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Vol. 20, Issue 5, 1533-1544, March 1, 2009
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Department of Neurology, Caritas St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, MA 02135
Submitted July 29, 2008;
Revised November 20, 2008;
Accepted January 5, 2009
Monitoring Editor: M. Bishr Omary
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Levels of insulin-like growth factor I (IGF-I), insulin, and/or cognate receptors are dysregulated in AD brain (Messier and Teutenberg, 2005; Moloney et al., 2008). In normal brain, IGF-I and insulin promote glucose utilization, energy metabolism, and neuronal survival (Hoyer, 2004), largely through PI3K/Akt/GSK-3β signaling (Bondy and Cheng, 2004). Insulin receptors (IRs) populate neuronal synapses and astrocytes in memory-processing brain regions (Lee et al., 2005). Acute insulin treatment increased memory function in rats on a passive-avoidance task (Park et al., 2000) and in small studies involving normal adults and AD patients (Craft and Watson, 2004), consistent with positive effects on synaptic plasticity (Horwood et al., 2006). How β-amyloid, a major culprit neurotoxin in AD, could cause central insulin resistance is unknown.
Intraneuronal β-amyloid (Aβi) in particular, is a significant factor in the early pathogenesis of AD (Gouras et al., 2005; LaFerla et al., 2007). Inclusion body myositis (IBM), another disorder associated with intracellular β-amyloid (Aβ) deposits, is a major cause of skeletal muscle inflammation and degeneration in the elderly. Cytoplasmic Aβ induces programmed cell death (apoptosis) in a number of experimental and transgenic models (Magrane et al., 2005; Oakley et al., 2006).
Interference with or alteration of the Akt signaling pathway has emerged as an important feature in several neurodegenerative diseases characterized by neuronal attrition including AD and schizophrenia (Emamian et al., 2004; Griffin et al., 2005). In previous studies of the effects of intracellular Aβ on this pathway using primary cortical neurons as well as Tg2576 mice that overexpress human Aβ precursor protein bearing the familial AD Swedish double mutation (KM-670/671-NL; Hsiao et al., 1996), we found that Aβi expression leads to a decrease in levels of p-Akt, an increase in the activation state of GSK-3β, and induction of apoptosis (Magrane et al., 2005). We hypothesize that extra- and intracellular Aβ have different targets in the insulin-signaling path and that the latter may well exert its effect distal to the immediate receptor and its adaptor protein.
The PI3K-Akt signaling pathway is responsive to trophic factors, metabolic signals, and environmental stress and regulates survival, growth, differentiation, and other homeostatic functions. This pathway is pivotally affected in the opposing processes of tumorigenesis and apoptosis (Martelli et al., 2006). Stimulated insulin- and IGF-receptor tyrosine kinases phosphorylate the insulin receptor substrate (IRS), an adaptor that initiates the PI3K-Akt signaling cascade (Asano et al., 2005). PI3K activation, in turn, catalyzes the 3'-phosphorylation of second messenger, membrane-bound signaling inositol lipids. The lipid phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3 or PIP3] brings together phosphoinositide-dependent kinase (PDK) and Akt in a submembrane complex through interaction with each of their pleckstrin homology (PH) domains (Andjelkovic et al., 1997). After two sequential activating phosphorylations, first on a regulatory hydrophobic domain and then by PDK on a T loop of the catalytic domain, p-Akt phosphorylates a number of cellular targets, regulating their function (Brunet et al., 2001; summarized in Figure 8). For instance, certain proapoptotic mediators, such as the transcription factor forkhead (FOXO), the tau kinase GSK-3β, and the Bcl2 antagonist BAD proteins, are inactivated by Akt (Cross et al., 1995; Datta et al., 1997; Zheng et al., 2000).
