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Elevated Membrane Cholesterol Disrupts Lysosomal Degradation to Induce β-Amyloid Accumulation

The Potential Mechanism Underlying Augmentation of β-Amyloid Pathology by Type 2 Diabetes Mellitus
  • Shingo Takeuchi
    Affiliations
    Section of Cell Biology and Pathology, Department of Alzheimer's Disease Research, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology, Obu, Japan
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  • Naoya Ueda
    Affiliations
    Section of Cell Biology and Pathology, Department of Alzheimer's Disease Research, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology, Obu, Japan
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  • Keiko Suzuki
    Affiliations
    Section of Cell Biology and Pathology, Department of Alzheimer's Disease Research, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology, Obu, Japan
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  • Nobuhiro Shimozawa
    Affiliations
    Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition, Tsukuba, Japan
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  • Yasuhiro Yasutomi
    Affiliations
    Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition, Tsukuba, Japan
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  • Nobuyuki Kimura
    Correspondence
    Address correspondence to Nobuyuki Kimura, D.V.M., Ph.D., Section of Cell Biology and Pathology, Department of Alzheimer's Disease Research, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology, Morioka 7-430, Obu, Aichi 474-8511, Japan.
    Affiliations
    Section of Cell Biology and Pathology, Department of Alzheimer's Disease Research, Center for Development of Advanced Medicine for Dementia, National Center for Geriatrics and Gerontology, Obu, Japan
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Open ArchivePublished:November 15, 2018DOI:https://doi.org/10.1016/j.ajpath.2018.10.011
      The endocytic membrane trafficking system is altered in the brains of early-stage Alzheimer disease (AD) patients, and endocytic disturbance affects the metabolism of β-amyloid (Aβ) protein, a key molecule in AD pathogenesis. It is widely accepted that type 2 diabetes mellitus (T2DM) is one of the strongest risk factors for development of AD. Supporting this link, experimentally induced T2DM enhances AD pathology in various animal models. Spontaneous T2DM also enhances Aβ pathology with severe endocytic pathology, even in nonhuman primate brains. However, it remains unclear how T2DM accelerates Aβ pathology. Herein, we demonstrate that cholesterol metabolism–related protein levels are increased and that membrane cholesterol level is elevated in spontaneous T2DM-affected cynomolgus monkey brains. Moreover, in vitro studies that manipulate cellular cholesterol reveal that elevated membrane cholesterol disrupts lysosomal degradation and enhances chemical-induced endocytic disturbance, resulting in great accumulation of Aβ in Neuro2a cells. These findings suggest that an alteration of cerebral cholesterol metabolism may be responsible for augmentation of Aβ pathology in T2DM-affected brains, which, in turn, may increase the risk for developing AD.
      Alzheimer disease (AD) is a progressive neurodegenerative disorder that is histopathologically characterized by the formation of senile plaques and neurofibrillary tangles.
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      In the brains of patients with early-stage AD, endocytic pathology is manifested as intracellular accumulation of abnormally enlarged endosomes. Intracellular accumulation of APP and Aβ is commonly observed in such enlarged endosomes, suggesting that the endocytic membrane trafficking system is altered in the brain of AD patients.
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      Moreover, recent genome-wide association studies have revealed that several variants of the endocytosis-related gene are associated with AD,
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      and the studies using Fluor-labeled ligands demonstrated that endocytosis mediates the internalization of extracellular Aβ in neurons.
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      These findings suggest that alterations in the endocytic membrane trafficking system are involved in AD pathogenesis. Even with all this evidence, it remains unclear why endocytic disturbance is enhanced in AD patient brains.
      Several epidemiologic and clinical studies showed that type 2 diabetes mellitus (T2DM) patients have high susceptibility to AD in their later life.
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      Moreover, experimental induction of T2DM enhances AD pathology in various animal models.
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      Diabetes mellitus induces Alzheimer's disease pathology: histopathological evidence from animal models.
      Spontaneous T2DM enhances Aβ pathology even in nonhuman primate brains, and severe endocytic pathology is also observed in T2DM-affected monkey brains.
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      • Yasutomi Y.
      • Yanagisawa K.
      • Kimura N.
      Diabetes mellitus accelerates Aβ pathology in brain accompanied by enhanced GAβ generation in nonhuman primates.
      However, it remains unclear how T2DM accelerates Aβ pathology. Thus, we hypothesized that T2DM may alter the endocytic membrane trafficking system in a way that enhances endocytic disturbance.
      Herein, we demonstrate that cholesterol metabolism–related protein levels are increased and that the membrane cholesterol level is elevated in the brains of spontaneous T2DM-affected monkeys. Moreover, in vitro studies by using methyl-β-cyclodextrin (MβCD)–cholesterol reveal that elevated membrane cholesterol disrupts lysosomal degradation and enhances chloroquine- and ciliobrevin D–induced endocytic disturbance, resulting in great accumulation of Aβ in Neuro2a cells. These findings suggest that an alteration in cerebral cholesterol metabolism may be responsible for the severe endocytic disturbance in T2DM-affected brains, which, in turn, may increase the risk for developing AD accompanied by augmentation of Aβ pathology.

      Materials and Methods

      Antibodies

      For Western blot analyses, the following primary antibodies were used: rabbit polyclonal anti-Akt antibody (1:5000; Cell Signaling Technology, Danvers, MA; catalog number 9272); rabbit monoclonal anti–phosphorylated Akt antibody (1:5000; Cell Signaling Technology; 4060); rabbit monoclonal anti-GSK3β antibody (1:10,000; Cell Signaling Technology; 12,456); rabbit monoclonal anti–phosphorylated GSK3β antibody (1:10,000; Cell Signaling Technology; 5558); mouse monoclonal anti–β-actin antibody (1:200,000; Sigma, St. Louis, MO; A5441); rabbit polyclonal anti–sterol regulatory element–binding protein 2 (SREBP2) antibody (1:5000; Cayman Chemical, Ann Arbor, MI; 10007663); goat polyclonal anti–apolipoprotein E (ApoE) antibody (1:5000; Millipore, Temecula, CA; AB947); rabbit polyclonal anti–ATP-binding cassette subfamily A member 1 antibody (1:2000; Abcam, Cambridge, UK; ab7360); goat polyclonal anti–low-density lipoprotein receptor–related protein 1 antibody (1:2000; Santa Cruz Biotechnology, Dallas, TX; sc-16168); rabbit monoclonal anti-APP antibody (1:10,000; Abcam; ab32136); rabbit monoclonal anti-Rab5 antibody (1:10,000; Cell Signaling Technology; 3547); rabbit polyclonal anti-Rab7 antibody (1:6000; Sigma; R4779); mouse monoclonal anti–glyceraldehyde-3-phosphate dehydrogenase antibody (1:200,000; GeneTex, Irvine, CA; GTX28245); rabbit polyclonal anti-SQSTM1/p62 antibody (1:2000; Cell Signaling Technology; 5114); mouse monoclonal anti–light chain 3 (LC3) antibody (1:2000; Nanotools, Teningen, Germany; 0231-100/LC3-5F10); mouse monoclonal anti-APP clone 22C11 (1:4000; Millipore; MAB348); rabbit polyclonal anti-sAPP-β (1:4000; BioLegend, San Diego, CA; 813401); rabbit polyclonal anti-clathrin heavy chain antibody (1:2000; Santa Cruz Biotechnology; sc-9069); and rabbit polyclonal anti-dynamin antibody (1:2000; Santa Cruz Biotechnology; sc-11362).
      For immunocytochemistry, the following primary antibodies were used: rabbit polyclonal anti-APP antibody (1:2000; IBL, Gunma, Japan; 28,053); mouse monoclonal anti-Rab5 antibody (1:200; Santa Cruz Biotechnology; sc-46692); mouse monoclonal anti-Rab7 antibody (1:4000; Abcam; ab50533); mouse monoclonal anti-clathrin heavy chain (1:500; Santa Cruz Biotechnology; sc-12734); and rabbit polyclonal anti-dynamin antibody (1:1000; Santa Cruz Biotechnology; sc-11362).

