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Originally published In Press as doi:10.1074/jbc.M906994199 on April 10, 2000

J. Biol. Chem., Vol. 275, Issue 24, 18495-18502, June 16, 2000
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Inositol Stereoisomers Stabilize an Oligomeric Aggregate of Alzheimer Amyloid beta  Peptide and Inhibit Abeta -induced Toxicity*

JoAnne McLaurinDagger §, Rivka GolombDagger , Anna Jurewicz||, Jack P. Antel||, and Paul E. FraserDagger **

From the Dagger  Centre for Research in Neurodegenerative Diseases, § Department of Laboratory Medicine and Pathobiology, ** Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M5S 3H2, and || Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada

Received for publication, August 26, 1999, and in revised form, March 2, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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- and scyllo-inositol are also present. myo-Inositol complexes with Abeta 42 in vitro to form a small stable micelle. The ability of inositol stereoisomers to interact with and stabilize small Abeta 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 beta -structure in Abeta 42. Alternatively, none of the stereoisomers were able to induce a structural transition in Abeta 40. Electron microscopy demonstrated that inositol stabilizes small aggregates of Abeta 42. We demonstrate that inositol-Abeta 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 Abeta -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 Abeta 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 Abeta and attenuation of Abeta neurotoxic effects.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease is characterized neuropathologically by amyloid deposits, neurofibrillary tangles, and selective neuronal loss. The major component of the amyloid deposits is amyloid-beta (Abeta ), a 39-43 residue peptide. Soluble forms of Abeta generated from cleavage of amyloid precursor protein are normal products of metabolism (1, 2). The importance of residues 1-42 (Abeta 42) 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 Abeta 42 over Abeta 1-40 (Abeta 40; Refs. 3-5). These results in conjunction with the presence of Abeta 42 in both mature plaques and diffuse amyloid (6) 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 Abeta 42 deposition precedes that of Abeta 40 in Down's syndrome (7, 8), in PS1 mutations (9) and in hereditary cerebral hemorrhage with amyloidosis (10).

Many in vitro studies have demonstrated that Abeta can be neurotoxic or enhance the susceptibility of neurons to excitotoxic, metabolic, or oxidative insults (11-14). Initially it was thought that only the fibrillar form of Abeta was toxic to neurons (15-18) but more thorough characterization of Abeta structures demonstrated that dimers and small aggregates of Abeta are also neurotoxic (19, 20). These data suggested that prevention of Abeta oligomerization would be a likely strategy to prevent AD-related neurodegeneration. Several studies have demonstrated that in vitro Abeta -induced neurotoxicity can be ablated by compounds that can increase neuronal resistance by targeting cellular pathways involved in apoptosis (21), block downstream pathways after Abeta induction of destructive routes (14, 22, 23), or compounds that block Abeta oligomerization and ultimately fibril formation (16, 24-26). The site at which Abeta acts to induce neurotoxicity has yet to be elucidated but its toxic effects have been blocked by a variety of disparate agents.

Docking of Abeta -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 Abeta with molecules containing sugar moieties. Previous studies have demonstrated that Abeta interaction with glycosaminoglycans results in aggregation of Abeta possibly adding to their insolubility and plaque persistence (27-29). Glycosaminoglycans have also been implicated in neuronal toxicity (30) and microglial activation (31, 32). Alternatively, interaction with glycolipids such as gangliosides results in the stabilization and prevention of Abeta fibril formation, as well as, the site of Abeta production (33-37). The family of phosphatidylinositols, on the other hand, results in acceleration of fibril formation (38). 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 Abeta 42 (38). The interaction of Abeta with small sulfated compounds, antibiotics, and glycosaminoglycans has been shown to vary as the charge distribution across the compound is varied (12, 38, 39). 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 Abeta 40 and Abeta 42. The resultant Abeta -inositol complexes were subsequently examined for their ability to modulate Abeta -induced toxicity of nerve growth factor (NGF)-differentiated PC-12 cells and primary human fetal neuronal cultures.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inositol stereoisomers: myo-, epi-, and scyllo-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 and D-glucose, were purchased from Sigma.

