Báo cáo khoa học: Modulation of amyloid-b aggregation and toxicity by inosose stereoisomers pot

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Modulation of amyloid-b aggregation and toxicity by inosose stereoisomers Mark Nitz 1 , Daniela Fenili 2,3 , Audrey A. Darabie 2 , Ling Wu 2 , Julian E. Cousins 2 and JoAnne McLaurin 2,3 1 Department of Chemistry, University of Toronto, Canada 2 Centre for Research in Neurodegenerative Diseases, University of Toronto, Canada 3 Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada Inositol is a simple polyol with eight naturally occurring stereoisomers, the most common of which are myo-inositol, chiro-inositol, epi-inositol, and scyllo-inositol [1]. myo-Inositol is the most abundant isomer and is a ubiquitous component of all eukaryotic cells. We have shown that scyllo-inositol may represent a potential therapeutic agent for amyloid disorders such as Alzheimer’s disease (AD) [2,3]. We showed that myo- inositol complexes with amyloid-b (Ab)42 in vitro to form a small, stable micelle [4]. The ability of inositol stereoisomers to interact with and stabilize small Ab complexes was subsequently addressed [5]. CD spectroscopy demonstrated that epi-inositol and scyllo- inositol, but not chiro-inositol, were able to induce a structural transition from a random to a b-structure in Ab42. Furthermore, electron microscopy demonstrated that scyllo-inositol stabilizes small aggregates of Ab42 that are nontoxic to nerve growth factor (NGF)-differentiated PC-12 cells and primary human neuronal cultures. We then examined the in vivo effects of the inositol stereoisomers. myo-Inositol, epi-inositol and scyllo-inositol were administered to the TgCRND8 mouse model of AD, which demonstrates age-associated cognitive deficits and AD-like pathology [2,6]. myo-Inositol was effective in vitro but was ineffective in vivo [2,5]. epi- Inositol had some initial positive results as a prophy- lactic treatment, but these positive effects were not sustained with disease advancement [2]. scyllo-Inositol treatment, however, resulted in a significant improve- ment in cognitive function, synaptic function, and life- span. Significant decreases in Ab40 and Ab42 levels, vascular amyloid levels, plaque size and area were also observed [2]. Therefore, scyllo-inositol is effective at Keywords aggregation; Alzheimer’s disease; amyloid; fibrillogenesis; inosose Correspondence J. McLaurin, Centre for Research in Neurodegenerative Diseases, University of Toronto, 6 Queen’s Park Crescent West, Toronto, ON, Canada M5S 3H2 Fax: +1 416 978 1878 Tel: +1 416 978 1035 E-mail: j.mclaurin@utoronto.ca (Received 21 November 2007, revised 28 January 2008, accepted 5 February 2008) doi:10.1111/j.1742-4658.2008.06321.x Amyloid-b (Ab) aggregation and amyloid formation are key pathological features of Alzheimer’s disease, and are considered to be two of the major contributing factors to neurodegeneration and dementia. Identification of small molecule inhibitors that are orally available, have low toxicity and high central nervous system bioavailability is one approach to the potential development of a disease-modifying treatment for Alzheimer’s disease. We have previously identified inositol stereoisomers as exhibiting stereospecific inhibition of A b aggregation and toxicity in vitro and in vivo. We report here the effects of inosose versus inositol stereoisomers on Ab fibrillogenesis as determined using CD and fluorescence spectroscopy and negative- stain electron microscopy. The inososes differ from inositols by the oxidation of one of the hydroxyl groups to a ketone. These molecules help in the further elucidation of the structure–activity relationships of inositol– Ab interactions and identify both allo-inositol and epi-2-inosose as in vitro inhibitors of Ab aggregation. Abbreviations AD, Alzheimer’s disease; Ab, amyloid-b; HMIT, hydrogen myo-inositol transporter; NGF, nerve growth factor; SMIT, sodium myo-inositol transporter; TFE, trifluoroethanol. FEBS Journal 275 (2008) 1663–1674 ª 2008 The Authors Journal compilation ª 2008 FEBS 1663 decreasing the pathological and behavioral correlates of AD in the TgCRND8 model [2]. Our in vivo and in vitro results were recently confirmed using stable, soluble Ab oligomers [7]. In vitro and in vivo data demonstrate that inositols can inhibit the toxic effects of amyloid peptides through the formation of nontoxic species. The effectiveness of a specific inositol is highly dependent on the stereochemistry of the hydroxyl groups. scyllo-Ino- sitol is the most potent inositol, and a single epimerization to form myo-inositol renders the compound less active in vivo [1]. This dependence on subtle changes in the orientation of hydroxyl groups suggests that a specific interaction with the amyloid peptides is necessary. These results further suggest that the hydroxyl groups may represent key recognition