We embarked on a systematic study of the effect of Aβi on the complex series of steps leading to Akt activation. The following results were collected on human brain, and from cultures of neuronal and skeletal muscle cell lines and using in vitro assays. We conclude that Aβi specifically inhibits the PDK-dependent activation of Akt through a mechanism preventing their direct interaction. The inhibition of a defined step within this essential pathway suggests a target for the identification of a specific Aβ antagonist designed to reestablish Akt signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture and Reagents
Mouse C2C12 cells (ATCC, Manassas, VA) were grown in DMEM, and 20% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and maintained for passage below 60% confluence. Cultures at or above 90% confluence were differentiated in DMEM, 2% adult horse serum (Differentiation Medium, DM). SH-SY5Y, and HEK-293, or NIH/3T3 cells were grown in DMEM, 10% FBS, at below 80% confluence. Recombinant PTEN (R&D Systems, Minneapolis, MN), PP2A (Upstate Biotechnology, Lake Placid, NY), protein kinase A (PKA; Promega, Madison, WI), and phospholipase A2 (PLA2; Sigma, St. Louis, MO) were stored at –20°C and used freshly. PKA inhibitor (PKI, BioSource Technologies, Camarillo, CA) and PI3K inhibitor, LY294002 (Cell Signaling, Beverly, MA) were diluted to 20 or 10 µM, respectively, in kinase buffer. The following antibodies were used: anti-cdk4, Akt, Actin, neuron-specific enolase (NSE), and IR
(Santa Cruz Biotechnology, Santa Cruz, CA); p-Akt (Ser473 and Thr308), p-GSK-3/β (Ser21/9), GSK-3β, IRβ, p-Tyrosine, p-IR, PKA, and p-PDK (Ser241; Cell Signaling); IRS-1 and PDK (BD Biosciences, San Jose, CA); PI3K (p85) and SGK (Upstate Biotechnology); Rictor (Bethyl Laboratories, Montgomery, TX); 6E10 (Covance, Madison, WI); R1282 (gift from Dr. D. Selkoe, Harvard Medical School, Boston, MA). The lipids, dipalmitoyl-PIP3 (Matreya, State College, PA) and D-myo-PtdIns (PI; Echelon Biosciences, Salt Lake City, UT) were diluted into water or CHCl3:MeOH:H2O (1:2:0.8), aliquoted into 100-µg vials, and stored at –20°C. ATP (Cell Signaling), [
-32P]ATP (300 µCi/ml; Perkin Elmer, Boston, MA) and thin-layer chromatography (TLC) silica gel plate (no. 105553, Merck, Rahway, NJ) coated with potassium oxalate (Sigma) were used in PI3K activity assays. The GSK-3 fusion peptide, a substrate for phosphorylations, was purchased from Cell Signaling.
Infection of C2C12 Myotubes with Adenoviruses
Adv Tet-On and TRE-Aβ42 viruses (Magrane et al., 2005) and Adv WT-Akt, expressing wild-type Akt (Fujio and Walsh, 1999) were described previously. C2C12 were switched to DM and on day 3 infected with Adv Aβ/TetOn (4:1 ratio, 24–36 h) before doxycycline induction (1 µg/ml) for an additional 24–36 h. Some cultures were infected with Adv WT-Akt for 48 h, before harvest. Cell extracts were prepared in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1% NP-40, 10% glycerol, 1 mM Na4P2O7, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 µg/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail; Roche) and were stored at –80°C until use.
Cell Viability
C2C12 were grown in eight-chamber slides (Nalge Nunc, Rochester, NY) and infected/induced with Adv Aβ/TetOn. Cell profiles were captured and quantified on a Nikon TE200 microscope (10x; Melville, NY) coupled to a SPOT cooled CCD camera. Myotube thickness and length were measured in each of five unbiased, random fields. The cells were fixed using 4% paraformaldehyde in 0.1N Phosphate buffer, pH 7.4 and incubated with bis-benzimide (Hoechst 33258). Nuclear pyknosis was quantified by manual counting.