Abeta Peptides

Abeta 40 and Abeta 42 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. Abeta 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 (40). 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. Abeta was either used directly or aged 3 days at room temperature to induce fibrillogenesis. The Abeta solutions were then incubated with various inositols for 20-24 h at room temperature. Abeta with and without inositols was added to cultures at a final Abeta 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 (41). 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 Abeta 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 Abeta -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, Abeta 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 beta -counter.

Inositol Inhibitor Studies

NGF-differentiated PC-12 cells were cultured in glucose-free media for inositol competition assays. 1 mM D-Glucose was added to PC-12 cells immediately prior to the addition of Abeta /inositol mixtures. Similarly, 100 µM phloridzin was added to NGF-differentiated PC-12 cells in the presence and absence of Abeta /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 Abeta on the cell surface of NGF-differentiated PC-12 cells was examined between 30 min and 3 h of incubation in the presence of Abeta with and without inositols. Abeta was visualized using Abeta -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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Structural Characteristics of Abeta -Inositol Complexes-- In order to determine the interaction of the inositol stereoisomers (Fig. 1) with Abeta , we investigated the effect of these compounds in the random coil to beta -structure transition necessary for Abeta -micelle and fibril formation. Examination of Abeta structure immediately upon incubation with inositol will lend evidence for the effect of inositols on fibril nucleation. Previously, we have shown that Abeta 42-myo-inositol interactions result in an immediate conformational change from random coil to beta -sheet, whereas, Abeta 40 does not undergo this transition (38). The structural details of Abeta 40 and Abeta 42-inositol isomer interactions were investigated by CD spectroscopy (Fig. 2). Abeta 40 and Abeta 42 stored in 40% trifluoroethanol displayed CD spectra indicative of partially alpha -helical structures as previously reported (34, 42). At a concentration of 10 µM, both Abeta 40 and Abeta 42 became unstructured after dilution in PBS (pH 7.0) (Fig. 2, A and D).


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  		Fig. 1.   Inositol stereoisomer structures. The positioning of hydroxyl groups on the ring structure of myo-, epi-, scyllo-, and chiro-inositol are shown. Hydroxyl groups important for Abeta interactions are shown in bold.


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  		Fig. 2.   Circular dichroism spectra of Abeta 40 and Abeta 42 in the presence and absence of the inositol stereoisomers. Inositol isomers were present at a 1:20 peptide:inositol ratio with a final peptide concentration of 10 µM. CD spectra of Abeta in PBS (pH 7.0) (solid line) and in the presence of the following inositols (dotted line); epi-inositol (A and D), scyllo-inositol (B and E), and chiro-inositol (C and F).

When incubated in the presence of epi-, scyllo-, and chiro-inositol, Abeta 40 remained random as seen with myo-inositol (Fig. 2; Ref. 38). Variation of the charge density on the inositol ring did not induce a conformational switch in Abeta 40. We have previously shown that Abeta 40-GAG interactions are inhibited in buffers containing phosphate counterions (29). Therefore, we examined the interactions of Abeta 40 with inositol in dH2O (pH 6.8). None of the stereoisomers were able to induce a conformational transition in Abeta 40, suggesting that charge shielding was not the limiting factor in Abeta 40-inositol interactions. Increasing the concentration of myo-inositol to 1 M induced a beta -structural transition in Abeta 40. At this concentration, myo-inositol functions as a chemical chaperone to stabilize the peptide through osmotic remediation and not peptide binding (42, 43).

In contrast, Abeta 42 was immediately induced to form beta -structure in the presence of both epi-inositol (Fig. 2D) and scyllo-inositol (Fig. 2E) but not in the presence of chiro-inositol at peptide:inositol ratio of 1:20 (w/w) (Fig. 2F). The stereoisomers differ from myo-inositol in the number of hydroxyl groups extending on either side of the inositol ring (Fig. 1). myo-Inositol has four hydroxyl groups on one surface of the ring and two hydroxyl groups on the other. If we consider only the more highly charged surface of the ring, then epi-inositol increases the surface charge by one hydroxyl group, whereas, scyllo-inositol decreases the hydroxyl groups from 4 to 3 (Fig. 1). chiro-Inositol also decreases the number of hydroxyl groups to 3 but unlike scyllo-inositol, the spacing of the charge density is uneven (Fig. 1). The structure-activity relationship between inositol stereoisomers and Abeta elucidate the necessity for hydroxyl groups with the same orientation at either positions 1, 3, and 5 or 2, 4, and 6 of the inositol ring to stabilize the Abeta 42-inositol complex. The concentration dependence of Abeta 42-inositol structural transition was examined to determine the stoichiometry of this interaction. Both epi- and scyllo-inositol were able to induce a transition from random to beta -structure in Abeta 42 at a 1:1 ratio (by weight). This corresponds to one molecule of Abeta 42 to 25 molecules of inositol. Although inositol is in excess amounts with respect to Abeta 42, this is not disparate with inositol concentrations in the central nervous system of young adults.

