phenyl)[1,3,5]triazine-2,4-diamine (RS-0466), does not inhibitA
fibril formation but retains the ability to prevent A
-mediated toxicity (Nakagami et al., 2002b).Here, we show that both RS-0406 and RS-0466 inhibit theintracellularformationofcell-derivedA
oligomersandthatCMfrom cells treated with RS-0406 or RS-0466 contained reducedoligomerlevelsandnolongerblockedLTP.Usingnondenaturingsize exclusion chromatography, we physically separated A
monomer from low-
n
oligomers and unambiguously identifiedA
oligomers as the species responsible for perturbing synapticactivity.Usingthisorthogonalapproach,weconfirmedthatbothRS-0406 and RS-0466 inhibit natural oligomer formation. Weconcludethatthefractionationprotocoldescribedherewillbeof use in further investigating the biological activity of natural A
oligomers and that compounds such as RS-0406 and RS-0466mayhaveutilityintreatingtheearlieststagesofsynapticdysfunc-tion in AD.
MaterialsandMethods
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) un-less otherwise stated. RS-0406 (Nakagami et al., 2002a) and RS-0466(Nakagami et al., 2002b) were obtained from Dr. Yasuhiro Nakagami(Sankyo,Tokyo,Japan)andDr.RussellHagan(BTGInternational,Lon-don, UK), respectively. Peptides OR1 and OR2 were synthesized usingconventional Fmoc [
N
-(9-fluorenyl) methoxycarboxyl] chemistry andpurified on a Vydac (Columbia, MD) C4 column. Peptide purity (
95%) and identity were confirmed by analytical HPLC and electros-pray mass spectrometry.
A
peptides.
A
(1-40) and A
(1-42) were synthesized and purifiedessentially as described previously (Walsh et al., 1997). Peptide mass,purity, and quantity were determined by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,analytical HPLC, and quantitative amino acid analysis.
Thioflavin T-binding assay.
A
peptides were dissolved in dimethyl-sulfoxide (DMSO) to yield protein concentrations of
2 m
M
and thendiluted with milliQ water to yield a
200
M
stock solution. To initiatereactions, 142.5
l of 100 m
M
Tris-HCl, pH 7.4, was added to 150
l of A
stock solution with 7.5
l of either 10 m
M
test compound in 10%DMSO or 10% DMSO vehicle alone. Samples were vortexed briefly,aliquots were assayed immediately, and the remainder was incubated at37°C. At intervals of 24 and 48 h, samples were removed, vortexed for10 s, and then assayed. Electron microscopy (EM) was performed onsamples after 48 h of 37°C incubation.Thioflavin T (ThT) binding was assessed as described previously (Walshetal.,1999).Briefly,25
lofsamplewasaddedtoa1cm
2
cuvettecontaining425
l of waterand500
l of 100m
M
glycine-NaOH,pH 8.5.The cuvette was vortexed, and then 50
l of 100
M
ThT was added andthe solution was vortexed again. Fluorescence was measured at 90, 100,110, and 120 s after ThT addition. Measurements were made using aHitachi(Tokyo,Japan)F-4500fluorescencespectrophotometerwithex-citationandemissionat446nm(slitwidth,5nm)and490nm(slitwidth,10 nm), respectively.
APP-expressing cells.
Chinese hamster ovary (CHO) cells stably trans-fected with a cDNA encoding APP
751
containing the Val717Phe familialAD mutation (referred to as 7PA2 cells) were cultured in DMEM with10% fetal bovine serum (Hyclone, Logan, UT), as described previously (Koo and Squazzo, 1994).
Antibodies.
AntibodiestoAPPanditsproteolyticderivativeshavebeendescribedpreviously(Walshetal.,2000).Monoclonalantibody2G3wasraised to A
33-40 and specifically recognizes A
species ending at resi-due 40, whereas monoclonal antibody 21F12 was raised to A
33-42 andspecifically recognizes A
species ending at residue 42. Both 2G3 and21F12 were kindly provided by Drs. P. Seubert and D. Schenk (ElanPharmaceuticals, San Francisco, CA). Monoclonal antibodies 6E10 and4G8(SignetPathologySystems,Dedham,MA)recognizeepitopeswithinthe human A
sequence corresponding to residues 6–10 and 17–24,respectively; R1282 is a high-titer polyclonal antiserum raised to syn-theticA
1-40(Haassetal.,1992).PolyclonalantiserumC7wasraisedtoa peptide encompassing residues 676–695 of the APP sequence and wasused to immunodeplete cell lysates of full-length APP and its C-terminalfragments (Walsh et al., 2000).
Treatmentofcellswithtestcompounds.