      Animals

      Fourteen cynomolgus monkey (M. fascicularis) brains were used in this study. Of these, seven brains were from normal monkeys [age: 18 years (n = 1), 19 years (n = 1), 20 years (n = 2), 22 years (n = 1), 26 years (n = 1), and 28 years (n = 1)]; and seven were from spontaneous T2DM-affected moneys [age: 18 years (n = 1), 19 years (n = 1), 20 years (n = 1), 21 years (n = 2), 26 years (n = 1), and 27 years (n = 1)]. Both sexes were used. Information of all monkeys used for this study is shown in Table 1.
      Table 1Cynomolgus Monkeys Used in the Present Study
      NumberAge, yearsSexGlu, mg/dL
      The last value taken from each monkey.
      TG, mg/dL
      The last value taken from each monkey.
      Experimental purpose
      Control
       118Female478WB
       219Male6633WB
       320Female4663WB
       420Female3447MC
       522Male5676MC
       626Female7785MC
       728Female50100MC
      T2DM
       118Female154766WB
       219Female443937WB
       320Female341425WB
       421Female147131MC
       521Female144252MC
       626Female96380MC
       727Female255192MC
      Glu, blood glucose levels; MC, analysis of membrane cholesterol; TG, blood triglyceride levels; WB, Western blot analysis.
      The last value taken from each monkey.
      The cerebral cortices of 6 of the 14 monkeys were used for Western blot analyses. Of these six, three were from normal monkeys [age: 18, 19, and 20 years (n = 1 each)]; three were from spontaneous T2DM-affected monkeys [age: 18, 19, and 20 years (n = 1 each)]. The cerebral cortices of 8 of the 14 monkeys were used for analysis of membrane cholesterol levels. Of these eight, four were from normal monkeys [age: 20, 22, 26, and 28 years (n = 1 each)]; four were from spontaneous T2DM-affected monkeys [age: 21 years (n = 2), 26 years (n = 1), and 27 years (n = 1)].
      All brains were obtained from the Tsukuba Primate Research Center (TPRC), National Institutes of Biomedical Innovation, Health, and Nutrition (Osaka, Japan). The maintenance and care of animals were performed according to the rules for animal care of the TPRC at the National Institutes of Biomedical Innovation, Health, and Nutrition for the care, use, and biohazard countermeasure of laboratory animals. All monkeys were bred and maintained in an air-conditioned room at the TPRC with controlled illumination (12 hours light/12 hours dark), temperature (23°C to 27°C), humidity (50% to 70%), and ventilation (12 air changes/hour). Each monkey was given 70 g of commercially available pellet monkey chow (CMK-2; CLEA Japan, Inc., Tokyo, Japan); 100 g of apples; and unlimited access to tap water every day. Every morning, their health status (eg, assessment of viability, appetite, and coat appearance) was monitored. When any abnormality was found, a veterinarian examined the monkey promptly and applied the appropriate treatment, such as fluid replacement.
      In the TPRC colony, some adult monkeys are spontaneously affected with T2DM for various reasons, such as pregnancy history and environmental factors. The TPRC has accumulated clinical data for >40 years. On the basis of these data, the normal blood glucose level for female monkeys is in the range of 24 to 74 mg/dL; and for male monkeys, the range is from 24 to 76 mg/dL. Normal blood triglyceride levels are in the range of 8 to 85 mg/dL for females and of 6 to 52 mg/dL for males. The animals were diagnosed with T2DM by periodic health examination, and the blood glucose and triglyceride levels of monkeys are used to outline DM criteria for this study (Table 1 and Supplemental Figure S1). T2DM-affected monkeys used in this study have not received any DM-related treatments before experiments.
      This study was performed in strict accordance with the rules for animal care and management of the TPRC,
      • Honjo S.
      The Japanese Tsukuba Primate Center for Medical Science (TPC): an outline.
      the Guiding Principles for Animal Experiments Using Nonhuman Primates formulated by the Primate Society of Japan,
      • Honjo S.
      Guiding principles for animal experiments using nonhuman primates.
      and the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals. The research protocol was approved by the Animal Care and Use Committee of the National Institutes of Biomedical Innovation, Health, and Nutrition (DS17-001R1). When a monkey presented clinical symptoms due to injury or illness, and it was not expected to recover from pain or morbidity, it was judged to have a poor prognosis. The animals used in this study either died of natural causes or were euthanized when they reached end points determined as poor prognosis. For euthanasia, the monkeys were deeply anesthetized with a lethal dose of pentobarbital, and all efforts were made to minimize suffering.

      Biochemical Analysis of Monkey Brains

      For Western blot analyses, frozen monkey brain tissue was homogenized in a glass homogenizer with homogenate buffer solution containing 0.32 mol/L sucrose, 10 mmol/L Tris-HCL (pH 7.6), 1 mmol/L EDTA, and cOmpleteMini proteinase inhibitor cocktail (Roche Molecular Biochemicals, Penzberg, Germany), and then the tissue was centrifuged at 1000 × g for 10 minutes to obtain the supernatant fraction. The supernatant was centrifuged at 100,000 × g for 1 hour to obtain the pellet fraction. The resulting pellets were resuspended in homogenization buffer and then subjected to Western blot analyses. The proteins were adjusted to 10 μg and then subjected to SDS-PAGE by using 12.8% acrylamide gels. Separated proteins were blotted onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% nonfat dried milk in phosphate-buffered saline (pH 7.0) and 0.1% Tween-20 for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. They were then incubated with either horseradish peroxidase–conjugated goat anti-mouse IgG (1:10,000) or goat anti-rabbit IgG (1:10,000; Cell Signaling Technology) for 1 hour at room temperature. Immunoreactive elements were visualized using enhanced chemiluminescence (Immobilon Western Detection Reagents; Millipore). Each experiment was duplicated.
      To analyze membrane cholesterol level, frozen monkey brain tissue was homogenized and then centrifuged at 1000 × g for 10 minutes to obtain a supernatant fraction, as mentioned above. The supernatant (S1) was centrifuged at 13,000 × g for 15 minutes to yield a pellet 2 fraction. The supernatant (S2) from the pellet 2 fraction was centrifuged at 100,000 × g for 1 hour to obtain the microsomal (pellet) fractions. The pellet was resuspended in homogenization buffer and sonicated. This sonicated fraction was layered over homogenization buffer containing 0.85 or 1.2 mol/L sucrose and then subjected to discontinuous equilibrium sucrose density gradient centrifugation at 50,000 × g for 30 minutes using a swing-out MLS-50 Beckman rotor (Beckman Coulter, Fullerton, CA). Then, a band was obtained at the 0.32 to 0.85 mol/L sucrose interface (myelin), and a band was obtained at the 0.85 to 1.2 mol/L sucrose interface fraction (membranes). Free cholesterol level in the membrane fraction was measured by a free cholesterol test E Wako kit (Wako, Osaka, Japan) and normalized by protein concentrations, as measured by bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA). Each experiment was duplicated.