elements in the interaction with Ab. Furthermore, the dependence on the hydroxyl group positioning also suggests that hydrogen bonding is important for stabilizing this interaction. Therefore, we hypothesized that investigating the interactions of structurally related, naturally occurring inosose compounds with Ab may provide insights into the importance of hydrogen bonding and the position of the hydroxyl groups for the formation of an Ab–inositol complex. Furthermore, small molecule inhibitors of aggregation may act on Ab oligomer formation and ⁄ or fibril formation; as these two assembly pathways are independent and distinct [8], alterations in the structure of the small molecule may then affect which pathway is targeted. Results Structural characteristics of inosose–Ab complexes To investigate the interaction of inosose compounds with Ab, we examined the effects of these compounds on the random coil to b-sheet transition that is necessary for Ab-nucleation, oligomer formation and fiber formation. Examination of transitions in b-structure over time gives an indication of the effect of the compounds on the initiation step of either oligomerization or fibrillogenesis. Previously, we have shown that incubation with myo-inositol, epi-inositol and scyllo-inositol induces an immediate structural transition in Ab42 but not in Ab40 [5]. CD spectroscopy was used to evaluate structural transitions from randomly structured Ab40 and Ab42 diluted from 40% trifluoroethanol (TFE) into NaCl ⁄ P i . The following compounds were examined: chiro-inositol, chiro-1-inosose, scyllo- inositol, scyllo-inosose, epi-inositol, epi-1-inosose, epi- 2-inosose, allo-inositol and allo-3-inosose (Fig. 1). We had previously shown the efficacy of scyllo-inositol and epi-inositol in inducing structural transitions, but had not examined allo-inositol; all inositol stereoisomers were included for comparison with the related inosose compounds, and chiro-inositol was used as a negative control. We chose to use the same conditions as in our original studies in order to have a direct comparison with previously published data [5]. As previously reported by numerous laboratories, in the absence of any compounds, Ab40 remains mostly randomly structured [5,9]. As was seen for the inositol stereoisomers, none of the inosose compounds induced an immediate structural transition in Ab40 (data not shown). However after 2 days, allo-3-inosose induced a b-structural transition. By 3 days, scyllo-inosose, epi-1- inosose, epi-2-inosose and allo-inositol induced a strong b-structure in Ab40; as expected, the results obtained with chiro-inositol and chiro-1-inositol were similar to those obtained with Ab40 alone, consisting of a mixture of random coil and b-structured peptide (data not shown). In order to rule out Ab batch or Fig. 1. Structures of inositol and inosose compounds. Modulating amyloid-b aggregation M. Nitz et al. 1664 FEBS Journal 275 (2008) 1663–1674 ª 2008 The Authors Journal compilation ª 2008 FEBS day effects, experiments were repeated three separate times on two different Ab preparations, and gave consistent, reproducible data. These results suggest that none of the compounds are strong inducers of Ab40 structural transitions, but some may affect the kinetics of this transition. In contrast to Ab40, the inosose compounds induced immediate transitions to b-structure when incubated with Ab42 (Fig. 2). Ab42 adopted a b-structure in the presence of scyllo-inositol (Fig. 2C), epi-inositol (Fig. 2E), epi-1-inosose (data not shown), epi-2-inosose (Fig. 2F), allo-inositol (Fig. 2G), and allo-3-inosose (Fig. 2H), but not in the presence of chiro-inositol (Fig. 2A), chiro-1-inosose (Fig. 2B), and scyllo-inosose (Fig. 2D). These results suggest that for epi-inositol and allo-inositol, replacement of a single hydroxyl with a ketone does not abolish their ability to induce Ab42 structural transitions, and that the position of this replacement is not critical, as both epi-1-inosose and epi-2-inosose were equally effective. On the other hand, replacement of a single hydroxyl group on scyllo-inositol by a ketone abolished activity towards Ab42. Similarly, our previous results sug- gested that the epimerization of a single hydroxyl group from scyllo-inositol to myo-inositol substan- tially decreased efficacy [2,4]. These results also suggest that the all-equatorial position of hydroxyl groups, as is the case for scyllo-inositol, is optimal for interaction with Ab and that subtle changes decrease potency. In order to determine the affinity of this interaction, we examined the concentration dependence of Ab42– inosose structural transitions. We chose to compare epi-inositol and epi-2-inosose, as both induced a strong b-structure and their activity is retained upon modifi- cation to a single ketone group. We have previously shown that epi-inositol induces a transition to b-structure