REDUCTION OF BETA-AMYLOID LEVELS BY NOVEL PKC EPSILON

ACTIVATORSThomas J. Nelson 1, Changhai Cui 1, Yuan Luo 2, and Daniel L. Alkon 1

From the Blanchette Rockefeller Neurosciences Institute1, Morgantown WV 26506, USA and the Departmentof Pharmaceutical Sciences, University of Maryland School of Pharmacy2, Baltimore, MD 21201

Running head: Reduction of beta-amyloid by PKC epsilon

Address correspondence to: Thomas J. Nelson, Blanchette Rockefeller Neurosciences Institute, 8 Medical Cen-ter Drive, Morgantown WV 26506, USA; Email: tjnelson@brneurosci.org

ABSTRACT

Isoform-specific protein kinase C (PKC) activatorsmay be useful as therapeutic agents for the treat-ment of Alzheimer’s disease. Three new epsilon-specific PKC activators, made by cyclopropana-tion of polyunsaturated fatty acids, have beendeveloped. These activators, AA-CP4, EPA-CP5,and DHA-CP6, activate PKC epsilon in a dose-dependent manner. Unlike PKC activators thatbind to the 1,2-diacylglycerol binding site, suchas bryostatin and phorbol esters, which produceprolonged downregulation, the new activators pro-duced sustained activation of PKC. When appliedto cells expressing human APPSwe/PS1Delta, whichproduce large quantities of Abeta, DCP-LA andDHA-CP6 reduced the intracellular and secretedlevels of Abeta by 60-70%. In contrast to themarked activation of alpha-secretase produced byPKC activators in fibroblasts, the PKC activatorsproduced only a moderate and transient activationof alpha-secretase in neuronal cells. However, theyactivated endothelin-converting enzyme (ECE) to180% of control levels, suggesting that the Abeta-lowering ability of these PKC epsilon activators iscaused by increasing the rate of Abeta degradationby ECE, and not by activating nonamyloidogenicAPP metabolism.

Alzheimer’s disease (AD) is characterized by theaccumulation of aggregatedb-amyloid (Ab), which isa 4 kDa peptide produced by the proteolytic cleav-age of amyloid precursor protein (APP) byb- andg-secretases. Oligomers of Ab are the most toxic, whilefibrillar Ab is largely inert. Monomeric Ab is found innormal patients and has an as-yet undetermined func-

tion. The earliest consistent cytopathological change inAD is loss of synapses [1, 2]. Finding a way to protectagainst the loss of synapses is a major therapeutic goal.

PKC activators have demonstrated neuroprotectiveactivity in animal models of Alzheimer’s disease [3],depression [4], and stroke [5]. Bryostatin, a potentPKC activator, also increases the rate of learning in ro-dents, rabbits, and invertebrates [4, 6, 7]. This effect isaccompanied by increases in levels of synaptic proteinsspinophilin and synaptophysin and structural changesin synaptic morphology [8]. PKC activators also canreduce the levels of Ab and prolong the survival ofAlzheimer’s disease transgenic mice [3].

Evidence suggests that PKCa ande are the mostimportant PKC isoforms in eliciting these changes. An-tisense inhibition of PKCa blocks secretion of sAPPa,while indirect activators of PKC, such as carbachol,increase sAPPa secretion [9]. Experiments with spe-cific PKC isozyme inhibitors also point to PKCe asthe isozyme that most effectively suppresses Ab pro-duction [10]. Thus, isoform-specific PKC activatorsare highly desirable as potential anti-Alzheimer drugs.Specific activators are preferable to compounds suchas bryostatin that show less specificity among conven-tional and novel forms of PKC, because nonspecific ac-tivation of PKCd or b could produce undesirable sideeffects.