      Cell Cultures and Chemical Treatments

      A neuronal cell line, mouse neuroblastoma Neuro2a cells from ATCC (Manassas, VA), was grown in Dulbecco's modified Eagle's medium (Sigma) with 5% fetal calf serum. Cells were cultured under humidified air containing 5% CO2 at 37°C. Cells were plated at a density of 2.5 × 104 cells onto 6- or 12-well culture plates (Thermo Fisher Scientific) and coverslips (Matsunami, Osaka, Japan) coated with 0.1% polyethyleneimine (Wako).
      For chemical treatment studies, cells were incubated with 10 μmol/L chloroquine (Wako), 50 μmol/L ciliobrevin D (Millipore), 50 nmol/L insulin (Wako), or a combination of 10 μmol/L pepstatin A (Sigma), 10 μmol/L E64d (Cayman Chemical), and 10 μmol/L leupeptin (Sigma) for 24 hours. To manipulate membrane cholesterol level, cells were incubated with 75 μmol/L MβCD-cholesterol complex (Sigma) for 1 hour to increase membrane cholesterol levels. For transferrin uptake, cells were pretreated with 75 μmol/L MβCD-cholesterol for 1 hour. After washing, cells were cultured for another 24 hours and then treated with 50 nmol/L transferrin-biotin (Sigma) for 20 minutes.
      Cells that were plated in 12-well plates were lysed in a sample buffer solution containing 62.5 mmol/L Tris-HCl (pH 6.8), 2.3% SDS, 0.5% Triton X-100, 2 mmol/L EGTA, 2.5% 2-mercaptoethanol, and cOmplete Mini protease inhibitor cocktail to extract total cellular proteins. Total proteins were adjusted to 10 μg and then subjected to Western blot analyses, as described above. For detection of transferrin-biotin, the membranes were incubated with VECTASTAIN ABC reagent (Vector Laboratories, Burlingame, CA) for 1 hour, and then antigen was detected using enhanced chemiluminescence. Three independent experiments (n = 6 for each experimental group) were performed, and each experiment was duplicated.
      Neuro2a cells that were plated in 6-well plates, as well as tissue from monkey brains, were subjected to measurement of membrane cholesterol levels. Cells were homogenized in homogenate buffer solution and then centrifuged at 1000 × g for 10 minutes to obtain a supernatant fraction. The supernatant (S1) was centrifuged at 100,000 × g for 1 hour to obtain the microsomal (pellet) fractions. The pellet was resuspended in homogenization buffer and sonicated. Then, the free cholesterol level in the membrane fraction was measured by a free cholesterol test E Wako kit and normalized by protein concentrations, as measured by bicinchoninic acid assay. Two independent experiments were performed.

      Immunofluorescence

      Neuro2a cells that were plated on coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 10 minutes. After blocking with 3% bovine serum albumin in phosphate-buffered saline and 0.1% Tween-20, cells were incubated with primary antibodies overnight at 4°C. After thorough washing, cells were then incubated with AlexaFluor 488–conjugated anti-mouse IgG (1:1000; Jackson ImmunoResearch, Laboratories, Inc., West Grove, PA) and AlexaFluor 568–conjugated anti-rabbit IgG (1:1000; Abcam) for 2 hours at room temperature, and the excess antibody solution was washed out. The stained cells were imaged with an LSM700 confocal laser-scanning microscope (Zeiss, Oberkochen, Germany).
      For labeling late endosomes/lysosomes, cells were pretreated with 50 nmol/L LysoTracker Red DND-99 (Invitrogen, Carlsbad, CA) for 30 minutes before fixation, and then counterstained with DAPI. The particle number and size of endosomes/lysosomes were analyzed by using ImageJ software version 1.49v (NIH, Bethesda, MD; http://imagej.nih.gov/ij; 20 independent cells were analyzed for each group). Cells were also stained with 100 μg/mL Filipin (70,440; Cayman Chemical) for 1 hour to label cholesterol. Three independent experiments were performed.

      Aβ Enzyme-Linked Immunosorbent Assay

      Aβ level was determined using a sandwich enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay kit for Human/Rodent Aβ1-40 was obtained from Wako. Cells plated on 6-well plates were lysed in 70% formic acid. After incubation at room temperature for 10 minutes, the lysates were further diluted with 1 mol/L Tris-HCl (pH 8.0) and centrifuged at 15,000 × g for 20 minutes. The resulting supernatants were then subjected to enzyme-linked immunosorbent assay. Two independent experiments (n = 6 for each experimental group) were performed, and all samples were measured in duplicate.

      Statistical Analysis

      To bolster reproducibility, immunoreactive bands of the Western blots were quantified using commercially available software (Quantity One version 4.6.6; PDI, Inc., Upper Saddle River, NJ). Data are expressed as means ± SD. For statistical analyses, data were analyzed by t-test to compare between two groups. For comparison of more than three groups, one-way analyses of variance were used, followed by the Tukey honestly significant difference post hoc tests. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Tochigi, Japan), which is a graphical user interface for R version 2.13.0 (The R Foundation for Statistical Computing, Vienna, Austria).
      • Kanda Y.
      Investigation of the freely available easy-to-use software “EZR” for medical statistics.
      More precisely, it is a modified version of R commander version 1.6-3 that includes statistical functions that are frequently used in biostatistics. Because the number of spontaneous T2DM-affected monkeys was limited, sample calculation or an assessment of the normality of data was not performed.

      Results

      Aberrant Insulin Signal Transduction in T2DM-Affected Monkey Brains

      T2DM is characterized by peripheral insulin resistance and aberrant insulin signal transduction.
      • Saltiel A.R.
      New perspectives into the molecular pathogenesis and treatment of type 2 diabetes.
      When binding to insulin receptors, insulin induces signal transduction of various downstream molecules, such as those in the phosphatidylinositide 3-kinase (PI3K)/Akt pathway.
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      Insulin signaling meets mitochondria in metabolism.
      Several studies showed that alteration in PI3K activity can affect endosome trafficking.
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      Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3'-kinase-dependent machinery.
      Although there are limitations such that the number of available animals is relatively small, signal transduction was analyzed in the PI3K/Akt pathway to assess whether PI3K activity is altered in spontaneous T2DM-affected monkey brains. Akt and GSK3β, the major molecules of the PI3K/Akt pathway, are highly phosphorylated in spontaneous T2DM-affected monkey brains compared with age-matched normal monkey brains (Figure 1).
      Figure thumbnail gr1
      Figure 1Phosphatidylinositide 3-kinase (PI3K)/Akt pathway is activated in spontaneous T2DM-affected monkey brains. A: Western blot analyses showing the amounts of Akt, p-Akt, GSK3β, p-GSK3β, and β-actin in the brains of normal (CT) and T2DM-affected (DM) monkeys. Monkeys were 18, 19, and 20 years of age (y). B: Relative expression of p-Akt/Akt and p-GSK3β/GSK3β in brains from normal and T2DM-affected monkeys. PI3K/Akt pathway was enhanced in spontaneous T2DM-affected monkey brains. p-Akt level was normalized to total Akt level. p-GSK3β level was normalized to total GSK3β level. Data are expressed as means ± SD (B). n = 3 (B). P < 0.05, ∗∗P < 0.01 versus control.