Aβ Peptides, Aβ-derived Diffusible Ligands, and Fibril Preparation
Aβ peptides were obtained from BioSource as dried trifluoroacetic acid salts. The scrambled Aβ42 sequence peptide was from rPeptide (Bogart, GA). Monomeric Aβ peptides were prepared by solubilization in 5% dimethyl sulfoxide (DMSO); 25 mM Tris-HCl, pH 7.4, and used fresh or flash-frozen. Aβ-derived diffusible ligands (ADDL) were prepared according to Lambert et al. (1998) and Klein (2002). Briefly, Aβ peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (Sigma) and evaporated on a Speedvac (Savant Instruments, Ramsey, MN). The Aβ film was resuspended in 100% anhydrous DMSO, diluted to 5 mM in F12 medium lacking phenol red (BioSource), and incubated at 4°C for 24–48 h. After incubation and centrifugation at 14,000 x g for 10 min at 4°C, the supernatant containing ADDL-enriched Aβ was transferred to a new tube. Fibrillar Aβ was made by diluting Aβ42 in DMSO to 100 µM in 10 mM HCl, immediate vortexing for 30 s, and incubation at 37°C for 24 h (Stine et al., 2003). All peptide preparations were flash-frozen and stored at –80°C until use.
Thioflavin T Fluorescence Assay
Fibril-enriched preparations of Aβ were verified according to Conway et al. (2000). Thioflavin T (100 µM; ThT; Sigma) was filtered through a 0.22-µm filter unit (Nalge Nunc). To construct a standard curve, 90 µl of each Aβ sample (0, 0.5, 1, 2, and 4 µg) was added to 100 µl glycine-NaOH (pH 8.5) solution, to which 10 µl ThT solution was added to a total volume of 200 µl per well in a C96 White Maxisorp plate (Nalge Nunc). After mixing, fluorescence was measured with a CytoFluor Multiwell plate reader (excitation at 450 nm, bandwidth 50; emission at 508 nm, bandwidth 20; PerSeptive Biosystems, Framingham, MA).
Immunoprecipitations and Western Blot Analysis
Cell lysates (see above) were incubated on ice and centrifuged at 14,000 x g for 10 min. IPs on 100
300 µg of extract protein commenced with an overnight incubation at 4°C using 2–3 µg of primary antibody. Protein G-Sepharose was added for an additional 1.5 h. IPs were harvested at 5000 x g for 1 min at 4°C and washed two or three times in PBS containing 0.5% NP-40 and 0.1 mM Na3VO4. For kinase and phosphatase reactions, the samples were washed two more times. Whole cell extracts were used directly for Western blot analysis (20–30 µg for cell cultures or 60–80 µg for human samples). Extracts or IPs from either cultured cells or human brain samples, prepared in lysis buffer, were diluted into Laemmli sample buffer, heated at 95°C for 10 min, cleared by centrifugation, separated on SDS-PAGE, and transferred to PVDF membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were blocked in TBS containing 0.3% Tween-20 and 5% (wt/vol) nonfat dry milk. After incubation with primary antibodies (18 h at 4°C in buffer containing 5% BSA and 0.05% NaN3), blots were washed and incubated in HRP-conjugated secondary antibodies (1:2000 dilution; Cell Signaling). Signals were detected using ECL reagents and quantified using a Kodak Image Station 4000R (Eastman Kodak, Rochester, NY).
Akt1 Activity Assay
Akt1 was immunoprecipitated overnight from 100 µg of human brain sample or extract from C2C12 myotubes infected with adenovirus encoding Aβ, using a 1:100 dilution of goat anti-Akt1 antibody. The following morning, 40 µl of 50% slurry of protein G-agarose (Roche) was added for 1.5 h. The collected beads were washed twice in buffer (1x PBS, 0.5% NP-40, and 0.1 mM Na3VO4) and twice in kinase buffer (25 mM Tris, pH 7.5, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, and 200 µM ATP). Finally GSK-3 fusion protein (1 µg/50 µl) was added in the presence of kinase buffer, and the reaction (50 µl) was incubated for 30 min at 30°C. The reaction was stopped by adding 40 µl of Laemmli buffer. Twenty microliters of sample was loaded onto a 10% polyacrylamide gel.
In Vitro p-Akt and Activity Levels
IPs of PDK and Akt1 were prepared from 100 µg of either SH-SY5Y, C2C12 myotubes or from insulin-treated cultures. ATP (200 µM), GSK-3 fusion protein (1 µg/50 µl), kinase buffer, and Aβ peptides were added (0.01, 0.1, 1, 5, 10, 20 µM; final). Akt activity was determined as above.