One compound known to activate PKCe specifi-cally is DCP-LA (8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid), a derivative of linoleicacid in which the double bonds are replaced bycyclopropane groups [11]. Like the polyunsaturatedfatty acids docosahexaenoic acid and arachidonic acid,DCP-LA binds to the phosphatidylserine binding siteand specifically activates PKCe [11]. However, DCP-

∗Abbreviations: APP amyloid precursor protein, AD Alzheimer’s disease, Aβbeta-amyloid peptide, ECE endothelin-convertingenzyme, PMA phorbol 12-myristate 13-acetate, TACE tumor necrosis factor-α-converting enzyme, DAG 1,2-diacyl-sn-glycerol, PKCprotein kinase C

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http://www.jbc.org/cgi/doi/10.1074/jbc.M109.016683The latest version is at JBC Papers in Press. Published on October 22, 2009 as Manuscript M109.016683

Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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LA requires relatively high concentrations to produceits maximal effect. Therefore, we synthesized a varietyof cyclopropanated fatty acid derivatives and comparedtheir ability to activate PKCe. Two such compounds,DHA-CP6 and EPA-CP5, were found to be highly spe-cific for the PKCe and were effective at 1000- and 100-× lower concentrations than DCP-LA. DHA-CP6 alsoreduced the levels of Ab by 60-70% in cells expressingAPP with the Swedish mutation. This effect was notcaused by activation ofa-secretase, but may have beencaused by increased Ab degradation by endothelin-converting enzyme.

Experimental Procedures

Materials—Culture media were obtained from K-D Medical (Columbia, MD) or Invitrogen (Carlsbad,CA). Ab1-42 was purchased from Anaspec (San Jose,CA). Polyunsaturated fatty acid methyl esters were ob-tained from Cayman Chemicals, Ann Arbor, MI. Otherchemicals were obtained from Sigma-Aldrich Chemi-cal Co. (St. Louis, MO).

Cell culture—Human SH-SY5Y neuroblastomacells (ATCC) were cultured in 45% F12K / 45% MEM/ 10% FCS. Mouse N2A neuroblastoma cells werecultured in DMEM/10%FCS without glutamine. Rathippocampal neurons from from 18-day-old embry-onic Sprague Dawley rat brains were plated on 24-well plates coated with poly-D-lysine (Sigma-Aldrich,St. Louis, MO) in B-27 neurobasal medium contain-ing 0.5 mM glutamine and 25µM glutamate (Invitro-gen, Carlsbad, CA) and cultured for three days in themedium without glutamate. The neuronal cells weregrown under 5% CO2 for 14 days in an incubator main-tained at 37◦ C.

All experiments on cultured cells were carried outin triplicate unless otherwise stated. All data points aredisplayed as mean±SE. DCP-LA was used as its freeacid in all experiments, while DHA-CP6, EPA-CP5,and AA-CP4 were used as their methyl esters.

TACE—TACE was measured [12] by incubating 5µl cell homogenate, 3µl buffer (50 mM Tris-HCl 7.4+ 25 mM NaCl + 4% glycerol), and 1µl of 100 µMTACE substrate IV (Abz-LAQAVRSSSR-DPa) (Cal-biochem) for 20 min at 37◦ in 1.5-ml polypropylenecentrifuge tubes. The reaction was stopped by coolingto 4◦ . The samples were diluted to 1 ml and the flu-orescence was rapidly measured (ex=320 nm, em=420nm) in a Spex Fluorolog 2 spectrofluorometer.

Protein kinase C assay—After removal of culturemedium, cells were scraped in 0.2 ml homogenizationbuffer (20 mM Tris-HCl, pH 7.4, 50 mM NaF, 1µg/mlleupeptin, and 0.1 mM PMSF) and immediately ho-mogenized in the cell culture plate by sonication in a

Marsonix microprobe sonicator (5 sec, 10W). Aliquotswere transferred immediately after sonication to 0.5-mlcentrifuge tubes and frozen at -80◦ . To measure PKC,10µl of cell homogenate or purified PKC isozyme wasincubated for 15 min at 37◦ in the presence of 10µMhistones, 4.89 mM CaCl2, 1.2µg/µl phosphatidyl-L-serine, 0.18µg/µl 1,2-dioctanoyl-sn-glycerol, 10 mMMgCl2, 20 mM HEPES (pH 7.4), 0.8 mM EDTA, 4mM EGTA, 4% glycerol, 8µg/ml aprotinin, 8µg/mlleupeptin, and 2 mM benzamidine. 0.5µCi [γ32P]ATPwas added and32P-phosphoprotein formation wasmeasured by adsorption onto phosphocellulose as de-scribed previously [13]. For measurements of activa-tion by DCP-LA and similar compounds, PKC activ-ity was measured in the absence of diacylglycerol andphosphatidylserine, as described by Kannoet al. [11],and PKCd and e were measured in the absence ofadded EGTA and CaCl2, as described by Kannoet al.[11]. Low concentrations of Ca2+ are needed becausehigh Ca2+ interacts with the PKC phosphatidylserinebinding site and prevents activation. For measurementsof bryostatin activation, 1,2-diacylglycerol was omit-ted unless otherwise stated. Freeze-thawing of the sam-ples more than once was avoided because it was foundto greatly reduce the PKC activity and the degree ofactivation.

