Morphology-Specific Inhibition of b-Amyloid Aggregatesby 17b-Hydroxysteroid Dehydrogenase Type 10Laura Aitken+,[a] Steven D. Quinn+,[b, c, e] Cibran Perez-Gonzalez,[c, d] Ifor D. W. Samuel,[b]

J. Carlos Penedo,*[c, d] and Frank J. Gunn-Moore*[a]

Introduction

Alzheimer’s disease (AD) is the most common form of demen-tia, defined clinically as a progressive loss of declarativememory, leading to complete social dependence and eventual-ly death. It is characterised by cerebral extracellular amyloidplaques and intracellular neurofibrillar tangles.[1] The molecularmechanisms of AD pathogenesis are still not fully under-stood[2, 3] because of its intrinsic complexity. No effective treat-ment has been developed. Nevertheless, it has been shownthat polymerisation of the b-amyloid peptide (Ab) into amyloidfibrils and other morphologies, including plaques and oligo-meric species, constitutes a major hallmark of AD.[4] The devel-opment of Ab inhibitors has thus received much attention,

with peptides[5–7] and small organic molecules[8–10] now estab-lished as the two main classes of amyloid inhibitor. Ab produc-tion, aggregation and accumulation within the brain is sum-marised in Figure 1.

Since the discovery that Ab peptides are found within themitochondria of AD brains,[11] several attempts have beenmade to comprehend the mechanisms underpinning Ab-induced mitochondrial dysfunction,[12–20] and to identify recep-

A major hallmark of Alzheimer’s disease (AD) is the formationof toxic aggregates of the b-amyloid peptide (Ab). Given thatAb peptides are known to localise within mitochondria and in-teract with 17b-HSD10, a mitochondrial protein expressed athigh levels in AD brains, we investigated the inhibitory poten-tial of 17b-HSD10 against Ab aggregation under a range ofphysiological conditions. Fluorescence self-quenching (FSQ) ofAb(1–42) labelled with HiLyte Fluor 555 was used to evaluate

the inhibitory effect under conditions established to grow dis-tinct Ab morphologies. 17b-HSD10 preferentially inhibits theformation of globular and fibrillar-like structures but has noeffect on the growth of amorphous plaque-like aggregates atendosomal pH 6. This work provides insights into the depend-ence of the Ab-17b-HSD10 interaction with the morphology ofAb aggregates and how this impacts enzymatic function.

Figure 1. Production, aggregation and accumulation of Ab peptides withinthe brain is associated with neural dysfunction. Ab peptides are formed inthe cell membrane by cleavage of the amyloid precursor protein (APP) by b-secretase cleaving enzymes (BACEs). Soluble Ab oligomers interact with cell-surface membranes and receptors, as well as intracellular components tolead to neuronal dysfunction. In particular, Ab interaction with 17b-HSD10 inthe mitochondria can lead to the inhibition of 17b-HSD10 activity, in turnleading to mitochondrial dysfunction. Ab can self-assemble into pathogenicfibrils, plaques and other higher-order structures, thereby displacing vitalcellular components and leading to their malfunction.

[a] Dr. L. Aitken,+ Prof. F. J. Gunn-MooreSchool of Biology, University of St. AndrewsMedical and Biological Sciences BuildingNorth Haugh, St. Andrews, Fife, KY16 9TF (UK)E-mail : fjg1@st-andrews.ac.uk

[b] Dr. S. D. Quinn,+ Prof. I. D. W. SamuelOrganic Semiconductor Centre, SUPASchool of Physics and Astronomy, University of St. AndrewsNorth Haugh, St. Andrews, Fife, KY16 9SS (UK)

[c] Dr. S. D. Quinn,+ C. Perez-Gonzalez, Dr. J. C. PenedoSUPA, School of Physics and Astronomy, University of St. AndrewsNorth Haugh, St. Andrews, Fife, KY16 9SS (UK)E-mail : jcp10@st-andrews.ac.uk

[d] C. Perez-Gonzalez, Dr. J. C. PenedoBiomedical Sciences Research Complex, University of St. AndrewsBiomolecular Sciences BuildingNorth Haugh, St. Andrews, Fife, KY16 9ST (UK)

[e] Dr. S. D. Quinn+

Present address: WestCHEM, School of ChemistryJoseph Black Building, University of GlasgowUniversity Avenue, Glasgow, G12 8QQ (UK)

[+] These authors contributed equally to this work.

Supporting information and ORCID(s) from the author(s) for this article isavailable on the WWW under http ://dx.doi.org/10.1002/cbic.201600081.

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tors that might be involved in this process.[21] The interactionbetween mitochondrial proteins and aggregated Ab peptideshas been suggested as a pathogenic mechanism contributingto Ab neurotoxicity in AD.[17–22] For instance, 17b-hydroxyste-roid dehydrogenase type 10 (17b-HSD10; also known as amy-loid-binding alcohol dehydrogenase (ABAD))[21, 23–26] is one suchmitochondrial Ab-interacting protein.[6, 27] 17b-HSD10, a 27 kDamultifunctional enzyme expressed in all cell types, is thoughtto play a central role in the b-oxidation of fatty acids,[24, 28] iso-leucine degradation, the catalysis and oxidation of alcoholsand the reduction of aldehydes and ketones.[22] In transgenicmouse models for AD and in human AD sufferers, 17b-HSD10has been shown to have increased expression; it has gainedconsiderable attention as a result of its ability to bind Ab andto suppress Ab-induced apoptosis and free-radical generationin neurons.[11] 17b-HSD10 interacts with Ab(1-40), Ab(1–42) andAb(1–20) in the nanomolar range (Kd 40–80 nm),[11] which re-flects the low expected cellular concentration of Ab peptide inthe early stages of AD. Mutagenesis and inhibition studieshave suggested that the LD loop of 17b-HSD10 (residues C91–D119) plays a critical role in Ab binding.[11] However, the lack ofelectron density for Ab and the LD loop of 17b-HSD10 in thecrystal structure (PDB ID: 1SO8) of the complex precludes de-tailed characterisation of the binding interface. A recent NMRstudy suggested that the 17b-HSD10–Ab interaction takesplace mostly by contacts between Ab residues 17–20 (LVFF)and hydrophobic residues in LD.[29] Importantly, saturationtransfer difference (STD) NMR experiments suggested that thebinding of Ab and nicotinamide adenine dinucleotide (NAD) to17b-HSD10 is mutually exclusive, thus providing an explana-tion for how Ab-binding alters the activity of 17b-HSD10.[29]

