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γ-Secretase Activity Is Associated with Braak Senile Plaque Stages

  • Nobuto Kakuda
    Correspondence
    Address correspondence to Nobuto Kakuda, Ph.D., or Yasuo Ihara, M.D., Department of Neuropathology, Faculty of Life and Medical Sciences, Doshisha University, 1-3, Tatara-miyakodani, Kyotanabe, Kyoto 610-0394, Japan.
    Affiliations
    Department of Neuropathology, Doshisha University, Kyoto, Japan

    Center for Neurologic Research in Neurodegenerative, Doshisha University, Kyoto, Japan

    Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo, Japan
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  • Haruyasu Yamaguchi
    Affiliations
    Faculty of Medicine School of Health Sciences, Gunma University, Maebashi, Japan
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  • Kohei Akazawa
    Affiliations
    Department of Medical Informatics, Niigata University Medical and Dental Hospital, Niigata University, Niigata, Japan
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  • Saori Hata
    Affiliations
    Department of Neuropathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
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  • Toshiharu Suzuki
    Affiliations
    Department of Neuropathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
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  • Hiroyuki Hatsuta
    Affiliations
    Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan
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  • Shigeo Murayama
    Affiliations
    Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido University, Hokkaido, Japan
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  • Satoru Funamoto
    Affiliations
    Department of Neuropathology, Doshisha University, Kyoto, Japan

    Center for Neurologic Research in Neurodegenerative, Doshisha University, Kyoto, Japan

    Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo, Japan
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  • Yasuo Ihara
    Correspondence
    Address correspondence to Nobuto Kakuda, Ph.D., or Yasuo Ihara, M.D., Department of Neuropathology, Faculty of Life and Medical Sciences, Doshisha University, 1-3, Tatara-miyakodani, Kyotanabe, Kyoto 610-0394, Japan.
    Affiliations
    Department of Neuropathology, Doshisha University, Kyoto, Japan

    Center for Neurologic Research in Neurodegenerative, Doshisha University, Kyoto, Japan

    Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo, Japan

    Graduate School of Brain Science and Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan
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Open AccessPublished:March 19, 2020DOI:https://doi.org/10.1016/j.ajpath.2020.02.009
      Amyloid β-proteins (Aβs) Aβ1-42 and Aβ1-43 are converted via two product lines of γ-secretase to Aβ1-38 and Aβ1-40. This parallel stepwise processing model of γ-secretase predicts that Aβ1-42 and Aβ1-43, and Aβ1-38 and Aβ1-40 are proportional to each other, respectively. To obtain further insight into the mechanisms of parenchymal Aβ deposition, these four Aβ species were quantified in insoluble fractions of human brains (Brodmann areas 9 to 11) at various Braak senile plaque (SP) stages, using specific enzyme-linked immunosorbent assays. With advancing SP stages, the amounts of deposited Aβ1-43 in the brain increased proportionally to those of Aβ1-42. Similarly, the amounts of deposited Aβ1-38 correlated with those of Aβ1-40. Surprisingly, the ratios of deposited Aβ1-38/Aβ1-42 and Aβ1-40/Aβ1-43 were proportional and discriminated the Braak SP stages accurately. This result indicates that the generation of Aβ1-38 and Aβ1-40 decreased and the generation of Aβ1-42 and Aβ1-43 increased with advancing SP stages. Thus, Aβs deposition might depend on γ-secretase activity, as it does in the cerebrospinal fluid. Here, the extracted γ-secretase from Alzheimer disease brains generates an amount of Aβ1-42 and Aβ1-43 compared with cognitively normal brains. This refractory γ-secretase localized in detergent-solubilized fractions from brain cortices. But activity modulated γ-secretase, which decreases Aβ1-42 and Aβ1-43 in the cerebrospinal fluid, localized in detergent-insoluble fractions. These drastic alterations reflect Aβ situation in Alzheimer disease brains.
      γ-Secretase is assumed to have two amyloid β-protein (Aβ) product lines that successively convert Aβ1-49 and Aβ1-48, which are generated by ε-cleavage from the carboxyl-terminal fragment of the β amyloid protein precursor, generating conventional Aβs by trimming tripeptides or tetrapeptides in a stepwise manner. Aβ1-49 is successively cleaved into mostly Aβ1-40 via Aβ1-46 and Aβ1-43, whereas Aβ1-48 is cleaved similarly into Aβ1-38 via Aβ1-45 and Aβ1-42.
      • Takami M.
      • Nagashima Y.
      • Sano Y.
      • Ishihara S.
      • Morishima-Kawashima M.
      • Funamoto S.
      • Ihara Y.
      γ-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of β-carboxyl terminal fragment.
      ,
      • Matsumura N.
      • Takami M.
      • Okochi M.
      • Wada-Kakuda S.
      • Fujiwara H.
      • Tagami S.
      • Funamoto S.
      • Ihara Y.
      • Morishima-Kawashima M.
      γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of β-carboxyl terminal fragment.
      It is noteworthy that the most abundant species, Aβ1-40, is derived not from Aβ1-42, but from Aβ1-43. Moreover, Aβ1-38 is derived largely from Aβ1-42 and Aβ1-43.
      • Takami M.
      • Nagashima Y.
      • Sano Y.
      • Ishihara S.
      • Morishima-Kawashima M.
      • Funamoto S.
      • Ihara Y.
      γ-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of β-carboxyl terminal fragment.
      • Matsumura N.
      • Takami M.
      • Okochi M.
      • Wada-Kakuda S.
      • Fujiwara H.
      • Tagami S.
      • Funamoto S.
      • Ihara Y.
      • Morishima-Kawashima M.
      γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of β-carboxyl terminal fragment.
      • Okochi M.
      • Tagami S.
      • Yanagida K.
      • Takami M.
      • Kodama T.S.
      • Mori K.
      • Nakayama T.
      • Ihara Y.
      • Takeda M.
      γ-Secretase modulators and presenilin 1 mutants act differently on presenilin/γ-secretase function to cleave Aβ42 and Aβ43.
      Herein, we measured these four Aβ species (Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-43), which are the major end products of stepwise β amyloid protein precursor processing, in human brains at various senile plaque (SP) stages to assess SP stage-related alterations of γ-secretase activity and to obtain further insight into the mechanisms of parenchymal Aβ deposition in the human brain.
      Previously, we reported the unusual relationship between the four Aβs (Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-43) in the cerebrospinal fluid (CSF)
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      ; the levels of Aβ1-42 and Aβ1-43, and Aβ1-38 and Aβ1-40 are proportional in the CSF, which is consistent with a parallel stepwise processing via the two product lines of γ-secretase; and decreased levels of Aβ1-42 and Aβ1-43 in the CSF of patients affected by mild cognitive impairment (MCI) and Alzheimer disease (AD) may not be caused by selective deposition of Aβ1-42 and Aβ1-43 in pre-existing SPs, but may be caused by the modulation of γ-secretase activity, which enhances the conversion of Aβ1-42 and Aβ1-43 to Aβ1-38 and Aβ1-40, respectively. As a result, the levels of Aβ1-42 and Aβ1-43 in the CSF are reduced, whereas, reciprocally, the levels of Aβ1-38 and Aβ1-40 are increased.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      We assessed the γ-secretase activity in rafts isolated from autopsied cognitively normal and MCI/AD brains. The activity of raft-associated (detergent-insoluble) γ-secretase from MCI/AD brains to generate Aβ1-38 and Aβ1-40 from Aβ1-42 and Aβ1-43, respectively, was significantly modulated compared with that from control brains.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      However, this observation raises a new question. Why do Aβ1-42 and Aβ1-43 continue to accumulate in the brain (as assessed by amyloid positron emission tomography) despite significant modulation of γ-secretase activity?
      • Klunk W.E.
      • Engler H.
      • Nordberg A.
      • Wang Y.
      • Blomqvist G.
      • Holt D.P.
      • Bergström M.
      • Savitcheva I.
      • Huang G.F.
      • Estrada S.
      • Ausén B.
      • Debnath M.L.
      • Barletta J.
      • Price J.C.
      • Sandell J.
      • Lopresti B.J.
      • Wall A.
      • Koivisto P.
      • Antoni G.
      • Mathis C.A.
      • Långström B.
      Imaging brain amyloid in Alzheimer's disease with Pittsburgh compound-B.
      ,
      • Mintun M.A.
      • Larossa G.N.
      • Sheline Y.I.
      • Dence C.S.
      • Lee S.Y.
      • Mach R.H.
      • Klunk W.E.
      • Mathis C.A.
      • DeKosky S.T.
      • Morris J.C.
      [11C]PIB in a nondemented population: potential antecedent marker of Alzheimer disease.
      It was particularly puzzling to observe that the modulation appears as early as the beginning of Aβ depositions in the cerebral parenchyma, which is decades before the development of clinical symptoms,
      • Kakuda N.
      • Akazawa K.
      • Hatsuta H.
      • Murayama S.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Suspected limited efficacy of γ-secretase modulators.
      and that, nevertheless, Aβ deposition continues to progress. To address this issue, we used the same approach as in the CSF study.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      On the basis of the levels of the four Aβs in the brain tissues, we sought to deduce the alteration in the activity of γ-secretase that is involved in Aβ deposition in the interstitial fluid (ISF) compartment of the brain. We found that the amount of Aβ1-43 deposited in the brain increased proportionally to that of Aβ1-42 with advancing Braak SP stages. Similarly, the amount of Aβ1-38 deposition increased proportionally to that of Aβ1-40 with advancing SP stages. Surprisingly, the ratios of insoluble Aβ1-38/Aβ1-42 levels versus Aβ1-40/Aβ1-43 levels were proportional and discriminated the Braak SP stages accurately, as did the levels in the CSF.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      The plots representing SP stage O and A subjects were located distant from the origin on the regression line, whereas those representing SP stage B and C and AD subjects were close to the origin on the regression line or its surroundings. However, these plots were different from the patterns of the previous CSF study, in which the ratios of Aβ1-38/Aβ1-42 versus Aβ1-40/Aβ1-43 were plotted close to the origin for cognitively normal subjects and far from the origin for MCI/AD patients.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      Consequently, in this study, amyloid-free (SP stage O) subjects and less subjects (SP stage A) plotted far from the origin, and amyloid-bearing subjects (SP stages B, C, and AD) plotted close to the origin. These ratios suggest that ISF compartment-associated γ-secretase activity is altered for active Aβ1-42 and Aβ1-43 generation after the subjects of amyloid-bearing SP stage B. Herein, the in vitro γ-secretase activity assay allowed that ISF compartment-associated (detergent-soluble raft-nonassociated) γ-secretase from the brain cortices generates large amounts of Aβ1-42 and Aβ1-43 in AD brains compared with cognitively normal control (SP stage O) brains; thus, the two γ-secretases, raft-associated and raft-nonassociated γ-secretase, generate Aβs in the brain. Both γ-secretase activities have been altered to modulation and refractory modulation each other in the amyloid-bearing brain.

