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

We had previously shown the efficacy of scyllo-inositol and epi-inositol in inducing structural transitions, but had not examined allo-inositol; all inositol stereoiso-mers were included

by inosose stereoisomers

Mark Nitz1, Daniela Fenili2,3, Audrey A Darabie2, Ling Wu2, Julian E Cousins2

and JoAnne McLaurin2,3

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

occur-ring stereoisomers, the most common of which are

myo-inositol, chiro-inositol, epi-inositol, and

scyllo-ino-sitol [1] myo-Inoscyllo-ino-sitol is the most abundant isomer and

is a ubiquitous component of all eukaryotic cells We

have shown that scyllo-inositol may represent a

poten-tial 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

spec-troscopy demonstrated that epi-inositol and

scyllo-inositol, but not chiro-scyllo-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)-differ-entiated 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-ino-sitol 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 Ab aggregation and toxicity in vitro and in vivo We report here the effects of inosose versus inositol stereoisomers on Ab fibrillogene-sis 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.

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

effec-tiveness of a specific inositol is highly dependent on

the stereochemistry of the hydroxyl groups

scyllo-Ino-sitol is the most potent inoscyllo-Ino-sitol, and a single

epimeriza-tion 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

spe-cific interaction with the amyloid peptides is necessary

These results further suggest that the hydroxyl groups

may represent key recognition elements in the

inter-action with Ab Furthermore, the dependence on the

hydroxyl group positioning also suggests that

hydro-gen bonding is important for stabilizing this

interac-tion 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

for-mation 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

neces-sary for Ab-nucleation, oligomer formation and fiber

formation Examination of transitions in b-structure

over time gives an indication of the effect of the

com-pounds on the initiation step of either oligomerization

or fibrillogenesis Previously, we have shown that

incu-bation with myo-inositol, epi-inositol and

scyllo-inosi-tol induces an immediate structural transition in Ab42

but not in Ab40 [5] CD spectroscopy was used to

evaluate structural transitions from randomly

struc-tured Ab40 and Ab42 diluted from 40%

trifluoroetha-nol (TFE) into NaCl⁄ Pi The following compounds

were examined: chiro-inositol, chiro-1-inosose,

scyllo-inositol, scyllo-inosose, scyllo-inositol, 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 stereoiso-mers 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.

day effects, experiments were repeated three separate

times on two different Ab preparations, and gave

con-sistent, 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

sug-gest 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-struc-ture 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

sug-gests 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

com-pounds for 48 h, and the tyrosine fluorescence of each

solution was measured prior to and directly after cen-trifugation 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 solu-tion 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-inosi-tol 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 nucle-ation 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 fibrillogen-esis 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 com-pounds 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 com-pounds 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-inosi-tol, chiro-1-inosichiro-inosi-tol, scyllo-inosichiro-inosi-tol, epi-1-inositol and allo-3-inosose, no difference could be detected from

Ab40 alone (data not shown) In contrast, when

epi-2-inosose (Fig 4C) and allo-inositol (Fig 4E) were

incu-bated 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 Wavelength (nm)

A

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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

D C

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 struc-tured 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-chiro-1-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.

incubated for 14 days (Fig 4) After 14 days, the

num-ber and structure of Ab40 finum-bers 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

fibrillogene-sis but does not inhibit fiber formation In contrast,

only a few fibers could be detected in samples

incu-bated 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 vari-ous 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-inosi-tol, scyllo-inosichiro-1-inosi-tol, 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-inos-ose 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 (supplemen-tary 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, Ab42 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 for-mation 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 fibril-logenesis 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 disaggrega-tion was monitored over a 2 week period using nega-tive-stain electron microscopy (Fig 5) As was seen for the inhibition of aggregation, inositol, chiro-1-inositol, epi-1-inositol and allo-3-inosose had no effect on preformed Ab40 and Ab42 fibers (supple-mentary 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 Tyr fluorescence/Total fluorescence x 100)

A β40

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

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

exper-iments *P < 0.05, Fisher’s PLSD.