The interaction between 17b-HSD10 and Ab in vivo has beendemonstrated within mitochondria by using co-localisationand co-immunoprecipitation techniques,[11] but many aspectsof this interaction remain poorly understood.

Here, we employed a fluorescence self-quenching (FSQ) ap-proach to investigate the interaction between 17b-HSD10 andamyloid aggregates. FSQ was based on the site-specific posi-tioning of a fluorescent dye HiLyte Fluor 555 (Figure S1 in theSupporting Information) at the N terminus of Ab. In FSQ, thefluorescence of the dye is highest for non-aggregated Ab(1–42) but becomes progressively quenched as monomers aggre-gate, thereby resulting in a combination of partially quenchedand non-emissive fluorophores (Figure 2), thus decreasing theemission as aggregation progresses. The advantages of FSQover extrinsic dyes such as thioflavin-T (ThT) to Ab aggregateshave been discussed.[29, 33–35]

There are many reasons for the present work. Firstly, wewant to assess the applicability of FSQ for investigating the in-teraction between amyloid and 17b-HSD10, as has been previ-ously shown for the interaction between Ab and Hsp20 (heatshock protein, 20 kDa).[33] Secondly, despite in vivo evidencefor an association of 17b-HSD10 and Ab, it is not knownwhether 17b-HSD10 inhibits all amyloid morphologies equallyor whether it has selectivity for some types of aggregate. Inaddition to inhibiting amyloid aggregation, it has been shownthat 17b-HSD10 when in complex with Ab becomes dysfunc-

tional, and that its enzymatic inactivation leads to an increasein mitochondrial stress and ultimately loss of neuron viabili-ty.[36] However, no in vitro data has been reported to confirmthis, and it is unclear how the two consequences of the inter-action between 17b-HSD10 and Ab (inhibition of aggregationand enzymatic inactivation of 17b-HSD10) are related.

Our results with FSQ demonstrate that 17b-HSD10 specifical-ly inhibits the formation of NaCl-induced fibrils and globularaggregates, but that this has no effect on the formation ofplaque-like structures grown at endosomal pH. Furthermore,by monitoring the conversion of NADH into NAD+ through anabsorbance assay, we investigated the impact of these mor-phologies on the enzymatic function of 17b-HSD10. Althoughthe formation of fibrils and globules was inhibited to similarextents by 17b-HSD10, their influences on 17b-HSD10 functionwere significantly different, with fibrillar structures showing thehighest enzymatic inhibition. Lastly, a comparison betweenFSQ and the widely used ThT-based assay revealed that theThT reporter dye exhibits, even in the absence of amyloid,a significant time-dependent fluorescence enhancement underthe aggregation conditions used to promote the formation ofglobular amyloid structures. Thus, our findings highlight thelimitations of ThT-based methods to monitor aggregationacross different conditions and confirm that the FSQ-basedassay is a superior analytical tool to investigate amyloid self-as-

Figure 2. FSQ assay to monitor aggregation of Ab peptides. A) HiLyte Fluor555 fluorescence is progressively quenched by FSQ as monomers aggregate.This can lead to the combination of partially quenched and completelyquenched (non-emissive) states (illustrative purposes only; not drawn toscale). B) Variation in the emission spectra of HiLyte Fluor 555 attached tothe N terminus of Ab(1–42) as a function of aggregation time. C) Representa-tive fluorescence intensity profile (lex = 547 nm) during aggregation of 1 mm

Ab555 over 30 min.

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sembly, and for searching for small-molecule or protein-basedinhibitors of this process.

Results

Monitoring amyloid aggregation and inhibition by FSQ

Aggregation of Ab(1–42) functionalised at the N terminus withHiLyte Fluor 555 (Ab555) into three morphologies (globules, fi-brils and plaques) was studied in real-time by FSQ, with Ab/17b-HSD10 molar ratios of 1:1, 2:1 and 1:0 (control). The ra-tionale for choosing these morphologies was twofold. Firstly,they constitute representative examples of the vast array ofamyloid morphologies than can be generated as a function ofenvironmental conditions.[43] Secondly, there are well-estab-lished aggregation protocols to reproducibly promote the for-mation of Ab(1–42) aggregates, and these aggregates havebeen extensively characterised by structural imaging methods,including atomic-force microscopy (AFM) and electron micros-copy (EM).[32–51] We previously demonstrated by EM that thepresence of HiLyte Fluor 555 at the N terminus of the Ab(1–42)does not affect the aggregation kinetics or the morphology ofthe aggregates.[34] Indeed, we observed amyloid structures ofAb555 with diameters of ~4 nm (fibrils) and ~22 nm (globules),identical to those obtained in the absence of N-terminal func-tionalisation.[32, 33, 52] At pH 6 and pH 4 we observed the forma-tion of amorphous plaque-like aggregates, as previously re-ported under similar acidic conditions.[52]