      Insulin Treatment Neither Induces nor Enhances Endocytic Disturbance

      To assess whether up-regulation in the PI3K/Akt pathway enhances endocytic disturbance, insulin was applied to cultured Neuro2a cells, a mouse neuroblastoma cell line, and the expression of molecules in the PI3K/Akt pathway, endosome proteins, and APP was quantified. Insulin treatment in vitro clearly induces phosphorylation of Akt in Neuro2a cells (Figure 2, A and B ). Rab GTPase is a good indicator of alterations in intracellular endosome trafficking.
      • Kimura N.
      • Inoue M.
      • Okabayashi S.
      • Ono F.
      • Negishi T.
      Dynein dysfunction induces endocytic pathology accompanied by an increase in Rab GTPases: a potential mechanism underlying age-dependent endocytic dysfunction.
      • Kimura N.
      • Okabayashi S.
      • Ono F.
      Dynein dysfunction disrupts intracellular vesicle trafficking bidirectionally and perturbs synaptic vesicle docking via endocytic disturbances a potential mechanism underlying age-dependent impairment of cognitive function.
      • Kimura N.
      • Samura E.
      • Suzuki K.
      • Okabayashi S.
      • Shimozawa N.
      • Yasutomi Y.
      Dynein dysfunction reproduces age-dependent retromer deficiency: concomitant disruption of retrograde trafficking is required for alteration in β-amyloid precursor protein metabolism.
      Herein, insulin treatment alone did not affect APP levels or subcellular distribution of Rab5- and Rab7-positive endosomes (Figure 2, A–C). Chloroquine, a lysosomotropic reagent, disturbs endosome/lysosome trafficking and lysosomal degradation by alkalinizing endosomal pH, leading to significant endocytic disturbance.
      • Kimura N.
      • Inoue M.
      • Okabayashi S.
      • Ono F.
      • Negishi T.
      Dynein dysfunction induces endocytic pathology accompanied by an increase in Rab GTPases: a potential mechanism underlying age-dependent endocytic dysfunction.
      • de Duve C.
      • de Barsy T.
      • Poole B.
      • Trouet A.
      • Tulkens P.
      • Van Hoof F.
      Commentary: lysosomotropic agents.
      • Nixon R.A.
      • Cataldo A.M.
      • Mathews P.M.
      The endosomal-lysosomal system of neurons in Alzheimer's disease pathogenesis: a review.
      • Ueda N.
      • Tomita T.
      • Yanagisawa K.
      • Kimura N.
      Retromer and Rab2-dependent trafficking mediate PS1 degradation by proteasomes in endocytic disturbance.
      Consistent with previous studies,
      • Nixon R.A.
      • Cataldo A.M.
      • Mathews P.M.
      The endosomal-lysosomal system of neurons in Alzheimer's disease pathogenesis: a review.
      • Ueda N.
      • Tomita T.
      • Yanagisawa K.
      • Kimura N.
      Retromer and Rab2-dependent trafficking mediate PS1 degradation by proteasomes in endocytic disturbance.
      chloroquine treatment increased APP levels in Neuro2a cells (Figure 2, A and B). Immunocytochemistry also confirmed that chloroquine treatment reproduced endocytic pathology, such as APP accumulation, in enlarged Rab5- and Rab7-positive endosomes (Figure 2C). Quantitative image analyses confirmed that chloroquine treatment increased the number of both Rab5- and Rab7-positive endosomes, and their size was also enlarged (Figure 2, D and E). On the other hand, no additional effects of chloroquine-induced endocytic disturbance were observed with insulin treatment (Figure 2).
      Figure thumbnail gr2
      Figure 2Insulin (Ins) treatment does not alter chloroquine (Cq)–induced endocytic disturbance in Neuro2a cells. Quantitation of molecules in the phosphatidylinositide 3-kinase (PI3K)/Akt pathway, endosome proteins, and β-amyloid precursor protein (APP) after chloroquine and/or insulin treatment. Neuro2a cells were treated with 10 μmol/L chloroquine and/or 50 nmol/L insulin for 24 hours. A: Western blot analyses showing the amounts of APP, Rab5, Rab7, p-Akt, Akt, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in extracts derived from Neuro2a cells 24 hours after chloroquine and/or insulin treatment. B: Relative amounts of p-Akt/Akt, APP, Rab5, and Rab7 in Neuro2a cells after chloroquine and/or insulin treatment. Insulin treatment activated PI3K/Akt pathway in Neuro2a cells; however, it does not affect APP and Rab GTPases levels. p-Akt level was normalized to total Akt level. APP, Rab5, and Rab7 were normalized to GAPDH. C: Photomicrographs of treated and control (CT) Neuro2a cells immunostained for Rab5, Rab7, and APP 24 hours after chloroquine and/or insulin treatment. Chloroquine treatment induces endocytic pathology in Neuro2a cells. However, insulin treatment does not show any changes. D: Quantitative image analyses of relative number of Rab5- and Rab7-positive endosomes for each treatment group. No additional effects are observed by insulin on chloroquine-treated cells. E: Quantitative image analyses of relative area of Rab5- and Rab7-positive endosomes for each treatment group. No additional effects are observed by insulin on chloroquine-treated cells. Data are expressed as means ± SD (B, D, and E). n = 6 (B); n = 20 (D and E). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 versus control. Scale bars = 40 μm (C, all images).

      Cholesterol Metabolism–Related Proteins Are Increased, and Membrane Cholesterol Level Is Elevated, in T2DM-Affected Monkey Brains

      Dyslipidemia is also frequently observed in T2DM patients.
      • Brunzell J.D.
      • Ayyobi A.F.
      Dyslipidemia in the metabolic syndrome and type 2 diabetes mellitus.
      • Adeli K.
      • Lewis G.F.
      Intestinal lipoprotein overproduction in insulin-resistant states.
      • Ginsberg H.N.
      • MacCallum P.R.
      The obesity, metabolic syndrome, and type 2 diabetes mellitus pandemic, part I: increased cardiovascular disease risk and the importance of atherogenic dyslipidemia in persons with the metabolic syndrome and type 2 diabetes mellitus.
      • Markgraf D.F.
      • Al-Hasani H.
      • Lehr S.
      Lipidomics: reshaping the analysis and perception of type 2 diabetes.
      Alterations in cholesterol homeostasis have been suggested to be associated with AD pathology.
      • Di Paolo G.
      • Kim T.W.
      Linking lipids to Alzheimer's disease: cholesterol and beyond.
      This observation prompted us to analyze cholesterol metabolism–related proteins in spontaneous T2DM-affected monkey brains.
      When cellular membrane cholesterol level is decreased, SREBP2 is translocated from endoplasmic reticulum to Golgi by SREBP cleavage–activating protein and cleaved into a mature form, which induces cholesterol synthesis.
      • Hua X.
      • Nohturfft A.
      • Goldstein J.L.
      • Brown M.S.
      Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein.
      • DeBose-Boyd R.A.
      • Brown M.S.
      • Li W.P.
      • Nohturfft A.
      • Goldstein J.L.
      • Espenshade P.J.
      Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi.
      Mature SREBP2 was clearly increased in spontaneous T2DM-affected monkey brains (Figure 3, A and B ). Moreover, cholesterol trafficking–related protein levels, such as ApoE, ATP-binding cassette subfamily A member 1, and low-density lipoprotein receptor–related protein 1, were also increased in spontaneous T2DM-affected monkey brains (Figure 3, A and B).
      Figure thumbnail gr3
      Figure 3Cholesterol metabolism–related proteins are increased and membrane cholesterol is elevated in T2DM-affected monkey brains. A: Western blot analyses showing the amounts of mature sterol regulatory element–binding protein 2 (mSREBP2), apolipoprotein E (ApoE), ATP-binding cassette subfamily A member 1 (ABCA1), low-density lipoprotein receptor–related protein 1 (LRP1), and β-actin in normal (CT) and T2DM-affected (DM) monkey brains. B: Relative expression of mSREBP2, ApoE, ABCA1, and LRP1 in brains from normal and T2DM-affected monkeys. mSREBP2, ApoE, ABCA1, and LRP1 levels were normalized to β-actin levels. Not only mSREBP2 but also cholesterol trafficking–related protein levels in the brains of spontaneous T2DM-affected monkeys are clearly elevated. C: Concentration of free cholesterol levels in membrane fractions derived from the brains of T2DM-affected and control monkeys. Membrane cholesterol levels are significantly elevated in T2DM-affected monkey brains compared with normal control brains. Data are expressed as means ± SD (B and C). n = 3 (B); n = 4 (C). P < 0.05, ∗∗P < 0.01 versus control. y, years.
      Much of the cholesterol in the brain is found in myelin, where cholesterol turnover is slow.
      • Dietschy J.M.
      • Turley S.D.
      Thematic review series: brain lipids: cholesterol metabolism in the central nervous system during early development and in the mature animal.
      To assess whether cholesterol level is abnormally altered in T2DM-affected brains, the brain-derived membrane fraction was purified by removing myelin using sucrose gradient centrifugation. Noteworthy, the amount of membrane cholesterol was significantly elevated in spontaneous T2DM-affected monkey brains compared with normal control brains (Figure 3C).
      Previous study showed that insulin regulates cholesterol level via modifying SREBP2 level.
      • Suzuki R.
      • Lee K.
      • Jing E.
      • Biddinger S.B.
      • McDonald J.G.
      • Montine T.J.
      • Craft S.
      • Kahn C.R.
      Diabetes and insulin in regulation of brain cholesterol metabolism.
      Although insulin treatment slightly increased mature SREBP2 level in Neuro2a cells, other cholesterol trafficking–related protein levels were not affected (Supplemental Figure S2, A and B). Insulin treatment also failed to increase the level of membrane cholesterol in Neuro2a cells (Supplemental Figure S2C).