at a 1 : 1 (w ⁄ w) ratio [4]. epi-2-Inosose was also able to induce a structural transition in Ab42 from a random to a b-structure at a 1 : 1 ratio (w ⁄ w; data not shown). This corresponds to a peptide ⁄ inosose molar ratio of 1 : 27 (peptide concentration 10 lm) and suggests a dissociation constant in the high millimolar range for the inosose–peptide complex. These results also suggest that the hydroxyl at position 2 of the epi- inositol ring is not crucial for interaction with Ab. To determine whether the inosose ⁄ inositol-induced transition to b-structure corresponds to an increase in the formation of insoluble Ab species, we utilized the inherent tyrosine fluorescence of Ab in the presence and absence of inosose compounds (Fig. 3). Ab40 was incubated at a 1 : 20 (w ⁄ w) ratio with inosose compounds for 48 h, and the tyrosine fluorescence of each solution was measured prior to and directly after centrifugation to remove insoluble Ab species (Fig. 3A). The percentage fluorescence after centrifugation in comparison to total fluorescence is an indication of the amount of soluble Ab species. We measured samples at a 48 h time point, as preliminary aggregation assays demonstrated that approximately 60% of Ab40 and Ab42 was pelleted at this time, which allowed us to distinguish between compounds that increase and decrease insoluble Ab species. Ab40 remained in solution only when incubated with epi-2-inosose and allo- inositol, suggesting that epi-2-inosose and allo-inositol promote a b-structural transition, leaving the complex in a soluble form (Fig. 3A). In contrast, solutions trea- ted with epi-1-inosose had less soluble Ab species than Ab40 alone, suggesting that the strong b-structural transition detected with epi-1-inosose promotes further aggregation and increased insoluble Ab (Fig. 3A). As previously reported, both epi-inositol and scyllo-inositol maintain Ab42 in a soluble state (Fig. 3B). Of the stereoisomers, only allo-inositol maintained Ab42 as a soluble species; in contrast, with epi-2-inosose, there was a reduced level of Ab42 in solution (Fig. 3B). These results, in combination with the CD data, suggest that allo-inositol may exhibit antiaggregant properties similar to those seen for other inositol stereoisomers, and that epi-2-inosose may represent an inducer of Ab42 but not Ab40 aggregation. Effect of inosose compounds on Ab fibril structure The CD and tyrosine fluorescence studies suggest that the inositol ⁄ inosose compounds may affect Ab nucleation and assembly. However, they may have variable effects on oligomerization versus fibril assembly and growth [8]. Using a large panel of compounds, Glabe et al. reported that Ab oligomerization and fibrillogenesis are not mutually dependent and that compounds can affect one pathway without inhibiting the other [8]. The characteristics of Ab40 and Ab42 assembly products in the presence and absence of inosose compounds were examined by negative-stain electron microscopy, using two stocks of Ab40 and Ab42 to ensure that the results obtained were consistent and representative [5,9]. Unseeded samples of both Ab40 and Ab42 were incubated in the presence of all compounds and alone for up to 96 h. When Ab40 was incubated alone, it formed fibers of varying length, as shown by representative electron microscopy images (Fig. 4A). When Ab40 was incubated with chiro-inositol, chiro-1-inositol, scyllo-inositol, epi-1-inositol and allo-3-inosose, no difference could be detected from M. Nitz et al. Modulating amyloid-b aggregation FEBS Journal 275 (2008) 1663–1674 ª 2008 The Authors Journal compilation ª 2008 FEBS 1665 Ab40 alone (data not shown). In contrast, when epi-2- inosose (Fig. 4C) and allo-inositol (Fig. 4E) were incubated with Ab40, very few fibers were detected after 72 h of incubation. In order to differentiate between compounds that inhibit fiber formation and those that decrease the kinetics of fiber formation, samples were Wavelen g th (nm) Theta A 500 500 0 –500 –1000 –1500 500 0 –500 –1000 –1500 –2000 4000 2000 0 –2000 –4000 –6000 –8000 1000 500 0 –500 –1000 –1500 –2000 –2500 –3000 1000 500 0 –500 –1000 –1500 –2000 0 –500 –1000 –1500 –2000 0 –100 –200 –300 –400 –500 0 –500 –1000 –1500 –2000 –2500 190 200 210 220 230 240 250 260 190 200 210 220 230 240 250 260 180 190 200 210 220 230 240 250 260 190 200 210 220 230 240 250 260 190 200 210 220 230 240 250 260 190 200 210 220 230 240 250 260 190 200 210 220 230 240 250 260 190 200 210 220 230 240 250 260 B DC E F GH chiro-inositol chiro-inosose scyllo-inositol scyllo-inosose epi-inositol epi-2-inosose allo-inositol allo-3-inosose Fig. 2. Determination of the secondary structure of Ab42 in the presence and absence of inosose compounds using CD spectroscopy. Ab42 was randomly structured in distilled H 2 O after dilution from 40% TFE stock solutions to a final peptide concentration of 10 m M (solid line). Repre- sentative