ECE assay—Endothelin-converting enzyme wasmeasured fluorometrically using the method of John-son and Ahn [14]. A sample of cell homogenate (20µl)was incubated in 50 mM MES-KOH, pH 6.0, 0.01%C12E10 (polyoxyethylene-10-lauryl ether), and 15µMMcaBK2 (7-Methoxycoumarin-4-acetyl [Ala7-(2,4-Dinitrophenyl)Lys9]-bradykinin trifluoroacetate salt)(Sigma-Aldrich). After 60 min at 37◦, the reaction wasquenched by adding trifluoroacetic acid to 0.5%. Thesample was diluted to 1.4 ml with water and the fluo-rescence was measured at ex=334 nm, em=398 nm.

Other assays—Ab in cell extracts was measuredusing an Abeta 1-42 human fluorometric ELISA kit(Biosource/Invitrogen). Ab in culture medium was iso-lated by adsorption onto reversed-phase columns (Wa-ters C18 Sep-Pak cartridges). The columns were con-ditioned with acetonitrile and equilibrated with water.The sample was applied and washed with 2 ml of 5%acetonitrile and 2 ml of 25% acetonitrile containing0.1% trifluoroacetic acid (TFA). The Ab was elutedwith 1 ml 50% acetonitrile + 0.1% TFA. The acetoni-trile was evaporated by heating under a stream of ni-trogen, and the solution was lyophilized to dryness andredissolved in a minimum volume of water, followedby ELISA measurements. Fluorescence was measuredin a Biotek Synergy HT microplate reader (ex=460nm, em=560 nm). AlamarBlue and CyQuant NF (In-vitrogen) were used according to the manufacturer’s

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instructions.Synthesisof cyclopropanated fatty acids—Methyl

esters of polyunsaturated fatty acids were cyclo-propanated using the modified Simmons-Smith reac-tion using chloroiodomethane and diethylzinc [15–17].All apparatus was baked at 60◦ for 1 hr and flame-driedwhile passing dry nitrogen through the apparatus. A100 ml 3-neck round bottom flask with a stirring barand a temperature probe was surrounded by an ice-dryice mixture and filled with 1.25 g (4.24 mmol) linoleicacid methyl ester or docosahexaenoic acid methyl es-ter in 25 ml dichloromethane and bubbled with N2.A 1M solution of diethylzinc (51 ml, 54.94 mmol) inhexane was added anaerobically using a 24-inch-long20-gauge needle and the solution was cooled to -5◦ .Chloroiodomethane (ClCH2I, 8.02 ml, 109.88 mmol)was added dropwise, one drop per second, with con-stant stirring. The rate of addition was decreased if nec-essary to maintain the reaction mixture below 2◦ C.The reaction mixture became cloudy during the reac-tion and an insoluble white zinc product was produced.The flask was sealed and the mixture was allowed to re-act further for 1 hr and then allowed to come to roomtemperature gradually over 2 hr.

To prevent the formation of an explosive residuein the hood, diethylzinc was not evaporated off. Themixture was slowly poured into 100 ml of water understirring to decompose any excess diethylzinc. Ethanewas evolved. The mixture was centrifuged at 5000rpm in glass centrifuge tubes and the upper aq. layerdiscarded. The white precipitate was extracted withCH2Cl2 and combined with the organic phase. The or-ganic phase was washed with water and centrifuged.The product was analyzed by silica gel G TLC usinghexane + 1% ethyl acetate and purified by chromatog-raphy on silica gel using increasing concentrations of1-10% ethyl acetate in n-hexane and evaporated undernitrogen, leaving the methyl ester as a colorless oil.