Effect of Inositols on Abeta Fibril Structure-- Our CD studies show that inositols induce the structural transition necessary for fibrillogenesis but this may not correlate with increased fibril growth. The characteristics of Abeta 40 and Abeta 42 fibrils in the presence and absence of inositol stereoisomers were examined by electron microscopy. Unseeded samples of both Abeta 40 and Abeta 42 were incubated in the presence of epi-, scyllo-, chiro-inositol, and alone for 96 h. Negative stain electron microscopy demonstrated that when Abeta 40 was incubated in buffer alone, it formed fibrils of varying lengths with some apparent intertwining of fibrils (Fig. 3A). When Abeta 40 was incubated in the presence of epi-inositol (Fig. 3B), scyllo-inositol, or chiro-inositol the fibrils formed were indistinguishable from those of Abeta 40 alone.


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  		Fig. 3.   Negative stain electron microscopy of Abeta 40 in the presence and absence of epi-inositol. Abeta 40 incubated in buffer alone (A) demonstrates long thin fibers and fibers that are laterally aggregated. When incubated in the presence of epi-inositol (B) no differences could be detected in the structure of the fibrils formed. Scale represents 100 nm.

Negative stain electron microscopy analysis of Abeta 42 demonstrated that when Abeta 42 is incubated in buffer, fibrils were of varying lengths (Fig. 4A). In the presence of chiro-inositol, the Abeta 42 fibers were indistinguishable from Abeta 42 alone (Fig. 4B). In contrast, no fibrils could be detected in the presence of epi-inositol (Fig. 4C) and scyllo-inositol (Fig. 4D) demonstrating an activity similar to myo-inositol (38). The fine thread-like structures detected in the Abeta 42-epi- and scyllo-inositol samples were present in the inositol solutions alone and therefore not Abeta fibrils. These results demonstrate that although epi- and scyllo-inositol are able to induce beta -structure in Abeta 42, the Abeta 42-inositol complex does not progress to form fibrils. Similar to that reported for myo-inositol, Abeta 42 can form stable beta -structured, non-fibrillar complexes with epi- and scyllo-inositol.


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  		Fig. 4.   Negative stain electron microscopy of Abeta 42 in the presence and absence of inositol stereoisomers. Abeta 42 incubated in buffer alone demonstrated long thin fibers (A) with very little aggregation detected at lower magnification (C). When incubated in the presence of chiro-inositol fibers were indistinguishable from Abeta 42 alone but were less abundant (B). Similar to that seen with myo-inositol, no fibers could be detected when Abeta 42 was incubated in the presence of epi-inositol (C) and scyllo-inositol (D). Scale represents 50 nm.

Abeta -induced Neurotoxicity-- Abeta -induced toxicity of neuronal cell lines and primary cultures is well established (15, 17, 44). Not only is the Abeta -fibril toxic to neuronal populations but smaller Abeta aggregates and dimers also induce toxicity (19, 20, 45). The ability of inositol stereoisomers to induce beta -structure and stabilize a small Abeta complex appears to have all the requirements so far described as necessary for Abeta -induced toxicity. Light microscopy demonstrated that addition of aggregated Abeta 40 or Abeta 42 to NGF-differentiated PC-12 cells resulted in decreased cell number and retraction of neurites (Fig. 5B) as compared with PC-12 cells alone (Fig. 5A). NGF-differentiated PC-12 cells retained their morphology when incubated in the presence of Abeta 42-myo-inositol complexes (1:20; Fig. 5C). Abeta has been shown to kill neurons through both apoptotic and necrotic pathways, therefore Abeta -induced toxicity in the presence of inositol stereoisomers was examined using the SRB and LDH assays. The SRB assay measures total cell death whereas the LDH assay measures cell death that is associated with membrane damage. The SRB assay demonstrated that Abeta 40 treatment resulted in a 61% cell survival that was not significantly changed by preincubation of Abeta 40 with myo-inositol (Table I), whereas, Abeta 42 resulted in a 55% cell survival which increased to 80% by preincubation with myo-inositol (Table I). Finally supernatants from the toxicity assays were assessed for the release of LDH, Abeta 40-induced LDH release was decreased slightly by preincubation with myo-inositol, whereas Abeta 42-myo-inositol decreased the amount of LDH released to levels of myo-inositol treatment alone (data not shown). These results suggest that incubation of Abeta 42 with myo-inositol either stabilizes a small non-toxic oligomer, blocks Abeta -neuronal cell surface interactions, or alternatively that myo-inositol alone blocks Abeta -induced toxicity.


