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Mechanism and prevention of neurotoxicity caused by β-amyloid peptides: relation to Alzheimer's disease
Brain Research 776 Ž1997. 40–50
Research report
Mechanism and prevention of neurotoxicity caused by b-amyloid peptides: relation to Alzheimer’s disease Barbara J. Blanchard, Genevieve Konopka, Margaret Russell, Vernon M. Ingram
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Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Accepted 29 July 1997
Abstract In Alzheimer’s disease, neurotoxic b-amyloid peptides cause a deleterious influx of calcium ions into neurons. This increase in wCa2q x int is expected to trigger intracellular events that eventually cause cell dysfunction and cell death. We find that the aggregated b-amyloid peptide bAP25 – 35 opens irreversibly a Ca2q-carrying channel, as does aggregated bAP1 – 42 . The opening of this channel is unaffected by DL-AP5, but it is blocked by Mg 2q, CNQX and DNQX, suggesting a non-NMDA channel. External calcium enters and cytosolic calcium levels rise several-fold, as measured by fura-2 ratiometric analysis. Our findings illustrate a very early molecular event in the neurotoxicity of Alzheimer’s disease. To combat the neurotoxic effect of aggregated b-amyloid peptides, we have devised a series of very short antagonistic peptides. Using a combinatorial library of hexapeptides made from D-amino acids, we have selected peptides by their ability to complex with the tagged b-amyloid peptide bAP25 – 35. Certain of these so-called ‘decoy peptides’, as well as some modified decoy peptides, are able to abolish the calcium influx caused by aggregated, probably fibrillar, b-amyloid peptides bAP25 – 35 and bAP1 – 42 . q 1997 Elsevier Science B.V. Keywords: b-Amyloid peptide; b-Amyloid fibril; Calcium ion channel; Calcium homeostasis; Calcium ion concentration; Decoy peptide; hNT neuron; Non-NMDA channel
1. Introduction The development of Alzheimer’s disease ŽAD. is an age-related process involving the dysfunction and eventual death of certain types of neurons in particular regions of the brain. It is important to realize that the differences between the slow neurodegenerative changes associated with ‘normal’ aging and the much more rapid changes in AD are driven by additional factors, genetic or environmental.Thus, there may be several preconditions leading to AD. However, the neuropathology of AD is remarkably similar in all cases. Apparently, different root causes lead to the same or very similar molecular scenarios. It has been proposed w5,10x that the molecular basis of Alzheimer pathogenesis is the appearance in the brain of abnormally high concentrations of the neurotoxic bamyloid peptides that are themselves abnormal degradation products of the large transmembrane precursor protein, APP. Neurotoxicity occurs through the ability of aggregates of these peptides to interact with sites on neurons
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Corresponding author.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 0 0 3 - 2
that carry the appropriate receptors. The result of this interaction is an influx of external calcium into these neurons that is too large for the cell to control by sequestration or by pumping out. Neurons of older subjects are especially prone to this attack, because they suffer from an age-related deficiency in mitochondrial function and ATP production, both needed to sequester unwanted calcium. There can be many end effects of the increased calcium: activation of protein kinases Že.g. ERKs., consequent hyperphosphorylation of t proteins and formation of NFTs, activation of immediate early gene products, possible induction of apoptosis, etc. Certainly the normal response of the affected neurons to stimuli will be much altered. The early findings of Yankner’s group in 1990 w20x indicated that the b-amyloid peptides bAP25 – 35 and bAP1 – 42 were neurotoxic towards cultured hippocampal neurons in micromolar concentration. To investigate the immediate molecular mechanism of the neurotoxicity, we exposed cultured human hNT neuronal cells to the aggregated b-amyloid peptide bAP25 – 35 as a simple model system. In each experiment the peptide was diluted from a DMSO stock solution into the aqueous external solution and thereby aggregated. We also used aggregated bAP1 – 42 ,
B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
which gave similar results. These treatments cause a large persistent influx of Ca2q from outside of the cell through channels that are described in this paper. It is known that the b-amyloid peptides are neurotoxic when they are in aggregated Žfibrillar. form w13x and that an amorphous precipitate is not toxic to cultured cells. We undertook the present study in order to interfere with b-sheet aggregation that is involved in fibril formation by selecting and designing ‘decoy peptides’ ŽDPs.. These have themselves sufficient b-sheet forming potential to associate with the multimer-forming b-amyloid peptide, and either block the usual aggregation or incorporate into the multimer so as to make it inactive. We describe a number of peptides that have these properties. The successful DP should be resistant to proteolytic digestion, if it is to be useful in therapy. Therefore, we chose to use peptides composed of D-amino acids.