CD spectra of Ab42 immediately upon addition of chiro-inositol (A), chiro-1- inosose (B), scyllo-inositol (C), scyllo-inosose (D), epi-inositol (E), epi-2-inosose (F), allo- inositol (G) and allo-3-inosose (H) (dashed line) are presented from three separate experiments. Modulating amyloid-b aggregation M. Nitz et al. 1666 FEBS Journal 275 (2008) 1663–1674 ª 2008 The Authors Journal compilation ª 2008 FEBS incubated for 14 days (Fig. 4). After 14 days, the number and structure of Ab40 fibers in the presence and absence of epi-2-inosose (Fig. 4D) were not different from those of Ab40 alone (Fig. 4B), suggesting that this compound alters the kinetics of Ab40 fibrillogenesis but does not inhibit fiber formation. In contrast, only a few fibers could be detected in samples incubated with allo-inositol after 14 days, suggesting that this compound inhibits fiber formation (Fig. 4F). Although Ab40 fibrillogenesis was altered by inosose stereoisomers, Ab42 aggregates at a much higher rate; depending on the strength of inosose–Ab interactions, the efficacy of inosose stereoisomers may be altered. When Ab42 is incubated in buffer alone, it forms various long fibers that begin to self-assemble over time (supplementary Fig. S1). Similar to what was found for Ab40, incubation with chiro-inositol, chiro-1-inositol, scyllo-inositol, epi-1-inositol and allo-3-inosose did not alter the number and structure of fibers formed in comparison with Ab42 alone. Very few fibers were detected after a 3 day incubation with epi-2-inosose and allo-inositol. These results suggest that epi-2-inosose and allo-inositol are either inhibitors of oligomer formation or inhibitors of both oligomer and fiber formation. To differentiate between these two pos- sibilities, we incubated Ab with both epi-2- inosose and allo-inositol for 14 days. In contrast to the 3 day incubation, when Ab42 was incubated for 14 days with epi-2-inosose, small protofibril species could be detected but were not abundant (supplementary Fig. S1). These results suggest that epi-2-inosose is more effective at decreasing the kinetics of Ab42 fiber formation than of Ab40. In vitro examination of increasing molar ratios of epi-2-inosose to peptide from 1 : 1 to 1 : 100 were investigated by negative- stain electron microscopy. In the presence of all molar ratios, epi-2-inosose decreased the kinetics of Ab40 and Ab42 fiber formation by a greater extent than that of Ab42 alone (supplementary Fig. S1). Similarly, after 14 days of incubation with allo-inositol, A b42 fibers were less abundant but were of similar structure and length to those seen with Ab42 alone. These results suggest that allo-inositol is a kinetic inhibitor of Ab42 fiber formation but is unable to fully inhibit fiber formation. These results further suggest that allo-inositol binds to and inhibits fibrillogenesis for Ab40 more effectively than for Ab42. Compounds that inhibit Ab aggregation and fibrillogenesis can also disaggregate preformed fibers. The latter is important not only for decreasing plaque load but also for increasing the pool of soluble, potentially toxic Ab species. To investigate the disassembly of fibers, three separate preparations of preformed Ab40 and Ab42 fibers were incubated in the presence of varying ratios of inosose compounds, and disaggregation was monitored over a 2 week period using negative-stain electron microscopy (Fig. 5). As was seen for the inhibition of aggregation, chiro-inositol, chiro- 1-inositol, epi-1-inositol and allo-3-inosose had no effect on preformed Ab40 and Ab42 fibers (supplementary Fig. S2). Although scyllo-inosose did not disaggregate preformed Ab40 fibers, it appeared to laterally aggregate fibers into thicker, rope-like fibers (Fig. 5B). The fiber bundles had a cumulative dia- meter of 200 A ˚ , and exhibited an apparent helical Percentage soluble Aβ (Soluble T y r fluorescence/Total fluorescence x 100) Aβ40 Chiro-inositol Scyllo-inosose Epi-2-inosose Chiro-1-inosose Allo-inositol Epi-1-inosose Allo-3-inosose Scyllo-inositol Epi-inositol Aβ42 Chiro-inositol Scyllo-inosose Epi-2-inosose Chiro-1-inosose Allo-inositol Epi-1-inosose Allo-3-inosose Scyllo-inositol Epi-inositol * * * * * A B 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Fig. 3. A tyrosine fluorescence assay was used to determine the extent of Ab40 (A) and Ab42 (B) aggregation in the presence and absence of inosose compounds. Ab40 and Ab42 were incubated with various inosose compounds at a ratio of 1 : 20 (w ⁄ w) or a 1 : 500 molar ratio for 48 h. The extent of Ab aggregation was determined using the ratio of tyrosine fluorescence before and after centrifugation, with n = 3 per experiment and three separate experiments. *P < 0.05, Fisher’s PLSD. M. Nitz et al. Modulating amyloid-b aggregation FEBS Journal 275 (2008) 1663–1674 ª 2008 The Authors Journal compilation ª 2008 FEBS 1667 twisting with a 150 nm period. The