H.B.
        • Coffey E.E.
        • Gunther E.C.
        • Laurán J.
        • Gimbel Z.A.
        • Strittmatter S.M.
        Memory impairment in transgenic Alzheimer mice requires cellular prion protein.
        J. Neurosci. 2010; 30 (20445063): 6367-6374
        • Kostylev M.A.
        • Kaufman A.C.
        • Nygaard H.B.
        • Patel P.
        • Haas L.T.
        • Gunther E.C.
        • Vortmeyer A.
        • Strittmatter S.M.
        Prion-protein-interacting amyloid-β oligomers of high molecular weight are tightly correlated with memory impairment in multiple Alzheimer mouse models.
        J. Biol. Chem. 2015; 290 (26018073): 17415-17438
        • Larson M.
        • Sherman M.A.
        • Amar F.
        • Nuvolone M.
        • Schneider J.A.
        • Bennett D.A.
        • Aguzzi A.
        • Lesná S.E.
        The complex PrPc-Fyn couples human oligomeric Aβ with pathological Tau changes in Alzheimer’s disease.
        J. Neurosci. 2012; 32 (23175838): 16857-16871a
        • Um J.W.
        • Nygaard H.B.
        • Heiss J.K.
        • Kostylev M.A.
        • Stagi M.
        • Vortmeyer A.
        • Wisniewski T.
        • Gunther E.C.
        • Strittmatter S.M.
        Alzheimer amyloid-β oligomer bound to postsynaptic prion protein activates Fyn to impair neurons.
        Nat. Neurosci. 2012; 15 (22820466): 1227-1235
        • Rushworth J.V.
        • Griffiths H.H.
        • Watt N.T.
        • Hooper N.M.
        Prion protein-mediated toxicity of amyloid-β oligomers requires lipid rafts and the transmembrane LRP1.
        J. Biol. Chem. 2013; 288 (23386614): 8935-8951
        • Chong Z.Z.
        • Li F.
        • Maiese K.
        Oxidative stress in the brain: novel cellular targets that govern survival during neurodegenerative disease.
        Prog. Neurobiol. 2005; 75 (15882775): 207-246
        • Barry A.E.
        • Klyubin I.
        • Mc Donald J.M.
        • Mably A.J.
        • Farrell M.A.
        • Scott M.
        • Walsh D.M.
        • Rowan M.J.
        Alzheimer’s disease brain-derived amyloid-β-mediated inhibition of LTP in vivo is prevented by immunotargeting cellular prion protein.
        J. Neurosci. 2011; 31 (21593310): 7259-7263
        • Freir D.B.
        • Nicoll A.J.
        • Klyubin I.
        • Panico S.
        • Mc Donald J.M.
        • Risse E.
        • Asante E.A.
        • Farrow M.A.
        • Sessions R.B.
        • Saibil H.R.
        • Clarke A.R.
        • Rowan M.J.
        • Walsh D.M.
        • Collinge J.
        Interaction between prion protein and toxic amyloid β assemblies can be therapeutically targeted at multiple sites.
        Nat. Commun. 2011; 2 (21654636): 336
        • Klyubin I.
        • Nicoll A.J.
        • Khalili-Shirazi A.
        • Farmer M.
        • Canning S.
        • Mably A.
        • Linehan J.
        • Brown A.
        • Wakeling M.
        • Brandner S.
        • Walsh D.M.
        • Rowan M.J.
        • Collinge J.
        Peripheral administration of a humanized anti-PrP antibody blocks Alzheimer’s disease Aβ synaptotoxicity.
        J. Neurosci. 2014; 34 (24790184): 6140-6145
        • Risse E.
        • Nicoll A.J.
        • Taylor W.A.
        • Wright D.
        • Badoni M.
        • Yang X.
        • Farrow M.A.
        • Collinge J.
        Identification of a compound that disrupts binding of amyloid-β to the prion protein using a novel fluorescence-based assay.
        J. Biol. Chem. 2015; 290 (25995455): 17020-17028
        • Um J.W.
        • Kaufman A.C.
        • Kostylev M.
        • Heiss J.K.
        • Stagi M.
        • Takahashi H.
        • Kerrisk M.E.
        • Vortmeyer A.
        • Wisniewski T.
        • Koleske A.J.
        • Gunther E.C.
        • Nygaard H.B.
        • Strittmatter S.M.
        Metabotropic glutamate receptor 5 is a coreceptor for Alzheimer Aβ oligomer bound to cellular prion protein.