PDK and Akt1 Interaction Assay
For cell-based interaction studies, intracellular Aβ42 was induced in C2C12 myotubes exposed to Adv Aβ/TetOn. Cell extracts (100 µg) were used to IP endogenous Akt and determine the levels of associated PDK by Western blot. The in vitro interaction study involved first enriching for Akt through viral-mediated expression of WT-Akt. "Akt-enriched/PDK-depleted" extract was then prepared by removing the PDK through IP. Akt-depleted extracts were prepared from control extracts by pre-IP Akt1 and were relatively enriched with endogenous PDK. Enrichments and depletions were confirmed by Western blot. The interaction assay consisted of mixing 100 µg Akt-enriched and 100 µg control cell extract in the presence or absence of Aβ42 (up to 10 µM). These were incubated for 30 min at 30°C, followed by an overnight incubation at 4°C with goat anti-Akt1. Protein G beads were added for 1.5 h. PDK and Akt1 levels were determined by Western blot. In other experiments, 100 µg of Akt-enriched/PDK-depleted extract and 100 µg of a control extract depleted for Akt were mixed in the presence or absence of Aβ42 (up to 5 µM).
PI3K Activity Assay
IP PI3K from the NIH/3T3 cell lysates with rabbit anti-p85 subunit was used. The IPs were washed twice with cold PBS buffer containing 0.5% NP-40 and 0.1 mM Na3VO4 and then twice with TNE buffer (20 mM Tris.HCl, pH 7.4, 100 mM NaCl, and 0.5 mM EGTA). PI3K activity was determined by incubating the beads with kinase buffer (6.5 mM HEPES, pH 7.4, 10 mM MgCl2), 50 µM ATP, 2 µCi [-32P]ATP, 4 µg of 3-sn-phosphatidyl-L-serine and 2 µg of D-myo-PI for 10 min at 25°C. The reactions were stopped with 50 µl of 4N HCl. Phospholipids were extracted using 100 µl of CHCl3/methanol (1:1). Products were separated by TLC as described previously (Whitman et al., 1985). The conversion of PI to PI3P was quantified on a Storm Phosphorimager (GE Healthcare).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Toker and Newton, 2000). Their interactions were decreased in the same AD brain samples (Figure 1B, top). The reverse pulldown experiment also showed less associated total PDK in the AD samples. IP samples were also tested for nonspecific and non-disease–related interactions. PKA (another AGC kinase) did not pull down with PDK or Akt1. Moreover, a known Akt-1–binding partner, HSP27 (Vandermoere et al., 2007), showed no changes in interaction level with disease state (Figure 1C, top).
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), phosphorylation of tau and neurofilament proteins are elevated in our AD specimens (Supplemental Figure S1C). Most other tyrosine-phosphorylated proteins do not significantly change with respect to disease state or PMI (Supplemental Figure S1D), thus excluding nonspecific phosphatase effects on p-Akt in the AD cases. Finally, when cases with similar mean PMIs are subanalyzed (control, 17.7 h, n = 3; AD, 18.8 h, n = 3), the effect of AD to reduce Akt phosphorylation (p-Akt/total: control, 0.81; AD, 0.56; p < 0.05) and activity (arbitrary density units: control, 0.11; AD, 0.05, p < 0.05) remains consistent.
Aβ42 Toxicity and Akt1 Inhibition in Cultured Cells
The specific inhibition of Akt activation in AD brain was explored mechanistically in neuronal (SH-SY5Y human neuroblastoma) and skeletal muscle cell line cultures. Previous studies have characterized infection of neurons with an inducible adenovirus construct encoding Aβ42 and signal peptide domain (Adv Aβ/TetOn). These show Aβ accumulation into intracellular membrane compartments that is contemporaneous with the exclusion of p-Akt immunocytolocalization (Magrane et al., 2005). After exposure to doxycycline, C2C12 cells expressing Aβ were treated for 30 min with insulin (0.1 µg/ml) or vehicle. Both monomers and oligomers of Aβ were inducibly expressed in this system (Figure 2A). Insulin treatment had no effect on expressed Aβ levels per se. Aβ-expressing myotubes exhibited significant reductions in both length and thickness (Figure 2B) and contained increased number of condensed nuclei (Figure 2B, graphs) consistent with apoptotic cell death.