The Simmons-Smith reaction preserves the stere-ochemistry of the starting materials [16]. Docosahex-aenoic acid methyl ester was converted into DHA-CP6in 90-95% yield. The product was a colorless oil with asingle absorbance maximum at 202 nm in ethanol andno reaction with I2. The IR spectrum showed cyclo-propane ring absorption at 3070 and 1450 cm−1. Un-der the same conditions, eicosapentaenoic acid methylester was converted to EPA-CP5, and arachidonic acidmethyl ester was converted to AA-CP4. Linoleic acidmethyl ester was converted to DCP-LA methyl esterwhich was identical to a known sample.

Hydrolysis of methyl ester—The methyl ester (0.15g) was dissolved in 1 ml 1N LiOH and 1 ml diox-ane. Dioxane and methanol were added until it be-came homogeneous and the solution was stirred 60◦

overnight to 3 days. The product was extracted inCH2Cl2 and centrifuged. The aqueous layer and whiteinterface were re-extracted with water and washed un-til the white layer no longer formed. The product wasevaporated under N2 and purified by chromatographyon silica gel. The product, a colorless oil, eluted in 20%EtOAc in n-hexane. Its purity was checked by TLC in10% EtOAc/hexane and by C18 RP-HPLC with UVdetection at 205 nm, using 95% acetonitrile as the mo-bile phase.

RESULTS

The structures of the new PKC activators are shownin Fig. 1. To determine their PKC isozyme specificity,the new compounds were preincubated with purifiedisoforms of PKC for five minutes and the PKC activ-ity was measured radiometrically. DHA-CP6 and EPA-CP5, which are cyclopropanated derivatives of docosa-hexaenoic acid and eicosapentaenoic acid, preferen-tially activated PKCe to a similar extent. The maximaleffect for DHA-CP6 and EPA-CP5 was observed at 10nM and 100 nM, respectively (Fig. 2a, b). AA-CP4, thecyclopropanated derivative of arachidonic acid, pro-duced only modest activation (Fig. 2c), and cyclo-propanated linolenyl and linoleyl alcohols, epoxys-tearic acid, and vernolic acid methyl ester had little orno effect on PKC (Fig. 2e). Cyclopropanated verno-lic acid methyl ester inhibited PKCe at concentrationsabove 1µM (Fig. 2e). DCP-LA also activated PKCe,as reported by Kannoet al. [11], but required 1000×higher concentrations than DHA-CP6 for a maximaleffect (Fig. 2d). At higher concentrations, the com-pounds were weak inhibitors of PKCg,d, andbII.

Incubation of DHA-CP6, EPA-CP5, or AA-CP4with cultured human neuroblastoma cells produced ac-tivation of PKC that remained elevated for up to 1hour (Fig. 3a). Sustained activation was even more pro-nounced in primary neurons, with PKC activation last-ing up to 24 hours (Fig. 3b). In contrast to conventionalPKC activators such as phorbol ester, no PKC down-regulation to below control levels was observed. How-ever, inhibition could be produced at extremely highconcentrations (>100µM) (Fig. 2).

In contrast, PKC activators that bind to PKC’sdiacylglycerol-binding site, including bryostatin, gni-dimacrin, and phorbol esters, produce a transient (5-10 min) activation of PKC activity, followed by a pro-longed downregulation (not shown). Following appli-cation of PMA (phorbol 12-myristate 13-acetate) at itseffective concentration of 10-100 nM, PKC is rapidlydownregulated and remains at 20-30% of control levelsfor up to 24 hours, while downregulation by bryostatin

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is milder and relatively short-lasting ( [18–20] and Nel-son,unpublished results). These differences have beenattributed to differences in the diacylglycerol-bindingC1 domain of PKC [19].

A growing body of evidence suggests that PKCdownregulation, particularly of PKCδ, may be respon-sible for the tumor-promoting effects of phorbol es-ters [18, 21]. Downregulation of PKC therefore com-plicates any proposed usage of DAG-binding PKC ac-tivators as therapeutic agents. Downregulation has alsobeen implicated as a factor in increasing Ab produc-tion [22]. Thus, the ability to produce sustained acti-vation of PKC without inducing downregulation is ahighly desirable characteristic.

