Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

γ-Secretase Activity Is Associated with Braak Senile Plaque Stages

2020; Elsevier BV; Volume: 190; Issue: 6 Linguagem: Inglês

10.1016/j.ajpath.2020.02.009

ISSN

1525-2191

Autores

Nobuto Kakuda, Haruyasu Yamaguchi, Kohei Akazawa, Saori Hata, Toshiharu Suzuki, Hiroyuki Hatsuta, Shigeo Murayama, Satoru Funamoto, Yasuo Ihara,

Tópico(s)

Pharmacological Effects of Medicinal Plants

Resumo

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. 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 Aβ1-42.1Takami 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.J Neurosci. 2009; 29: 13042-13052Crossref PubMed Scopus (381) Google Scholar,2Matsumura 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.J Biol Chem. 2014; 289: 5109-5121Crossref PubMed Scopus (65) Google Scholar 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 Aβ1-43.1Takami 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.J Neurosci. 2009; 29: 13042-13052Crossref PubMed Scopus (381) Google Scholar, 2Matsumura 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.J Biol Chem. 2014; 289: 5109-5121Crossref PubMed Scopus (65) Google Scholar, 3Okochi 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.Cell Rep. 2013; 3: 42-51Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar 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)4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar; 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.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar 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.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar 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?5Klunk 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.Ann Neurol. 2004; 55: 306-319Crossref PubMed Scopus (3541) Google Scholar,6Mintun 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.Neurology. 2006; 67: 446-452Crossref PubMed Scopus (923) Google Scholar 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,7Kakuda N. Akazawa K. Hatsuta H. Murayama S. Ihara Y. Japanese Alzheimer's Disease Neuroimaging InitiativeSuspected limited efficacy of γ-secretase modulators.Neurobiol Aging. 2013; 34: 1101-1104Crossref PubMed Scopus (12) Google Scholar and that, nevertheless, Aβ deposition continues to progress. To address this issue, we used the same approach as in the CSF study.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar 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.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar 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.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar 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. 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.8Braak H. Braak E. Neuropathological stageing of Alzheimer-related changes.Acta Neuropathol. 1991; 82: 239-259Crossref PubMed Scopus (11609) Google Scholar,9Braak H. Alafuzoff I. Arzberger T. Kretzschmar H. Del Tredici K. Staging of Alzheimer disease-associated neurofibrillary pathology using paraffin sections and immunocytochemistry.Acta Neuropathol. 2006; 112: 389-404Crossref PubMed Scopus (1841) Google Scholar 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).10Hughes C.P. Berg L. Danziger W.L. Coben L.A. Martin R.L. A new clinical scale for the staging of dementia.Br J Psychiatry. 1982; 140: 566-572Crossref PubMed Scopus (5688) Google Scholar 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.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar 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. 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 Aβ38,4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar Aβ40 (M40),11Yamaguchi 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.J Neuropathol Exp Neurol. 2001; 60: 731-739Crossref PubMed Scopus (54) Google Scholar and Aβ42 (M42),11Yamaguchi 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.J Neuropathol Exp Neurol. 2001; 60: 731-739Crossref PubMed Scopus (54) Google Scholar and a polyclonal antibody specific for Aβ43.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar,7Kakuda N. Akazawa K. Hatsuta H. Murayama S. Ihara Y. Japanese Alzheimer's Disease Neuroimaging InitiativeSuspected limited efficacy of γ-secretase modulators.Neurobiol Aging. 2013; 34: 1101-1104Crossref PubMed Scopus (12) Google Scholar M42 did not cross-react with Aβ43, and the Aβ43 polyclonal antibody did not cross-react with Aβ42 on the blot.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar The antibodies were visualized using the ABC Elite kit (Vector Laboratories, Burlingame, CA) using 3, 3′-diaminobenzidine as a chromogen. The sucrose density gradient method was described previously.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar 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). Microsomal fractions from cortical pieces were obtained, as described previously,12Kakuda 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.J Biol Chem. 2006; 281: 14776-14786Crossref PubMed Scopus (130) Google Scholar 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 Aβ43,4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar 9C3 for nicastrin,13Acx 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.J Biol Chem. 2013; 289: 4346-4355Crossref PubMed Scopus (62) Google Scholar 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,13Acx 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.J Biol Chem. 2013; 289: 4346-4355Crossref PubMed Scopus (62) Google Scholar,14Serneels 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.Science. 2009; 324: 639-642Crossref PubMed Scopus (206) Google Scholar 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). A previously reported procedure was used to quantify GSM-1 efficacy.7Kakuda N. Akazawa K. Hatsuta H. Murayama S. Ihara Y. Japanese Alzheimer's Disease Neuroimaging InitiativeSuspected limited efficacy of γ-secretase modulators.Neurobiol Aging. 2013; 34: 1101-1104Crossref PubMed Scopus (12) Google Scholar The ratios of Aβ1-38/Aβ1-42 in the presence of GSM-1 minus in the absence of GSM-1 were calculated. 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). SPs in the brain cortices probably consist of Aβ1-42.15Hardy J. Selkoe D.J. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.Science. 2001; 297: 353-356Crossref Scopus (11002) Google Scholar 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.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar 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). 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,4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar because these proportionalities are characteristic of the parallel stepwise processing of β amyloid protein precursor by γ-secretase.4Kakuda 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 InitiativeAltered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.EMBO Mol Med. 2012; 4: 344-352Crossref PubMed Scopus (49) Google Scholar 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. 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.4Kakuda N. Shoji M. Arai H. Furukawa K. Ikeuchi T.

Referência(s)