      Increase in Membrane Cholesterol Levels Enhances Endocytic Disturbance to Augment Intracellular Accumulation of APP and Aβ

      To assess whether elevated membrane cholesterol enhances endocytic disturbance, Neuro2a cells were treated with MβCD-cholesterol, a chemical commonly used to load membrane cholesterol. The cellular membrane cholesterol was increased after MβCD-cholesterol treatment (Figure 4, A and B ). Although chloroquine treatment alone did not affect membrane cholesterol level, it significantly enhanced membrane cholesterol when combined with MβCD-cholesterol treatment (Figure 4, A and B). MβCD-cholesterol treatment alone did not affect APP or Rab GTPase levels (Figure 4, C and D). However, MβCD-cholesterol in combination with chloroquine significantly enhanced the increase in APP and Rab7 levels even more (Figure 4, C and D). Rab5 level also tended to increase when MβCD-cholesterol and chloroquine treatment were combined (Figure 4, C and D).
      Figure thumbnail gr4
      Figure 4Elevated membrane cholesterol aggravates chloroquine (Cq)–induced endocytic disturbance, leading to enhanced intracellular accumulation of APP and Aβ. Neuro2a cells were treated in vitro with 75 μmol/L MβCD-cholesterol (CHO) for 1 hour to experimentally load membrane cholesterol. After washing, cells were cultured for another 24 hours with 10 μmol/L chloroquine to disrupt endosome/lysosome trafficking. A: Photomicrographs of Neuro2a cells stained with 100 μg/mL Filipin 24 hours after chloroquine and/or MβCD-cholesterol treatment. MβCD-cholesterol treatment increases Filipin-positive fluorescence, and combined treatment of MβCD-cholesterol and chloroquine strongly enhances it. B: Concentration of free cholesterol in membrane fraction derived from Neuro2a cells after chloroquine and/or MβCD-cholesterol treatment. C: Western blot analyses showing the amounts of β-amyloid precursor protein (APP), Rab5, Rab7, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in extracts derived from Neuro2a cells after chloroquine and/or MβCD-cholesterol treatment. D: Relative expression of APP, Rab5, and Rab7 in Neuro2a cells, showing the effect of MβCD-cholesterol and chloroquine treatment. APP, Rab5, and Rab7 levels were normalized to GAPDH levels. E: Photomicrographs of MβCD-cholesterol– and chloroquine-treated Neuro2a cells immunostained for Rab5 and APP or Rab7 and APP. F: Quantitative image analyses of relative number of Rab5- and Rab7-positive endosomes for each treatment group. G: Quantitative image analyses of relative area of Rab5- and Rab7-positive endosomes for each treatment group. H: Relative amounts of Aβ in Neuro2a cells after MβCD-cholesterol and chloroquine treatment. Aβ levels were measured by enzyme-linked immunosorbent assay. Data are expressed as means ± SD (B, D, and FH). n = 6 (B, D, and H); n = 20 (F and G). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001; P < 0.05, ††P < 0.01, and †††P < 0.001 versus control. Scale bar = 40 μm (A and E, all images). CT, control cell.
      Immunocytochemistry of cells in this experiment showed that enlarged Rab5- and Rab7-positive endosomes appeared to increase, and large amounts of APP accumulated in the enlarged endosomes when combining MβCD-cholesterol and chloroquine treatment (Figure 4E). Quantitative image analyses confirmed that MβCD-cholesterol treatment increased the number of Rab5- and Rab7-positive endosomes by itself, and MβCD-cholesterol in combination with chloroquine strongly enhanced the size of both endosomes (Figure 4, F and G). Intracellular Aβ level was also significantly increased in cells treated with both MβCD-cholesterol and chloroquine compared with chloroquine treatment alone (Figure 4H). Intriguingly, MβCD-cholesterol treatment solely induced intracellular accumulation of Aβ (Figure 4H).
      Aging disrupts the ability of dynein-mediated transport in brain, and dynein dysfunction reproduces endocytic pathology, resulting in the intracellular accumulation of Aβ.
      • Kimura N.
      • Imamura O.
      • Ono F.
      • Terao K.
      Aging attenuates dynactin-dynein interaction: down-regulation of dynein causes accumulation of endogenous tau and amyloid precursor protein in human neuroblastoma cells.
      • Kimura N.
      • Inoue M.
      • Okabayashi S.
      • Ono F.
      • Negishi T.
      Dynein dysfunction induces endocytic pathology accompanied by an increase in Rab GTPases: a potential mechanism underlying age-dependent endocytic dysfunction.
      Ciliobrevin D is a well-known cytoplasmic dynein inhibitor, and it can induce modest endocytic disturbance compared with chloroquine (Supplemental Figure S3). Evidently, MβCD-cholesterol treatment also enhanced ciliobrevin D–induced endocytic disturbance to augment intracellular accumulation of Aβ (Supplemental Figure S3).