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  		Fig. 5.   Morphologies of NGF-differentiated PC-12 cells after treatment with Abeta 42 in the presence of the inositol stereoisomers. PC-12 cells alone had long neuritic processes (A), which retracted when treated with Abeta 42 (B). Preincubation of Abeta 42 with myo-inositol (C) and scyllo-inositol (D) attenuated the survival and maintained the morphological properties of the PC-12 cells. myo-Inositol alone did not effect the condition of the PC-12 cells (F). Cells were stained with sulfhydryl rhodamine B. Scale represents 10 µM.

                              
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Table I
Concentration dependence of inositol rescue of Abeta -induced neurotoxicity
Percent cell survival is determined using the SRB assay and using NGF-differentiated PC-12 cells alone as our control. Abeta was incubated in the presence of increasing concentrations of inositol for 3 days prior to determining cell survival. Values are reported as mean ± S.D. of at least three separate experiments.

It was surprising to find that when Abeta 40 was preincubated in the presence of epi- and scyllo-inositol, these mixtures increased the cell survival of PC-12 cells from 56 to 93% and 83%, respectively (Table I). Preincubation of Abeta 42 with epi- and scyllo-inositol increased cell survival from 54 to 89% and 83%, respectively. Preincubation of Abeta 40 with various concentrations of epi- and scyllo-inositol resulted in attenuation of toxicity as low as 1:1 Abeta :inositol ratio (by weight; Table I). Similar results were seen when Abeta 42 was preincubated with the stereoisomers (Table I). In contrast, chiro-inositol preincubation did not rescue PC-12 cells from either Abeta 40- or Abeta 42-induced toxicity with neuritic dystrophy similar to Abeta treatment alone (Table I). The ability of epi- and scyllo-inositol to attenuate toxicity when preincubated at low concentrations suggests that the Abeta -inositol interaction is specific.

In order to more closely mimic in vivo conditions, we examined the ability of the inositol stereoisomers to protect primary human fetal neuronal cultures from Abeta -induced toxicity. Primary human cultures contain a small population of astrocytes and microglia, both of which are present in brain milieu. Both cell types have been proposed to either enhance neuronal toxicity in the presence of Abeta through the production of cytokines and neurotoxins (31, 46, 47) or attenuate toxicity by removal of Abeta from the extracellular milieu (48-50). Preincubation of Abeta 42 but not Abeta 40 with myo-inositol (Fig. 6, A and B), epi-inositol, and scyllo-inositol (data not shown) at a 1:20 ratio (by weight) protected the primary neuronal cultures from death. Attenuation of toxicity was also detected at 72 h after addition of Abeta -inositol complexes. LDH assay confirmed the decreased toxicity upon preincubation of Abeta 42 with inositols (data not shown). Ratios of as low as 1:1 Abeta 42 to myo-inositol resulted in an increase in the cell survival of primary human neuronal cultures (Fig. 6A). LDH release was decreased in the presence of increasing concentrations of myo-inositol for both PC-12 cells and primary human neuronal cultures (data not shown). The decreased amount of myo-inositol necessary to induce cell survival in the primary cultures may result from the presence of astrocytes and microglia both of which contain receptors for Abeta and inositol removal.


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  		Fig. 6.   Concentration dependence of inositol attenuation of Abeta -induced toxicity was investigated on primary human neuronal cultures (A and B). Abeta 40 and Abeta 42 were incubated in increasing ratios of peptide to inositol and the subsequent cell survival was calculated with respect to primary cultures alone. Values are expressed as mean ± S.D. for at least three experiments. Paired t test demonstrated that myo-inositol treatment was significantly different for Abeta 42 (p < 0.01) but not for Abeta 40.

