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OMMUNICATION

40 Protects Non-toxic A

42 Monomer fromAggregation

Yilin Yan and Chunyu Wang

⁎

Center for Biotechnologyand Interdisciplinary Studies,Biology Department,Rensselaer Polytechnic Institute,Troy, NY 12180, USA

40 and A

42 are the predominant A

species in the human body. ToxicA

42 oligomers and fibrils are believed to play a key role in causingAlzheimer's disease (AD). However, the role of A

40 in AD pathogenesisis not well established. Emerging evidence indicates a protective role forA

40 in AD pathogenesis. Although A

40 is known to inhibit A

42fibril formation, it is not clear whether the inhibition acts on the non-toxicmonomer or acts on the toxic A

42 oligomers. In contrast to conventionalmethods that detect the appearance of fibrils, in our study A

42aggregation was monitored by the decreasing NMR signals from A

42monomers. In addition, differential NMR isotope labelling enabled theselective observation of A

42 aggregation in a mixture of A

42 and A

40.We found A

40 monomers inhibit the aggregation of non-toxic A

42monomers, in an A

42/A

40 ratio-dependent manner. NMR titrationrevealed that A

40 monomers bind to A

42 aggregates with higher affinitythan A

42 monomers. A

40 can also release A

42 monomers from A

42aggregates. Thus, A

40 likely protects A

42 monomers by competing forthebindingsitesonpre-existingA

42aggregates.Combiningourdatawithgrowing evidence from transgenic mice and human genetics, we proposethat A

40 plays a critical, protective role in Alzheimer's by inhibiting theaggregation of A

42 monomer. A

40 itself, a peptide already present in thehuman body, may therefore be useful for AD prevention and therapy.

*Corresponding author

Keywords:

Alzheimer's disease; A

40; A

42; NMR; aggregationAlzheimer's disease (AD) is the most commontype of senile dementia, but its etiologyandpathogenesis remain poorly understood.

ADpathology is characterized by extracellular senileplaques and intracellular neurofibrillary tangles inaffected brains. Amyloid

-peptides (A

) are themajor components of the senile plaques, generatedfrom the sequential cleavage of the amyloid pre-cursor protein (APP) by the

and

-secretases. Asmall percentage of AD cases are hereditary and areknown as familial Alzheimer's disease (FAD). Thecritical role of A

in AD has been underscored bythe fact that FAD mutations occur only in genesinvolved directly in A

production: APP, presenilin1(PS1)andpresenilin2(PS2)(theactivesiteresiduesof

-secretase are in PS1 and PS2). A

peptidesrange mostly from 39 to 43 residues in length. A

40and A

42, composed of 40 and 42 amino acidresidues, respectively, constitute the bulk of A

species in plasma, cerebrospinal fluid and plaques.

In the popular amyloid cascade hypothesis for ADpathogenesis, toxic A

42 aggregates such as A

42oligomers andfibrils play a dominant role incausing AD.

3,4

This is supported by the increasedratio of A

42/A

40 with many FAD mutations,

–

the enhanced aggregation of A

42,

–

and neuro-toxicity of A

42 aggregates.

–

Controversy existsconcerning the roles of A

42 fibrils and A

42oligomersincausing AD. A

42 fibrils

12,16,17

orprotofibrils

are toxic to neurons. However, thereis evidence showing a correlation between theamount of soluble A

oligomer and dementia inAD patients.

24,25

Transgenic mouse models of AD

Abbreviations used: AD, Alzheimer's disease; A

,amyloid

-peptide; APP, amyloid precursor protein; FAD,familial Alzheimer's disease; PS1, presenilin 1; PS2,presenilin 2; HSQC, heteronuclear single quantumcoherence.E-mail address of the corresponding author:wangc5@rpi.edu

doi:10.1016/j.jmb.2007.04.014

J. Mol. Biol.

(2007)

369

, 909

–

916

show functional changes before the appearance of plaques.

26,27

Recently, the neurotoxic effects of A

42 soluble oligomers have been characterizedextensively.

–

15,19,28,29

Many forms of the A

42oligomer, such as dimer,

trimer

and 12mer,

14,22

were shown to be toxic but the actual A

42oligomers that play a causative role in AD havenot been defined.

14,19,20,22,23

In contrast, the A

42monomer is not toxic.

–

Thus, any agent thatkeeps A

42 monomers from aggregation can have aprotective role against the development of AD byreducing the generation of toxic A

42 species.The role for A

40 in AD pathogenesis has not been well-established. Recently, several studieshave suggested a protective role for A

40 in AD.There was no plaqueformation in transgenic miceoverexpressing A

40.

Amyloid deposition wasreduceddramaticallybycrossing A

40over-expres-sing mice with AD mouse modelTg2576 or A

42over-expressing transgenic mice.