fibers also assem- bled into large masses in the presence of scyllo-inosose, rather than the disorganized distribution seen with Ab40 alone (Fig. 5B). epi-2-Inosose and allo-inositol disaggregate preformed Ab40 fibers, as evidenced by a decrease in Ab fibers and an increase in small oligomers (Fig. 5C,D). Similar to the effects on inhibition of fibrillogenesis, allo-inositol was more effective than epi-2-inosose at disaggregating Ab40 fibers (Fig. 5). Similar effects were seen for inosose- and inositol-induced Ab42 fiber disaggregation (data not shown), with the exception that allo-inositol and epi-2- inosose had similar potency. These results suggest that allo-inositol and epi-2-inosose may alter the equilib- Day 3 Day 14 Aβ40 Αβ40 + epi-2-inosose Αβ40 + allo-inositol A B C D E F Fig. 4. Negative-stain electron microscopy of Ab40 in the presence of inosose compounds. Ab40 incubated in buffer alone demonstrates long fibers with some small aggregates at day 3 (A), whereas only long fibers are detected after 14 days (B). After incubation in the presence of epi-2-inosose, no fibers could be detected at day 3 (C), whereas no difference was seen at day 14 (D) in comparison to Ab40 incubated alone. In contrast, in the presence of allo-inositol, very few small aggregates and protofibrils could be detected at either day 3 (E) or day 14 (F). Images are representative of three separate experiments. Scale bars: 50 nm. Modulating amyloid-b aggregation M. Nitz et al. 1668 FEBS Journal 275 (2008) 1663–1674 ª 2008 The Authors Journal compilation ª 2008 FEBS rium between the formation of Ab aggregates and disaggregation of preformed fibers. Effects on Ab-induced cytotoxicity The effect of inosose compounds on Ab -induced cytotoxicity was investigated because disaggregation of plaques and inhibition of the kinetics of Ab fibrillogenesis could increase the pool of toxic Ab species. Ab-induced toxicity against cell lines and primary neuronal cultures is well established, with Ab oligomers and protofibrils representing the toxic species [10]. NGF-differentiated PC-12 cells were incubated in the presence and absence of Ab40 and Ab42 with increasing ratios of compounds for 24 h. Toxicity was measured using two well-established assays for Ab- induced toxicity, Alamar blue and sulfhydryl rhoda- mine B assays [5]. When Ab40 and Ab42 were preincubated with chiro-inositol, chiro-1-inositol, scyllo-inositol, epi-1-inositol and allo-3-inosose, their toxicity was either unaffected or increased at high inosose concentrations, whereas the compounds alone were not toxic (Fig. 6; data not shown). These results suggest that at high concentrations, the compounds may subtly increase the level of toxic species of Ab. epi-2-Inosose rescued PC-12 cells from Ab40 ⁄ Ab42- induced toxicity at all molar ratios (1 : 38, 1 : 380, 1 : 760), suggesting that when epi-2-inosose binds to Ab it forms a noncytotoxic complex at molar ratios similar to that seen previously for epi -inositol, 1 : 25 Ab ⁄ epi-inositol [5] (Fig. 6). allo-Inositol also rescued cells from Ab40-induced cytotoxicity to the same extent as epi-2-inosose; however, it was less effective at rescuing them from Ab42-induced cytotoxicity (Fig. 6). These results are consistent with the structural data, in that allo-inositol has a stronger effect on Ab40 than on Ab42 in all regards. Transport of compounds by the sodium myo-inositol transporter (SMIT)-1 and SMIT-2 Inositol enters the brain by passive diffusion and by constitutively active stereospecific transport systems [11–15]. SMIT-1 and SMIT-2 are expressed at the blood–brain barrier and choroid plexus, and thus represent the main transport systems for inositol and related compounds [11–15]. Furthermore, these transport systems are expressed by neurons and astrocytes [12–16], and the latter can be used as a model system to examine inositol uptake. The astrocytoma cell line A C D B Fig. 5. Negative-stain electron microscopy of preformed Ab40 fibers in the presence and absence of inosose compounds. Ab40 formed large masses of long fibers that were often interwoven (A). scyllo-Inosose caused aggregation of Ab40 fibers into lar- ger, ordered ropes without evidence of disaggregation (B). epi-2-Inosose (C) and, to a greater extent, allo-inositol (D) disaggregat- ed fibers into short protofibrils and small aggregates. Images are representative of three separate experiments. Scale bars: 50 nm. M. Nitz et al. Modulating amyloid-b aggregation FEBS Journal 275 (2008) 1663–1674 ª 2008 The Authors Journal compilation ª 2008 FEBS 1669 1321 N1 was shown by RT-PCR to express both SMIT-1 and SMIT-2 (data not shown), and therefore we modified a competition uptake assay previously used in Xenopus oocytes [17]. We examined the transport of the two most effective compounds, allo-inositol and epi-2-inosose, using the 1321 N1 astrocytoma cell line (Fig. 7A). In order to silence