      Materials and Methods

       Braak SP Stages and Tissue Preparation

      In the present study, the extent of Aβ deposition detected by an anti-Aβ monoclonal antibody (12B2; 1:50 dilution; IBL, Gunma, Japan) was defined by the Braak SP (amyloid) stages.
      • Braak H.
      • Braak E.
      Neuropathological stageing of Alzheimer-related changes.
      ,
      • Braak H.
      • Alafuzoff I.
      • Arzberger T.
      • Kretzschmar H.
      • Del Tredici K.
      Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry.
      At SP stage O, there were almost no SPs throughout the isocortex. At stage A, a low density of Aβ deposits was found in the isocortex, particularly in the basal portions of the frontal, temporal, and occipital lobes. In addition, some SPs were found in the presubiculum and pre-β and pre-γ layers of the entorhinal complex. Stage B showed an increase in Aβ deposits in almost all isocortical-associated areas, and only the primary sensory areas and the primary motor field remained almost devoid of deposits. Some deposits were found in the hippocampal formation, and Aβ deposits may be found in the entorhinal cortex. At stage C, virtually all isocortical areas were affected, whereas deposits in the hippocampal formation showed the same pattern as that of stage B. AD brains were invariably at stage C.
      Human cortical specimens were obtained from the prefrontal cortex (Brodmann areas 9 to 11) within 12 hours postmortem (patients were placed in a room at 4°C within 2 hours after death) and stored at −80°C until use. Cortical pieces, approximately 200 mg each, were sampled from the following frozen brains at the Brain Bank of the Tokyo Metropolitan Institute of Gerontology: Braak SP stage O [Braak neurofibrillary tangle (NFT) stage < I, Clinical Dementia Rating (CDR) = 0, 68 to 94 years old, n = 10], SP stage A (Braak NFT stage < I, CDR < 0.5, 71 to 84 years old, n = 10), SP stage B (Braak NFT stage < III, CDR < 0.5, 66 to 89 years old, n = 10), SP stage C (Braak NFT stage < IV, CDR > 0.5, 70 to 91 years old, n = 10), and AD (Braak NFT stage > IV, CDR > 1, 75 to 87 years old, n = 10).
      • Hughes C.P.
      • Berg L.
      • Danziger W.L.
      • Coben L.A.
      • Martin R.L.
      A new clinical scale for the staging of dementia.
      The attached leptomeninges and small blood vessels were carefully dissected. Each sample was homogenized with a motor-driven Teflon/glass homogenizer in four volumes of Tris-saline (TS) [TS: 50 mmol/L Tris-HCl (pH 7.6) and 0.15 mol/L NaCl] containing a cocktail of protease inhibitors. Each homogenate was then centrifuged at 540,000 × g for 20 minutes in a TLX centrifuge (Beckman, Brea, CA). The resultant supernatant was saved as the TS-soluble fraction and subjected to an enzyme-linked immunosorbent assay (ELISA) to qualify TS-soluble Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-43, as described previously.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      The resulting pellet was washed with TS and extracted with 6 mol/L guanidine-HCl in 50 mmol/L Tris-HCl (pH 7.6). The homogenate was centrifuged at 540,000 × g for 20 minutes. The supernatant was diluted to 0.5 mol/L in guanidine-HCl. An ELISA for the four TS-insoluble Aβ species, Aβ1-38, Aβ1-40, Aβ1-42, and Aβ1-43, was performed.
      The human cortical specimens used for the quantification of raft-nonassociated γ-secretase activity were obtained from those brains that were removed, processed, and placed in a container at −80°C within 12 hours postmortem (patients were placed in a room at 4°C within 2 hours after death) at the Brain Bank of the Tokyo Metropolitan Institute of Gerontology. Written informed consent from the patient or the patient's family was obtained before the present study, which was approved by the ethics committees of the Doshisha University and Tokyo Metropolitan Institute of Gerontology.