To measure the effects of PKCe activation on Abproduction, we used cells transfected with human APP-Swe/PS1D [23,24], which produce large quantities ofAb. Incubation of these cells for 24h with PKC acti-vators markedly reduced the levels of both intracellu-lar (Fig. 4a) and secreted (Fig. 4b) Ab. For bryostatin,which activates PKC by binding to the diacylglycerol-binding site, the inhibition was biphasic, with concen-trations of 20 nM or higher producing no net effect.This may be explained by the ability of this class ofPKC activators to downregulate PKC when used athigh concentrations. In contrast, DCP-LA and DHA-CP6, which bind to PKC’s phosphatidylserine site,showed monotonically increasing inhibition at concen-trations up to 10 to 100µM with no evidence of down-regulation at higher concentrations.

To determine whether the reduced levels of Abcaused by PKC activators were due to inhibition of Absynthesis or activation of Ab degradation, we appliedDHA-CP6 and low concentrations (100 nM) of exoge-nous monomeric Ab1-42 to cultured SH-SY5Y cells.This concentration of Ab is too low to produce measur-able toxicity or cell death. Since SH-SY5Y cells pro-duce only trace amounts of Ab, this experiment wasan effective test of the ability of PKC activators to en-hance Ab degradation. By 24h, most of the Ab hadbeen taken up by the cells and the concentration of Abin the culture medium was undetectable. Addition of0.01 to 10µM DHA-CP6 to the cells reduced the cellu-lar levels of Ab by 45-63%, indicating that the PKCeactivator increased the rate of degradation of exoge-nous Ab (Fig. 5).

Previous researchers reported that PKC activatorssuch as phorbol 12-myristate 13-acetate produce largeincreases in TACE/ADAM17 activity [25, 26] whichcorrelated with increasd sAPPa and decreased Ab,suggesting that TACE and BACE1 compete for avail-ability of APP substrate, and that PKC activators shiftthe competition in favor of TACE. However, many ofthese earlier studies were carried out in fibroblasts and

other non-neuronal cell types, which may respond dif-ferently to PKC activators than neurons. For example,Etcheberrigarayet al. found that activation of PKC inhuman fibroblasts by 10 pM to 100 pM bryostatin in-creased the initial rate ofa-secretase activity by 16-fold and 132-fold, respectively [27]. In contrast, in cul-tured human neuroblastoma cells (Fig. 6a) PKC activa-tors produced no detectable increase in TACE activity.A transient increase in TACE activity was observed inprimary neurons after EPA-CP5 (Fig. 6b). Phorbol es-ter (PMA) also did not activate TACE in these cells (notshown). This suggests that any reduction of Ab levelsin neuronal cells by PKC activators is caused by someother mechanism besides activation of TACE.

Ab can be degradedin vivo by a number ofenzymes, including insulin degrading enzyme (in-sulysin), neprilysin, and endothelin-converting enzyme(ECE). Because PKCe overexpression has been re-ported to activate ECE [28], we examined the effectof PKC activators on ECE. Bryostatin, DCP-LA, andDHA-CP6 all produced a sustained increase in ECEactivity (Fig. 7a). A similar effect was observed in pri-mary cultured neurons (Fig. 7b). Since ECE does notpossess a diacylglycerol-binding C1 domain or a PKC-like phosphatidylserine-binding C2 domain, this sug-gests that the activation was not due to direct activationof ECE, but must have resulted from indirect activationof ECE or some ECE-activating intermediate by PKC.DHA-CP6, EPA-CP5, and AA-CP4 had no effect onthe enzyme activity of purified ECE (Fig. 8).

DHA-CP6, bryostatin, and DCP-LA had no effecton cell survival or on proliferation as measured by ala-marBlue and CyQuant staining (Fig. 9), indicating thatthe reduction in Ab production did not result from cellproliferation or a change in cell survival.

DISCUSSION

DHA-CP6, EPA-CP5, and DCP-LA represent anew class of PKC activators that are relatively specificfor the PKCe isoform. PKCe is expressed at very lowlevels in all normal tissues except for brain [29, 30].Wetsel et al. [31] reported that PKCe and PKCe′are predominantly expressed in brain, with very weakstaining in other tissues. The high abundance of PKCein presynaptic nerve fibers suggests a role in neuriteoutgrowth or neurotransmitter release [32]. Therefore,effects of specific PKCe activators would be largelyrestricted to brain, and unlikely to produce unwantedperipheral side effects [18].