Nearlyconfluent(95–100%)10cm
2
dishesof7PA2cellsandtheircorrespondinguntransfectedparentalCHO cell line were washed with serum-free medium (4 ml
1) andincubated with or without compound in serum-free medium for
15 h.CM was then removed and cleared of cells by centrifugation at 200
g
for 10 min at 4°C. Protease inhibitors (final concentration: 5
g/mlleupeptin, 5
g/ml aprotinin, 2
g/ml pepstatin, 120
g/ml Pefabloc)were added to the supernatant, and this CM was used for immunopre-cipitation(IP)orconcentrated
10-foldusingCentriprepYM-3centrif-ugal filter devices (Millipore, Waltham, MA). Protease inhibitors, many of which are biologically active, were not added to samples to be used forelectrophysiology; however, the absence of protease inhibitors did notsubstantially alter the detection or the size exclusion chromatography (SEC) elution pattern of A
monomer and oligomers.The concentrations of test compounds were determined empirically;inthecaseofRS-0406andRS-0466,theconcentrationsused(102and9.3
M
, respectively) were the maximum tolerated without inducing cyto-toxicity. For OR-1 and OR-2, the concentration used (18.5
M
) wasdetermined by the solubility of these peptides. In all cases, the ratios of compoundtoA
werefarinexcessofthoseusedinexperimentsinwhichRS-0406, OR-1, and OR-2 were shown to inhibit
in vitro
fibrillogenesis(see Fig. 1).
Immunoprecipitation/Western blot analysis.
A highly sensitive IP/Westernblot(wblot)protocolthatcanreadilydetectaslittleas200pgof naturally secreted A
(Walsh et al., 2000) was used for the detection of natural,secretedA
insamplesthatwerenotradiolabeled.Sampleswereimmunoprecipitated with the high-affinity antibody R1282 at a dilutionof 1:75. After IP, samples were electrophoresed on 10–20% tricine gelsand transferred to 0.2
M
nitrocellulose membranes at 400 mA for 2 h.Filters were boiled for 10 min in PBS (Ida et al., 1996) and then blockedovernight at 4°C with 5% fat-free milk in 20 m
M
Tris-HCl, pH 7.4,containing 150 m
M
NaCl and 0.05% Tween 20 (TBS-T). After washingthe membranes in TBS-T, they were probed with monoclonal 6E10 (1
g/ml). Bound antibody was visualized with horseradish peroxidase-conjugated donkey anti-mouse Ig (at 1:25,000) (Jackson ImmunoRe-search, West Grove, PA) and the ECL
detection system (AmershamBiosciences, Arlington Heights, IL).
Radiolabeling, immunoprecipitation, and gel fluorography.
Nearly con-fluent (95–100%) 10 cm
2
dishes of cells were starved of methionine for30 min and were then labeled with 750
Ci [
35
S]methionine for
15 hwith or without the test compounds used above. Media were harvestedandimmunoprecipitated(Podlisnyetal.,1995).Afterelectrophoresison10–20% tricine gels, bands were visualized by gel fluorography.
Size exclusion chromatography.
A Superdex 75 10/30 HR column at-tached to an AKTA FPLC system (Amersham Biosciences, Piscataway,NJ) was used for all experiments and calibrated using unbranched dex-tran standards of molecular masses: 123,600; 66,700; 43,800; 21,400;9890;and4440(Pharmacosmos,Holbaek,Denmark).ConcentratedCM(1 ml) was injected onto the Superdex 75 column and eluted with 50 m
M
ammonium acetate, pH 8.5, at a flow rate of 1 ml/min. One milliliterfractionswerelyophilized,resuspendedin2
SDS-tricinesamplebuffer(20
l), boiled for 10 min, and then electrophoresed on 10–20% tricinegels. Proteins were transferred onto 0.2
M
nitrocellulose and used forWestern blotting as described above.
Preparation of brain slices.
Coronal sections of mouse hippocampus(males and females; 14–35 d of age) were used for all experiments. Afterasphyxiation with CO
2
, the brain was rapidly removed and placed inice-cold oxygenated artificial CSF (ACSF) containing sucrose that con-tained the following (in m
M
): 206 sucrose, 2 KCl, 2 MgSO
4
, 1.25NaH
2
PO
4
, 1 CaCl
2
, 1 MgCl
2
, 26 NaHCO
3
, 10
D
-glucose, pH 7.4,
315mOsm(MoyerandBrown,1998).Transverseslices(350
mthick)werecut and transferred to a holding chamber containing artificial CSF thatcontained the following (in m
M
): 124 NaCl, 2 KCl, 2 MgSO
4
, 1.25NaH
2
PO
4
, 2 CaCl
2
, 26 NaHCO
3
, 10
D
-glucose, pH 7.4,
310 mOsm, inwhich they were allowed to recover for at least 1 h. The slices were then
2456
•
J. Neurosci., March 9, 2005
•
25(10):2455–2462 Walsh et al.
•
Inhibition of A
Oligomer Formation and Rescue of LTP
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