        Neuron. 2013; 79 (24012003): 887-902
        • Haas L.T.
        • Salazar S.V.
        • Kostylev M.A.
        • Um J.W.
        • Kaufman A.C.
        • Strittmatter S.M.
        Metabotropic glutamate receptor 5 couples cellular prion protein to intracellular signalling in Alzheimer's disease.
        Brain. 2016; 139 (26667279): 526-546
        • Beraldo F.H.
        • Ostapchenko V.G.
        • Caetano F.A.
        • Guimaraes A.L.
        • Ferretti G.D.
        • Daude N.
        • Bertram L.
        • Nogueira K.O.
        • Silva J.L.
        • Westaway D.
        • Cashman N.R.
        • Martins V.R.
        • Prado V.F.
        • Prado M.A.
        Regulation of amyloid β oligomer binding to neurons and neurotoxicity by the prion protein-mGluR5 complex.
        J. Biol. Chem. 2016; 291 (27563063): 21945-21955
        • Hamilton A.
        • Vasefi M.
        • Vander Tuin C.
        • McQuaid R.J.
        • Anisman H.
        • Ferguson S.S.
        Chronic pharmacological mGluR5 inhibition prevents cognitive impairment and reduces pathogenesis in an Alzheimer disease mouse model.
        Cell Rep. 2016; 15 (27210751): 1859-1865
        • Haas L.T.
        • Salazar S.V.
        • Smith L.M.
        • Zhao H.R.
        • Cox T.O.
        • Herber C.S.
        • Degnan A.P.
        • Balakrishnan A.
        • Macor J.E.
        • Albright C.F.
        • Strittmatter S.M.
        Silent allosteric modulation of mGluR5 maintains glutamate signaling while rescuing Alzheimer's mouse phenotypes.
        Cell Rep. 2017; 20 (28683325): 76-88
        • Kaufman A.C.
        • Salazar S.V.
        • Haas L.T.
        • Yang J.
        • Kostylev M.A.
        • Jeng A.T.
        • Robinson S.A.
        • Gunther E.C.
        • van Dyck C.H.
        • Nygaard H.B.
        • Strittmatter S.M.
        Fyn inhibition rescues established memory and synapse loss in Alzheimer mice.
        Ann. Neurol. 2015; 77 (25707991): 953-971
        • Andrew R.J.
        • Kellett K.A.
        • Thinakaran G.
        • Hooper N.M.
        A Greek tragedy: the growing complexity of Alzheimer amyloid precursor protein proteolysis.
        J. Biol. Chem. 2016; 291 (27474742): 19235-19244
        • Postina R.
        • Schroeder A.
        • Dewachter I.
        • Bohl J.
        • Schmitt U.
        • Kojro E.
        • Prinzen C.
        • Endres K.
        • Hiemke C.
        • Blessing M.
        • Flamez P.
        • Dequenne A.
        • Godaux E.
        • van Leuven F.
        • Fahrenholz F.
        A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model.
        J. Clin. Invest. 2004; 113 (15146243): 1456-1464
        • Ben Halima S.
        • Mishra S.
        • Raja K.M.P.
        • Willem M.
        • Baici A.
        • Simons K.
        • Brüstle O.
        • Koch P.
        • Haass C.
        • Caflisch A.
        • Rajendran L.
        Specific inhibition of β-secretase processing of the Alzheimer disease amyloid precursor protein.
        Cell Rep. 2016; 14 (26923602): 2127-2141
        • Caporaso G.L.
        • Gandy S.E.
        • Buxbaum J.D.
        • Ramabhadran T.V.
        • Greengard P.
        Protein phosphorylation regulates secretion of Alzheimer β/A4 amyloid precursor protein.
        Proc. Natl. Acad. Sci. U.S.A. 1992; 89 (1557413): 3055-3059
        • Parvathy S.
        • Hussain I.
        • Karran E.H.
        • Turner A.J.
        • Hooper N.M.
        Alzheimer’s amyloid precursor protein α-secretase is inhibited by hydroxamic acid-based zinc metalloprotease inhibitors: similarities to the angiotensin converting enzyme secretase.
        Biochemistry. 1998; 37 (9484239): 1680-1685
        • Parkin E.T.
        • Trew A.
        • Christie G.
        • Faller A.
        • Mayer R.
        • Turner A.J.
        • Hooper N.M.
        Structure-activity relationship of hydroxamate-based inhibitors on the secretases that cleave the amyloid precursor protein, angiotensin converting enzyme, CD23, and pro-tumor necrosis factor-α.