The concentration of DHA-CP6 (10 nM) requiredfor activation of PKC is comparable to that of PMA(10-100 nM) [33] but higher than that of bryostatin(0.2-1 nM). Further modifications of the structure

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of DHA-CP6, based on modeling studies of PKC’sphosphatidylserine-bindingsite, could create deriva-tives with even higher affinity. Furthermore, becausethese new compounds are closely related toω-3 andω-6 polyunsaturated acids, they are likely to possesslower toxicity than many of the existing terpenoid sec-ondary metabolite PKC activators. Thus, a requirementfor higher concentrations might be less of a disadvan-tage.

There are several mechanisms by which activa-tion of PKC could affect Ab production. One mech-anism is by activating TACE/a-secretase and shiftingthe competition for APP away from the amyloidogenicb-secretase. This appears to be an important mecha-nism in non-neuronal cells such as fibroblasts; how-ever, our data show that PKCe activators have muchless effect on TACE activity in neuronal cell types thanin fibroblasts. Specific inhibitors of PKCe also blockPMA-induced suppression of Ab levels [34], suggest-ing that PKCe is the principal isoform responsible fordepleting Ab. However, da Cruz e Silvaet al. foundthat long-term application of phorbol ester increasesthe synthesis of PKCe [22]. Thus, part of the effectcould also be mediated by induction of PKCe by PMA.

Decreased expression of PKCe also reducescholinergic regulation of sAPPa levels [35], which isconsistent with the theory that sAPPa and Ab com-pete for a limited pool of APP. Thus, in cells in whichAPP is rate-limiting, direct or indirect activation ofa-secretase by PKCe could also reduce Ab levels.

Another possible mechanism is by increasing therate of degradation. A recent report [28] presented ev-idence that PKCe increases the activity of endothelin-converting enzyme (ECE), an enzyme that degradesAb. These authors reported that in APP/PKCe trans-genic mice, which overexpress PKCe, levels of Abwere lower than in control animals, but there wasno change in other Ab-degrading enzymes, such asinsulin-degrading enzyme or neprilysin, and no changein APP metabolism. However, it was unclear whetherthis effect can be produced by activation of PKCe innon-transgenic, adult animals. Our results showing thatAb degradation is enhanced in cells treated with spe-

cific PKCe activators suggest that this may be the case.A third mechanism is by stimulation of PKC-

coupled M1 and M3 muscarinic receptors, which isreported to increase nonamyloidogenic APP process-ing bya-secretase (TACE/ADAM17 and/or ADAM10)[36]. Muscarinic agonists rescue 3x-transgenic ADmice from cognitive deficits and reduce Ab andtau pathologies [37], in part by activating theTACE/ADAM17 nonamyloidogenic pathway. Mus-carinic receptor signaling is closely tied to PKC: mus-carinic receptor mRNA is regulated by PKC [38] andneuronal differentiation produced by M1 muscarinicreceptor activation is mediated by PKC [39].

Activation of PKC can also have other neu-rotrophic or synaptogenic effects that complement theeffect of lowering Ab. For example, PKC activatorscan reverse some of the neurological damage in an is-chemia/hypoxia model for stroke [5]. PKC activatorsare also reported to induce expression of tyrosine hy-droxylase [40] and induce neuronal survival and neu-rite outgrowth [8, 41]. These neurotrophic effects maybe mediated by neurotrophins such as BDNF [41, 42].Neurotrophin signaling and APP metabolism pathwaysintersect at many points. For example, p75 and TrkANGF receptor signaling increase APP mRNA expres-sion [43]. Both APP and p75 are cleaved bya- andg-secretase [44]. Cleavage of the p75 extracellular do-main enables fibroblast growth factor 2 to stimulateneurite outgrowth and branching [45].

An advantage of compounds such as DCP-LAwhich specifically activate PKCe is that, in contrastto phorbol esters and similar 1,2-diacylglycerol (DAG)analogues, they do not produce detectable downregu-lation. The biphasic response of PKC to DAG-basedactivators means that a PKC activator may produce op-posite effects at different time points, such as reduc-ing Ab levels at one time point and increasing them atanother (e.g.,[22]). Careful dosing and monitoring ofpatients would be required to avoid effects opposite tothose that are intended. Because of the relative inabil-ity of this new class of PKC activators to downregulatePKC, this problem can be avoided.

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FIGURE LEGENDS

Fig. 1. Structures of DCP-LA, DHA-CP6, EPA-CP5, and AA-CP4.