      Elevated Membrane Cholesterol Disturbs Lysosomal Degradation

      Because MβCD-cholesterol treatment alone induced the intracellular accumulation of Aβ (Figure 4H), it was considered that elevated membrane cholesterol might alter APP metabolism. Several studies showed that Aβ generation is affected by alterations in endocytosis of APP and that endocytosis-related proteins, such as clathrin and dynamin, are involved in APP metabolism.
      • LaFerla F.M.
      • Green K.N.
      • Oddo S.
      Intracellular amyloid-beta in Alzheimer's disease.
      • Ovsepian S.V.
      • O'Leary V.B.
      • Zaborszky L.
      • Ntziachristos V.
      • Dolly J.O.
      Synaptic vesicle cycle and amyloid β: biting the hand that feeds.
      • Xiao Q.
      • Gil S.C.
      • Yan P.
      • Wang Y.
      • Han S.
      • Gonzales E.
      • Perez R.
      • Cirrito J.R.
      • Lee J.M.
      Role of phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) in intracellular amyloid precursor protein (APP) processing and amyloid plaque pathogenesis.
      • Thomas R.S.
      • Lelos M.J.
      • Good M.A.
      • Kidd E.J.
      Clathrin-mediated endocytic proteins are upregulated in the cortex of the Tg2576 mouse model of Alzheimer's disease-like amyloid pathology.
      MβCD-cholesterol treatment did not affect the levels of clathrin and dynamin in Neuro2a cells (Supplemental Figure S4, A and B). Immunocytochemistry showed that the subcellular distribution of clathrin and dynamin remained unchanged with the increase in membrane cholesterol levels (Supplemental Figure S4C). Also, transferrin uptake was not affected by MβCD-cholesterol treatment (Supplemental Figure S4, D and E).
      Previous studies showed that the manipulation of membrane cholesterol level alters APP metabolism to increase β-site cleavage, resulting in the increase of Aβ generation.
      • Bodovitz S.
      • Klein W.L.
      Cholesterol modulates alpha-secretase cleavage of amyloid precursor protein.
      • Simons M.1
      • Keller P.
      • De Strooper B.
      • Beyreuther K.
      • Dotti C.G.
      • Simons K.
      Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons.
      • Wahrle S.
      • Das P.
      • Nyborg A.C.
      • McLendon C.
      • Shoji M.
      • Kawarabayashi T.
      • Younkin L.H.
      • Younkin S.G.
      • Golde T.E.
      Cholesterol-dependent gamma-secretase activity in buoyant cholesterol-rich membrane microdomains.
      • Xiong H.
      • Callaghan D.
      • Jones A.
      • Walker D.G.
      • Lue L.F.
      • Beach T.G.
      • Sue L.I.
      • Woulfe J.
      • Xu H.
      • Stanimirovic D.B.
      • Zhang W.
      Cholesterol retention in Alzheimer's brain is responsible for high beta- and gamma-secretase activities and Abeta production.
      • Marquer C.
      • Laine J.
      • Dauphinot L.
      • Hanbouch L.
      • Lemercier-Neuillet C.
      • Pierrot N.
      • Bossers K.
      • Le M.
      • Corlier F.
      • Benstaali C.
      • Saudou F.
      • Thinakaran G.
      • Cartier N.
      • Octave J.N.
      • Duyckaerts C.
      • Potier M.C.
      Increasing membrane cholesterol of neurons in culture recapitulates Alzheimer's disease early phenotypes.
      In the present study, however, no changes were observed in α- and β-site cleaved products of APP with MβCD-cholesterol treatment (Supplemental Figure S5).
      The endolysosomal system is also important for APP metabolism, and lysosomal dysfunction is closely associated with the development of Aβ pathology in the brains of AD patients.
      • Nixon R.A.
      • Mathews P.M.
      • Cataldo A.M.
      The neuronal endosomal-lysosomal system in Alzheimer's disease.
      Especially, autophagy mediates not only Aβ generation but also Aβ clearance, and the clearance of autophagic vacuoles is significantly impaired in AD patient brains.
      • Nixon R.A.
      Autophagy, amyloidogenesis and Alzheimer disease.
      Therefore, we hypothesized that elevated membrane cholesterol might disturb the lysosomal degradation. The morphology of late endosomes/lysosomes was analyzed by using LysoTracker. Combined treatment with MβCD-cholesterol and chloroquine greatly increased the number of abnormally enlarged late endosomes/lysosomes compared with chloroquine treatment alone (Figure 5, A and B ). The size of LysoTracker-positive late endosomes/lysosomes was also enhanced by combined treatment with MβCD-cholesterol and chloroquine (Figure 5, A and C).
      Figure thumbnail gr5
      Figure 5Lysosomal dysfunction is exacerbated by MβCD-cholesterol (CHO) treatment. Neuro2a cells were treated with 75 μmol/L MβCD-cholesterol for 1 hour. After washing, cells were cultured for another 24 hours with 10 μmol/L chloroquine (Cq) to disrupt endosome/lysosome trafficking. A: Photomicrographs of Neuro2a cells stained with 50 nmol/L LysoTracker Red DND-99 for 30 minutes before fixation and counterstained for labeling cell nuclei with DAPI. Combined treatment of MβCD-cholesterol and chloroquine enhances the enlargement of LysoTracker-positive lysosomes/late endosomes. B: Quantitative image analyses of relative number of LysoTracker-positive lysosomes/late endosomes for each treatment group. C: Quantitative image analyses of relative area of LysoTracker-positive lysosomes/late endosomes for each treatment group. D: Western blot analyses showing the amounts of p62, light chain 3 (LC3)-II, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in extracts derived from Neuro2a cells after MβCD-cholesterol and/or chloroquine treatment. E: Relative expression of p62 and LC3-II, substrates of lysosomal degradation, in Neuro2a cells, showing the effect of MβCD-cholesterol and chloroquine treatment. p62 and LC3-II levels were normalized to GAPDH levels. Data are expressed as means ± SD (B, C, and E). n = 20 (B and C); n = 6 (E). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001; P < 0.05, ††P < 0.01, and †††P < 0.001 versus control. Scale bars = 40 μm (A). CT, control cell.
      Lysosomal degradation was also assessed biochemically. p62/SQSTM1, a ubiquitin binding protein, and microtubule-associated protein 1 LC3 are involved in autophagic clearance, and the levels of p62 and LC3-II are good indicators of lysosomal degradation.
      • Nixon R.A.
      Autophagy, amyloidogenesis and Alzheimer disease.
      • Bjørkøy G.
      • Lamark T.
      • Brech A.
      • Outzen H.
      • Perander M.
      • Overvatn A.
      • Stenmark H.
      • Johansen T.
      p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.
      Noteworthy, MβCD-cholesterol treatment increased both p62 and LC3-II levels, and it dramatically enhanced chloroquine-induced p62 and LC3-II levels (Figure 5, D and E, and Supplemental Figure S6). MβCD-cholesterol treatment also enhanced ciliobrevin D–induced p62 and LC3-II levels (Supplemental Figure S3). On the other hand, insulin treatment did not affect the levels of p62 and LC3-II, and it did not enhance chloroquine-induced p62 and LC3-II levels either (Supplemental Figure S7).

      Inhibition of Lysosomal Degradation also Enhances Chloroquine-Induced Endocytic Disturbance to Augment Intracellular Accumulation of Aβ

      To assess whether additional lysosomal dysfunction enhances chloroquine-induced endocytic disturbance, cultured Neuro2a cells were treated with a mixture of inhibitors of various subtypes of cathepsins, which are typical lysosomal hydrolases. This mixture consisted of pepstatin A, E64d, and leupeptin (PEL). Although PEL treatment did not alter APP levels when applied alone in the absence of chloroquine-induced endocytic disturbance, it significantly enhanced the increase of APP after chloroquine treatment. The same was true for p62 and LC3-II in chloroquine-treated Neuro2a cells (Figure 6, A and B ). It is noteworthy that PEL treatment also augmented Aβ accumulation in chloroquine-treated cells (Figure 6C).
      Figure thumbnail gr6
      Figure 6Inhibition of lysosomal degradation enhances intracellular accumulation of β-amyloid precursor protein (APP) and Aβ induced by chloroquine (Cq). Neuro2a cells were treated with 10 μmol/L chloroquine to induce endocytic dysfunction and/or a mixture of 10 μmol/L pepstatin A, 10 μmol/L E64d, and 10 μmol/L leupeptin (PEL) for 24 hours to induce lysosomal dysfunction. A: Western blot analyses showing the amounts of APP, p62, light chain 3 (LC3)-II, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in extracts derived from Neuro2a cells after chemical treatment. B: Relative expression of APP, p62, and LC3-II in Neuro2a cells, showing the effect of chloroquine and/or PEL. APP, p62, and LC3-II levels were normalized to GAPDH levels. PEL treatment aggravates chloroquine-induced endocytic disturbance. C: Relative amounts of Aβ in Neuro2a cells, showing the effect of chloroquine and/or PEL. Aβ levels were measured by enzyme-linked immunosorbent assay. Combined treatment of chloroquine and PEL strongly enhances intracellular accumulation of Aβ in Neuro2a cells. Data are expressed as means ± SD (B and C). n = 6 (B and C). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001; P < 0.05, ††P < 0.01, and †††P < 0.001 versus control.