The concept of the FSQ-based assay should be summarisedbefore describing our findings from its application to the inter-action between Ab and 17b-HSD10. FSQ methods rely on closepositioning of the dyes as a result of the tight packing of amy-loid chains in the aggregated structures. This induces very effi-cient quenching, which results in the observed time-depen-dent decrease in fluorescence emission of HiLyte Fluor 555[34]

as aggregation progresses, as this depends on the proximityand local density of identical dyes in the neighbourhood of agiven fluorophore (Figure 2). An in-register parallel beta-sheetpacking that placed the N-terminal domains next to eachother was recently demonstrated by solid-state NMR,[43] andthis provided a molecular-level explanation of how the organi-sation of the amyloid fibrils enables very efficient self-quench-ing. As suggested in that work, the hydrophobic assembly oftwo or more of these protofibrils might be possible, and thiscould also contribute to placing the dyes in close proximity.

17b-HSD10 inhibits the formation of fibril-like structures

The kinetics of amyloid aggregation and the predominant mor-phologies arising from this process are known to be extremelysensitive to environmental conditions including pH, tempera-ture and ionic strength.[42, 52] For instance, we have previouslydemonstrated by TEM, FSQ and ThT assays that fibrillar struc-tures constitute the predominant morphology at pH 7.9 andmoderate concentrations of NaCl (150 mm).[33] Here, we usedsimilar conditions to determine the impact of 17b-HSD10 onthe formation of these amyloid aggregates. In the absence of17b-HSD10, when a non-aggregated solution of 1 mm Ab555

was incubated in 50 mm Tris·HCl (pH 7.9) with 150 mm NaCl at37 8C, we observed a fluorescence decrease of 23.1�1.2 % (Fig-ure 3 A). When 1 mm Ab555 was induced to aggregate in thepresence of 17b-HSD10 under the same conditions, significantinhibition was detected at the two Ab555/17b-HSD10 molarratios: 7.7�0.5 % quenching at 2:1 (Figure 3 B) and undetecta-ble at 1:1 (Figure 3 C).

In order to validate and compare our FSQ assay against anestablished method, we used ThT as a sensor of amyloid fibrilaggregation.[38] ThT (1.5 mm) acting on unlabelled Ab(1–42) in50 mm Tris·HCl (pH 7.9) with 150 mm NaCl underwent a 60.4�0.7 % enhancement of fluorescence emission and exhibitedtime-dependence of the aggregation process, similarly to thatobserved by FSQ; both methods reached a plateau at ~50 min(Figure 3 A). At an Ab555/17b-HSD10 ratio of 2:1, the ThT fluo-rescence enhancement was just 8.1�0.2 % (Figure 3 B), and at1:1 no enhancement was detected (Figure 3 C). Thus, in agree-ment with the data obtained by FSQ, the ThT-based assayshowed that 17b-HSD10 interacts with Ab(1–42) and efficientlyinhibits the formation of amyloid fibrils in a concentration-dependent manner (spectra of Ab555 at these Ab/17b-HSD10ratios in Figure S2; quenching rates obtained from global fitsof the aggregation profiles at these ratios by FSQ and ThT inTable S1).

The dependence of inhibitory effect on 17b-HSD10 with theAb555/17b-HSD10 molar ratio agrees well with the observationthat 0.5 mm “in vitro aged” Ab(1–42) had no effect on 17b-HSD10 dehydrogenase activity, whereas at tenfold higher con-centration Ab(1–42) caused substantial inhibition (~50 %).[23]

The inhibitory effect observed here for 17b-HSD10 against fi-brillar aggregates is similar to that observed for other proteinswith no reported chaperone activity, such as b-lactoglobulin,a-lactalbumin and lysozyme.[58] These proteins have been

Figure 3. Representative aggregation time courses obtained for 1 mm Ab555 by FSQ (*) and for unlabelled Ab(1–42) by ThT enhancement (&) under fibril-form-ing conditions in the presence of 17b-HSD10 at Ab/17b-HSD10 molar ratios of A) 1:0, B) 2:1 and C) 1:1. Solid lines are fits to monoexponential decay functionsby nonlinear squares fitting; solid lines in (C) are fits to straight lines.

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shown to delay the formation of fibrillar aggregates at 1:1molar ratio, whereas the enzymes catalase and pyruvate kinasecompletely suppressed amyloid aggregation at a much lowerratio (protein/Ab, 1:100).[58] Several mechanisms have been pro-posed to explain the inhibitory effect of these proteins at sub-stoichiometric amounts, including interactions with monomericpeptides in the b-sheet conformation and the formation ofcomplexes with pre-fibrillar oligomers to slow or prevent thegrowth into fibrils. It is unclear whether the inhibitory effectwe observed for 17b-HSD10 towards the formation of fibrillaroligomers in both the FSQ and ThT assays involve similarmechanisms to those when acting on non-chaperone proteins.However, the fact that no interaction between 17b-HSD10 andmonomeric Ab was observed by surface-plasmon resonance(SPR)[11] and that no binding was observed in the NMR titrationof 17b-HSD10 into amyloid monomers[29] suggests that 17b-HSD10 might act by forming “dead-end” Ab–17b-HSD10 aggre-gates, as observed for b-lactoglobulin and catalase, as deter-mined by 1H,15N HSQC NMR spectroscopy.