Mechanism of Inositol Attenuation of Neurotoxicity-- The CD studies suggest that the mechanism by which the inositol stereoisomers protect neuronal populations from Abeta -induced toxicity is through binding of peptide, thereby sequestering it from cellular interactions or by stabilizing a non-toxic oligomer. A contributing factor may be that inositol competes for Abeta -binding sites on primary neurons or that inositol stimulates second messenger systems and therefore enhances neuronal survival. Preincubation of NGF-differentiated PC-12 cells with inositols for up to 12 h prior to Abeta treatment was investigated to examine the ability of inositol to compete for Abeta -binding sites (Table II). The pretreatment of PC-12 cells with all four stereoisomers of inositol had no significant effect on PC-12 survival (Table II). Abeta 40 was added to inositol pretreated PC-12 cells and at higher inositol concentrations a slight attenuation could be detected in comparison to untreated cells. In contrast, when Abeta 42 was added to myo-, epi-, or scyllo-inositol pretreated PC-12 cells the survival was increased even at the lowest concentrations. Pretreatment of cells with chiro-inositol had no effect on the Abeta 40 or Abeta 42-induced toxicity (Table II). These results suggest that inositol treatment attenuates toxicity at least in part by blocking Abeta -cell interactions. Proliferation assays in the presence and absence of inositols failed to show any significant difference in the amount of [methyl-3H]thymidine incorporation over 18 h demonstrating that the PC-12 cells do not de-differentiate (data not shown).

                              
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Table II
Inositol preincubation study
NGF-differentiated PC-12 cells were incubated overnight in the presence of inositols before the addition of Abeta . Percent cell survival was calculated with respect to PC-12 cells alone for inositol treatment alone or with respect to Abeta treatment alone for co-culture experiments. Cell survival was determined using the SRB assay. Values are reported as the mean ± S.D. of three independent experiments. Paired t test was used to determine significance of the presence of inositol pretreatment to the absence of inositol.

To examine the effect of inositol stereoisomers on Abeta -neuronal interactions, we used immunofluorescence localization of Abeta . In order to examine cell surface binding in the absence of internalization, Abeta accumulation on the surface of PC-12 cells was examined over 3 h and at 4 °C. Abeta 42 accumulation on the cell body and processes of PC-12 cells was evident at 30 min (Fig. 7A). In contrast, in the presence of myo-inositol (Fig. 7B), epi-, and scyllo-inositol (Fig. 7C), Abeta 42 accumulation was decreased. No difference could be detected in the amount of Abeta 42 accumulation in the presence of chiro-inositol, although the immunofluorescence had a more punctate appearance (Fig. 7D). Abeta 40 accumulated on the cell surface of PC-12 cells when incubated alone (Fig. 7E) which was partially blocked in the presence of epi- and scyllo-inositol (Fig. 7F). These results demonstrate that Abeta -inositol interactions decrease the interaction of Abeta with neuronal membranes which may contribute to attenuation of toxicity.


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  		Fig. 7.   Immunofluorescence localization of Abeta 42 on PC-12 cells in the presence and absence of inositol stereoisomers. Abeta 42 was incubated with PC-12 cells for up to 3 h at 4 °C to examine cell surface binding. Abeta 42 (A) and Abeta 40 (E) alone or in the presence of myo-inositol (B), scyllo-inositol (C and F), and chiro-inositol (D) were visualized using anti-Abeta IgG, 6E10, followed by secondary antibody conjugated to Cy3. Scale represents 5 µM.

