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Inositol Stereoisomers Stabilize an Oligomeric Aggregate of Alzheimer Amyloid β Peptide and Inhibit Aβ-induced Toxicity*

Open AccessPublished:June 16, 2000DOI:https://doi.org/10.1074/jbc.M906994199
      Inositol has 8 stereoisomers, four of which are physiologically active. myo-Inositol is the most abundant isomer in the brain and more recently shown that epi- andscyllo-inositol are also present. myo-Inositol complexes with Aβ42 in vitro to form a small stable micelle. The ability of inositol stereoisomers to interact with and stabilize small Aβ complexes was addressed. Circular dichroism spectroscopy demonstrated that epi- and scyllo- but not chiro-inositol were able to induce a structural transition from random to β-structure in Aβ42. Alternatively, none of the stereoisomers were able to induce a structural transition in Aβ40. Electron microscopy demonstrated that inositol stabilizes small aggregates of Aβ42. We demonstrate that inositol-Aβ interactions result in a complex that is non-toxic to nerve growth factor-differentiated PC-12 cells and primary human neuronal cultures. The attenuation of toxicity is the result of Aβ-inositol interaction, as inositol uptake inhibitors had no effect on neuronal survival. The use of inositol stereoisomers allowed us to elucidate an important structure-activity relationship between Aβ and inositol. Inositol stereoisomers are naturally occurring molecules that readily cross the blood-brain barrier and may represent a viable treatment for AD through the complexation of Aβ and attenuation of Aβ neurotoxic effects.
      AD
      Alzheimer's disease
      NGF
      nerve growth factor
      PBS
      phosphate-buffered saline
      SRB
      sulfhydryl rhodamine B
      LDH
      lactate dehydrogenase
      Alzheimer's disease is characterized neuropathologically by amyloid deposits, neurofibrillary tangles, and selective neuronal loss. The major component of the amyloid deposits is amyloid-β (Aβ), a 39–43 residue peptide. Soluble forms of Aβ generated from cleavage of amyloid precursor protein are normal products of metabolism (
      • Esch F.
      • Keim P.S.
      • Beattie E.C.
      • Blacher R.W.
      • Culwell A.R.
      ,
      • Haass C.
      • Schlossmacher M.G.
      • Hung A.Y.
      • Vigo-Pelfrey C.
      • Mellon A.
      • Ostaszewski B.L.
      • Lieberburg I.
      • Koo E.H.
      • Schenk D.
      • Teplow D.B.
      • Selkoe D.J.
      ). The importance of residues 1–42 (Aβ42) in Alzheimer's disease was highlighted in the discovery that mutations in codon 717 of the amyloid precursor protein gene, presenilin 1 and presenilin 2 genes result in an increased production of Aβ42 over Aβ1–40 (Aβ40; Refs.
      • Suzuki N.
      • Cheung T.T.
      • Cai X.D.
      • Odaka A.
      • Otvos Jr., L.
      • Eckman C.
      • Golde T.E.
      • Younkin S.G.
      ,
      • Citron M.
      • Westaway D.
      • Xia W.
      • Carlson G.
      • Diehl T.
      • Levesque G.
      • Johnson-Wood K.
      • Lee M.
      • Seubert P.
      • Davis A.
      • Kholodenko D.
      • Motter R.
      • Sherrington R.
      • Perry B.
      • Yao H.
      • Strome R.
      • Lieberburg I.
      • Rommens J.
      • Kim S.
      • Schenk D.
      • Fraser P.
      • St. George-Hyslop P.
      • Selkoe D.J.
      ,
      • Xia W.
      • Zhang J.
      • Kholodenko D.
      • Citron M.
      • Podlisny M.B.
      • Teplow D.
      • Haass C.
      • Seubert P.
      • Koo E.H.
      • Selkoe D.J.
      ). These results in conjunction with the presence of Aβ42 in both mature plaques and diffuse amyloid (
      • Gravina S.A.
      • Ho L.
      • Eckman C.B.
      • Long K.E.
      • Otvos L.
      • Younkin L.H.
      • Suzuki N.
      • Younkin S.G.