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In Vitro Akt Activity Levels: Effects of Aβ Peptides
To confirm the down-regulation of both Akt activation and activity and test the amyloid species most accountable for the effect, we performed cell-free, in vitro reactions. These were done on precipitates from three cell lines, chosen for low, medium, and high levels of resting Akt phosphorylation. A mixture of immunopurified PDK and Akt1 is shown to induce maximal Akt phosphorylation and activity at 30 min, the time point chosen for all subsequent experiments (see Supplemental Figure S2A). When the effect of adding freshly prepared, synthetic Aβ42 was compared between the cells types, Aβ42 inhibited all PDK1-stimulated Akt activity levels (Figure 3A). Levels of activity in the presence of Aβ resemble basal levels corresponding to reactions in which only Akt precipitates are added. Thus, Aβ42 appears to interfere with the activation that is attributable to PDK1. The in vitro inhibition of Akt phosphorylation and activation is more specific to the forward peptide sequence (F) than to the reverse (R) and shows dose dependency (Figure 3B).
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). We freshly solubilized synthetic Aβ monomers and prepared Aβ oligomer—as ADDL- (Lambert et al., 1998) and fibril-containing aliquots (Supplemental Figure S2B). These crude preparations (ADDL+, aged 24 and 48 h and fibril+) were tested against Akt activity derived from SH-SY5Y cells (Figure 3C). In vitro assays of PDK1-stimulated Akt activity demonstrate that oligomer-containing preparations (ADDL+) are more potent inhibitors than either monomers or fibrillar Aβ. A near total inhibition of PDK-stimulated Akt1 activity occurred at 10 µM (conservatively estimated 4.5 µM oligomeric content; Figure 3C, top). In comparing monomer or ADDL- to fibril-containing preparations, the fibrillated ones appeared least active (Figure 3C, bottom, and Supplemental Figure S2C, respectively).
In Vitro p-Akt and Activity Levels: Roles of PIP3 and AGC Kinases
The dose-dependent, inhibitory effects of monomeric Aβ42 on both the phosphorylation and activation of Akt by PDK are summarized for the in vitro data in Figure 4A. Although 10 µM Aβ42 monomer produced up to 50% inhibition of Akt phosphorylation, only 1 µM was sufficient to inhibit Akt activation in the coupled assay to a similar degree (IC50 
1–5 µM). The reverse peptide (Aβ42-1, R) at 5 µM (or 10, not shown) had a significantly smaller yet measurable effect to inhibit Akt activation. Thus, the functional degree of Akt activation, as judged by enzyme activity level, is the more sensitive measure of impairment by Aβ. The ADDLs, compared with the same starting material of monomeric Aβ, are additively more injurious (Figure 4A, right; p < 0.05).
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Another possibility is that Aβ acts like the lipid phosphatase and tensin homolog (PTEN) in dephosphorylating PIP3. In Figure 4C, we tested if preincubation of the kinase-bearing beads with PTEN, expected to remove endogenous coprecipitated PIP3, would eliminate PDK-stimulated Akt activity. Indeed, there is no activation in lane 6 versus 4, as there is in lane 3 versus 1. Moreover, preincubation with Aβ1-42, followed by washing of beads before setting up the activity reaction, had no effect on GSK phosphorylation (lane 8 vs. 7). The inference is that Aβ does not act like PTEN to remove 3'-phosphate from lipid cofactors or prevent their attachment.
Another AGC kinase, serum/glucocorticoid-regulated kinase (SGK), is activated by PDK. Similar to Akt, SGK has a PH domain and phosphorylates GSK-3/β (Tessier and Woodgett, 2006). If Aβ inhibits the catalytic function of PDK to activate PH-containing kinase substrates in general, it is predicted to affect this reaction. Indeed, ADDL preparations suppressed PDK-stimulated SGK activity (Supplemental Figure S3A). We tested the role of PDK's PH domain by using another kinase that activates Akt but lacks a PH domain, Rictor (Hresko and Mueckler, 2005; Sarbassov et al., 2005). In Supplemental Figure S3B, we added IP Rictor to show that Akt activity is up-regulated. The enhanced activity was not, however, interrupted by Aβ (10 µM). Lastly, another AGC kinase that converges to phosphorylate GSK, PKA (Li et al., 2000), was tested to determine specificity. In Supplemental Figure S3B, c-AMP–dependent PKA (2U), although very active in phosphorylating the peptide substrate, was not inhibited by Aβ. Thus, the mechanism of Aβ inhibition of Akt activation is specific to PDK-like kinases and their substrate.