In order to rule out any direct effects of inositol on neuronal cell survival, we included inositol transport inhibitors in our assay system. Inositol is taken up by cells through both passive diffusion and active transport (51, 52). Prior to inhibition assays, PC-12 cells were incubated in glucose-free media. Previous studies have demonstrated that 1 mM D-glucose inhibits passive diffusion of inositol into cells (53). The addition of 1 mM D-glucose to cells resulted in enhanced survival of PC-12 cells, and in turn resulted in a slightly decreased Abeta -induced toxicity (Table III). D-Glucose was not able to compete with myo-, epi-, and scyllo-inositol attenuation of toxicity, suggesting that inositol alone does not contribute to attenuation of toxicity. Active transport of inositol has been shown to be a Na+-dependent, stereoisomer-specific, saturable mechanism that is active in the brain and at the blood-brain barrier (51, 54). A known inhibitor of active uptake is phloridzin (52, 53). Phloridzin had no effect on the cell survival of PC-12 cells that were treated with Abeta 40/42 in the presence or absence of all inositol stereoisomers in the range of 0-100 µM (Table III). These results suggest that inositol uptake through the Na+-dependent transporter does not contribute to the attenuation of Abeta toxicity.

                              
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Table III
Inhibitor studies
NGF-differentiated PC-12 cells were incubated in the presence of Abeta -inositol complexes and either diffusion or active transport inhibitors. Diffusion inhibitor cell survival was calculated with respect to PC-12 cells in the presence of the D-glucose and the absence of Abeta -inositol complexes. Active transport inhibition was calculated with respect to cell survival in the presence of Abeta -inositol complexes but the absence of phloridzin. Cell survival was determined using the SRB assay. Data is reported as the mean ± S.D. of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -structure in Abeta 42 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 Abeta to form either a template for fibril growth or for inhibition (55). 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 Abeta 42 fibrillogenesis. Many molecules that bind Abeta in vitro and in vivo have been tested for their ability to effect fibrillogenesis. Congo red has been shown to have variable ability to inhibit Abeta 40 and Abeta 42 fibrillogenesis with inhibition seen for Abeta 40 only (16). Alternatively, Abeta interaction with ApoJ results in the formation of slowly sedimenting oligomers of Abeta 42 but not Abeta 40 (45). These results demonstrate some inherent differences in the interaction with Abeta 40 and Abeta 42, which results in variations in the formation of aggregates and fibrils.

Clusterin, alpha 2-macroglobulin, and glycosaminoglycans have all been shown to attenuate Abeta -induced toxicity presumably by binding Abeta and thereby preventing interaction with the cell (24-26). The interaction of Abeta with inositol stereoisomers is reminiscent of these molecules in that inositol prevents Abeta interactions with the cell membrane. It was previously demonstrated that Abeta dimers are only neurotoxic in the presence of microglial cells (19) and that soluble oligomers of Abeta -clusterin are also toxic to neurons but are sufficient on their own (45). Our results suggested the formation of a small complex of Abeta -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 (19, 31). The interaction of Abeta with inositol may allow for more efficient clearance of the complex than Abeta 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 (56) and Trisomy 16 mice (57). These results were later shown to be the result of increased number of myo-inositol transporter, which is present on human chromosome 21 and mouse chromosome 16. It is also of interest that large amounts of Abeta 42 are present in Down's syndrome central nervous system prior to the deposition of plaques (7, 8). It would be interesting to postulate that the presence of high cerebral myo-inositol in young Down's syndrome patients without dementia (58) and the ability to tolerate an increased Abeta 42 load might be due to Abeta -inositol interactions. The stability of a non-toxic Abeta 42 complex would allow the high Abeta load without detrimental effects. In AD, it is well established that phosphoinositide levels are reduced (59) 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 Abeta -deposition and Abeta -induced toxicity. The use of inositol stereoisomers may represent a therapeutic benefit over myo-inositol, since these isomers are present in very low concentrations in the brain, are incorporated poorly into phosphoinositides but have similar mechanisms of uptake (54, 60).

    ACKNOWLEDGEMENT

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

    FOOTNOTES

* This work was supported by grants from the Ontario Mental Health Foundation (to J. M. and P. E. F), the Alzheimer Society of Ontario (to J. M. and P. E. F.), and the Kevin Burke Memorial Amyloid Fund (to J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Centre for Research in Neurodegenerative Diseases, Tanz Neuroscience Building, 6 Queens Park Crescent West, Toronto, Ontario, M5S 3H2, Canada. Tel.: 416-946-3703; Fax: 416-978-1878; E-mail: j.mclaurin@utoronto.ca.

Published, JBC Papers in Press, April 10, 2000, DOI 10.1074/jbc.M906994199

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; NGF, nerve growth factor; PBS, phosphate-buffered saline; SRB, sulfhydryl rhodamine B; LDH, lactate dehydrogenase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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