17b-HSD10 inhibits the formation of rapidly growingglobular aggregates

The formation of soluble amyloid oligomers and spherical ag-gregates is known to induce rapid cell degeneration, thus sug-gesting that soluble Ab aggregates have higher toxicity thanplaques and Ab fibrils.[51] The formation of these globular spe-cies is known to be accelerated in the presence of lipids andother interfaces (a process that, under certain conditions, canlead to the formation of Ab pores that disrupt the cellularmembrane).[48] The addition of small amounts of fluorinatedsolvents such as 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) and2,2,2-tri-fluoroethanol (TFE) as co-solvents to a solution ofmonomeric Ab has been demonstrated as a method to inducethe formation of amyloid aggregates.[46] For instance, HFIP at�4 % (v/v) induced the formation of globular structures invitro.[42] HFIP droplets formed at these concentrations in anaqueous solution have been hypothesised to act as a growinginterface where amyloid monomers can nucleate and acceler-ate growth, as observed at the interface of biomimetic mem-branes, including ganglioside micelles and lipid-rafts.[48] More-over, it has been proposed that the toxicity of these globular

structures is responsible for the cognitive problems associatedwith the use of polyfluorinated anaesthetic compounds.[55]

In order to investigate the interaction and potential inhibito-ry properties of 17b-HSD10 under these globular-forming con-ditions, we carried out FSQ experiments with 1.5 % (v/v) HFIP.We previously showed that, at this concentration, HFIP in-duced Ab aggregation over several minutes, easily followed inreal-time by FSQ.[33–34] In the absence of 17b-HSD10, the addi-tion of 1.5 % (v/v) HFIP to a non-aggregated solution of 1 mm

Ab555 in 50 mm Tris·HCl buffer (pH 7.9) at 4 8C with continuousagitation induced a decrease in fluorescence intensity of 60�4 % over 30 min (Figure 4 A). When 1 mm Ab555 was induced toaggregate under the same conditions but with 17b-HSD10 at2:1 and 1:1 Ab555/17b-HSD10, self-quenching was 19�4 % and13�3 %, respectively (Figure 4 A). Importantly, no quenchingwas observed over 30 min when Ab555 and 17b-HSD10 were in-cubated at a 1:1 molar ratio in the absence of HFIP (Figure S3),thus confirming that under these conditions, 17b-HSD10 inter-acts with Ab during HFIP-induced self-assembly.

For comparative purposes, we also explored the use of ThTfluorescent enhancement to monitor the formation of theseglobular structures as well as its inhibition by 17b-HSD10. In-terestingly, injection of <4 % (v/v) HFIP into 1.5 mm ThT (in50 mm Tris·HCl, pH 7.9, 4 8C) induced a significant (~ fourfold)increase in fluorescence over 5–10 min, even in the absence ofamyloid (Figure 4 B). ThT emission enhancement has been ex-tensively used as a reporter of HFIP-induced aggregation;[41, 45]

the emission behaviour of ThT in different restricted media, in-cluding b-sheet and non-b-sheet cavities, such as cyclodextrin,polymer films, and micelles, has been discussed.[45] In contrast,no detailed investigation of ThT in mixtures of water and poly-fluorinated solvents, which are known to form microdropletsand solvent clusters,[47] has been reported. However, the forma-tion of HFIP micro-droplets acting as adsorption platforms forBSA by TEM analysis has been reported,[42] and micro-heteroge-neities in HFIP–water mixtures have been studied in detail byNMR and small-angle neutron scattering (SANS); these re-vealed a correlation in lengths ranging from 7 to 10 �.[54] Inter-estingly, these lengths match the diameter (8–9 �) of g-cyclo-dextrin cavities, which are known to promote the characteristicenhancement in ThT emission.[45] From NMR and SANS data itwas suggested that these micro-heterogeneities are maximisedat HFIP concentrations of 30–35 % (v/v), and that the clusters

Figure 4. A) Representative aggregation time courses obtained by FSQ for HFIP-induced aggregation of Ab555 in the presence of 17b-HSD10 at the indicatedmolar ratios at 4 8C after injection of 1.5 % (v/v) HFIP. Solid lines are fits to exponential decay functions. B) ThT fluorescence enhancement in the absence ofAb(1–42) at the indicated concentrations of HFIP.

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organise with the trifluoromethyl (CF3) groups pointing insidethe core and hydroxy (OH) groups forming hydrogen bondswith the surrounding water molecules.[54] At lower concentra-tions of HFIP (<5 % v/v), oligomers were detected by MS, buttheir size and stoichiometry were unknown.[54]

The influence of HFIP on the photophysical properties ofThT in aqueous solution was further investigated by monitor-ing its absorption spectrum as a function of HFIP concentra-tion. We observed a significant decrease in absorption after ad-dition of 1.5 % and 4 % (v/v) HFIP (Figure S4), whereas higherconcentrations (~30 %) reversed this behaviour and shifted theabsorption maximum (Figure S4). Similar features were foundin the absorption (Figure S5 A) and emission spectra (Fig-ure S5 B) when using TFE in place of HFIP, although this wasless pronounced. These results suggest that the observed dif-ferences in photophysical properties of ThT might arise froman interaction with the droplet–water interface, which alters itsmolecular-rotor properties, or from local changes in the dielec-tric constant, which is known to influence ThT emission.[47] Thisclearly indicates that care should be taken when using it asa reporter of amyloid aggregation in mixtures of water andpolyfluorinated solvents.

In view of the uncertainties associated with the use of ThTto monitor HFIP-induced aggregation and to take into accountthe spherical organisation of the globular aggregates, we em-ployed dynamic light scattering (DLS) as an alternative to FSQ,and compared the results. We wished to determine how theseHFIP-induced aggregates are affected by the presence of 17b-HSD10. In the absence of 17b-HSD10, the values for the hydro-dynamic radius (RH) were broadly distributed between 60 and400 nm (Table S2). These values are in good agreement withthose previously reported by DLS and multi-angle light scatter-ing (200–300 nm) for globule growth at similar HFIP concentra-tions.[41] Upon incubation with 17b-HSD10, a marked decreasein RH was observed at both 2:1 and 1:1 Ab/17b-HSD10: 44.9–108.0 and 36–150 nm, respectively, correspond to a decreasein mass of close to an order of magnitude (Table S2). We inter-pret the decrease in average RH as additional evidence for theinhibitory effect of 17b-HSD10 on globular aggregation.