      ) lead to the hypothesis that this more amyloidogenic species may be the critical element in plaque formation. This hypothesis was supported by the fact that Aβ42 deposition precedes that of Aβ40 in Down's syndrome (
      • Iwatsubo T.
      • Mann D.M.
      • Odaka A.
      • Suzuki N.
      • Ihara Y.
      ,
      • Teller J.K.
      • Russo D.
      • DeBusk L.M.
      • Angelini G.
      • Saccheo D.
      • Dagna-Bricarelli F.
      • Scartezzini P.
      • Bertolini S.
      • Mann D.M.
      • Tabaton M.
      • Gambetti P.
      ), in PS1 mutations (
      • Mann D.M.
      • Iwatsubo T.
      • Cairns N.J.
      • Lantos P.L.
      • Nochlin D.
      • Sumi S.M.
      • Bird T.D.
      • Poorkaj P.
      • Hardy J.
      • Hutton M.
      • Prihar G.
      • Crook R.
      • Rossor M.N.
      • Haltia M.
      ) and in hereditary cerebral hemorrhage with amyloidosis (
      • Castano E.M.
      • Prelli R.
      • Soto C.
      • Beavis R.
      • Matsubara E.
      • Shoji M.
      • Frangione B.
      ).
      Many in vitro studies have demonstrated that Aβ can be neurotoxic or enhance the susceptibility of neurons to excitotoxic, metabolic, or oxidative insults (
      • Weiss J.H.
      • Pike C.J.
      • Cotman C.W.
      ,
      • Tomiyama T.
      • Shoji A.
      • Kataoka K.
      • Suwa Y.
      • Asano S.
      • Kaneko H.
      • Endo N.
      ,
      • Goodman Y.
      • Mattson M.P.
      ,
      • Goodman Y.
      • Bruce A.J.
      • Cheng B.
      • Mattson M.P.
      ). Initially it was thought that only the fibrillar form of Aβ was toxic to neurons (
      • Pike C.J.
      • Burdick D.
      • Walencewicz A.J.
      • Glabe C.G.
      • Cotman C.W.
      ,
      • Lorenzo A.
      • Yankner B.
      ,
      • Simmons L.K.
      • May P.C.
      • Tomaselli K.J.
      • Rydel R.E.
      • Fuson K.S.
      • Brigham E.F.
      • Wright S.
      • Lieberburg I.
      • Becker G.W.
      • Brems D.N.
      • Li W.Y.
      ,
      • Ueda K.
      • Fukui Y.
      • Kageyama H.
      ) but more thorough characterization of Aβ structures demonstrated that dimers and small aggregates of Aβ are also neurotoxic (
      • Roher A.E.
      • Chaney M.O.
      • Kuo Y-M.
      • Webster S.D.
      • Stine W.B.
      • Haverkamp L.J.
      • Woods A.S.
      • Cotter R.J.
      • Tuohy J.M.
      • Krafft G.A.
      • Bonnell B.S.
      • Emmerling M.R.
      ,
      • Lambert M.P.
      • Barlow A.K.
      • Chromy B.A.
      • Edwards C.
      • Freed R.
      • Liosatos M.
      • Morgan T.E.
      • Rozovsky I.
      • Trommer B.
      • Viola K.L.
      • Wals P.
      • Zhang C.
      • Finch C.E.
      • Krafft G.A.
      • Klein W.L.
      ). These data suggested that prevention of Aβ oligomerization would be a likely strategy to prevent AD-related neurodegeneration. Several studies have demonstrated that in vitro Aβ-induced neurotoxicity can be ablated by compounds that can increase neuronal resistance by targeting cellular pathways involved in apoptosis (
      • Paradis E.
      • Douillard H.
      • Koutroumanis M.
      • Goodyer C.
      • LeBlanc A.
      ), block downstream pathways after Aβ induction of destructive routes (
      • Goodman Y.
      • Bruce A.J.
      • Cheng B.
      • Mattson M.P.
      ,
      • McGeer P.L.
      • McGeer E.G.
      ,
      • Iverson L.L.
      • Mortishire-Smith R.J.
      • Pollack S.J.
      • Shearman M.S.
      ), or compounds that block Aβ oligomerization and ultimately fibril formation (
      • Lorenzo A.
      • Yankner B.
      ,
      • Pollack S.J.
      • Sadler H.
      • Hawtin S.R.
      • Tailor V.J.
      • Shearman M.S.
      ,
      • Boggs L.N.
      • Fuson K.S.
      • Baez M.
      • Churgay L.
      • McClure D.
      • Becker G.
      • May P.C.
      ,
      • Du Y.
      • Bales K.R.
      • Dodel R.C.
      • Liu X.
      • Glinn M.A.