       Immunohistochemistry

      Small blocks of frontal and temporal cortices from five AD and five non-AD (aged) subjects were fixed with 4% formaldehyde in a buffer for 2 days and embedded in paraffin. After pretreatment with 100% formic acid for 5 minutes to enhance immunolabeling, dewaxed serial tissue sections (4 μm thick) were incubated with monoclonal antibodies specific for Aβ38,
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      Aβ40 (M40),
      • Yamaguchi H.
      • Sugihara S.
      • Ogawa A.
      • Oshima N.
      • Ihara Y.
      Alzheimer β amyloid deposition enhanced by apoE ε4 gene precedes neurofibrillary pathology in the frontal association cortex of nondemented senior subjects.
      and Aβ42 (M42),
      • Yamaguchi H.
      • Sugihara S.
      • Ogawa A.
      • Oshima N.
      • Ihara Y.
      Alzheimer β amyloid deposition enhanced by apoE ε4 gene precedes neurofibrillary pathology in the frontal association cortex of nondemented senior subjects.
      and a polyclonal antibody specific for Aβ43.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      ,
      • Kakuda N.
      • Akazawa K.
      • Hatsuta H.
      • Murayama S.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Suspected limited efficacy of γ-secretase modulators.
      M42 did not cross-react with Aβ43, and the Aβ43 polyclonal antibody did not cross-react with Aβ42 on the blot.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      The antibodies were visualized using the ABC Elite kit (Vector Laboratories, Burlingame, CA) using 3, 3′-diaminobenzidine as a chromogen.

       Isolation of γ-Secretase Complexes Using Sucrose Density Gradient Centrifugation

      The sucrose density gradient method was described previously.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      Briefly, after careful removal of the leptomeninges and blood vessels, small (<0.5 g) blocks from the prefrontal cortices (Brodmann areas 9 to 11) were homogenized in approximately 10 volumes of 10% sucrose in 2-morpholinoethanesulfonic acid (MES)-buffered saline (25 mmol/L MES, pH 6.5, and 0.15 mol/L NaCl) containing 1% 3-[(3-cholamidopropyl) dimethylammonio]-2-hydroxypropanesulfonate (CHAPSO) and various protease inhibitors. The homogenate was adjusted to 40% sucrose by the addition of an equal volume of 70% sucrose in MES-buffered saline, placed at the bottom of an ultracentrifuge tube, and overlaid with 4 mL of 35% sucrose and 4 mL of 5% sucrose in MES-buffered saline. The discontinuous gradient was centrifuged at 260,800 × g for 20 hours at 4°C with an SW 41 Ti rotor (Beckman). After ultracentrifugation, each 1-mL fraction was recovered from the top of ultracentrifuge tube to the bottom (Figure 1A).
      Figure thumbnail gr1
      Figure 1Distinct raft-nonassociated γ-secretase complex fractionated by sucrose density gradient centrifugation and the CHAPSO-solubilizing method. A: After sucrose density gradient centrifugation, each fraction (F) was collected from the top and subjected to 12% SDS-PAGE. F2 contained raft-associated γ-secretase (CHAPSO-resistant) components, whereas F4 contained raft-nonassociated γ-secretase (CHAPSO-soluble) components. B: Human brain microsomal fractions were solubilized with 1% CHAPSO. After centrifugation, supernatants were collected (raft-nonassociated γ-secretase). The fractionated γ-secretase contained nicastrin (NCT), presenilin 1/2 (PS1/2), pen-2, and Aph1A/B. AD, Alzheimer disease; βCTF, β amyloid protein precursor; Sup, supernatant.