The Interaction of PDK with Akt1 Is Interrupted by Aβ42
If Aβ interferes with the action of PDK to activate Akt, we predict their physical interaction should be lessened, as shown in AD brain. In Figure 5A, the expression of Aβ42 in C2C12 myotube cells resulted in less pulldown of PDK with Akt. The direct association of PDK with Akt was similarly affected in the presence of synthetic Aβ in cell-free extracts (Figure 5B). We used extracts from C2C12 myotubes enriched for Akt after infection by Adv encoding WT-Akt1 and from control cell cultures, as the source of additional PDK, for starting material. In Figure 5B, the addition of Aβ42 to a mixture of Akt-enriched and control extracts resulted in the inhibited pulldown of Akt-PDK complexes (lane 4 vs. 3). Next, an Akt-enriched/PDK-depleted and a control/Akt-depleted extract were combined before Aβ treatment and IP. When tested for Akt pulldown after IP of PDK or in reverse, Aβ42 again inhibited the signals (Figure 5C, lane 2 vs. 1, left panel).
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). Neither scrambled nor forward Aβ peptide affected Akt p-Ser473 levels or activity (Figure 6B, lanes 2 and 3 vs. 1), whereas PP2A treatment decreased both as expected (lane 4 and graph below). Thus, Aβ does not have intrinsic phosphatase action.
The prior cell-based and in vitro experiments are based on intracellular Aβ effects. To determine this specificity, cell cultures were pretreated with extracellular Aβ peptides for 24 h. In the last 30 min before harvest, we added insulin (0.1 µg/ml). Although p-Akt (Ser473) and p-GSK-3/β levels were increased by insulin treatment (Figure 6C, lane 2 vs. 1), they were not affected by extracellular Aβ treatments. Moreover, when the insulin response was measured in detail at varying doses, Aβ concentrations in the media up to 25 µM did not affect p-Akt (Thr308) levels (Figure 6D). Constitutive levels of PDK phosphorylated on S241 also remain unchanged by Aβ (Figure 6E, bottom lane). These experiments suggest that Aβi specifically interferes with the action of PDK to phosphorylate Akt.
In Vitro PI3K Activity Is Not Affected by the Addition of Aβ
Akt and PDK interactions are hosted by the second-messenger lipid PIP3. Results of Figure 4, B and C, suggest tethered and added PIP3 concentrations that although helpful to activation, are not limiting in the presence of Aβ. However, it remained possible that Aβ-induced interference with Akt activation involves a reduction in its critical availability. To test this, we grew NIH-3T3 cells to confluence and immunoprecipitated PI3K activity. PI was used as substrate in a PI3K activity assay (Figure 7A). We chose a 10-min incubation for subsequent experiments. Aβ reverse (42-1) and forward (1-42) peptides were tested in doses shown in Figure 7B. Aβ additions had no effect on PI3K activity, whereas the compound LY294002 completely abolished it (Figure 7B). Addition of PLA2, an enzyme that converts phospholipids (including PIP3 and PIP) into constituent fatty acids and other lipophilic substances, also showed the expected decrease in PI3P levels, whereas Aβ42 had no effect (Figure 7C). Recombinant PTEN blocked PI3K activity by 50%, whereas Aβ42 (10 µM) and scrambled Aβ (10 µM) did not (Figure 7D). We conclude that Aβ does not interfere with the activation or activity of PI3K to produce PI3P.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Steen et al., 2005). Defects in IGF-I receptor and an increase in the phospho-inactive form of IRS-1/2 in AD have recently been found, further indicating resistance to IGF-I signaling (Moloney et al., 2008). We however, did not find any change in levels of either IR or in protein interactions involving its adaptor-substrate, IRS-1, in whole brain tissue lysates. Apart from these differences, whether insulin deregulation is a causative factor in AD pathogenesis or a sideshow is debated (Gasparini et al., 2002), and the mechanism by which Aβ might cause this has received little attention.