        Biochemistry. 2002; 41 (11939793): 4972-4981
        • Canet-Aviles R.M.
        • Anderton M.
        • Hooper N.M.
        • Turner A.J.
        • Vaughan P.F.
        Muscarine enhances soluble amyloid precursor protein secretion in human neuroblastoma SH-SY5Y by a pathway dependent on protein kinase Cα, src-tyrosine kinase and extracellular signal-regulated kinase but not phospholipase C.
        Brain Res. Mol. Brain Res. 2002; 102 (12191495): 62-72
        • Tippmann F.
        • Hundt J.
        • Schneider A.
        • Endres K.
        • Fahrenholz F.
        Up-regulation of the α-secretase ADAM10 by retinoic acid receptors and acitretin.
        FASEB J. 2009; 23 (19144697): 1643-1654
        • Taylor D.R.
        • Parkin E.T.
        • Cocklin S.L.
        • Ault J.R.
        • Ashcroft A.E.
        • Turner A.J.
        • Hooper N.M.
        Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein.
        J. Biol. Chem. 2009; 284 (19564338): 22590-22600
        • Altmeppen H.C.
        • Prox J.
        • Puig B.
        • Kluth M.A.
        • Bernreuther C.
        • Thurm D.
        • Jorissen E.
        • Petrowitz B.
        • Bartsch U.
        • De Strooper B.
        • Saftig P.
        • Glatzel M.
        Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo.
        Mol. Neurodegener. 2011; 6 (21619641): 36
        • Altmeppen H.C.
        • Prox J.
        • Krasemann S.
        • Puig B.
        • Kruszewski K.
        • Dohler F.
        • Bernreuther C.
        • Hoxha A.
        • Linsenmeier L.
        • Sikorska B.
        • Liberski P.P.
        • Bartsch U.
        • Saftig P.
        • Glatzel M.
        The sheddase ADAM10 is a potent modulator of prion disease.
        eLife. 2015; 4 (25654651)
        • Hussain I.
        • Hawkins J.
        • Shikotra A.
        • Riddell D.R.
        • Faller A.
        • Dingwall C.
        Characterization of the ectodomain shedding of the β-site amyloid precursor protein-cleaving enzyme 1 (BACE1).
        J. Biol. Chem. 2003; 278 (12857759): 36264-36268
        • Vincent B.
        • Paitel E.
        • Saftig P.
        • Frobert Y.
        • Hartmann D.
        • De Strooper B.
        • Grassi J.
        • Lopez-Perez E.
        • Checler F.
        The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein.
        J. Biol. Chem. 2001; 276 (11477090): 37743-37746
        • Perera W.S.
        • Hooper N.M.
        Proteolytic fragmentation of the murine prion protein: role of Tyr-128 and His-177.
        FEBS Lett. 1999; 463 (10606736): 273-276
        • Endres K.
        • Fahrenholz F.
        • Lotz J.
        • Hiemke C.
        • Teipel S.
        • Lieb K.
        • Tüscher O.
        • Fellgiebel A.
        Increased CSF APPs-α levels in patients with Alzheimer disease treated with acitretin.
        Neurology. 2014; 83 (25344383): 1930-1935
        • Criddle D.N.
        • Gillies S.
        • Baumgartner-Wilson H.K.
        • Jaffar M.
        • Chinje E.C.
        • Passmore S.
        • Chvanov M.
        • Barrow S.
        • Gerasimenko O.V.
        • Tepikin A.V.
        • Sutton R.
        • Petersen O.H.
        Menadione-induced reactive oxygen species generation via redox cycling promotes apoptosis of murine pancreatic acinar cells.
        J. Biol. Chem. 2006; 281 (17088248): 40485-40492
        • Shi Y.
        • Kirwan P.
        • Livesey F.J.
        Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks.
        Nat. Protoc. 2012; 7 (22976355): 1836-1846
        • Tomic J.L.
        • Pensalfini A.
        • Head E.
        • Glabe C.G.
        Soluble fibrillar oligomer levels are elevated in Alzheimer's disease brain and correlate with cognitive dysfunction.
        Neurobiol. Dis. 2009; 35 (19523517): 352-358
        • Chabrier M.A.
        • Blurton-Jones M.
        • Agazaryan A.A.
        • Nerhus J.L.
        • Martinez-Coria H.
        • LaFerla F.M.
        Soluble Aβ promotes wild-type tau pathology in vivo.
        J. Neurosci. 2012; 32 (23197725): 17345-17350
        • Chung E.