       Isolation and Quantification of Raft-Nonassociated γ-Secretase Activity

      Microsomal fractions from cortical pieces were obtained, as described previously,
      • Kakuda N.
      • Funamoto S.
      • Yagishita S.
      • Takami M.
      • Osawa S.
      • Dohmae N.
      • Ihara Y.
      Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from β−carboxyl-terminal fragment by γ-secretase.
      with modifications. Briefly, small pieces of the cortices were homogenized in buffer A [20 mmol/L piperazine-1, 4-bis(2-ethanesulfonic acid) (PIPES), pH 7.0, 0.14 mol/L KCl, 0.25 mol/L (8.56%) sucrose, and 5 mmol/L EGTA] using a Teflon/glass homogenizer. The homogenates were centrifuged at 800 × g for 10 minutes. The postnuclear supernatants were centrifuged at 100,000 × g for 1 hour. The resulting pellets, which represented the microsomal fractions, were suspended in buffer (20 mmol/L PIPES, pH 7.0, 0.25 mol/L sucrose, and 1 mmol/L EGTA). Their protein concentrations were adjusted to 10 mg/mL. The membranes were solubilized by the addition of an equal volume of 2× Nobuto Kakuda (NK) buffer (20 mmol/L PIPES, pH 7.0, 0.25 mol/L sucrose, 1 mmol/L EGTA, 2% CHAPSO, and protease inhibitors) and incubated on ice for 1 hour. After centrifugation at 100,000 × g for 1 hour, the supernatants were saved (1% CHAPSO lysate). Each 1% CHAPSO-solubilized fraction, adjusted to 0.25% CHAPSO, was incubated with 1 μmol/L C99FLAG for 1 hour at 37°C in the presence or absence of 10 μmol/L GSM-1. The produced Aβs were separated using SDS-PAGE and subjected to quantitative Western blot analysis using the following antibodies: 3B1 for Aβ38, BA27 for Aβ40, 44A3 for Aβ42, anti-Aβ43 polyclonal antibody for Aβ43,
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      9C3 for nicastrin,
      • Acx H.
      • Chávez-Gutiérrez L.
      • Serneels L.
      • Lismont S.
      • Benurwar M.
      • Elad N.
      • De Strooper B.
      Signature Aβ profiles are produced by different γ-secretase complexes.
      monoclonal antibodies 1563 and 5232 for presenilin 1 (Millipore, Burlington, MA), D30G3 for presenilin 2 (Cell Signaling, Danvers, MA), B126 for pen-2 and B80 for Aph1A,
      • Acx H.
      • Chávez-Gutiérrez L.
      • Serneels L.
      • Lismont S.
      • Benurwar M.
      • Elad N.
      • De Strooper B.
      Signature Aβ profiles are produced by different γ-secretase complexes.
      ,
      • Serneels L.
      • Van Biervliet J.
      • Craessaerts K.
      • Dejaegere T.
      • Horré K.
      • Van Houtvin T.
      • Esselmann H.
      • Paul S.
      • Schäfer M.K.
      • Berezovska O.
      • Hyman B.T.
      • Sprangers B.
      • Sciot R.
      • Moons L.
      • Jucker M.
      • Yang Z.
      • May P.C.
      • Karran E.
      • Wiltfang J.
      • D'Hooge R.
      • De Strooper B.
      γ-Secretase heterogeneity in the Aph1 subunit: relevance for Alzheimer's disease.
      O2E2 for Aph1B (Covance, Princeton, NJ), 82E1 for β amyloid protein precursor (IBL, Gunma, Japan), caveolin-1 for caveolin (Santa Cruz Biotechnology, Dallas, TX), and flotillin-1 for flotillin (BD, Franklin Lakes, NJ).

       Quantification of GSM-1–Induced Shifts for the Ratios of Aβ38/Aβ42

      A previously reported procedure was used to quantify GSM-1 efficacy.
      • Kakuda N.
      • Akazawa K.
      • Hatsuta H.
      • Murayama S.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Suspected limited efficacy of γ-secretase modulators.
      The ratios of Aβ1-38/Aβ1-42 in the presence of GSM-1 minus in the absence of GSM-1 were calculated.

       Statistical Analysis

      All statistical analyses were performed using SPSS software version 20 (SPSS, Chicago, IL) and GraphPad Prism 8 (GraphPad, San Diego, CA). An analysis of variance with a Kruskal-Wallis test was used to assess the equality of mean values of continuous variables among five groups (ie, SP stage O, SP stage A, SP stage B, SP stage C, and AD). A Dunn's multiple comparisons test for TS-soluble and TS-insoluble Aβ amounts showed a significant difference between SP stage O, SP stage A, SP stage B, SP stage C, and AD (P < 0.005). A U-test for raft-nonassociated γ-secretase activity showed a significant difference between normal controls and AD (P < 0.05). A paired t-test was used to examine raft-associated γ-secretase activity, which was recovered from the CHAPSO insoluble pellet fraction, and the effect of GSM-1 (P < 0.05).

      Results

       The Levels of Insoluble Aβ1-42 and Aβ1-43 Are Proportional

      SPs in the brain cortices probably consist of Aβ1-42.
      • Hardy J.
      • Selkoe D.J.
      The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.
      To assess deposited Aβs in the cerebral cortex, Aβs were extracted from autopsied frozen brain cortices (SP stages O, A, B, and C, and AD) with guanidine hydrochloride after soluble Aβs were discarded. Aβs in these extracts were measured using specific ELISA.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      The concentration of guanidine-soluble Aβ1-42 (termed as TS-insoluble Aβ1-42) was exactly proportional to that of TS-insoluble Aβ1-43, and the ratio of Aβ1-42/Aβ1-43 was approximately 20:1 (Figure 2A) (Aβ1-43 = 0.03746 × Aβ1-42 + 378.8; R = 0.9463). The concentration of TS-insoluble Aβ1-42 and Aβ1-43 significantly differed between advanced SP stages (Supplemental Tables S1 and S2). In this TS-insoluble Aβ1-42 ELISA measurement, all of SP stage O and nine of SP stage A subjects had levels of Aβ1-42 that were below the limit of detection (data not shown).
      Figure thumbnail gr2
      Figure 2Relationships between the levels of Aβ1-42 and Aβ1-43 (A) and Aβ1-38 and Aβ1-40 (B) in the Tris-saline (TS)–insoluble fractions from Braak senile plaque (SP) stages O, A, B, and C, and Alzheimer disease (AD) brains. All fractions were appropriately diluted, and Aβ1-38, Aβ1-40, Aβ1-42, or Aβ1-43 was measured using enzyme-linked immunosorbent assay. The levels of Aβs in the TS-insoluble fractions were correlated with the SP stages.
      On the other hand, the concentration of TS-insoluble Aβ1-38 and Aβ1-40 also correlated, except for some AD brains. The ratio of Aβ1-38/Aβ1-40 was approximately 1:6 (Figure 2B) (Aβ1-40 = 6.009 × Aβ1-38 − 1671; R = 0.4705). The level of TS-insoluble Aβ1-38 and Aβ1-40 significantly differed between advanced SP stages, with the exception of Aβ1-38 for some AD brains (Supplemental Tables S1 and S2). It is possible that the relatively weak correlation between Aβ38 and Aβ40 is caused by their preferential accumulation in leptomeningeal blood vessels and lower insolubilities of their aggregates in the cerebral parenchyma (Supplemental Figure S1). In contrast, Aβ42 and Aβ43 are preferentially deposited in the cerebral parenchyma rather than in the vessels (Supplemental Figure S1). These observed proportionalities between TS-insoluble Aβ1-42 and Aβ1-43 reminded us of the relationships between the four Aβs in the CSF, as reported previously,
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      because these proportionalities are characteristic of the parallel stepwise processing of β amyloid protein precursor by γ-secretase.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      Thus, Figure 2A may indicate that as SP stages advance, γ-secretase activity to generate Aβ1-38 and Aβ1-40 from Aβ1-42 and Aβ1-43 decelerates, and, therefore presumably enhances Aβ1-42 and Aβ1-43 accumulation in the cerebral parenchyma.