Taken together, the FSQ and DLS data provide clear evi-dence of 17b-HSD10 inhibiting the formation of globular ag-gregates, and suggest that the interaction of 17b-HSD10 withAb(1–42) must be at a rate that competes with the relativelyfast average aggregation rate reported for the globular aggre-gate (<2 min�1).[34] It has been shown that freshly prepared

HFIP-induced globules are highly unstable after dilution andevolve over time into more stable and still soluble fibrillar ag-gregates.[34, 42] Based on this, we hypothesise that the inhibitoryaction of 17b-HSD10 towards globular species might involvethe interaction and sequestering of Ab material that is indynamic exchange with the globular structures. Equilibriumbetween globules and low-molecular-weight Ab structures isstrongly supported by the evolution of these globules intomore-stable structures over time. Remarkably, the ability of17b-HSD10 to disrupt the formation of globular aggregateswas significantly more pronounced than that reported forchaperone proteins aB-crystallin[57] and Hsp20,[33] which consti-tute examples of natural defence against protein misfolding.[33]

For instance, wild-type Hsp20 did not interfere with the forma-tion of globular aggregates even at an Ab/Hsp20 molar ratioof 1:2 (higher than used here for 17b-HSD10).[33]

17b-HSD10 does not inhibit amorphous plaque-likeaggregate formation under acidic conditions

Ab peptides are found within the mitochondria of AD brains,[11]

and it has been suggested that soluble Ab aggregates (mono-meric and oligomeric structures) enter the mitochondria viathe translocase of the outer membrane (TOM) machinery.[12] As17b-HSD10 is a mitochondrial protein, it is unlikely to interactwith large plaque-like material usually found to aggregate inlysosomes under slightly acidic conditions (pH 6); however, nodirect experimental evidence had been reported to confirmthis. Therefore, we decided to take advantage of the robust-ness of FSQ over a wide pH range to test the potential interac-tion between 17b-HSD10 and plaque-like aggregates formedat Ab555/17b-HSD10 molar ratios similar to those used to inves-tigate the interaction with globules and fibrils. In the controlexperiment, 1 mm Ab555 in 2-(N-morpholino)ethanesulfonic acid(MES) buffer (50 mm, pH 6) at 37 8C induced a decrease in fluo-rescence of 21.1�0.5 % over 50 min (Figure 5 A). However, nosignificant inhibition was detected for Ab555/17b-HSD10 molarratios of 2:1 and 1:1 (FSQ largely unchanged: 21.1�0.9 % and14.4�0.7 %, respectively; Figure 5 B, C; Ab555 quenching underthese conditions in the presence and absence of 17b-HSD10 isin Table S3; spectra at the different molar ratios are in Fig-ure S6).

Although amyloid staining methods based on extrinsicprobes, such as ThT, are known to suffer from a decrease in af-finity (<30-fold) when the pH is decreased from pH 8.5 to 6,[40]

Figure 5. Representative aggregation time courses obtained for 1 mm Ab555 by FSQ (*) and for unlabelled Ab(1–42) by ThT enhancement (&) at pH 6 in thepresence of 17b-HSD10 at Ab/17b-HSD10 molar ratios of A) 1:0, B) 2:1 and C) 1:1. Solid lines are fits to a monoexponential decay functions by nonlinearsquares fitting.

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they have been used to monitor aggregation at endosomalpH. For comparison with the FSQ data, we carried out an iden-tical set of experiments for ThT emission. For unlabelled Ab(1–42) in the absence of 17b-HSD10 we observed a 32.0�0.2 %enhancement in fluorescence over 50 min (Figure 5 A). AtAb(1–42)/17b-HSD10 molar ratios of 1:1 and 2:1, ThT enhance-ment remained largely unchanged (emission enhancements of23.6�0.3 and 22.8�0.2 %, respectively; Figure 5 B, C). The ob-servation with ThT that 17b-HSD10 does not inhibit aggrega-tion at pH 6 is therefore entirely consistent with our analysisby FSQ.

Amyloid-induced inactivation of 17b-HSD10 enzymaticfunction

17b-HSD10 was the only human protein found to interact withAb in a yeast two-hybrid screen,[21] thus suggesting that Ab-in-duced cytotoxicity occurs mostly by its interaction with 17b-HSD10.[18, 36, 50] The current hypothesis is that HSD10 in complexwith Ab becomes dysfunctional and leads to increased mito-chondrial stress and ultimately loss of neuron viability.[50] Theinhibitory effect of Ab(1–42) on 17b-HSD10 binding to theNADH/NAD+ cofactor has already been confirmed by SPR andSTD NMR.[29] However, the precise morphological state ofAb(1–42) that inactivates 17b-HDS10 has not been established.Our FSQ data indicate that 17b-HDS10 interacts with fibrillarand globular amyloid structures but not with plaques; there-fore, we decided to investigate the enzymatic activity of 17b-HDS10 under fibril- and globule-growing conditions.