        • Ji Y.
        • Sun Y.
        • Kascsak R.J.
        • Kascsak R.B.
        • Mehta P.D.
        • Strittmatter S.M.
        • Wisniewski T.
        Anti-PrPC monoclonal antibody infusion as a novel treatment for cognitive deficits in an Alzheimer's disease model mouse.
        BMC Neurosci. 2010; 11 (20946660): 130
        • Guillot-Sestier M.V.
        • Sunyach C.
        • Ferreira S.T.
        • Marzolo M.P.
        • Bauer C.
        • Thevenet A.
        • Checler F.
        α-Secretase-derived fragment of cellular prion, N1, protects against monomeric and oligomeric amyloid β (Aβ)-associated cell death.
        J. Biol. Chem. 2012; 287 (22184125): 5021-5032
        • Fluharty B.R.
        • Biasini E.
        • Stravalaci M.
        • Sclip A.
        • Diomede L.
        • Balducci C.
        • La Vitola P.
        • Messa M.
        • Colombo L.
        • Forloni G.
        • Borsello T.
        • Gobbi M.
        • Harris D.A.
        An N-terminal fragment of the prion protein binds to amyloid-β oligomers and inhibits their neurotoxicity in vivo.
        J. Biol. Chem. 2013; 288 (23362282): 7857-7866
        • Alfa Cissá M.
        • Sunyach C.
        • Slack B.E.
        • Fisher A.
        • Vincent B.
        • Checler F.
        M1 and M3 muscarinic receptors control physiological processing of cellular prion by modulating ADAM17 phosphorylation and activity.
        J. Neurosci. 2007; 27 (17428986): 4083-4092
        • Guillot-Sestier M.-V.
        • Sunyach C.
        • Druon C.
        • Scarzello S.
        • Checler F.
        The α-secretase-derived N-terminal product of cellular prion, N1, displays neuroprotective function in vitro and in vivo.
        J. Biol. Chem. 2009; 284 (19850936): 35973-35986
        • Vaughan P.F.
        • Walker J.H.
        • Peers C.
        The regulation of neurotransmitter secretion by protein kinase C.
        Mol. Neurobiol. 1998; 18 (10065877): 125-155
        • Crews L.
        • Masliah E.
        Molecular mechanisms of neurodegeneration in Alzheimer's disease.
        Hum. Mol. Genet. 2010; 19 (20413653): R12-R20
        • Kim M.
        • Suh J.
        • Romano D.
        • Truong M.H.
        • Mullin K.
        • Hooli B.
        • Norton D.
        • Tesco G.
        • Elliott K.
        • Wagner S.L.
        • Moir R.D.
        • Becker K.D.
        • Tanzi R.E.
        Potential late-onset Alzheimer’s disease-associated mutations in the ADAM10 gene attenuate α-secretase activity.
        Hum. Mol. Genet. 2009; 18 (19608551): 3987-3996
        • Suh J.
        • Choi S.H.
        • Romano D.M.
        • Gannon M.A.
        • Lesinski A.N.
        • Kim D.Y.
        • Tanzi R.E.
        ADAM10 missense mutations potentiate β-amyloid accumulation by impairing prodomain chaperone function.
        Neuron. 2013; 80 (24055016): 385-401
        • Kuhn P.-H.
        • Wang H.
        • Dislich B.
        • Colombo A.
        • Zeitschel U.
        • Ellwart J.W.
        • Kremmer E.
        • Rossner S.
        • Lichtenthaler S.F.
        ADAM10 is the physiologically relevant, constitutive α-secretase of the amyloid precursor protein in primary neurons.
        EMBO J. 2010; 29 (20676056): 3020-3032
        • Colombo A.
        • Wang H.
        • Kuhn P.H.
        • Page R.
        • Kremmer E.
        • Dempsey P.J.
        • Crawford H.C.
        • Lichtenthaler S.F.
        Constitutive α- and β-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines.
        Neurobiol. Dis. 2013; 49 (22940630): 137-147
        • Hoey S.E.
        • Williams R.J.
        • Perkinton M.S.
        Synaptic NMDA receptor activation stimulates α-secretase amyloid precursor protein processing and inhibits amyloid-β production.
        J. Neurosci. 2009; 29 (19357271): 4442-4460
        • Moss M.L.
        • Powell G.
        • Miller M.A.
        • Edwards L.
        • Qi B.
        • Sang Q.-X.A.
        • De Strooper B.
        • Tesseur I.