       Insoluble Aβ1-38/Aβ1-42 versus Aβ1-40/Aβ1-43 Accurately Discriminates Various SP Stages

      To clarify whether not only Aβs in CSF or also Aβ deposits in the brain depend on γ-secretase activity, we plotted the two ratios of the product and precursor set for Aβ1-38/Aβ1-42 versus Aβ1-40/Aβ1-43 of the TS-insoluble fractions (Supplemental Figure S2). However, because of the large amount of Aβ1-42 and Aβ1-43 deposits in subjects with Braak SP stages B and C and AD, these subjects were plotted close to the origin without discrimination. To assess further the distribution for these plots, the values of TS-insoluble Aβs were plotted after being logarithmically transformed [ln(Aβ1-38/Aβ1-42) versus ln(Aβ1-40/Aβ1-43)] (Figure 3A). In these plots, an unusual proportion was allowed in Figure 3A [ln(Aβ1-38/Aβ1-42) = 1.465 × ln(Aβ1-40/Aβ1-43) − 4.446; R = 0.850], similar to plots of Aβs in the CSF.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      All subjects were on the regression line or its surroundings. In detail, AD- and SP-abundant subjects (SP stages B and C) were plotted close to the origin on the regression line and its surroundings, whereas SP-free (SP stage O) and SP stage A subjects were plotted far from the origin on the regression line and its surroundings (Figure 3A and Supplemental Table S3). Thus, most interestingly, the plot images showed opposite results from those obtained for Aβ1-38/Aβ1-42 versus Aβ1-40/Aβ1-43 in the CSF from normal controls and MCI/AD subjects.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      In our previous CSF measurement study, MCI/AD subjects were plotted far from the origin and normal controls were plotted close to the origin on the regression line for Aβ1-38/Aβ1-42 versus Aβ1-40/Aβ1-43.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      Thus, the plots of CSF and TS-insoluble Aβs are different. In the cerebral parenchyma, Aβs form SPs decades before the onset of AD. Previous studies have claimed that Aβs’ accumulation and deposition are caused by an imbalance of Aβs’ production and clearance associated with aging.
      • Hardy J.
      • Selkoe D.J.
      The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.
      ,
      • Mawuenyega K.G.
      • Sigurdson W.
      • Ovod V.
      • Munsell L.
      • Kasten T.
      • Morris J.C.
      • Yarasheski K.E.
      • Bateman R.J.
      Decreased clearance of CNS beta-amyloid in Alzheimer's disease.
      However, in addition to aging, this plot implies that γ-secretase activity also causes Aβs’ accumulation and deposition in the brain. In addition, γ-secretase generated Aβ1-38 not only from Aβ1-42 but also from Aβ1-43.
      • Matsumura N.
      • Takami M.
      • Okochi M.
      • Wada-Kakuda S.
      • Fujiwara H.
      • Tagami S.
      • Funamoto S.
      • Ihara Y.
      • Morishima-Kawashima M.
      γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of β-carboxyl terminal fragment.
      ,
      • Okochi M.
      • Tagami S.
      • Yanagida K.
      • Takami M.
      • Kodama T.S.
      • Mori K.
      • Nakayama T.
      • Ihara Y.
      • Takeda M.
      γ-Secretase modulators and presenilin 1 mutants act differently on presenilin/γ-secretase function to cleave Aβ42 and Aβ43.
      Therefore, this product and precursor set of TS-insoluble Aβs also assessed the relationship for TS-insoluble Aβs. As expected, the ratio of Aβ1-38/Aβ1-42 versus Aβ1-38/Aβ1-43 also allowed a correlation pattern as advanced SP stage [ln(Aβ1-38/Aβ1-42) = 1.204 × ln(Aβ1-38/Aβ1-43) − 2.2829] (Figure 3B). Thus, Aβ deposition in the cerebral parenchyma also possibly depends on γ-secretase activity. More important, these Aβs generated by γ-secretase differ from Aβs generated by raft-associated γ-secretase, which is reflected by the Aβs found in the CSF. Raft-associated γ-secretase has been modulated to actively generate Aβ1-38 and Aβ1-40 from Aβ1-42 and Aβ1-43, respectively, in MCI/AD brains; and it decreased the levels of Aβ1-42 and Aβ1-43 in the CSF.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      However, another subset of γ-secretase, which is reflected by the Aβ deposits in the cerebral parenchyma, suggests that the γ-secretase cleavage efficacy decreases and with it the generation of Aβ1-38 and Aβ1-40 from Aβ1-42 and Aβ1-43, respectively, by resistance to modulation, rather than by nonmodulation. This γ-secretase in the ISF compartment continues generating Aβ1-42 and Aβ1-43 to a greater extent and secretes Aβs in the ISF compartment, which may continue to accumulate in the brain. Thus, it is possible that there are two different types of γ-secretase in the brain. One is raft-associated γ-secretase, which secretes Aβs into the CSF. Another is γ-secretase, which undergoes resistance to modulation in amyloid-bearing brains and generates longer Aβ species and secretes them into the ISF compartment. These two distinct γ-secretases’ functions may be regulated in opposite directions as SP stages advance.
      Figure thumbnail gr3
      Figure 3Relationships between the ratios of product Aβ/precursor Aβ sets in the Tris-saline (TS)–insoluble fractions. A: Braak senile plaque (SP) stages were distinguishable precisely by the ratios of ln(Aβ1-38/Aβ1-42) and ln(Aβ1-40/Aβ1-43) in the TS-insoluble fractions. B: The ratios of ln(Aβ1-38/Aβ1-42) and ln(Aβ1-38/Aβ1-43) in the TS-insoluble fractions. AD, Alzheimer disease.