      • Horn J.W.
      • Little S.P.
      • Paul S.M.
      ). The site at which Aβ acts to induce neurotoxicity has yet to be elucidated but its toxic effects have been blocked by a variety of disparate agents.
      Docking of Aβ-fibrils to neuronal and glial cell membranes may be an early and intervenable step during the progression of AD.1 Formation of amyloid plaques, as well as, neurotoxicity and inflammation may be direct or indirect consequences of the interaction of Aβ with molecules containing sugar moieties. Previous studies have demonstrated that Aβ interaction with glycosaminoglycans results in aggregation of Aβ possibly adding to their insolubility and plaque persistence (
      • Gupta-Bansal R.
      • Frederickson R.C.A.
      • Brunden K.R.
      ,
      • Castillo G.M.
      • Ngo C.
      • Cummings J.
      • Wight T.N.
      • Snow A.D.
      ,
      • McLaurin J.
      • Franklin T.
      • Zhang X.
      • Deng J.
      • Fraser P.E.
      ). Glycosaminoglycans have also been implicated in neuronal toxicity (
      • Schulz J.G.
      • Megow D.
      • Reszka R.
      • Villringer A.
      • Einhaupl K.M.
      • Dirnagl U.
      ) and microglial activation (
      • Guilian D.
      • Haverkamp L.J., Yu, J.H.
      • Karshin W.L.
      • Li J.
      • Kirkpatrick J.
      • Kuo Y-M.
      • Roher A.E.
      ,
      • Guilian D.
      • Haverkamp L.J., Yu, J.
      • Karshin W.
      • Tom D.
      • Li J.
      • Kazanskaia A.
      • Kirkpatrick J.
      • Roher A.E.
      ). Alternatively, interaction with glycolipids such as gangliosides results in the stabilization and prevention of Aβ fibril formation, as well as, the site of Aβ production (
      • Bouillot C.
      • Prochiantz A.
      • Rougon G.
      • Alliquant B.
      ,
      • McLaurin J.
      • Chakrabartty A.
      ,
      • McLaurin J.
      • Franklin T.
      • Fraser P.E.
      • Chakrabartty A.
      ,
      • Choo-Smith L-P.
      • Surewicz W.K.
      ,
      • Lee S-J.
      • Liyanage U.
      • Bickel P.E.
      • Xia W.
      • Lansbury P.T.
      • Kosik K.S.
      ). The family of phosphatidylinositols, on the other hand, results in acceleration of fibril formation (
      • McLaurin J.
      • Franklin T.
      • Chakrabartty A.
      • Fraser P.E.
      ). The headgroup of phosphatidylinositol is myo-inositol a naturally occurring simple sugar involved in lipid biosynthesis, signal transduction, and osmolarity control.
      We have demonstrated that myo-inositol stabilizes a small micelle of Aβ42 (
      • McLaurin J.
      • Franklin T.
      • Chakrabartty A.
      • Fraser P.E.
      ). The interaction of Aβ with small sulfated compounds, antibiotics, and glycosaminoglycans has been shown to vary as the charge distribution across the compound is varied (
      • Tomiyama T.
      • Shoji A.
      • Kataoka K.
      • Suwa Y.
      • Asano S.
      • Kaneko H.
      • Endo N.
      ,
      • McLaurin J.
      • Franklin T.
      • Chakrabartty A.
      • Fraser P.E.
      ,
      • Kisilevsky R.
      • Lemieux L.J.
      • Fraser P.E.
      • Kong X.
      • Hultin P.G.
      • Szarek W.
      ).myo-Inositol has 8 stereoisomers that alter the distribution of hydroxyl groups across the surfaces of the sugar ring. In the present study, we examined the ability of four inositol isomers (Fig.1) to stabilize small aggregates of Aβ40 and Aβ42. The resultant Aβ-inositol complexes were subsequently examined for their ability to modulate Aβ-induced toxicity of nerve growth factor (NGF)-differentiated PC-12 cells and primary human fetal neuronal cultures.
      Figure thumbnail gr1
      Figure 1Inositol stereoisomer structures. The positioning of hydroxyl groups on the ring structure ofmyo-, epi-, scyllo-, andchiro-inositol are shown. Hydroxyl groups important for Aβ interactions are shown in bold.