        • Lichtenthaler S.F.
        • Taverna M.
        • Zhong J.L.
        • Dingwall C.
        • Ferdous T.
        • Schlomann U.
        • Zhou P.
        • et al.
        ADAM9 inhibition increases membrane activity of ADAM10 and controls α-secretase processing of amyloid precursor protein.
        J. Biol. Chem. 2011; 286 (21956108): 40443-40451
        • Caillá I.
        • Allinquant B.
        • Dupont E.
        • Bouillot C.
        • Langer A.
        • Müller U.
        • Prochiantz A.
        Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone.
        Development. 2004; 131 (15073156): 2173-2181
        • Mattson M.P.
        • Cheng B.
        • Culwell A.R.
        • Esch F.S.
        • Lieberburg I.
        • Rydel R.E.
        Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the β-amyloid precursor protein.
        Neuron. 1993; 10 (8094963): 243-254
        • Ring S.
        • Weyer S.W.
        • Kilian S.B.
        • Waldron E.
        • Pietrzik C.U.
        • Filippov M.A.
        • Herms J.
        • Buchholz C.
        • Eckman C.B.
        • Korte M.
        • Wolfer D.P.
        • Müller U.C.
        The secreted β-amyloid precursor protein ectodomain APPsα is sufficient to rescue the anatomical, behavioral, and electrophysiological abnormalities of APP-deficient mice.
        J. Neurosci. 2007; 27 (17634375): 7817-7826
        • Obregon D.
        • Hou H.
        • Deng J.
        • Giunta B.
        • Tian J.
        • Darlington D.
        • Shahaduzzaman M.
        • Zhu Y.
        • Mori T.
        • Mattson M.P.
        • Tan J.
        Soluble amyloid precursor protein-α modulates β-secretase activity and amyloid-β generation.
        Nat. Commun. 2012; 3 (22491325): 777
        • Saftig P.
        • Lichtenthaler S.F.
        The α secretase ADAM10: a metalloprotease with multiple functions in the brain.
        Prog. Neurobiol. 2015; 135 (26522965): 1-20
        • Kuhn P.H.
        • Colombo A.V.
        • Schusser B.
        • Dreymueller D.
        • Wetzel S.
        • Schepers U.
        • Herber J.
        • Ludwig A.
        • Kremmer E.
        • Montag D.
        • Müller U.
        • Schweizer M.
        • Saftig P.
        • Bräse S.
        • Lichtenthaler S.F.
        Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function.
        eLife. 2016; 5 (26802628)e12748
        • Perera W.S.
        • Hooper N.M.
        Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.
        Curr. Biol. 2001; 11 (11413003): 519-523
        • van Wilgenburg B.
        • Browne C.
        • Vowles J.
        • Cowley S.A.
        Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions.
        PLoS One. 2013; 8 (23951090)e71098
        • Baud A.
        • Wessely F.
        • Mazzacuva F.
        • McCormick J.
        • Camuzeaux S.
        • Heywood W.E.
        • Little D.
        • Vowles J.
        • Tuefferd M.
        • Mosaku O.
        • Lako M.
        • Armstrong L.
        • Webber C.
        • Cader M.Z.
        • Peeters P.
        • et al.
        Multiplex high-throughput targeted proteomic assay to identify induced pluripotent stem cells.
        Anal. Chem. 2017; 89 (28192931): 2440-2448
        • Watt N.T.
        • Taylor D.R.
        • Kerrigan T.L.
        • Griffiths H.H.
        • Rushworth J.V.
        • Whitehouse I.J.
        • Hooper N.M.
        Prion protein facilitates uptake of zinc into neuronal cells.
        Nat. Commun. 2012; 3 (23072804)1134
        • Taylor D.R.
        • Watt N.T.
        • Perera W.S.
        • Hooper N.M.
        Assigning functions to distinct regions of the N-terminus of the prion protein that are involved in its copper-stimulated, clathrin-dependent endocytosis.
        J. Cell Sci. 2005; 118 (16254249): 5141-5153
        • Cui L.
        • Gao Y.
        • Xie Y.
        • Wang Y.
        • Cai Y.
        • Shao X.
        • Ma X.
        • Li Y.
        • Ma G.
        • Liu G.
        • Cheng W.
        • Liu Y.
        • Liu T.
        • Pan Q.
        • Tao H.
        • et al.
        An ADAM10 promoter polymorphism is a functional variant in severe sepsis patients and confers susceptibility to the development of sepsis.
        Crit. Care. 2015; 19 (25888255): 73