the hydrogen myo-inositol transporter (HMIT), the assays were done in the presence of the protonophore 2,4-dinitrophenol. In our assay, cold myo-inositol effectively competes 75% of [2- 3 H]myo-inositol uptake over a 3 h time course, whereas epi-inositol only blocked 25%. Both allo-inositol and epi-2-inosose block greater than 50% of radioactive myo-inositol uptake, suggesting effective transport of these compounds by SMIT-1 and SMIT-2. When SMIT-1, SMIT-2 and HMIT transport was inhibited using 2,4-dinitrophenol and phloridizin, no radioactivity was detected with the astrocytoma cell line, and this was unaffected by the presence or absence of competitors (Fig. 7B). Although these studies are indicative of inositol and inosose uptake into astrocytoma cytoplasm, we cannot completely differentiate between competitor-induced transporter blockage and uptake. These results support the potential for high central nervous system bioavailability of allo-inositol and epi-2-inosose, as SMIT-1 and SMIT-2 maintain a high concentration of myo-inositol within the central nervous system against a concentration gradient of 100-fold lower concentrations in the serum [18]. Discussion Several therapeutic approaches have been developed for the treatment of amyloidogenic diseases, such as AD. The small molecule inhibitor approach was initially based on the finding that small aromatic molecules such as Congo red and thioflavin interact specifically with amyloid fibrils and inhibit their formation [10,19]. Evaluation of the more potent compounds demonstrated that at least three hydroxyl groups were present on the aromatic rings, and that the charge distribution across small molecules represents the limiting factor for Ab binding [20]. scyllo-Inositol, which was shown to possess potent antiamyloidogenic activity in vitro and in vivo, has an amphipathic surface. The all-equatorial orientation of the hydroxyl groups renders the faces of the molecule hydrophobic. Hydroxyl groups located at the edge of the ring are capable of hydrogen bonding with Ab. allo-Inositol and epi-2-inosose also have single hydrophobic surfaces and therefore may bind to similar sites as scyllo-inositol, thus accounting for the similar effects seen in vitro between these molecules (Table 1). Conversion of a single hydroxyl group contained within a reactive group in an all-equatorial orientation (scyllo-inositol) to a ketone (scyllo-inosose) renders the molecule inactive with respect to inhibition of Ab oligomerization and fiber formation. This, however, does not preclude any interaction with Ab. scyllo-Inosose is able to bind to Ab, as illustrated by the presence of A B Epi-1-inosose Epi-2-inosose Epi-2-inosose Epi-1-inosose Allo-inositol Allo-inositol 0 20406080100 Percent cell survival 120 0 20 40 60 80 100 120 * * * * * * * * * * * * * * * * Fig. 6. Concentration dependence of inosose rescue of Ab40- induced (A) and Ab42-induced (B) cytotoxicity. Percentage cell survival was determined using the sulfhydrylrhodamine B assay and NGF-differentiated PC-12 cells alone as a control. Ab was incubated in the presence of increasing concentrations of inosose for 3 days prior to determining cell survival: Ab alone (black bars); Ab ⁄ inosose molar ratios of 1 : 25–38 (gray bars), 1 : 250–380 (dotted bars), 1 : 500–760 (white bars); and inosose alone (hatched bars). Bars represent mean ± SEM of three separate experiments. *P < 0.05, Fisher’s PLSD. Modulating amyloid-b aggregation M. Nitz et al. 1670 FEBS Journal 275 (2008) 1663–1674 ª 2008 The Authors Journal compilation ª 2008 FEBS large, rope-like bundles of Ab when it is incubated with preformed Ab fibers. This suggests that binding of scyllo-inosose to Ab fibers is sufficient to stabilize the macromolecular structure of pre-existing fibers by interfiber stabilization. This is analogous to extended glycosaminoglycan polymer aggregation of pre-existing Ab40 and Ab42 fibers, where the minimum unit com- prises chondroitin sulfate-derived disaccharides [21]. Together, these studies highlight the importance of the extent and distribution of charge across the backbone for the interaction with and stabilization of Ab by sul- fates for glycosaminoglycans and hydroxyl groups for inositols [5,20]. However, scyllo-inosose is unique, as this small six-carbon ring is sufficient to laterally aggregate Ab, whereas monosaccharides derived from glycosaminoglycans cannot. In agreement with previous reports [4,5], we show that allo-inositol induces a structural transition in Ab and that replacement of a single hydroxyl for a ketone (allo-3-inosose) does not inhibit this structural transition. However, only allo-inositol inhibits Ab fiber formation, suggesting that, although the inosose compounds are able to bind Ab, this interaction is not sufficient to inhibit further assembly to mature fibers. These results are in agreement with our previous studies showing