      MATERIALS AND METHODS

      Inositol stereoisomers: myo-, epi-, andscyllo-inositol were purchased from Sigma,chiro-inositol from Wako Chemicals (Richmond, VA). PC-12 cells were from ATCC. NGF was purchased from Alamone Laboratories (Israel). Competitive inhibitors used in this study, phloridzin andd-glucose, were purchased from Sigma.

      Aβ Peptides

      Aβ40 and Aβ42 were synthesized by solid phase Fmoc chemistry by the Hospital for Sick Children's Biotechnology Center (Toronto, Ontario). Peptides were purified by reverse phase high performance liquid chromatography on a C18 μBondapak column. Peptides were initially dissolved in 0.5 ml of 100% trifluoroacetic acid (Aldrich, Milwakee, WI), diluted in distilled H2O and immediately lyophilized. Peptides were then dissolved in 40% trifluoroethanol (Aldrich) in H2O and stored at −20 °C until use. Alternatively, the lyophilized peptides were dissolved in distilled H2O at 10 mg/ml concentration and used immediately.

      Circular Dichroism

      CD spectra were recorded on a Jasco Circular Dichroism Spectrometer Model J-715 (Easton, MO) at 25 °C. Spectra were obtained from 200 to 260 nm, with a 0.5-nm step, 1-nm band width. Peptide:inositol ratios were varied from 1:1 to 1:20 (w/w) with a final peptide concentration of 10 μm. The effects of the inositols on peptide conformation were determined by adding an aliquot of stock peptide solutions to inositol suspended in PBS, 50 mm phosphate buffer or dH2O. The contribution of inositols to the CD signal was removed by subtracting the inositol only spectra. Aβ peptide conformations were determined in 40% trifluoroethanol/H2O and in buffer under the same conditions.

      Electron Microscopy

      Peptides were incubated with the inositol stereoisomers at a 1:1 ratio (w/w) in 50 mm phosphate buffer (pH 7.0) or in dH2O. For negative staining, carbon-coated pioloform grids were floated on aqueous solutions of peptides (100 μg/ml). After grids were blotted and air dried, the samples were stained with 1% (w/v) phosphotungstic acid (pH 7.0). The peptide assemblies were observed in a Hitachi H-7000 operated with an accelerating voltage of 75 kV.

      Primary Human Neuronal Cultures

      Neural cells are derived from human fetal central nervous system (cerebral hemispheres) tissue obtained at 12–16 weeks gestation as described previously (
      • McLaurin J.
      • D'Souza S.
      • Stewart J.
      • Blain M.
      • Beaudet A.
      • Nalbantoglu J.
      • Antel J.P.
      ). Cultures were obtained using MRC (Canada) approved guidelines. The cultures are prepared by dissociation of the fetal central nervous system tissue with 0.05% trypsin and 50 μg/ml DNase, passing the tissue through a 125-μm nylon mesh screen, and then through a 70-μm screen. After washing with PBS, the cells are suspended in minimal essential medium supplemented with 5% fetal bovine serum, 0.1% glucose, and 1 mm sodium pyruvate and placed onto poly-l-lysine-coated 96-well dishes. Cultures are treated on day 4 with 1 mm 5-fluorodeoxyuridine to deplete astrocytes. The treatment is repeated twice over a 2-week period.

      Toxicity Assays

      PC-12 cells were plated at 500 cells per well in a 96-well plate and suspended in 30 ng/ml NGF diluted in N2/Dulbecco's modified Eagle's medium (Life Technologies, Inc.). Cells were differentiated over 5–7 days to a final cell number of 10,000–15,000 per well. Aβ was either used directly or aged 3 days at room temperature to induce fibrillogenesis. The Aβ solutions were then incubated with various inositols for 20–24 h at room temperature. Aβ with and without inositols was added to cultures at a final Aβ concentration of 0.1 μg/μl and incubated for 24 or 72 h at 37 °C. Toxicity was assayed using the sulfhydryl rhodamine B (SRB) assay and the lactate dehydrogenase assay (LDH).

      SRB Assay

      Cells were fixed with trichloroacetic acid at a final concentration of 10%. Plates were washed with H2O and air-dried. Protein was stained with 0.4% SRB (Molecular Probes Inc) in 1% acetic acid for 30 min (
      • Skehan P.
      • Storeng R.
      • Scudiero D.
      • Monks A.
      • McMahon J.
      • Vistica D.
      • Warren J.T.
      • Bokesch H.
      • Kenney S.
      • Boyd M.R.
      ). Plates were washed with 1% acetic acid and air-dried. The dye was extracted in unbuffered 10 mm Tris and absorbance was assayed at 550 nm on a Bio-Rad Benchmark microtiter plate reader.