Exacerbated pla-quepathologywasobservedintransgenicmicewithselectively reduced levels of A

40.

Reduced levelsofA

40werefoundwith numerous FADmutations,and were correlated with accelerated onset of dementia.

35,36

However, it is not clear how A

40executes its protective function in AD on a mole-cular level. It has been shown that A

40 inhibitsA

42 fibril formation.

–

There can be two verydifferent scenarios for A

40 inhibition of A

42 fibrilformation. The inhibition may act on the non-toxicmonomer or act on the toxic A

42 oligomers. Thus,inhibition may either protect the benign A

42monomers from aggregation or it may trap A

42in oligomeric forms that may be highly toxic.Whether A

40 can inhibit toxic A

42 oligomerformationand,moreimportantly,whetherA

40cankeep A

42 in a non-toxic, monomeric state has not beenstudied.InthissolutionNMRstudy,weshowedthat A

40 monomers protect non-toxic A

42 mono-mers against aggregation in a A

42/A

40 ratio-dependent manner and we explored the mechanismand significance of the protective effect of A

40.

40 specifically inhibits the aggregation ofnon-toxic A

42 monomers

To probe whether A

40 monomers can inhibit theaggregation of A

42 monomers, we developed anA

42 monomer stability and aggregation assayusing solution NMR. NMR is the ideal tool fortracking the aggregation of specific A

species in amixture of A

(e.g. A

40 and A

42) by selectiveisotope labelling.

H-

N heteronuclear single quan-tum coherence (HSQC) was used to selectivelymonitor the aggregation of

N labelled A

42monomers in the presence of unlabeled A

40 mo-nomers. The HSQC experiments were exclusivelydetecting NMR signals from

N-labeled A

42monomers prepared by treatment with NaOHasestablished previously at low temperatures.

40,41

Inaddition, the monomeric state of A

42 detected byNMR in our aggregation experiments was demon-stratedbytranslationaldiffusioncoefficientbetween0 °C and 37 °C, and by glutaldehyde crossing-linking experiments (Supplementary Data Figure 1).The aggregation of A

42 was monitored by thedecrease of HSQC signal of A

42 monomers as theA

42 monomers aggregate into NMR-invisible,high molecular weight fibrils. A

42 fibril formationwas confirmed by thioflavin T assay and atomicforce microscopy (Supplementary Data figure 2).NMR signals from A

42 monomers decreasedquickly in 3 h at 25 °C (Figure 1(a) and (b)).Strikingly, signals from A

42 monomers did notdecrease in the presence of four molar equivalent of A

40 monomers at 25 °C over 3 h, indicating thatA

40 keeps A

42 in a monomeric state and inhibitstheaggregationofA

42(Figure1(c)and(d)).NativePAGE and Western blot were employed to furtherconfirm the anti-A

42 aggregation effect of A

40.ThedetectionofA

42signalinthepresenceofA

40was achieved by an A

42-specific antibody in aWestern blot of the native protein gel. As shown inFigure 1(e), without A

40, the band representingA

42 monomers decreased quickly in intensity at25 °C and disappeared overnight; while in thepresence of four molar equivalent of A

40 mono-mers, there was little change in the intensity of theA

42 monomer band even after incubation over-night at 25 °C (Figure 1(f)). It has been shown thatA

40 inhibits A

42 fibrilformation by monitoringthe appearance of fibrils

–

but it is not clearwhether the inhibition protects the benign A

42monomers from aggregation or the inhibition trapsA

42 in toxic oligomeric forms. If A

40 inhibition blocks A

42 fibril formation at the oligomer stage,A

40 would not have inhibited the decrease of thesignal from A

42 monomers during A

42 aggrega-tion. In addition, if significant amount of lowmolecular weight A

42 oligomers (e.g. dimers ortrimers) were formed by A

40 inhibition, suchspecies would have been detected by NMR. How-ever, NMR signals from low molecular mass A

42oligomers were never observed in our aggregationexperiments. Thus, our NMR results demonstratedthat A

40 inhibition of A

42 fibril formation acts onA

42monomersandprotects A

42inthenon-toxic,monomeric form instead of trapping A

40 inoligomeric forms.Aseriesof control experiments were carried out toensure that A

40 indeed specifically inhibits theaggregation of A

42 monomers (Figure 2(a)). Weused both recombinant (rPeptide

†

) and syntheticA

40 peptides from two companies (Bachem andBioSource) to demonstrate that the anti-aggregationeffect did not originate from particular preparationsor sources of A

40 (Figure 2(a)). We again used bothrecombinant(rPeptide)andsyntheticA

42peptides(Bachem and BioSource) to demonstrate by Western blot of native gels (data not shown) that A

40inhibited the aggregation of A

42 peptides from allthree different sources. Negative controls werecarried out with ubiquitin, BSA and sequence-reversed A

(40-1), none of which had any anti-

†

http://www.rpeptide.com

910

40 Protects A

42 Monomer

aggregation effect upon A

42 (Figure 2(a)). Thus,the inhibition of A

42 monomers aggregation isspecific to A

40.M35 oxidation is not a factor in our aggregationexperiments. A

42 and A

40 purchased from

Figure 1.