       Raft-Nonassociated γ-Secretase Is Refractory Modulated in Amyloid-Bearing Brains

      To clarify whether there is another subset of the γ-secretase, brain cortices were fractionated into raft-associated and CHAPSO-solubilized γ-secretase by sucrose density gradient centrifugation.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      Most of the γ-secretase fraction, consisting of four components (presenilin 1/2, nicastrin, Aph1A/B, and pen-2), was recovered in the CHAPSO-insolubilized raft fraction (Figure 1A). Previously, we reported that these raft-associated γ-secretase activities have been modulated in MCI/AD brains to advance the generation of Aβ1-38 and Aβ1-40 from Aβ1-42 and Aβ1-43, respectively, compared with cognitively normal control brains and possibly reflected to the CSF Aβs.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      However, low levels of another γ-secretase were also recovered in the CHAPSO-solubilized fraction (Figure 1A).
      We measured this CHAPSO-solubilized γ-secretase activity to validate our hypothesis on the distinct modulation of the γ-secretase activity. First, we could not measure it directly because the concentration and activity of CHAPSO-solubilized γ-secretase was much lower after sucrose density gradient centrifugation for 20 hours. Therefore, we changed the isolation protocol: high-concentration raft-nonassociated γ-secretase in the CHAPSO-soluble fraction was recovered within 2 hours from the membrane fractions of brain cortices solubilized with CHAPSO, as reported previously (CHAPSO-solubilizing method).
      • Kakuda N.
      • Funamoto S.
      • Yagishita S.
      • Takami M.
      • Osawa S.
      • Dohmae N.
      • Ihara Y.
      Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from β−carboxyl-terminal fragment by γ-secretase.
      This CHAPSO-solubilized γ-secretase is referred to as raft-nonassociated γ-secretase. Raft-nonassociated γ-secretase was recovered in the supernatant as the solubilized γ-secretase, but raft-associated γ-secretase was pelleted in this method because it cannot be solubilized with CHAPSO (Figure 1B).
      This CHAPSO-solubilized, raft-nonassociated γ-secretase activity was measured in normal controls (SP stage O) and AD brains using an in vitro cell-free assay system that was established previously.
      • Kakuda N.
      • Funamoto S.
      • Yagishita S.
      • Takami M.
      • Osawa S.
      • Dohmae N.
      • Ihara Y.
      Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from β−carboxyl-terminal fragment by γ-secretase.
      The ratios of generated Aβ1-38/Aβ1-42 and Aβ1-40/Aβ1-43 were quantified via immunoblotting and plotted (Figure 4). As expected, all plots were distributed as a whole in a diagonal manner. The plots representing AD subjects were located near the origin, whereas the plots representing normal controls were far from the origin in Figure 4 (P = 0.0002 for Aβ1-40/Aβ1-43; P = 0.0052 for Aβ1-38/Aβ1-42), as observed for the plots of the ELISA results of TS-insoluble fraction (Figure 3A). These results allowed both from Aβ1-43 to Aβ1-40 and from Aβ1-42 to Aβ1-38 cleavage efficacy significantly decreased in AD raft-nonassociated γ-secretase, suggesting that it actively produces Aβ1-42 and Aβ1-43 in AD. The image shown in Figure 4 is the opposite pattern plot of raft-associated γ-secretase in vitro assay.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      It would be more appropriate to call this species refractory-modulated γ-secretase. To assess the remaining raft-associated γ-secretase activity, it was recovered from once raft-nonassociated γ-secretase removed pellet fractions and measured these raft-associated γ-secretase activities with in vitro assay. As expected, the raft-associated γ-secretase was modulated in AD brains compared with normal controls, as in our previous study (P = 0.0232 for Aβ1-40/Aβ1-43; P < 0.0001 for Aβ1-38/Aβ1-42) (Supplemental Figure S3).
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      Thus, two distinct types of γ-secretase, raft-associated and raft-nonassociated γ-secretase, are active in the brain, but their activities have been altered depending on the SP stages.
      Figure thumbnail gr4
      Figure 4Aβ1-38/Aβ1-42 versus Aβ1-40/Aβ1-43 plot based on the direct quantification of raft-nonassociated γ-secretase activity. The raft-nonassociated γ-secretase prepared from the microsomal fractions of control (Braak senile plaque stage O) and Alzheimer disease (AD) brains was incubated with β amyloid protein precursor for 1 hour at 37°C. The produced Aβs were quantified using Western blot analysis with specific antibodies. The plots clearly discriminated between control and AD brains. AD plots (closed circles) are located near the origin, whereas control plots (open circles) are located distant from the origin. The original values for Aβs are in the pmol/L range. n = 10 AD and control plots.
      GSM-1, a strong γ-secretase modulator, has previously been shown to limit Aβ42-decreasing and Aβ38-increasing modulation efficacy toward AD, because it had already been modulated to generate shorter Aβs.
      • Kakuda N.
      • Akazawa K.
      • Hatsuta H.
      • Murayama S.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Suspected limited efficacy of γ-secretase modulators.
      It was hypothesized that, because of the raft-nonassociated γ-secretase activity described above, GSM-1 would cause a larger modulation of AD raft-nonassociated γ-secretase because its activity has been resisted. In normal control subjects, GSM-1 definitely allowed a large modulation shift from Aβ1-42 to Aβ1-38 (Aβ1-38/Aβ1-42 with dimethyl sulfoxide versus Aβ1-38/Aβ1-42 with GSM-1; P < 0.0001, paired t-test) (Supplemental Figure S4). However, raft-nonassociated γ-secretase in AD brains, which had been refractory modulated to generate longer Aβs, showed only a poor modulation shift value (Aβ1-38/Aβ1-42 with dimethyl sulfoxide versus Aβ1-38/Aβ1-42 with GSM-1; P < 0.0001, paired t-test) (Supplemental Figure S4). Moreover, the shift value in AD brains was smaller than that in control brains (control versus AD, P = 0.0013, paired t-test). Thus, GSM-1 is unlikely to affect the modulation efficacy toward AD brain raft-nonassociated γ-secretase (ie, likely involved in the parenchymal Aβ deposition).