Before assessing the influence of amyloid aggregation stateon the function of 17b-HSD10, it was important to verify thatthe enzyme was active in the buffers required to promotethese morphologies. For this, we employed an assay that mon-itors changes in NADH absorbance. During 17b-HSD10 conver-sion of acetoacetyl coenzyme A, the cofactor NADH is convert-ed into NAD+ . Thus, the loss of NADH absorbance (340 nm) isa measure of 17b-HSD10 activity (Figure 6 A). When incubatedat 5 mg mL�1, 17b-HSD10 showed a specific activity of 4 mmolmin�1 mg�1 (Figure 6 B); the negative control (no 17b-HSD10 inthe reaction mixture) showed minimal background (~0.2 mmolmin�1 mg�1). Under conditions that promote both oligomeric/fibril formation (50 mm Tris·HCl (pH 7.9) containing 150 mm

NaCl) and globular aggregation (50 mm Tris·HCl (pH 7.9) con-taining 1.5 % (v/v) HFIP) there was no change in specific activi-ty (Figure 6 B). Under plaque-growing conditions (50 mm MES,pH 6.0, 37 8C) there was a very slight decrease in activity (<4 %; Figure 6 B).

When the absorbance assay was repeated under conditionsthat induce amyloid aggregation we observed a morphology-dependent response. Under fibril-promoting conditions, enzy-matic inhibition depended on the Ab(1–42)/17b-HSD10 molarratio: the enzymatic activity decreased by 3.4�1.9 % at 1:1and 20.2�1.6 % at 2:1 (Figure 6 C). For conditions that pro-mote the formation of globular structures, we observed a verymoderate decrease in the enzymatic activity (5.4�1.7 %), evenat the highest ratio (5:1). These data suggest that differentamyloid morphologies exhibit different inhibition of 17b-

HSD10, and confirm that, in a rich amyloid environment, Ab(1–42) fibrils have a deleterious effect on 17b-HSD10 activity.

Discussion

A bar plot summarising relative fluorescence self-quenchingfor the aggregation of 1 mm Ab555 into globular, fibrils andamorphous plaque-like aggregates in the presence and ab-sence of 17b-HSD10 is shown in Figure 7 A; the results froma ThT-based assay for fibrils and plaques is shown in Figure 7 B.Both methods provide strong evidence for 17b-HSD10 prefer-entially interacting with certain amyloid morphologies. Specifi-cally, 17b-HSD10 demonstrated inhibitory potential towardsglobular and fibrillar structures formed at neutral pH, but hadno effect on the aggregation of plaque structures formed atpH 6. A degree of preferential interaction of 17b-HSD10 withsome amyloid aggregates could be anticipated, as the crystalstructure of the 17b-HSD10/Ab(1–42) complex (PDB ID: 1SO8)reveals a large cavity (~70 �), which could easily accommodatefibrillar structures (40–60 �), but not, for instance, plaque-likeaggregates.[11]

The stoichiometry of the Ab-17b–HSD10 complex has notbeen determined, but it has been hypothesised that 17b-HSD10 does not interact with Ab(1–42) monomers in SPR and

Figure 6. 17b-HSD10 enzyme activity assessed by NADH absorbance. A) Con-version of acetoacetyl coenzyme A uses NADH as a cofactor (conversion intoNAD+). The loss of absorbance of NADH (340 nm) gives the rate of 17b-HSD10 activity. Without 17b-HSD10 (c) there is minimal NADH consump-tion in comparison to when it is present (c). B) 17b-HSD10 enzymatic ac-tivity (mmol min�1 mg�1) in the absence of amyloid in different aggregationbuffers: A) fibril-growing buffer (10 mm Tris·HCl pH 7.5, 150 mm NaCl, 10 %(v/v) glycerol) ; B) pH6 aggregation buffer (50 mm MES pH 6.0) ; C) HFIP-in-duced aggregation buffer (50 mm Tris·HCl pH 7.9, 1.5 % HFIP (v/v)). C) Per-cent enzyme activity in the presence of 1 mm freshly prepared Ab(1–42)under fibril- and globule-growing conditions at the indicated molar ratios.

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NMR experiments.[11, 29] Therefore, it is unlikely that physicaland/or structural differences between the monomeric state ofAb(1–42) at different pH values can account for the observeddiscrimination between plaque-like structures formed at pH 6and fibrils and globules formed at neutral pH. Moreover, underconditions identical to those used to generate amorphousplaque-like aggregates, 17b-HSD10 showed enzymatic levelscomparable to that at neutral pH (Figure 6 B), thus confirmingthat the numerous interactions formed by the substrate withloops LF, LE and LD are retained under slightly acidic conditions.In particular, the LD loop region, which has been shown by mu-tagenesis studies and NMR analysis to form the interface withAb,[29] should remain accessible for Ab binding.

Because 17b-HSD10 was present in the aggregation mixturefrom the very early stages in all our assays, we hypothesisethat either differences in the structures of the aggregation in-termediates or in the kinetics of the aggregation between neu-tral and slightly acidic conditions might be responsible for theobserved differences. This is supported by evidence suggestingthat indeed both amyloid morphology and aggregation kinet-ics at pH 6 are remarkably different to those at neutral pH.[39, 60]

Recent in vivo studies have demonstrated that neuronal Ab(1–42) aggregates faster in lysosomes and without a detectablelag phase; this finding was attributed to the acidic environ-ment bringing the peptide closer to its isoelectric point.[61] Byusing a combination of fluorescence methods and structuraltechniques, it was shown that amyloid aggregation at pH 5.8 ischaracterised by the rapid formation (~10 s) of amorphous ag-gregates, with sizes ranging from 50 to 500 nm.[40] Importantly,as determined by CD, these aggregates did not contain a-heli-cal or b-structures, and they were unable to seed fibril forma-tion.[40] It was also demonstrated that the aggregates evolveinto smaller structures (30–80 nm) after 30 min, in good agree-ment with the time for aggregation observed in our FSQ andThT experiments (Figure 5). The time scale of the initial growthphase is within our mixing time, so it is likely that with thetime course of our assay, we were monitoring the disappear-ance of the initially formed aggregates as they slowly evolvedinto other morphologies. As reported by Gorman and co-work-ers,[39] these slow-forming morphologies also display very little(if any) b-structure. However, a lack of b-sheet structure atacidic pH cannot alone account for the observed lack of aggre-

gation inhibition, as it has been shown that 17b-HSD10 doesnot bind Ab(25–35), which has been shown to exhibit b-sheetconformation in CD and NMR studies.[63]