2. Materials and methods 2.1. Experimental treatment hNT cells were from Stratagene. DL-AP5 ŽDL-2-amino5-phosphonovaleric acid . , C N Q X Ž 6-cyano-7nitroquinoxaline-2,3-dione . and DNQX Ž 6,7-dinitroquinoxaline-2,3Ž1 H,4 H .-dione. were from Sigma. Fura-2-AM was from Molecular Probes. bAP25 – 35 was synthesized by the Biopolymers Laboratory at MIT and purified by HPLC. bAP1 – 42 was purchased from Quality Control Biochemicals. hNT cells are human post-mitotic NT2-N neurons, derived by retinoic acid treatment of a teratocarcinoma cell line w15x. They have good neuronal morphology and possess NMDA and non-NMDA receptors w21x and voltagegated Ca-channels w16x. hNT cells were plated 4–20 days before use on poly-D-lysine-coated acid-washed glass coverslips. Only single phase-bright cells, connected by extensive neurite extensions, were used. For wCa2q x int estimation, the coverslips were placed in a coverslip holder ŽMedical Systems Corp... We find bAP25 – 35 to be insoluble in millimolar concentrations in aqueous media at neutral pH. Stock solutions of bAP25 – 35 in DMSO at 20–100 mM tend to aggregate during frozen storage, as well as at room temperature, due to the presence of seed aggregates. We filter our DMSO stock solutions immediately w8x by spin-filtering with Ultrafree-MC filters of low-binding regenerated cellulose with cut-off at 30 000 Mr ŽMillipore cat. no. UFC3LTK00, Bedford, MA.. Concentration of the filtrate is determined by amino acid analysis w8x. Aliquots can be stored for weeks at y408C. To treat cells, the DMSO stock solution of the peptide is diluted into the aqueous Tyrode’s solution, with or without calcium, shortly before use. Final DMSO concentrations are kept near 0.1%.
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2.2. Fura-2 measurements of internal calcium concentration hNT cells were loaded with 3 mM fura-2-AM at room temperature in Tyrode’s solution containing 2 mM calcium, as described by the supplier ŽMolecular Probes.. Cells were subsequently washed and allowed to recover at 378C for 30 min either in Tyrode’sr2 mM Ca solution alone or with DMSO Ž< 1%. equivalent to the dilution of DMSO stock solutions of the peptides. Loaded cells were examined at room temperature in 400 ml of control Tyrode’sr2 mM Ca, containing the appropriate concentration of DMSO, the vehicle. Typically, wCa2q x int was determined for 6–12 cells per coverslip first in the control buffer Tyrode’sr2 mM Ca, then again after exchanging the buffer for the test solution. Calcium measurements were taken on a Nikon Diaphot inverted microscope with a Fluor-40 objective using Photon Technology International ŽPTI. ratiometric technology ŽRM-M system. and PTI Felix software. The emission fluorescence at 510 nm was measured over an excitation range of 320–400 nm. The ratio of 510 nm emissions at 340 nm and at 380 nm was used to calculate wCa2q x int from a standard calcium concentration curve made with fura-2–Na salt and standard Ca2q solutions ŽMolecular Probes, cat. no. C3009.. Control values of wCa2q x int were usually between 10 and 100 nM. The effects of externally adding bAP25 – 35 , bAP1 – 42, or other reagents, to the cell culture were recorded during the first 15 min of exposure; however, we often noticed a small decrease in wCa2q x int after 30–45 min. The presence of the small concentration of DMSO made little difference. 2.3. Statistics Individual hNT cells Ž; 10. were examined on a glass coverslip. We recorded the change in wCa2q x int of each individual cell as it became exposed to one or more treatments with peptides and reagents. Statistical evaluation was therefore based on the behavior of individual cells and the significance of differences was calculated using paired Student’s t-test analysis, two populations, when the wCa2q x int themselves are reported. When percent changes of wCa2q x int are described, we used the individual Student’s t-test, two populations, to assess the significance of differences between means. The mean and its standard deviation are recorded in the figures; n s number of individual cells that responded to the treatment. 