Fig. 2. Activation of purified PKCe by PKC activators. Purified PKCa, bII, g, d, or e (9 ng) was preincubatedfor 5 min at room temperature with DHA-CP6 (A), EPA-CP5 (B), AA-CP4 (C), or DCP-LA (D), followed byPKC activity measurement as described inExperimental Procedures. (E). Activation of purified PKCe by othercyclopropanated and epoxidized fatty acids, alcohols, and methyl esters. Samples were preincubated for 5 minat room temperature with purified PKCe (500 ng), followed by PKC activity measurement. Values arex ± SE,two-tailed Student’s t test. * p<0.02, ** p<0.001.

Fig. 3. Time course of PKC activation in cultured human SH-SY5Y cells (A) and cultured primary rat hip-pocampal cells (B). DHA-CP6, EPA-CP5, or AA-CP4 in 10µl ethanol was added to cells growing in 6-wellplates with 1 ml culture medium. At the indicated time, the cells were washed with PBS and scraped into 200µlof homogenization buffer, followed by PKC activity measurement as described inExperimental Procedures.p<0.02, ** p<0.001.

Fig. 4. Decreased intracellular levels of Ab in cells (A) and in culture medium (B) from cells exposed to PKCactivators. Neuro2a (N2a) neuroblastoma cells transfected with human APPSwe/PS1D [23, 24], growing in6-well plates, were incubated with low-serum (1%) medium for 24h. The medium was replaced with fresh low-serum medium and various concentrations of bryostatin, DCP-LA, or DHA-CP6 in 10µl ethanol or 10µl ethanolalone was added. After 18h, the cells were washed with PBS and scraped into 200µl homogenization buffer,homogenized, and Ab was measured by ELISA. The medium was collected and Ab was isolated by adsorptiononto reversed-phase columns (Waters C18 Sep-Pak cartridges), washed, and eluted with acetonitrile, followedby ELISA measurements.

Fig. 5. Effect of DHA-CP6 on degradation of exogenously applied Ab in human SH-SY5Y neuroblastoma cells.Various concentrations of DHA-CP6 in 10µl ethanol or 10µl ethanol alone were added to cells grown in 6-wellplates. After 5 min, fresh Ab1-42 (100 nM) or buffer was applied and the cells were incubated for 24h. Ab 1-42was then measured in the cells and culture medium by ELISA. * p<0.02.

Fig. 6. Effect of PKC activators on TACE activity in SH-SY5Y neuroblastoma cells (A) and cultured primary rathippocampal neurons (B). DHA-CP6, EPA-CP5, or AA-CP4 in 10µl ethanol or 10µl ethanol alone was addedat a final concentration of 1µM to cells grown in 12- or 24-well plates. After various periods of time, the cellswere collected and TACE activity was measured fluorometrically. * p<0.02.

Fig. 7. Activation of endothelin converting enzyme (ECE) by PKC activators in SH-SY5Y cells (A) or culturedneurons (B). Bryostatin (0.27 nM), DCP-LA (1µM), DHA-CP6 (1µM), EPA-CP5 (1µM), AA-CP4 (1µM), orethanol alone were added to cells growing on 12- or 24-well plates. After various periods of time, the cells werecollected and ECE activity was measured fluorometrically. * p<0.05, ** p<0.001.

Fig. 8. Lack of activation of purified endothelin converting enzyme (ECE) by PKC activators. ECE activitywas measured as described inExperimental Proceduresin the presence of recombinant ECE (R&D Systems1784ZN, 20 ng) and varying concentrations of DHA-CP6, EPA-CP5, or AA-CP4 for 60 min at 37◦ .

Fig. 9. Lack of effect of DCP-LA and DHA-CP6 on cell survival and cell proliferation. Various concentrationsof DCP-LA or DHA-CP6, or ethanol alone were added to SH-SY5Y cells growing on 6-well plates. Cell via-bility was measured by adding alamarBlue reagent (Invitrogen) to 10% of the culture volume. AlamarBlue ischemically reduced in viable cells to form a fluorescent product. The fluorescence was measured in a microplatereader at 1, 2, 3, and 4 hours and the slope of the curve of fluorescence intensity vs. time was calculated. Cellproliferation was measured by adding 100µl of CyQuant NF, which is a cell-permeable DNA-binding dye. After60 min, the fluorescence, which is proportional to the number of cells, was measured in a microplate reader.