In contrast, the conformation of residues 17–20 of Ab(1–20),which constitute the binding interface with 17b-HSD10,[11] isknown to exhibit a random-coil/a-helix/b-sheet equilibriumthat is highly dependent on pH.[61, 63, 64] At pH 7–8, it has beendemonstrated that residues 10–28 (containing the putative17b-HSD10-binding interface) are in equilibrium betweenrandom coil and a-helical conformations. Over pH 4–7, thisequilibrium includes a further b-sheet conformation. Taken to-gether, our data suggest that such pH-induced conformationalre-arrangement of the N terminus might be crucial to modulat-ing the interaction of 17b-HSD10 with particular amyloid mor-phologies.

We extended our analysis of the interaction between Ab(1–42) and 17b-HSD10 by exploring how different aggregationconditions affect enzymatic activity. Although 17b-HSD10 effi-ciently inhibited the formation of fibrils and globules, rich amy-loid conditions that promote the formation of fibrillar struc-tures constitute a more toxic environment, and resulted in a20.2 % decrease in enzymatic function (Figure 6 C). The crystalstructure of 17b-HSD10 bound to Ab indicates the presence ofa large solvent cavity involving the LD loop, with estimated di-mensions of 70 �. This dimension is relatively close to the di-ameter of amyloid fibrils (4–6 nm) but much smaller than theaverage diameter for globular structures (~22 nm) measuredby TEM and DLS.[34, 42] Therefore, it is possible that the higherenzymatic inhibition under fibril-forming conditions arises fromthe ability of these structures to drift and fit into this cavityand block the active site, thus inactivating the enzyme.

Our data also provide evidence for uncoupling the determi-nants that influence the formation of Ab–17b-HSD10 com-plexes from those that mediate suppression of enzymaticactivity by amyloid fibrils at much higher Ab concentrations.High amyloid Ab(1–42)/17b-HSD10 molar ratios were requiredfor a significant impact on activity; this agrees well with previ-ous studies where the concentration of Ab for half-maximal in-hibition was in the micromolar range (1–3 mm), rather than thenanomolar range (~40 nm) required for efficient binding, andmore likely representative of the intracellular environment.[18]

The need for a rich amyloid environment to impair activity isalso supported by in vivo studies with SH-SY5Y (human neuro-blastoma) cells, which showed a decrease in the efficiency of17b-HSD10-catalysed estradiol-to-oestrone conversion upon in-cubation with a high concentration of Ab.[50] It has been sug-gested that the N-terminal domain of Ab is involved in the ini-tial association to 17b-HSD10, and in a rich amyloid environ-ment the C-terminal portion it is free to interact and recruit ad-ditional Ab molecules, thereby resulting in a macromolecularcomplex that distorts the enzyme and alters its function.[18]

From all the evidence, we hypothesise that intracellular amy-loid toxicity arises not exclusively from the formation of Ab–17b-HSD10 complexes but from the recruitment of amyloidmolecules that might disrupt, for example, the tertiary struc-ture of the enzyme, thereby modifying substrate specificity orlocalisation, and thus increasing cell vulnerability.

Figure 7. Differences in A) fluorescence self-quenching of 1 mm Ab555, andB) ThT enhancement for Ab(1–42) aggregation into globules, fibrils andplaques at the indicated molar ratios, Data are mean�SEM (n�3). ND: noaggregation detected.

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Conclusion

Biophysical characterisation of the Ab–17b-HSD10 interactionis pivotal for a full understanding of the interaction betweenAb accumulated inside nerve cells and intracellular proteins.Unfortunately, the exact details of the Ab–17b-HSD10 interac-tion have not been elucidated by X-ray crystallography and, asdemonstrated here, ThT-based methods to monitor amyloidaggregation and its inhibition by 17b-HSD10 are not suitableunder all experimental conditions. Consequently, the range ofamyloid morphologies capable of interacting with 17b-HSD10has remained elusive. In this study, we took advantage of therobustness of fluorescence self-quenching to monitor the in-hibitory efficiency of 17b-HSD10 against a range of amyloidmorphologies. Our results demonstrate preferential inhibitionby 17b-HSD10 towards globular and fibril-like aggregationunder neutral conditions. The lack of inhibition towards amor-phous plaque-like aggregates generated under slightly acidicconditions was discussed in terms of the known influence ofpH on the conformational equilibrium of the Ab peptide N-ter-minal fragment, which contains the putative 17b-HSD10-bind-ing interface. From the morphologies that were found to inter-act with 17b-HSD10, inhibition of acetoacetyl-CoA reductionwas only detected under fibril-forming conditions and whenAb(1–42) was at concentrations much higher than those re-quired for efficient binding. This work provides insights intothe dependence of the Ab–17b-HSD10 interaction on the mor-phology of amyloid aggregates and suggests that the determi-nants that mediate Ab(1–42) binding to 17b-HSD10 are differ-ent from those that suppress 17b-HSD10 activity.

Experimental Section

All aqueous solutions were prepared in deionised water (Merck Mil-lipore) ; all chemicals were purchased from Sigma–Aldrich unlessstated otherwise.