2.4. Selection of ‘decoy peptides’ The MIT Biopolymer Lab. ŽDirector Richard Cook. made a library w12x of some 43 000 individual sequences, each from the D-amino acids Ala, Ile, Val, Ser, Thr and from Gly, all of which are represented in b-amyloid
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B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
peptides. The peptides are six amino acids long and originally did not have charged side chains. The peptides are attached by their C-terminus to the polystyrene bead via a linker of three Gly residues to facilitate interaction between a member of the peptide library and the dissolved FITC- or Dansyl-tagged bAP25 – 35 peptide. There are many copies of a unique peptide sequence per bead. We have used bAP25 – 35 tagged with a fluorescent label, FITC- or Dansyl-, FITC–GGGGSNKGAIIGLM–COOH and Dansyl–GGGGSNKGAIIGLM–COOH. There is a Gly3 linker separating the bAP-sequence from the fluorescent label. An aliquot Ž100 ml. of a 50 mM solution of FITC-Gly3bAP25 – 35 was added to a suspension of ; 35 mg of the resin-bound hexapeptide library in 0.5 ml PBS. The suspension was very briefly vortexed six times during the next 15 min at room temperature. The beads were washed three times in 1 ml of PBS, spread out in a dish, and examined under ultraviolet light. Out of thousands of
beads, only very few fluoresced brightly on their surface. Beads were transferred with fine forceps onto glass filters for sequencing at MIT’s Biopolymers Laboratory. Similarly, a 12 mM solution in PBS of DANSYL-Gly3bAP25 – 35 was used. Very few blue fluorescent beads were seen, and these were also sequenced.
3. Results 3.1. Aggregated [Ca 2 q ]i n t
b AP2 5 – 35
and
b AP1 – 4 2
increase
3.1.1. b AP2 5 – 35 Application of unfiltered solutionsrsuspensions of bAP25 – 35 to a group of hNT cells loaded with fura-2-AM causes a very marked increase in cytosolic calcium within a few minutes ŽFig. 1.. There is a strong trend of increas-
Fig. 1. An unfiltered suspension of bAP25 – 35 in Tyrode’sr2 mM Ca applied to hNT cells causes influx of Ca2q. A: hNT cells in Tyrode’sr2 mM Ca Žcolumn 1. have an average wCa2q x int of 80 nM. Exposure to bAP25 – 35 at 20 mM Žfrom unfiltered stock solution. causes a stable rise of wCa2q xint to approximately 200 nM Žcolumn 2., decreasing only slightly after 30 min Žcolumn 3.. Final DMSO concentration is at 0.04% in both conditions. Each column represents the mean wCa2q x int of 8 cells " the S.D. of the mean. The significance of the means from the mean of the control values is expressed as: )))) P - 0.0001. B: an unfiltered suspension of aggregated bAP1 – 42 in Tyrode’sr2 mM Ca applied to hNT cells causes influx of Ca2q. Control Žcolumn 1. hNT cells in control Tyrode’sr2 mM Ca have an average wCa2q x int of 50 nM. Exposure to unfiltered aggregated bAP1 – 42 Ž378C, 24 h.. at 20 mM causes a stable rise of wCa2q x int to 94 mM Žcolumn 2.; ) ) ) ) P - 0.0001. C: display of wCa2q xint in individual cells in A. Open squares, control; upward triangle, immediate bAP25 – 35 effect; inverted triangle, after 30 min. D: display of wCa2q x int in individual cells in B. Open squares, control; upward triangle, immediate bAP1 – 42 effect.
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Table 1 Effect of different concentrations of bAP25 – 35 on wCa2q x int in hNT cells w bAP25 – 35 x
% increase in wCa2q x int Žmean"S.D..