      Discussion

      It is widely known that insulin signaling is impaired in T2DM patients and that aberrant insulin signaling is also involved in AD pathology.
      • Steen E.
      • Terry B.M.
      • Rivera E.J.
      • Cannon J.L.
      • Neely T.R.
      • Tavares R.
      • Xu X.J.
      • Wands J.R.
      • de la Monte S.M.
      Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer's disease: is this type 3 diabetes?.
      • Moloney A.M.
      • Griffin R.J.
      • Timmons S.
      • O'Connor R.
      • Ravid R.
      • O'Neill C.
      Defects in IGF-1 receptor, insulin receptor and IRS-1/2 in Alzheimer's disease indicate possible resistance to IGF-1 and insulin signalling.
      • Talbot K.
      • Wang H.Y.
      • Kazi H.
      • Han L.Y.
      • Bakshi K.P.
      • Stucky A.
      • Fuino R.L.
      • Kawaguchi K.R.
      • Samoyedny A.J.
      • Wilson R.S.
      • Arvanitakis Z.
      • Schneider J.A.
      • Wolf B.A.
      • Bennett D.A.
      • Trojanowski J.Q.
      • Arnold S.E.
      Demonstrated brain insulin resistance in Alzheimer's disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline.
      PI3K is an important kinase that mediates insulin signaling, especially within the PI3K/Akt pathway.
      • Cheng Z.
      • Tseng Y.
      • White M.F.
      Insulin signaling meets mitochondria in metabolism.
      • Vieira O.V.
      • Bucci C.
      • Harrison R.E.
      • Trimble W.S.
      • Lanzetti L.
      • Gruenberg J.
      • Schreiber A.D.
      • Stahl P.D.
      • Grinstein S.
      Modulation of Rab5 and Rab7 recruitment to phagosomes by phosphatidylinositol 3-kinase.
      • Houle S.
      • Marceau F.
      Wortmannin alters the intracellular trafficking of the bradykinin B2 receptor: role of phosphoinositide 3-kinase and Rab5.
      • Kalia M.
      • Kumari S.
      • Chadda R.
      • Hill M.M.
      • Parton R.G.
      • Mayor S.
      Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3'-kinase-dependent machinery.
      In spontaneous T2DM-affected monkey brains, Akt phosphorylation level was clearly up-regulated, suggesting that PI3K activity is enhanced (Figure 1). However, the insulin treatment study failed to reproduce endocytic disturbance, and it did not show any additional effects on chloroquine-induced endocytic disturbance in Neuro2a cells (Figure 2). Although we acknowledge the limitations of the in vitro experimental model in this study, the up-regulation of PI3K may not be responsible for the enhancement of endocytic disturbance in T2DM-affected brains.
      Disrupted lipid metabolism is also an important feature of T2DM, and alterations in cholesterol homeostasis have been suggested to be associated with AD pathology.
      • Brunzell J.D.
      • Ayyobi A.F.
      Dyslipidemia in the metabolic syndrome and type 2 diabetes mellitus.
      • Adeli K.
      • Lewis G.F.
      Intestinal lipoprotein overproduction in insulin-resistant states.
      • Ginsberg H.N.
      • MacCallum P.R.
      The obesity, metabolic syndrome, and type 2 diabetes mellitus pandemic, part I: increased cardiovascular disease risk and the importance of atherogenic dyslipidemia in persons with the metabolic syndrome and type 2 diabetes mellitus.
      • Markgraf D.F.
      • Al-Hasani H.
      • Lehr S.
      Lipidomics: reshaping the analysis and perception of type 2 diabetes.
      • Di Paolo G.
      • Kim T.W.
      Linking lipids to Alzheimer's disease: cholesterol and beyond.
      In the present study, cholesterol metabolism–related protein levels, such as mature SREBP2, ApoE, ATP-binding cassette subfamily A member 1, and low-density lipoprotein receptor–related protein 1, were totally increased in spontaneous T2DM-affected monkey brains (Figure 3, A and B). Noteworthy, membrane cholesterol level is clearly elevated in spontaneous T2DM-affected monkey brain (Figure 3C). To our knowledge, this is the first evidence that spontaneous T2DM increases the amount of membrane cholesterol in the brain of nonhuman primates. This finding prompted us to analyze the impact of elevated membrane cholesterol level on endocytosis. In our study, experimental load of membrane cholesterol in cultured Neuro2a cells enhances chloroquine- and ciliobrevin D–induced endocytic disturbance, resulting in great accumulation of APP and Aβ (Figure 4 and Supplemental Figure S3). Interestingly, chloroquine treatment did not affect membrane cholesterol level; however, it increased membrane cholesterol level remarkably after MβCD-cholesterol treatment (Figure 4, A and B). This finding suggests that endocytic disturbance can be a risk factor for abnormal membrane cholesterol metabolism. On the other hand, insulin treatment did not affect cholesterol trafficking–related protein levels, and it failed to increase membrane cholesterol level in Neuro2a cells (Supplemental Figure S2). These findings suggest that the alteration of cholesterol metabolism in T2DM-affected brain would be independent of aberrant insulin signaling.
      MβCD-cholesterol applied by itself resulted in elevated intracellular Aβ levels, suggesting that the increase in membrane cholesterol levels alters APP metabolism (Figure 4H). In the present study, MβCD-cholesterol treatment did not affect the levels or subcellular distribution of clathrin and dynamin, and no changes were observed in transferrin uptake with experimental load of membrane cholesterol (Supplemental Figure S4). Moreover, APP cleavage also remained unchanged with MβCD-cholesterol treatment (Supplemental Figure S5). These findings suggest that elevated membrane cholesterol may not affect Aβ generation in the present study.
      Strikingly, MβCD-cholesterol treatment increased the levels of substrates for lysosomal degradation, p62 and LC3-II, and it significantly enhanced chloroquine-induced intracellular accumulation of p62 and LC3-II (Figure 5, D and E, and Supplemental Figure S6). The results of this study are consistent with the previous finding that LC3-II level was significantly increased in T2DM-affected monkey brains.
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      Lysosomal dysfunction can induce endocytic disturbance by itself, and several other studies support this, showing that the accumulation of cholesterol in late endosome and/or lysosome disrupts lysosomal degradation.
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      Intracellular accumulation of amyloidogenic fragments of amyloid-beta precursor protein in neurons with Niemann-Pick type C defects is associated with endosomal abnormalities.
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      Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer's-like axonal dystrophy.
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      Cholesterol-depletion corrects APP and BACE1 misstrafficking in NPC1-deficient cells.
      Moreover, a recent study showed that cholesterol enrichment impairs autophagy-mediated Aβ clearance even in vivo.
      • Barbero-Camps E.
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      Cholesterol impairs autophagy-mediated clearance of amyloid beta while promoting its secretion.
      These results suggest that the effect of MβCD-cholesterol treatment persists even in the lysosome, and an increase of plasma membrane cholesterol level may be enough to disrupt lysosomal degradation (Figure 5, D and E). Moreover, the PEL treatment study confirmed that lysosomal dysfunction can enhance chloroquine-induced endocytic disturbance (Figure 6).
      It remains unclear which is responsible for age-related Aβ pathology, endosome trafficking disturbance or lysosomal dysfunction. Chloroquine primarily alters endosome trafficking via alkalizing endosomal pH, leading to lysosomal dysfunction.
      • de Duve C.
      • de Barsy T.
      • Poole B.
      • Trouet A.
      • Tulkens P.
      • Van Hoof F.
      Commentary: lysosomotropic agents.
      In the present study, APP level was increased by chloroquine treatment but not by PEL treatment, suggesting that APP accumulation may be caused by endosome trafficking disturbance rather than lysosomal dysfunction (Figure 6, A and B). This idea is reasonable because the immunocytochemical results showed that the accumulated APP is abundant in Rab5- and Rab7-positive endosomes of chloroquine-treated cells (Figure 2C). On the other hand, Aβ level increased as a result of each treatment alone, and the combination of chloroquine and PEL treatment enhanced Aβ accumulation even more (Figure 6C). These findings suggest that both endosome trafficking disturbance and lysosomal dysfunction can induce Aβ pathology, and their additive combination could possibly lead to an even worse scenario in the unfolding of AD pathogenesis.
      In conclusion, we provided evidence that spontaneous T2DM leads to the increase of membrane cholesterol level in nonhuman primate brain, and such elevated membrane cholesterol can enhance endocytic disturbance via lysosomal dysfunction. Postmortem brain analysis of AD patients shows that cholesterol levels were elevated compared with age-matched normal controls.
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      ApoE is the most abundant lipoprotein in brain and is crucial for transporting cholesterol among various types of cells.
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      Of the three ApoE alleles—ApoE-ε2, ApoE-ε3, and ApoE-ε4 (ApoE4)—having ApoE4 is the strongest genetic risk factor for developing late-onset AD.
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      Hence, ApoE4 may also play out its role in AD pathogenesis via an increase of membrane cholesterol level. In other words, abnormally high membrane cholesterol levels could be a risk factor for developing AD, which could come about through having T2DM and/or harboring the ApoE4 allele. Several clinical studies have reported that statins, well-known cholesterol-lowering drugs, have some potential effects related to preventing or delaying the development of AD.
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      Supplemental Data