      LDH Assay

      Prior to addition of Aβ and inositols, fetal calf serum was added to NGF-differentiated PC-12 cells to a final concentration of 1% in order to stabilize LDH in the supernatant. Supernatants from the Aβ-treated cultures were removed and analyzed for LDH release using a commercial kit (Sigma). Results are expressed as B-B units/ml.

      Proliferation Assay

      The proliferative properties of NGF-differentiated PC-12 cells were determined using a [methyl-3H]thymidine incorporation assay. Briefly, cells were differentiated with NGF for 5 days at 37 °C. In order to determine the basal, Aβ and inositol-induced proliferation 1 mCi of [methyl-3H]thymidine (NEN Dupont, Mississauga, ON) was added to each well and incubated for 18 h. Cells were then harvested onto glass fiber filters and radioactivity determined as counts/min per well by liquid scintillation counting on a Beckman β-counter.

      Inositol Inhibitor Studies

      NGF-differentiated PC-12 cells were cultured in glucose-free media for inositol competition assays. 1 mmd-Glucose was added to PC-12 cells immediately prior to the addition of Aβ/inositol mixtures. Similarly, 100 μmphloridzin was added to NGF-differentiated PC-12 cells in the presence and absence of Aβ/inositol mixtures. Cells were then incubated for 24 h before toxicity was measured using both the SRB and LDH assays.

      Immunofluorescence Studies

      PC-12 cells were plated onto poly-l-lysine-coated glass coverslips at 1000 cells per slip and differentiated in 30 ng/ml NGF in N2/Dulbecco's modified Eagle's medium for 5 days. The presence of Aβ on the cell surface of NGF-differentiated PC-12 cells was examined between 30 min and 3 h of incubation in the presence of Aβ with and without inositols. Aβ was visualized using Aβ-specific antibodies, 6E10 and 4G8 (Senetek, St. Louis, MA) followed by goat anti-mouse Ig conjugated to Cy3 (Dako, Cerpinteria, CA). Cells were post-fixed in 2% paraformaldehyde prior to fluorescence visualization.