17 b-HSD10 purification : Cell pellets of Escherichia coli BL21-Co-donPlus (Agilent Technologies) containing Histev-17b-HSD10 pro-tein[65] were re-suspended for 30 min at 4 8C in lysis buffer (sodiumphosphate (20 mm, pH 7.5) containing imidazole (30 mm), NaCl(500 mm) and glycerol (10 %, v/v)) supplemented with completeEDTA-free protease inhibitor tablets (Roche), lysozyme (1 mg mL�1),DNase (20 mg mL�1) and Triton X-100 (0.1 %, v/v). Cells were lysedby passage at 20 6842.7 kPa through a cell disruptor (Constant Sys-tems Ltd, Daventry, UK), and the lysate was cleared by centrifuga-tion in a Sorvall Evolution RC (rotor S5–34, 55–34 angle,20 500 rpm, 30 min, 4 8C). Cleared lysate was filtered (0.44 mmmembrane; Whatman) then applied to a Ni-NTA column (GEHealthcare) equilibrated with lysis buffer, and the protein waseluted in lysis buffer containing 300 mm imidazole. Tobacco etchvirus (TEV) protease was added at a molar ratio of 1:10, to cleavethe histidine tag, and the protein was dialysed into Tris·HCl(20 mm, pH 7.5) with imidazole (30 mm), NaCl (500 mm) and glyc-erol (10 %, v/v), supplemented with EDTA (1 mm) and DTT (1 mm)to aid solubility. Protein digestion and dialysis was carried out at4 8C for 16 h.

Complete digestion was verified by SDS-PAGE, then 17b-HSD10protein was passed through a second Ni-column, and the flow-

through was concentrated to ~7 mL in a Vivaspin column (10 kDaMWCO, GE Healthcare) before final purification by gel filtration toremove imidazole (Hi-Load 16/60 Superdex 75 prep grade column,GE Healthcare; flow rate 1.5 mL min�1). The protein was eluted ingel filtration buffer (Tris·HCl (10 mm, pH 7.5) NaCl (150 mm), glycer-ol (10 %)) and concentrated in the same Vivaspin column to10 mg mL�1. Aliquots (10 mL) were taken and flash frozen in liquidnitrogen for storage at �80 8C.

17 b-HSD10 Activity Assay : 17b-HSD10 was diluted (0.2 mg mL�1)in assay buffer (Tris·HCl (10 mm, pH 7.5), NaCl (150 mm), glycerol10 % (v/v)) at 30 8C. Acetoacetyl-CoA (AcAcCoA) was prepared at4.8 mm in assay buffer; NADH was prepared at 10 mm in assaybuffer. Enzyme activity was measured in a Nunc 96-well plate withthe following concentrations: AcAcCoA (120 mm), NADH (250 mm),17b-HSD10 (8 mg mL�1). 17b-HSD10 was added to initiate the reac-tion, and the initial rate was recorded over 30 s in a FLUOstar platereader (BMG Labtech; l= 340 nm, T = 30 8C, 0.5 s orbital measuringintervals). Enzyme activity was calculated from e= 6220 m

�1 cm�1

(NADH absorption coefficient); the NADH rate of consumption isthe same as the AcAcCoA rate of reduction. Assays were per-formed in triplicate; data are mean�SEM.

HiLyte Fluor 555 : Synthetic dye labelled Ab1–42 peptides incorpo-rating HiLyte Fluor 555 were purchased from Anaspec (Fremont,CA) and used without further purification (see the Supporting In-formation).

Amyloid monomer preparation and fluorescence spectroscopyof Ab(1–42) aggregates : The methods for amyloid monomer prep-aration and fluorescence spectroscopy of Ab(1–42) aggregates canbe found in the Supporting Information.

Aggregation protocols : Aggregation protocols were as previouslyreported[33, 34, 52] (details in the Supporting Information).

Dynamic light scattering : see the Supporting Information.

Acknowledgements

This research is part-funded by the MSD Scottish Life Sciencesfund. As part of an on-going contribution to Scottish life sciences,MSD Limited, a global healthcare leader, has given substantialmonetary funding to the Scottish Funding Council (SFC) for distri-bution via the Scottish Universities Life Science Alliance (SULSA)to develop and deliver a high-quality drug discovery researchand training programme. All aspects of the programme havebeen geared towards attaining the highest value in terms of sci-entific discovery, training and impact. The opinions expressed inthis research are those of the authors and do not necessarily rep-resent those of MSD Limited, or its affiliates. This research wasalso kindly supported by Alzheimer’s Society, The Barcapel Foun-dation, EPSRC and BBSRC.

Keywords: Alzheimer’s disease · amyloid beta-peptides ·dehydrogenases · fluorescent probes · neurochemistry

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Manuscript received: February 11, 2016

Accepted article published: March 16, 2016

Final article published: && &&, 0000

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L. Aitken, S. D. Quinn, C. Perez-Gonzalez,I. D. W. Samuel, J. C. Penedo,*F. J. Gunn-Moore*

&& –&&

Morphology-Specific Inhibition of b-Amyloid Aggregates by 17b-Hydroxysteroid Dehydrogenase Type10

17 b-HSD10 interaction with Ab amy-loid: what type of amyloid? 17b-hy-droxysteroid dehydrogenase type 10 in-teracts with b-amyloid (Ab) aggregatesand suppresses Ab-induced apoptosis inneurons, but the aggregate morphologyinhibited by 17b-HSD10 remains un-known. Fluorescence self-quenchingdemonstrated that fibrils and globularaggregates, but not plaques, are target-ed by 17b-HSD10.

ChemBioChem 2016, 17, 1 – 10 www.chembiochem.org � 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim10&

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