Insulin and IGF-I signaling is transduced through the PI3K/Akt pathway and is vital to the metabolism and survival of neurons and muscle. Akt deactivation is a well-recognized mediator of oxidative and excitotoxic neuronal death (Luo et al., 2003). In a search for alternative mechanisms of insulin resistance to the defect in proximal components, we discovered that Akt activation itself is compromised. Furthermore, it is concluded here that this step is a target of intracellular Aβ.
With regard to the extracellular pool of Aβ, one study showed that application of soluble Aβ inhibits insulin-IR binding (Xie et al., 2002). In two other reports, extracellular Aβ oligomers (ADDL) caused losses in surface IR number and/or reductions in insulin-induced IR phosphorylation in neurons (Townsend et al., 2007; Zhao et al., 2008). These studies did not report on intracellular Aβ effects nor give evidence in brain as shown in Figure 1. Our results detail a novel mechanism through which intracellular Aβ disrupts insulin-Akt signaling in neurons and muscle. Notably, when we tested extracellular applied Aβ on insulin-stimulated myotubes in culture, it did not provoke any changes in Akt phosphorylation, Akt activity, or levels of activated PDK. The mechanisms by which applied Aβ induces apoptosis in cultured neuronal and nonneuronal cells are several including activation of cell death pathways (e.g., p53) and/or inhibition of other protective pathways, e.g., mitogen-activated protein kinase (Tong et al., 2004). One study overcame such toxicity with IGF-I treatments (Wei et al., 2002).
We first give evidence that p-Akt levels and activity are decreased in our AD brain specimens, consistent with the concept that Alzheimer's disease is a multifaceted insulin resistance state (Craft and Watson, 2004; de la Monte et al., 2006). Even on this point, there are studies indicating mixed or even opposite results. In AD temporal cortex, Griffin et al., 2005 showed that p-Akt (Ser473) protein levels were increased in particulate fractions and moderately decreased in cytosolic fractions, relative to total. Immunohistochemical levels of p-Akt were depleted in the cytosol of hippocampal neurons. Although Akt substrates were examined, no activity assay was reported. Rickle et al. (2004) did examine Akt enzyme activities and found them to be generally higher than control, but the amount of Akt precipitated was highly variable between cases. Finally, Pei et al. (2003) showed that p-Akt (Thr308) levels were increased in the frontal cortex of AD patients compared with normal and Huntington's disease controls, but total Akt levels were not given. Our results are most consistent with Steen et al. (2005), who also used detergent-soluble total brain lysates to show reductions in p-Akt and p-GSK-3 levels relative to total protein in AD brain. Differences in tissue handling and solubilization conditions, including detergent use, may thus contribute to the variable findings above. Additionally, we now show reductions in interaction between Akt and PDK (Figure 1B) to explain our insulin resistance results.
A search for other abnormalities more proximally found no major changes in the binding of IRS-1 to the IR 2β2 homodimer subunits or in the association of the SH2 domain–bearing PI3K p85 catalytic subunit with IRS-1. These end points reflect an unaltered IRS-1 activation state. Using a different endpoint, Moloney et al. (2008) found an increase in the inactive p-IRS form to total IRS ratio. However, this result does not prove that the transduction activity to PI3K is functionally lost (see Figure 8), and the activated phospho-IRS form was not reported. Phosphotyrosine modification of the IR also showed no major differences between AD and control in our hands, consistent with the expectation that changes in these proximal components should track together in the same direction if functionally linked (Steen et al., 2005).
To dissect the role of intracellular Aβ in the disruption of insulin signaling, we turned to experimental cell culture and in vitro conditions. Both skeletal muscle-like myotubes, because they are highly responsive to insulin, and SH-SY5Y cells were utilized. Aβ42 expression in C2C12 myotubes was cytotoxic (Figure 2B) and resulted in a modest inhibition of Akt phosphorylation, consistent with results in primary neurons (Magrané et al., 2005). The effect was more evident when Aβ expression preceded signal amplification with insulin (Figure 2C), suggesting interference with some forward step. Even more striking was the functional consequence, a severe inhibition of Akt activity. As in brain, no changes in phospho-tyrosine IR levels were produced in culture (Figure 2D).