      Discussion

      This manuscript demonstrates the following: i) the levels of deposited Aβ1-42 and Aβ1-43 in the brains of cognitively normal subjects and AD patients were proportional in the TS-insoluble fractions; ii) the levels of accumulated Aβ1-38 and Aβ1-40 correlated in the TS-insoluble fractions; iii) the plots of Aβ1-38/Aβ1-42 versus Aβ1-40/Aβ1-43 for the TS-insoluble fractions discriminated the various SP stages accurately: stage B and C and AD subjects were located close to the origin on the regression line, whereas SP stage O and A subjects were located distant from the origin on the regression line; iv) in sharp contrast to the plots of Aβ1-38/Aβ1-42 versus Aβ1-40/Aβ1-43 for raft-associated γ-secretase,
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      the plots for raft-nonassociated γ-secretase activity measured in vitro showed that AD subjects were located close to the origin on the regression line, whereas cognitively normal subjects were located far from the origin on the line. Thus, raft-nonassociated γ-secretase is modulated to refractory alteration. Finally, v) GSM-1 showed poor modulation efficacy toward raft-nonassociated γ-secretase in AD brains, because this activity has been refractory modulated against the raft-associated γ-secretase modulation.
      Compared with the previous CSF study,
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      an apparent contradiction seems to exist: if the levels of intracellular soluble Aβs are negligible, the levels of Aβ1-42 and Aβ1-43 in the TS-soluble fractions are not significantly decreased, as predicted by the profound modulation of raft-associated γ-secretase in SP-bearing brains (Supplemental Figure S5). A reasonable explanation for this is that the CSF and ISF compartments do not communicate freely, although leaky pathways seem to be present.
      • Weller R.O.
      How well does the CSF inform upon pathology in the brain in Creutzfeldt-Jakob and Alzheimer's diseases?.
      The ISF compartment drains through the perivascular drainage pathways to the lymphatic flow on the meningeal blood vessel walls and to the regional lymph nodes in the neck.
      • Zhang E.T.
      • Inman C.B.
      • Weller R.O.
      Interrelationships of the pia mater and the perivascular (Virchow-Robin) spaces in the human cerebrum.
      ,
      • Weller R.O.
      • Subash M.
      • Preston S.D.
      • Mazanti I.
      • Carare R.O.
      Perivascular drainage of amyloid-β peptides from the brain and its failure in cerebral amyloid angiopathy and Alzheimer's disease.
      The Virchow-Robin space is completely separated from the CSF compartment by the pia mater.
      • Weller R.O.
      How well does the CSF inform upon pathology in the brain in Creutzfeldt-Jakob and Alzheimer's diseases?.
      ,
      • Zekonyte J.
      • Sakai K.
      • Nicoll J.A.
      • Weller R.O.
      • Carare R.O.
      Quantification of molecular interactions between ApoE, amyloid-beta (Aβ) and laminin: relevance to accumulation of Aβ in Alzheimer's disease.
      Raft-associated γ-secretase activity is faithfully reflected in the CSF,
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      ,
      • Kakuda N.
      • Akazawa K.
      • Hatsuta H.
      • Murayama S.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Suspected limited efficacy of γ-secretase modulators.
      but not in the ISF, indicating that the two γ-secretase compartments are largely separated. A particular subset of ISF-linked γ-secretase, raft-nonassociated γ-secretase, undergoes the refractory modulation and actively continues to generate Aβ1-42 and Aβ1-43 in the ISF compartment. This results in continued Aβ deposition in the ISF compartment, although the CSF-linked raft-associated γ-secretase is significantly modulated and Aβ1-42 and Aβ1-43 levels may be extremely low in the CSF (Figure 5).
      Figure thumbnail gr5
      Figure 5Scheme of generated Aβs releasing into cerebrospinal fluid (CSF) and interstitial fluid (ISF). In the neurons, Aβs generated by raft-associated γ-secretase are released into the CSF via still unknown factors. Aβs generated by raft-nonassociated γ-secretase are released into the extracellular space and the ISF. Aβ1-38 and Aβ1-40 tend to accumulate in the vessel walls, and Aβ1-42 and Aβ1-43 are deposited preferably in the cerebellum parenchyma, which forms senile plaques (SPs). AD, Alzheimer disease; MCI, mild cognitive impairment.
      Nishimura et al
      • Nishimura M.
      • Nakamura S.
      • Kimura N.
      • Liu L.
      • Suzuki T.
      • Tooyama I.
      Age-related modulation of γ-secretase activity in non-human primate brains.
      have reported that a CHAPSO-solubilized γ-secretase from aged cynomolgus monkey brains showed a decrease of Aβ40 generation, but not Aβ42 generation. These aged monkey brains deposited large amounts of Aβ42, as do AD patients. These results are consistent with our observation of CHAPSO-solubilized raft-nonassociated γ-secretase activity measured by an in vitro assay (Figure 4). Although Aβ40 generation decreased, Aβ40 accumulation has increased in human brains with SP stages B and C and AD (Figure 2B). A reasonable explanation for this contradiction is that Aβ38 and Aβ40 are accumulated predominantly in leptomeningeal blood vessels, and their aggregates have lower insolubility (Supplemental Figure S1). We found that the accumulation of Aβ1-38 and Aβ1-41 in the leptomeningeal blood vessels occurred in the cognitive normal brains at first and followed accumulation of Aβ1-36 to Aβ1-40 in the leptomeningeal blood vessels in AD.
      • Kakuda N.
      • Miyasaka T.
      • Iwasaki N.
      • Nirasawa T.
      • Wada-Kakuda S.
      • Takahashi-Fujigasaki J.
      • Murayama S.
      • Ihara Y.
      • Ikegawa M.
      Distinct deposition of amyloid-β species in brains with Alzheimer's disease pathology visualized with MALDI imaging mass spectrometry.
      Aβ drains through the cerebrovascular basement membrane from the CSF to the lymphatic node with perivascular drainage pathway.
      • Ma Q.
      • Ineichen B.V.
      • Detmar M.
      • Proulx S.T.
      Outflow of cerebrospinal fluid is predominantly through lymphatic vessels and is reduced in aged mice.
      ,
      • Morris A.W.
      • Sharp M.M.
      • Albargothy N.J.
      • Fernandes R.
      • Hawkes C.A.
      • Verma A.
      • Weller R.O.
      • Carare R.O.
      Vascular basement membranes as pathways for the passage of fluid into and out of the brain.
      In AD subjects, the levels of Aβ1-38 and Aβ1-40 in the CSF increased compared with cognitively normal subjects.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      These generated Aβs accumulate in the leptomeningeal vessels, and it might have occurred cerebral amyloid angiopathy (CAA).
      • Mawuenyega K.G.
      • Sigurdson W.
      • Ovod V.
      • Munsell L.
      • Kasten T.
      • Morris J.C.
      • Yarasheski K.E.
      • Bateman R.J.
      Decreased clearance of CNS beta-amyloid in Alzheimer's disease.
      ,
      • Zhang E.T.
      • Inman C.B.
      • Weller R.O.
      Interrelationships of the pia mater and the perivascular (Virchow-Robin) spaces in the human cerebrum.
      ,
      • Selkoe D.J.
      Clearing the brain's amyloid cobwebs.
      Thus, Aβ38 and Aβ40 seem to increase in the AD brains.
      In addition, we assessed raft-associated and raft-nonassociated γ-secretase activity using alcadein-α, which is known as one substrate of γ-secretase.
      • Omori C.
      • Kaneko M.
      • Nakajima E.
      • Akatsu H.
      • Waragai M.
      • Maeda M.
      • Morishima-Kawashima M.
      • Saito Y.
      • Nakaya T.
      • Taru H.
      • Yamamoto T.
      • Asada T.
      • Hata S.
      • Suzuki T.
      Japanese Alzheimer’s Disease Neuroimaging Initiative
      Increased levels of plasma p3-alcα35, a major fragment of Alcadeinα by γ-secretase cleavage, in Alzheimer's disease.
      The generated species ratio of p3-Alc-α35/p3-Alc-α38 is prone to increase in the raft-associated γ-secretase from AD rather than normal controls (data not shown). The altered γ-secretase activity is reflected in the results of an in vitro assay using another substrate. Currently, we do not know the mechanisms behind the altered activity of both the raft-associated γ-secretase and the raft-nonassociated γ-secretase in the amyloid-bearing brains. Both γ-secretases exist in the plasma membrane and are surrounded by lipid bilayers. A change of the lipid composition in the AD brains compared with control brains could alter both γ-secretase activities in the plasma membrane.
      • Fabelo N.
      • Martín V.
      • Marín R.
      • Moreno D.
      • Ferrer I.
      • Díaz M.
      Altered lipid composition in cortical lipid rafts occurs at early stages of sporadic Alzheimer's disease and facilitates APP/BACE1 interactions.
      These lipid compositions (eg, cholesterol, phosphatidylcholine, and phosphatidylserine) are known to modify γ-secretase activity drastically.
      • Osenkowski P.
      • Ye W.
      • Wang R.
      • Wolfe M.S.
      • Selkoe D.J.
      Direct and potent regulation of gamma-secretase by its lipid microenvironment.
      • Abad-Rodriguez J.
      • Ledesma M.D.
      • Craessaerts K.
      • Perga S.
      • Medina M.
      • Delacourte A.
      • Dingwall C.
      • De Strooper B.
      • Dotti C.G.
      Neuronal membrane cholesterol loss enhances amyloid peptide generation.
      • Holmes O.
      • Paturi S.
      • Ye W.
      • Wolfe M.S.
      • Selkoe D.J.
      Effects of membrane lipids on the activity and processivity of purified γ-secretase.
      • Osawa S.
      • Funamoto S.
      • Nobuhara M.
      • Wada-Kakuda S.
      • Shimojo M.
      • Yagishita S.
      • Ihara Y.
      Phosphoinositides suppress gamma-secretase in both the detergent-soluble and -insoluble states.
      Previously, Dotti and colleagues
      • Abad-Rodriguez J.
      • Ledesma M.D.
      • Craessaerts K.
      • Perga S.
      • Medina M.
      • Delacourte A.
      • Dingwall C.
      • De Strooper B.
      • Dotti C.G.
      Neuronal membrane cholesterol loss enhances amyloid peptide generation.
      showed that cholesterol might affect γ-secretase activity in neuronal cells, but they and we also showed that cholesterol loss does not affect γ-secretase activity in nonneuronal cells.
      • Abad-Rodriguez J.
      • Ledesma M.D.
      • Craessaerts K.
      • Perga S.
      • Medina M.
      • Delacourte A.
      • Dingwall C.
      • De Strooper B.
      • Dotti C.G.
      Neuronal membrane cholesterol loss enhances amyloid peptide generation.
      ,
      • Wada S.
      • Morishima-Kawashima M.
      • Qi Y.
      • Misono H.
      • Shimada Y.
      • Ohno-Iwashita Y.
      • Ihara Y.
      Gamma-secretase activity is present in rafts but is not cholesterol-dependent.
      Thus, these lipid compositions are especially sensitive factors for the γ-secretase activity in the brain. Lipid alteration might be the reason that raft-associated γ-secretase actively generates Aβ1-38 and Aβ1-40 into the CSF. On the other hand, raft-nonassociated γ-secretase actively generates Aβ1-42 and Aβ1-43 in the ISF compartment (Figure 5). The vast major fraction of γ-secretase is found in the raft-associated γ-secretase, but a minor fraction is found in the raft-nonassociated γ-secretase. Thus, it might take a few decades to form SPs in the brain because these deposited Aβs are generated by raft-nonassociated γ-secretase in the brain, which is only a minor γ-secretase fraction.
      Another unexpected result of this study was that GSM-1 showed only a small modulation toward AD raft-nonassociated γ-secretase, which has been refractory modulated rather than cognitively normal brain. Previously, we showed that GSM-1 had limited modulation efficacy in AD raft-associated γ-secretase
      • Kakuda N.
      • Akazawa K.
      • Hatsuta H.
      • Murayama S.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Suspected limited efficacy of γ-secretase modulators.
      because raft-associated γ-secretase in AD brains already had been modulated to generate actively Aβ38 and Aβ40.
      • Kakuda N.
      • Shoji M.
      • Arai H.
      • Furukawa K.
      • Ikeuchi T.
      • Akazawa K.
      • Takami M.
      • Hatsuta H.
      • Murayama S.
      • Hashimoto Y.
      • Miyajima M.
      • Arai H.
      • Nagashima Y.
      • Yamaguchi H.
      • Kuwano R.
      • Nagaike K.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.
      ,
      • Kakuda N.
      • Akazawa K.
      • Hatsuta H.
      • Murayama S.
      • Ihara Y.
      Japanese Alzheimer's Disease Neuroimaging Initiative
      Suspected limited efficacy of γ-secretase modulators.
      Thus, we need more strong γ-secretase modulators to decrease the Aβ42 levels in the ISF compartment of amyloid-bearing brains, which might be involved in the parenchymal Aβ deposition.