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Fig. 1.

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0.000 0.001 0.010 0.100 1.000 10.000 100.000Concentration, µM

0

20

40

60

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100

120

140

160

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200

PK

C A

ctiv

ity, %

of C

ontr

ol

PKCαPKCβIIPKCγPKCδPKCε

* *

**

*

****

**

**

**

*

**

Activation of PKCε by DHA−CP6

Fig. 2a.

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0.000 0.001 0.010 0.100 1.000 10.000 100.000Concentration, µM

0

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240

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PK

C A

ctiv

ity, %

of C

ontr

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PKCαPKCβIIPKCγPKCδPKCε

* *

* **

*

*

**

**

*

Activation of PKCε by EPA−CP5

Fig. 2b.

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0.000 0.001 0.010 0.100 1.000 10.000 100.000Concentration, µM

0

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PK

C A

ctiv

ity, %

of C

ontr

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PKCαPKCβIIPKCγPKCδPKCε

**

* *

****

*

Activation of PKCε by AA−CP4

Fig. 2c.

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0.00 0.01 0.10 1.00 10.00 100.00[DCP−LA], µM

0

50

100

150

200

250

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PK

C a

ctiv

ity,

% o

f Con

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PKCαPKCγPKCδPKCε

*

*

* *

**

*** ** **

*

**

**

****

*

Fig. 2d.

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0.0 0.1 1.0 10.0 100.0Concentration, µM

0

100

200

300

PK

C A

ctiv

ity (

% o

f con

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)

Cyclopropanated linolenyl alcoholCyclopropanated linoleyl alcoholEpoxystearic acidVernolic acid methyl esterCyclopropanated vernolic acid methyl ester

**

Fig. 2e.

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0 1 2 3 4time, hr

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PK

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of c

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DHA−CP6 1µMEPA−CP5 1µM AA−CP4 1µM

Time course of ECE activation in primary neurons

**

*

*

**

*

24

Fig. 3a

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0 1 2 3 4time, hr

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PK

C a

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ity, %

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DHA−CP6 1µMEPA−CP5 1µM AA−CP4 1µM

*

*

*

*

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Fig. 3b

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BryostatinDCP−LADHA−CP6

Intracellular Aβ

0 0.0001 0.001 0.01 0.1 1 10 1000.00001

Concentration, µM

Aβ,

% o

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Fig. 4a.

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0

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40

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BryostatinDCP−LADHA−CP6

Secreted Aβ

0 0.00001 0.0001 0.001 0.01 0.1 1 10 100

Concentration, µM

Aβ,

% o

f Con

trol

Fig. 4b.

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0

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60

Aβ

Control 0.01 0.1 1 10 µM

No addition

0.01 0.1 10µM

Effect of DHA−CP6 on Aβ 100 nM Aβ No Aβ

**

Fig. 5.

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0.0 0.5 1.0 1.5 2.0Time, hr

020406080

100120140160

TA

CE

, % o

f Con

trol

DHA−CP6EPA−CP5AA−CP4

0 1 2 3 4Time, hr

0

50

100

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TA

CE

, % o

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DHA−CP6 1µMEPA−CP5 1µM AA−CP4 1µM

* **

24

A

B

Fig. 6.

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0 1 2 3 4time, hr

0

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EC

E, %

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Bryo 0.27 nMDCPLA 1µMDHA−CP6 1µM

**

*

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Fig. 7a

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0 1 2 3 4time hr

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Time course of ECE activation in primary neurons

* * * *

*

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Fig. 7b

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DHA−CP6EPA−CP5AA−CP4

0 0.01 0.1 1 10 100 1000 10000

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EC

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Fig. 8

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0

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0

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Cell survival (alamarBlue)

Proliferation (Cyquant)

Control 1µM 10µM 100µM

Control 1µM 10µM 100µM

% o

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Fig. 9

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Thomas J. Nelson, Changhai Cui, Yuan Luo and Daniel L. AlkonReduction of beta-amyloid levels by novel PKCepsilon activators

published online October 22, 2009J. Biol. Chem. 

  10.1074/jbc.M109.016683Access the most updated version of this article at doi:

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