Switching to an in vitro format and after validating the stimulation of Akt activity by PDK, we show a consistent reversal after additions of Aβ1-42. Oligomeric species of Aβ (ADDL) were more toxic to Akt activation than monomer predominant or fibril-containing preparations (summarized in Figure 4A). The IC50 for the monomer preparations of Aβ42 is 1–5 (2) µM in activity assays and 10 µM in Western blots of p-Akt levels. The more robust inhibition in the activity assay than when immunoblotting for phosphorylation levels was also shown in the cell-based experiments. This difference suggests that activity is steeply dependent on phosphorylation level. For the oligomer preparations, intracellular concentrations of Aβ42 in various AD models are difficult to come by; however, a range of 100 nM to a low micromolar concentration has been suggested, probably depending on the specific compartment (Gouras et al., 2000; Zhang et al., 2002; Magrane et al., 2005). Previous works on synaptic transmission and cell viability have detailed the heightened toxicity of oligomeric amyloid species (Lambert et al., 1998; Walsh et al., 2002; DeFelice et al., 2008). These studies used concentrations 0.5–5 µM, similar to those we estimate in our ADDL-based in vitro assays.
The lipid second-messenger PIP3 is necessary to the efficient activation of Akt by PDK. Importantly, Aβ addition greatly attenuated Akt activation in the presence or absence of added PIP3 (Figure 4, B and C), eliminating sequestration of/or competition with this lipid as a mechanism. To be clear on this, we tested oligomeric Aβ against the PDK-dependent, but PIP3-independent, stimulation of the SGK (Kobayashi and Cohen, 1999) and found a similar interruption (Supplemental Figure S3A). These together with control experiments using Rictor and PKA and the previously shown effect of myristoylated Akt to overcome Aβi (Magrane et al., 2005), all strongly suggest that Aβi inhibits the action of PDK to activate Akt independent of the lipid-PH domain modification.
Definitive evidence on the remaining possibility that Aβ inhibits p85-PI3K function to convert membrane phosphoinositides to 3' phosphorylated second messengers was sought by directly measuring PI3K activity. Aβ additions had no effect on PIP3 generation, making it unlikely that Aβ interferes with PIP3 production or PH binding in vivo.
From the AD brain sample studies, it appeared highly plausible that Aβ interferes with the activation reaction by directly preventing the interaction between PDK and Akt. We confirmed the decrease in their association in a cell-based, Aβ expression assay, as well as when cell extracts were mixed in vitro in the presence of synthetic Aβ42. Importantly, we established that Aβ does not dephosphorylate or otherwise inactivate Akt once this has occurred.
In conclusion, our data point to the specific action of intracellular Aβ to interrupt insulin signaling by inhibiting PDK activity. The likely mechanism is via interference of binding with its target, Akt (Figure 8). Intracellular Aβ accumulation, particularly oligomeric forms (Takahashi et al., 2004) is an early marker of AD pathology (Gouras et al., 2005; Oakley et al., 2006), predicting from these results that insulin resistance could soon follow. Prevention of resistance to insulin signaling in AD and even strategies to up-regulate it may have the added beneficial effects to limit cellular Aβ production by reducing secretase action or promoting IDE activity and APP transport mechanisms (Phiel et al., 2003; Ho et al., 2004). Two recent trials of rosiglitazone, a PPAR agonist and insulin sensitizer, showed positive trends in AD patients (Guo and Tabrizchi, 2006; Risner et al., 2006). Our findings suggest that a pharmacologic approach to reverse the blockade of Akt docking onto PDK or on PDK catalytic action may dramatically improve insulin signaling in AD brain.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Henry W. Querfurth (henry.querfurth{at}tufts.edu)
Abbreviations used: LY, LY294002; PP2A, protein phosphatase 2A; PLA2, phospholipase A2.
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, phosphorylated residues; 
, phosphorylated inositide (see inset).