      Acknowledgment

      We thank Dr. Bart De Strooper (VIB Center for the Biology of Disease, KU Leuven, Belgium, and UK Dementia Research Institute, University College London) for helpful suggestions and discussions.

      Supplemental Data

      • Supplemental Figure S1

        Immunohistochemistry for the detection of Aβs’ accumulation in the frontal cortex. Aβ carboxyl-terminal specific antibodies stained each of the Aβ fragment. Aβ42 and Aβ43 deposited in the senile plaques of Alzheimer disease (AD) subjects (A and B) but are less present in normal control subjects (E, F, I, and J). Aβ40 accumulation was less in AD brains (C) and normal controls (G and K), and Aβ38 accumulation occurred in the blood vessel wall (D, H, and L). Scale bars = 250 μm (AL).

      • Supplemental Figure S2

        Aβ38/Aβ42 versus Aβ40/Aβ43 for raft-nonassociated γ-secretase activity. Braak senile plaque (SP) stages O and A were precisely distinguished, but SP stages B and C and Alzheimer disease (AD) overlapped near the origin of the plots of the Tris-saline (TS)–insoluble fractions.

      • Supplemental Figure S3

        Aβ1-38/Aβ1-42 versus Aβ1-40/Aβ1-43 for raft-associated γ-secretase activity. These raft fractions were recovered from CHAPSO-solubilized fraction of the removed pellets. Raft-associated γ-secretase from control subjects and Alzheimer disease (AD) subjects was incubated with β amyloid protein precursor for 1 hour at 37°C. The produced Aβs were quantified using Western blot analysis with specific antibodies.

      • Supplemental Figure S4

        GSM-1 shift for Aβ38/Aβ42 in raft-nonassociated γ-secretase from control subjects and Alzheimer disease (AD) subjects. The raft-nonassociated γ-secretase prepared from control (Braak senile plaque stage O) and AD brains was incubated with β amyloid protein precursor for 1 hour at 37°C in the presence of dimethyl sulfoxide (DMSO) or 10 μmol/L GSM-1. The produced Aβ38 and Aβ42 were quantified using Western blot analysis with specific antibodies. Open circles indicate control plots, and closed circles indicate GSM-1–treated plots. GSM-1 shifts were smaller in AD patients (paired t-test, P = 0.0013).

      • Supplemental Figure S5

        Relationships between the levels of Aβ1-42 and Aβ1-43 in the Tris-saline (TS)–soluble fractions from Braak senile plaque (SP) stages O, A, B, and C, and Alzheimer disease (AD) brains. Ten SP stage O and nine SP stage A subjects in which Aβ1-42 was undetectable were not plotted.

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