that myo-inositol and scyllo-inositol stabilize small b-sheet-containing Ab conformers and inhibit progression to Ab fibers [4,5]. Further- more, a ketone at position 3 of the allo-conformation changes the activity of this molecule from an inhibitor of fibrillogenesis to a kinetic inhibitor of assembly. epi-2-Inosose accelerated the Ab structural transition and inhibited fibrillogenesis. The magnitude of the effect of epi-2-inosose was similar to that of epi-inositol [4]. l-epi-1-Inosose, as reported above, did not inhibit fiber formation, suggesting that a hydroxyl at position 1 but not at position 2 is important for this function. Inositol and inosose compounds have unique in vivo stability and toxicology as compared to compounds reported in the literature for treatment of AD [22– 24]. For example, the polyphenols are considered to be antioxidants and are rapidly degraded in the presence of reactive oxygen species [25] and by cytochrome P450 in the liver [26]. As inositols are saturated, they are very stable to oxidative conditions, whereas the inosose compounds may be susceptible to ketose reactions in vivo. Thus, these compounds, which probably bind to similar sites on Ab, have very different pharmacological profiles. In addition, the inositol and inosose compounds have other advanta- ges over other small molecule therapies, in that they are actively transported across the blood–brain barrier [14,27], have low toxicity, and are readily available orally. Only a small subpopulation of drugs has the low molecular weight and high lipophilicity that are necessary to allow diffusion across the blood–brain barrier. For these reasons, it has been estimated that over 98% of all small molecule drugs developed do not cross the blood–brain barrier. SMITs, present at the blood–brain barrier, transport ‡ ‡ ‡ ‡ ‡ A B Control ++ ++ ++ * Myo-inositol No inhibitors Control Myo-inositol Epi-inositol Epi-2-inosose Allo-inositol Epi-inositol Epi-2-inosose Allo-inositol 0 20 40 60 80 100 120 Active transport (% control) 0 20 40 60 80 100 120 Active trans p ort ( % control ) Fig. 7. Substrate specificity of the SMIT-1 and SMIT-2 transporters for inositol and inosose compounds. (A) Transport of 100 l M [2- 3 H]myo-inositol was measured in the presence or absence of 10 m M cold myo-inositol as a positive control or 10 mM other inositol and inosose compounds. (B) Inhibition of 100 l M [2- 3 H]myo-inositol in the presence of SMIT-1, SMIT-2 and HMIT inhibition, in the presence and absence of competitors. Scheffe test, a = 0.05, *P < 0.05, à P < 0.001. M. Nitz et al. Modulating amyloid-b aggregation FEBS Journal 275 (2008) 1663–1674 ª 2008 The Authors Journal compilation ª 2008 FEBS 1671 most of the inositol stereoisomers, as well as related small molecules, suggesting there is some breadth in tolerance with the substrate flexibility of these transporters [3,14,27]. Our results demonstrate that both epi-2-inosose and allo-inositol are effectively transported by SMIT-1 and SMIT-2. Collectively, our results demonstrate that the stereochemistry of inositol binding to Ab is unaffected by substitution of one hydroxyl group for a ketone at various positions around the carbon ring, but, depending on the position of the ketone, there are dramatic effects on the inhibition of fiber formation (Table 1). These results highlight the potential negative impact of subtle changes in charge distribution on small molecules such as inositol when using drug-based design of new compounds. Experimental procedures Peptides Ab40 and Ab42 were synthesized by solid-phase Fmoc- chemistry by the Hospital for Sick Children’s Biotechnol- ogy Centre (Toronto, Canada). They were purified by RP-HPLC on a C18 lbondapak column. Ab peptides were initially dissolved in 0.5 mL of 100% trifluoroacetic acid (Aldrich Chemicals, Milwaukee, WI, USA), to ensure that the peptide remained monomeric and free of fibril seeds, diluted in distilled H 2 O, and immediately lyophilized. Ab peptides were then dissolved at 1 mgÆmL )1 in 40% TFE (Aldrich Chemicals) in distilled H 2 O and stored at )20 °C until use [28]. CD CD spectra were recorded on a Jasco Circular Dichroism Spectrometer Model J-715 (Easton, MD, USA) at 25 °C, as previously described [9]. Spectra were obtained from 200 to 250 nm, with a 0.5 nm step, 1 nm bandwidth and 1 s col- lection time per step. Spectra were averaged from five repeat scans. Peptide ⁄ inositol ratios were 1 : 1 (w ⁄ w) or 1 : 25–38 by molar ratio, with a final peptide concentration of 10 lm. CD experiments were repeated three times on two separate Ab stocks. The effect of the inositol compounds on peptide conformation was determined by adding an aliquot of stock peptide solutions into cosolvent solutions suspended in 20 mm sodium phosphate buffer (pH 7.0) or distilled H 2 O (approximately pH 6.8). CD spectra were examined immediately after addition of Ab and over a 96 