twisting with a 150 nm period The fibers also

assem-bled into large masses in the presence of

scyllo-inos-ose, rather than the disorganized distribution seen

with Ab40 alone (Fig 5B) epi-2-Inosose and

allo-ino-sitol 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

inhibi-tion of fibrillogenesis, allo-inositol was more effective than epi-2-inosose at disaggregating Ab40 fibers (Fig 5) Similar effects were seen for inosose- and ino-sitol-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

A β40

Αβ40 +

epi-2-inosose

Αβ40 +

allo-inositol

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 pres-ence of epi-2-inosose, no fibers could be detected at day 3 (C), whereas no differpres-ence was seen at day 14 (D) in comparison to Ab40 incu-bated 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.

rium between the formation of Ab aggregates and

dis-aggregation of preformed fibers

Effects on Ab-induced cytotoxicity

The effect of inosose compounds on Ab-induced

cyto-toxicity was investigated because disaggregation of

plaques and inhibition of the kinetics of Ab

fibrillo-genesis could increase the pool of toxic Ab species

Ab-induced toxicity against cell lines and primary

neu-ronal 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

increas-ing 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 struc-tural 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 rep-resent the main transport systems for inositol and related compounds [11–15] Furthermore, these trans-port 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

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

dis-aggregation (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.

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

trans-port 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 [2-3H]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 poten-tial 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 ini-tially based on the finding that small aromatic mole-cules such as Congo red and thioflavin interact specifically with amyloid fibrils and inhibit their for-mation [10,19] Evaluation of the more potent com-pounds 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 simi-lar 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 olig-omerization 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

Percent cell survival

120

*

*

*

* *

*

*

*

*

*

*

*

* *

*

*

Fig 6 Concentration dependence of inosose rescue of

Ab40-induced (A) and Ab42-Ab40-induced (B) cytotoxicity Percentage cell

sur-vival 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.

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 previ-ous studies showing that myo-inositol and scyllo-inosi-tol 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 inhibi-tor of fibrillogenesis to a kinetic inhibiinhibi-tor of assem-bly 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 pres-ence of reactive oxygen species [25] and by cyto-chrome 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

Active transport (% control)

Active transport (% control)

Fig 7 Substrate specificity of the SMIT-1 and SMIT-2 transporters

for inositol and inosose compounds (A) Transport of 100 l M

[2-3H]myo-inositol was measured in the presence or absence of

10 m M cold myo-inositol as a positive control or 10 m M other

inosi-tol and inosose compounds (B) Inhibition of 100 l M [2- 3

H]myo-ino-sitol 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,  P < 0.001.

most of the inositol stereoisomers, as well as related

small molecules, suggesting there is some breadth in

tolerance with the substrate flexibility of these

trans-porters [3,14,27] Our results demonstrate that both

epi-2-inosose and allo-inositol are effectively

trans-ported by SMIT-1 and SMIT-2

Collectively, our results demonstrate that the

stereo-chemistry of inositol binding to Ab is unaffected by

substitution of one hydroxyl group for a ketone at

var-ious 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 H2O, and immediately lyophilized Ab

peptides were then dissolved at 1 mgÆmL)1 in 40% TFE

(Aldrich Chemicals) in distilled H2O 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 25C, 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 com-pounds on peptide conformation was determined by adding

an aliquot of stock peptide solutions into cosolvent solu-tions suspended in 20 mm sodium phosphate buffer (pH 7.0) or distilled H2O (approximately pH 6.8) CD spec-tra were examined immediately after addition of Ab and over a 96 h time course The contribution of inositol com-pounds to the CD signal was removed by subtracting the inositol-only spectra Ab peptide conformations were determined in 40% TFE⁄ H2O, in 20 mm phosphate buffer and distilled H2O under the same conditions

Tyrosine fluorescence assay Steady-state fluorescence was measured at 20C 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 centrifu-gation 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 deter-mined The fluorescence of the noncentrifuged samples was used as a measure of total fluorescence Steady-state fluo-rescence 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