      DISCUSSION

      Factors that alter amyloid aggregation or fibril formation may contribute to AD pathology. Molecules associated with neuritic plaques have been proposed to either enhance or decrease both plaque formation and neuronal loss. The ability of inositol stereoisomers to induce non-fibrillar β-structure in Aβ42 is a striking phenomena. It has been proposed that molecules with the appropriate pattern of polar and non-polar surfaces, including hydrogen donors and acceptors, may interact with Aβ to form either a template for fibril growth or for inhibition (
      • Lansbury P.T.
      • Costa P.R.
      • Griffiths J.M.
      • Simon E.J.
      • Auger M.
      • Halverson K.J.
      • Kocisko D.A.
      • Hendsch Z.S.
      • Ashburn T.T.
      • Spencer R.G.S.
      • Tidor B.
      • Griffin R.G.
      ). Inositol stereoisomers vary the charge distribution across the surfaces of the sugar ring, in effect varying the pattern of available hydrogen donors or acceptors, which may explain the differences in myo-, epi-, scyllo-, and chiro-inositol's ability to inhibit Aβ42 fibrillogenesis. Many molecules that bind Aβ in vitro andin vivo have been tested for their ability to effect fibrillogenesis. Congo red has been shown to have variable ability to inhibit Aβ40 and Aβ42 fibrillogenesis with inhibition seen for Aβ40 only (
      • Lorenzo A.
      • Yankner B.
      ). Alternatively, Aβ interaction with ApoJ results in the formation of slowly sedimenting oligomers of Aβ42 but not Aβ40 (
      • Oda T.
      • Wals P.
      • Osterburg H.H.
      • Johnson S.A.
      • Pasinetti G.M.
      • Morgan T.E.
      • Rozovsky I.
      • Stine W.B.
      • Snyder S.W.
      • Holzman T.F.
      ). These results demonstrate some inherent differences in the interaction with Aβ40 and Aβ42, which results in variations in the formation of aggregates and fibrils.
      Clusterin, α2-macroglobulin, and glycosaminoglycans have all been shown to attenuate Aβ-induced toxicity presumably by binding Aβ and thereby preventing interaction with the cell (
      • Pollack S.J.
      • Sadler H.
      • Hawtin S.R.
      • Tailor V.J.
      • Shearman M.S.
      ,
      • Boggs L.N.
      • Fuson K.S.
      • Baez M.
      • Churgay L.
      • McClure D.
      • Becker G.
      • May P.C.
      ,
      • Du Y.
      • Bales K.R.
      • Dodel R.C.
      • Liu X.
      • Glinn M.A.
      • Horn J.W.
      • Little S.P.
      • Paul S.M.
      ). The interaction of Aβ with inositol stereoisomers is reminiscent of these molecules in that inositol prevents Aβ interactions with the cell membrane. It was previously demonstrated that Aβ dimers are only neurotoxic in the presence of microglial cells (
      • Roher A.E.
      • Chaney M.O.
      • Kuo Y-M.
      • Webster S.D.
      • Stine W.B.
      • Haverkamp L.J.
      • Woods A.S.
      • Cotter R.J.
      • Tuohy J.M.
      • Krafft G.A.
      • Bonnell B.S.
      • Emmerling M.R.
      ) and that soluble oligomers of Aβ-clusterin are also toxic to neurons but are sufficient on their own (
      • Oda T.
      • Wals P.
      • Osterburg H.H.
      • Johnson S.A.
      • Pasinetti G.M.
      • Morgan T.E.
      • Rozovsky I.
      • Stine W.B.
      • Snyder S.W.
      • Holzman T.F.
      ). Our results suggested the formation of a small complex of Aβ-inositol that was non-toxic in both clonal cell lines and mixed human cultures. This suggested that this small complex was unable to induce activation of microglia and subsequent loss of neurons as previously reported (
      • Roher A.E.
      • Chaney M.O.
      • Kuo Y-M.
      • Webster S.D.
      • Stine W.B.
      • Haverkamp L.J.
      • Woods A.S.
      • Cotter R.J.
      • Tuohy J.M.
      • Krafft G.A.
      • Bonnell B.S.
      • Emmerling M.R.
      ,
      • Guilian D.
      • Haverkamp L.J., Yu, J.H.
      • Karshin W.L.
      • Li J.
      • Kirkpatrick J.
      • Kuo Y-M.
      • Roher A.E.
      ). The interaction of Aβ with inositol may allow for more efficient clearance of the complex than Aβ oligomers or fibrils alone.
      Inositol has been shown to be dysregulated in both AD and Down's syndrome. The uptake of myo-inositol was shown to be enhanced in Down's syndrome fibroblasts (
      • Fruen B.R.
      • Lester B.R.
      ) and Trisomy 16 mice (
      • Acevedo L.D.
      • Holloway H.W.
      • Rapoport S.I.
      • Shetty H.U.
      ). These results were later shown to be the result of increased number ofmyo-inositol transporter, which is present on human chromosome 21 and mouse chromosome 16. It is also of interest that large amounts of Aβ42 are present in Down's syndrome central nervous system prior to the deposition of plaques (
      • Iwatsubo T.
      • Mann D.M.
      • Odaka A.
      • Suzuki N.
      • Ihara Y.
      ,
      • Teller J.K.
      • Russo D.
      • DeBusk L.M.
      • Angelini G.
      • Saccheo D.
      • Dagna-Bricarelli F.
      • Scartezzini P.
      • Bertolini S.
      • Mann D.M.
      • Tabaton M.
      • Gambetti P.
      ). It would be interesting to postulate that the presence of high cerebralmyo-inositol in young Down's syndrome patients without dementia (
      • Shonk T.
      • Ross B.D.
      ) and the ability to tolerate an increased Aβ42 load might be due to Aβ-inositol interactions. The stability of a non-toxic Aβ42 complex would allow the high Aβ load without detrimental effects. In AD, it is well established that phosphoinositide levels are reduced (
      • Stokes C.E.
      • Hawthorne J.N.
      ) thereby effecting signal transduction. It is unclear whether inositol levels are increased or decreased. Our data suggest that inositol treatment for AD patients may help to prevent Aβ-deposition and Aβ-induced toxicity. The use of inositol stereoisomers may represent a therapeutic benefit overmyo-inositol, since these isomers are present in very low concentrations in the brain, are incorporated poorly into phosphoinositides but have similar mechanisms of uptake (
      • Spector R.
      ,
      • Michaelis T.
      • Helms G.
      • Merboldt K-D.
      • Hanicke W.
      • Bruhn H.
      • Frahm J.
      ).

      Acknowledgments

      We thank Dr. N. Wang at the Hospital for Sick Children's Biotechnology Center for the synthesis of all peptides used in this study.

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