40 protects A

42 monomers from aggre-gation. (a) and (b) The 2D

N-

H HSQC spectra of A

42NMR samples at 0 and 3 h at 25 °C, respectively, with the1D proton trace at I41

N frequency presented below the2D HSQC. After 3 h at 25 °C, almost all A

42 monomersignals disappeared. (c) and (d) The 2D

N-

H HSQCspectra of A

42 NMR samples in the presence of 4X A

40monomers at time zero and after 3 h at 25 °C, respectively,with the 1D proton trace at I41

N frequency presented below the 2D HSQC. In contrast to A

42 alone, in thepresence of four molar equivalents of A

40, the A

42monomer signal remained intact after 3 h of incubation at25 °C. (e) Native gel of A

42 with samples taken fromdifferent time-points at 25 °C. The intensity of A

42monomerbandsdecreasedrapidlyintheabsenceofA

40.(f) Westernblot ofthe native gel of A

42in the presence of four molar equivalents of A

40 at various time-points at25 °C. The A

42 monomer bands were stable even afterincubation overnight at 25 °C, demonstrating the protec-tive effect of A

40 against the aggregation of A

42monomers. The Western blot was performed with A

42from rPeptide, Bachem and BioSource and similar resultswere obtained. The A

42 NMR sample concentrationswere 20

M. A

40 andA

42peptides were obtained fromrPeptide (http://www.rpeptide.com; catalogue numbersA-1101-1, A-1102-1, A-1001-2 and A-1002-2 for

Nlabelled A

40,

N labelled A

42, natural abundanceA

40 and natural abundance A

42, respectively.),Bachem (catalogue numbers H-1194, H-1368 and H-2972for A

40, A

42 and sequence reversed A

(40-1) andBioSource (catalogue numbers 03136 and 03111 for A

40and A

42). The A

monomers were prepared using thesodium hydroxide method as described.

41,48

The lyophi-lized A

powder obtained from commercial sources wasdissolved in 10mM NaOHto 1 mg/mlandthen sonicatedfor 1 min in a waterbath to disaggregate A

. Naturalabundance A

40 and

N-labeled A

42 were mixed todesired ratios. A

was then diluted to the desiredconcentration using 20 mM potassium phosphate bufferand the pH was adjusted to 7.2 using TFA. A control

42 sample was always prepared by adding the sameamount of NaOH and adjusting the sample pH to 7.2using TFA. The 2D

N-

H NMR HSQC spectra of thefreshly prepared

N A

42 samples with different molarratios of A

40 were collected consecutively at the desiredtemperature (25 °C or 37 °C in this study) on an 800 MHzBruker Avance NMR spectrometer equipped with acryoprobe. The

N-

H HSQC and the 1D

N-selectedexperiments detected signals only from A

monomers.

The recycle delay was 1.5 s, and the number of scans was8. For 2D

N-

H HSQC, 64 complex points were taken inthe

N dimension and 1024 complex points were taken inthe

Hdimension. Thetotalacquisitiontimewas

∼

30min.The intensity of the strongest peak (A42) in A

42 wasused as an indication of relative A

42 monomer concen-tration. When the aggregation was too fast to be moni-tored by 2D NMR,

N-selected 1D was used instead. In1 D NMR spectra, areas under the peaks were integrated between 7.8 ppm and 8.5 ppm as an indication of re-lative monomer concentration. The sample temperaturewas carefully calibrated using 100% methanol. For

N-selected 1D, the number of scans was 256 with totalacquisition time of

∼

7 min. The data were processed withTopspin (Bruker Biospin), NMRPipe and Sparky (http://www.cgl.ucsf.edu/home/sparky/). For native gel analy-sis of A

aggregation, A

42 samples incubated with orwithout four molar equivalents of A

40 were analyzed bynative PAGE (15% (w/v) polyacrylamide gel; BioRadcatalogue number 161

–

1103). WesternBreeze Chromo-genic Immunodetection Kit (Invitrogen catalogue numberWB7105) was used for Western blots and A

42 specificantibody (Chemicon catalogue number AB5739,1:5000dilution) was used as the primary antibody.

42antibody did not recognize A

40 in the native gel (datanot shown).

911

40 Protects A

42 Monomer

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