      Figure thumbnail figs1
      Supplemental Figure S1Blood glucose (Glu) and triglyceride (TG) levels are increased in T2DM-affected (DM) monkeys. Histograms showing the levels of blood Glu and blood TG in cynomolgus monkeys used in the present study. Both Glu and TG are significantly increased in T2DM-affected monkeys. Data are expressed as means ± SD. n = 7. ∗∗P < 0.01, ∗∗∗P < 0.001 versus normal. CT, normal.
      Figure thumbnail figs2
      Supplemental Figure S2Insulin (Ins) treatment does not alter cholesterol metabolism. A: Neuro2a cells were treated with 50 nmol/L insulin for 24 hours. Western blot analyses showing the amounts of mature sterol regulatory element–binding protein 2 (mSREBP2), apolipoprotein E (ApoE), ATP-binding cassette subfamily A member 1 (ABCA1), low-density lipoprotein receptor–related protein 1 (LRP1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in extracts derived from Neuro2a cells 24 hours after insulin treatment. B: Relative amounts of cholesterol metabolism–related proteins in Neuro2a cells after insulin treatment. Insulin treatment slightly increases mSREBP2 level; however, other cholesterol trafficking–related protein levels remain unchanged. mSREBP2, ApoE, ABCA1, and LRP1 levels were normalized to GAPDH. C: Concentration of free cholesterol in membrane fraction derived from Neuro2a cells after insulin treatment. Insulin treatment does not affect membrane cholesterol level. Data are expressed as means ± SD (B and C). n = 6 (B and C).
      Figure thumbnail figs3
      Supplemental Figure S3Elevated membrane cholesterol (CHO) also enhances ciliobrevin D (CbD)–induced endocytic disturbance to augment intracellular accumulation of Aβ. Neuro2a cells were treated in vitro with 75 μmol/L MβCD-cholesterol for 1 hour to experimentally load membrane cholesterol. After washing, cells were cultured for another 24 hours with 50 μmol/L ciliobrevin D to disturb endosome trafficking. A: Western blot analyses showing the amounts of β-amyloid precursor protein (APP), Rab5, Rab7, p62, light chain 3 (LC3)-II, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in extracts derived from Neuro2a cells after MβCD-cholesterol and/or ciliobrevin D treatment. B: Relative expression of APP, Rab5, Rab7, p62, and LC3-II in Neuro2a cells, showing the effect of MβCD-cholesterol and ciliobrevin D treatment. APP, Rab5, Rab7, p62, and LC3-II levels were normalized to GAPDH levels. MβCD-cholesterol treatment aggravates ciliobrevin D–induced endocytic disturbance. C: Relative amounts of Aβ in Neuro2a cells after MβCD-cholesterol and/or ciliobrevin D treatment. Aβ levels were measured by enzyme-linked immunosorbent assay. Combined treatment of MβCD-cholesterol and ciliobrevin D significantly induces intracellular accumulation of Aβ. Data are expressed as means ± SD (B and C). n = 6 (B and C). P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
      Figure thumbnail figs4
      Supplemental Figure S4Elevated membrane cholesterol (CHO) does not affect endocytosis in Neuro2a cells. Neuro2a cells were treated in vitro with 75 μmol/L MβCD-cholesterol for 1 hour to experimentally load membrane cholesterol. After washing, cells were cultured for another 24 hours. A: Western blot analyses showing the amounts of clathrin, dynamin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in extracts derived from Neuro2a cells after MβCD-cholesterol treatment. B: Relative amounts of clathrin and dynamin in Neuro2a cells, showing the effect of MβCD-cholesterol treatment. Clathrin and dynamin levels were normalized to GAPDH levels. C: Photomicrographs of MβCD-cholesterol–treated Neuro2a cells immunostained for clathrin and dynamin. MβCD-cholesterol treatment did not affect subcellular distribution of clathrin or dynamin. D: Neuro2a cells were treated with 75 μmol/L MβCD-cholesterol for 1 hour to increase membrane cholesterol. After washing, cells were cultured for another 24 hours, and then treated with 50 nmol/L transferrin-biotin for 20 minutes. Western blot analysis showing the amount of transferrin and GAPDH in extracts derived from Neuro2a cells after the experimental treatments. E: Quantitation of transferrin uptake after MβCD-cholesterol application. Histograms showing the effect of elevated membrane cholesterol on the amount of transferrin in Neuro2a cells. Transferrin level was normalized to GAPDH level. MβCD-cholesterol treatment did not affect transferrin uptake. Data are expressed as means ± SD (B and E). n = 6 (B and E). Scale bar = 10 μm (C, all images). CT, control cell.
      Figure thumbnail figs5
      Supplemental Figure S5Elevated membrane cholesterol (CHO) levels do not change the amount of sAPP-α and sAPP-β cleavage products. A: Neuro2a cells were treated with 75 μmol/L MβCD-cholesterol for 1 hour. After washing, cells were cultured for another 24 hours. Western blot analysis showing the amount of full APP, sAPP-α, sAPP-β, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in extracts derived from Neuro2a cells after MβCD-cholesterol treatment. B: Quantitation of APP cleavage products after experimental elevation of membrane cholesterol. Histograms showing the effect of elevated membrane cholesterol on the amounts of full APP, sAPP-α, and sAPP-β in Neuro2a cells. Full-APP, sAPP-α, and sAPP-β levels were normalized to GAPDH. MβCD-cholesterol treatment does not affect APP cleavage in Neuro2a cells. Data are expressed as means ± SD (B). n = 6 (B). sAPP-α, secreted fragment of APP by alpha-secretase; sAPP-β, secreted fragment of APP by beta-secretase.
      Figure thumbnail figs6
      Supplemental Figure S6Elevated membrane cholesterol (CHO) increases p62 and light chain 3 (LC3)-II. A: Neuro2a cells were treated with 75 μmol/L MβCD-cholesterol for 1 hour. After washing, cells were cultured for another 24 hours. Western blot analysis showing the amounts of substrates of lysosomal degradation (p62 and LC3-II) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in extracts derived from Neuro2a cells after MβCD-cholesterol treatment. B: Quantitation of p62 and LC3-II levels after experimental load of membrane cholesterol. Histograms showing the effect of elevated membrane cholesterol on the amounts of p62 and LC3-II in Neuro2a cells. p62 and LC3-II levels were normalized to GAPDH. MβCD-cholesterol treatment significantly increases p62 and LC3-II levels in Neuro2a cells. Data are expressed as means ± SD (B). n = 6 (B). ∗∗∗P < 0.001 versus no treatment.
      Figure thumbnail figs7
      Supplemental Figure S7Insulin (Ins) treatment does not affect lysosomal degradation. A: Neuro2a cells were treated with 10 μmol/L chloroquine (Cq) and/or 50 nmol/L insulin for 24 hours. Western blot analyses showing the amounts of p62, light chain 3 (LC3), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in extracts derived from Neuro2a cells 24 hours after chloroquine and/or insulin treatment. B: Relative amounts of p62 and LC3-II in Neuro2a cells after chloroquine and/or insulin treatment. Insulin treatment does not alter p62 and LC3-II levels in Neuro2a cells, and it does not enhance chloroquine-induced effects either. p62 and LC3-II levels were normalized to GAPDH. Data are expressed as means ± SD (B). n = 6 (B). ∗∗∗P < 0.001 versus no treatment.

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