h time course. The contribution of inositol compounds to the CD signal was removed by subtracting the inositol-only spectra. Ab peptide conformations were determined in 40% TFE ⁄ H 2 O, in 20 mm phosphate buffer and distilled H 2 O under the same conditions. Tyrosine fluorescence assay Steady-state fluorescence was measured at 20 °C using an AM-1 fluorescence spectrophotometer (Photon Technology International, London, Canada) equipped with excitation intensity correction and a magnetic stirrer [4]. Tyrosine emission spectra from 290 nm to 340 nm were collected (excitation wavelength 281 nm, 0.5 sÆnm )1 , bandpass 4 nm). A cuvette with a 1 cm path length was used. For centrifugation studies, 50 lm Ab40 or Ab42 was incubated in the presence or absence of inosose compounds at a peptide ⁄ inosose ratio of 1 : 20 (w ⁄ w) for 24 h with continuous shaking. Tyrosine fluorescence was measured, and this was followed by a centrifugation step at 15 600 g for 30 min in order to sediment insoluble species. The relative amount of tyrosine fluorescence in the supernatant was then determined. The fluorescence of the noncentrifuged samples was used as a measure of total fluorescence. Steady-state fluorescence measurements were repeated in three separate experiments with a sample size of three per condition within each experiment. Table 1. Summary of Ab40 ⁄ Ab42 interaction with inositol and inosose compounds. Compound Secondary structure (CD spectroscopy) Fiber formation (electron microscopy) PC-12 cell toxicity Ab42 Ab40 Ab42 Ab40 Ab42 L-chiro-Inositol Random < Control < Control No effect No effect L-chiro-1-Inosose Random < Control No effect Inhibits No effect scyllo-Inositol b-Sheet No effect Inhibits Inhibits Inhibits scyllo-Inosose Random < Control No effect Increase Increase epi-Inositol b-Sheet No effect Inhibits Inhibits Inhibits L-epi-1-Inosose Partial b-sheet No effect No effect No effect Increase L-epi-2-Inosose Strong b-sheet < Control Inhibits Inhibits Inhibits allo-Inositol b-Sheet Inhibits < Control Inhibits Inhibits D-allo-3-Inosose Random < Control No effect No effect No effect Modulating amyloid-b aggregation M. Nitz et al. 1672 FEBS Journal 275 (2008) 1663–1674 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... microscopy of Ab42 in the presence of inosose compounds Fig S2 Negative-stain electron microscopy of preformed Ab42 fibers in the presence and absence of inosose compounds This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors... Modulating amyloid-b aggregation 8 9 10 11 12 13 14 15 16 17 18 19 20 M Nitz et al by the Alzheimer amyloid-oligomers Ann Neurol 60, 668–676 Necula M, Kayed R, Milton S & Glabe CG (2007) Small molecule inhibitors of aggregation indicate that amyloid b oligomerization and fibrillization pathways are independent and distinct J Biol Chem 282, 10311– 10324 McLaurin J & Fraser PE (2000) Effect of amino acid... concentration of 0.1 mgÆmL)1, and incubated for 24 h at 37 °C Toxicity was assayed using the Alamar Blue Assay (Biosource Inc., Camarillo, CA, USA) and the sulfhydrylrhodamine B assay [5] Toxicity assays were repeated three times with n = 4 per condition within an experiment Inositol transport assay 1321 N1 astrocytoma cells were plated onto 24-well plates at a density of 10 000 cellsÆmL)1 and grown for... Properties of scyllo-inositol as a therapeutic treatment of AD-like pathology J Mol Med 85, 603– 611 4 McLaurin J, Franklin T, Chakrabartty A & Fraser PE (1998) Phosphatidylinositol and inositol involvement in Alzheimer amyloid-beta fibril growth and arrest J Mol Biol 278, 183–194 5 McLaurin J, Goloumb R, Jurewicz A, Antel JP & Fraser PE (2000) Inositol stereoisomers stabilize an oligomeric aggregate of Alzheimer...M Nitz et al Electron microscopy Ab40 and Ab42 were incubated in the presence and absence of inositol compounds at a final peptide concentration of 100 lgÆmL)1 Ab was used directly after solubilization in water at a concentration of 10 mgÆmL)1, or after assembly into mature amyloid fibers Serial dilutions of inositol were added to Ab and incubated at room temperature for up to 2 weeks The... 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Center for synthesis of peptides, and Kevin Da Silva for helpful comments on the manuscript The authors acknowledge support from the Ontario Alzheimer’s Society (J McLaurin), Canadian Institutes of Health Research (J McLaurin, M Nitz), Natural Science and Engineering Research Council of Canada (J McLaurin, M Nitz), and Cryptic Rite Charitable Foundations (J McLaurin) Ab-induced toxicity PC-12 cells . Modulation of amyloid-b aggregation and toxicity by inosose stereoisomers Mark Nitz 1 , Daniela Fenili 2,3 ,. stereospecific inhibition of A b aggregation and toxicity in vitro and in vivo. We report here the effects of inosose versus inositol stereoisomers on Ab fibrillogenesis

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