P
a
10 mM 20 mM 50 mM 112–114 mM 130 mM 172 mM 334 mM
170" 85 286"179 343"245 366"260 623"555 367"108 508"561
- 0.05 -10y9 -10y5 -10y5 - 0.05 -10y6 - 0.05
nb 9 70 22 16 11 8 9
hNT cells in Tyrode’sr2 mM Ca have an average wCa2q x int of 80 nM. Exposure to unfiltered dilutions of bAPbAP25 – 35 in Tyrode’sr2 mM Ca for ;10 min causes a stable rise of wCa2q xint . a Difference from Tyrode’sr2 mM Ca control; by paired Student’s t-test. b Number of responding cells 145r163s89%.
ing cytosolic calcium with increasing peptide concentration ŽTable 1.. The effect decreases only slightly during the first hour when peptide is present. It is not readily reversible for as long as 1 h after the peptide is removed from the external medium Ždata not shown; see also Fig. 2.. We often observed that hNT cells treated with the peptide will detach when a second change of external medium is attempted; there is no obvious change in the well-differentiated neuronal morphology of the treated cells before lift-off. The data reported here are derived from experiments in which the designated treatment immediately followed the control Tyrode’s solution. Within each experiment involving 6–12 cells on one coverslip, there is considerable variability from cell to cell in the magnitude
Fig. 2. The increased wCa2q x int is derived from the external medium only. Each column represents wCa2q xint "S.D. after the external medium was changed as indicated; ns6. The same cells were exposed to Tyrode’sr0 mM Ca plus 50 mM bAP25 – 35 Žfrom unfiltered stock., Tyrode’sr2 mM Ca without bAP25 – 35 , and Tyrode’sr2 mM Ca plus 50 mM bAP25 – 35 . Some measurements were repeated after 20 or 15 min, as indicated. The mean control value for cells in Tyrode’sr0 mM Ca was 44 nM. The results are shown"S.D. The significance of the means of the last 3 columns from the initial Tyrode’sr0 mM Ca condition was calculated by the paired Student’s t-test: ) ) ) P - 0.001; ) ) ) ) P - 0.0001.
Fig. 3. Mg 2q blocks the influx of Ca2q caused by bAP25 – 35 . Each column represents wCa2q x int "S.D. for separate experiments in which hNT cells were first exposed to the control solution of Tyrode’sr2 mM Ca. The external solution was then replaced by a test solution containing bAP25 – 35 Ž ; 20 mM. and also MgCl 2 at either 0.5 mM Ž ns18. or 1 mM Ž ns 5., in Tyrode’sr2 mM Ca, as indicated.
of the effect on cytosolic calcium, as expressed by the standard deviation of the mean displayed in the graphs and the table. To determine the origin of the increased internal calcium, cells were exposed to the usual concentrations of peptide in an external Tyrode’s solution that had no calcium ŽFig. 2.. There was no increase in internal calcium, even after 15 min. In fact, there was a small decrease in
Fig. 4. Response of bAP25 – 35-induced calcium influx to NMDA and non-NMDA channel blockers. hNT cells were first exposed to the control solution of Tyrode’sr2 mM Ca. The external solution was then replaced by a test solution containing bAP25 – 35 Ž21 mM. and also DL-AP5 at 50 mM Ž ns10 cells., or CNQX at 22 mM Ž ns14 cells., or DNQX at 8 mM Ž ns6 cells., as indicated. As an exception, included among the CNQX-treated cells were four cells that had first been exposed to bAP25 – 35 Ž21 mM. and had shown the expected 270% increase in wCa2q x int before becoming blocked by CNQX. The results are shown" S.D. The significance of the mean of the DL-AP5 column from the initial Tyrode’sr2 mM Ca condition was calculated by the paired Student’s t-test: ) ) ) ) P - 0.0001.
B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
44 Table 2 D-Amino acid decoy peptides C-Terminus –COOH
–CONH 2
FITC-bAP-Selected I.A.A.G.I.T.G.G.G T.V.I.G.T.I.G.G.G T.G.I.I.A.S.G.G.G
DP1 DP2 DP4
DP3 DP5
Dansyl-bAP-selected T.T.I.V.S.T.G.G.G A.G.V.I.S.I.G.G.G
DP6 DP7
Decoy peptide derived from DP3 T.V.I.Rq.T.I.A.A.A DP8
bAP25 – 35 G.S.N.Kq.G.A.I.I.G.L.M b-Sheet-forming potentials of regions of peptide, wsx s relative b-sheetforming potential, using the Chou–Fasman algorithm: Ts wsx barely above 1; T s wsx )1; Ts wsx 41.
wCa2q x int at 15 min which could indicate the outflow of calcium into the zero calcium external solution. Restoring external calcium to 2 mM, even in the absence of peptide,
at once caused wCa2q x int to rise substantially to almost 200% of control. This was increased slightly by further addition of external peptide. Evidently, the calcium increase comes from external sources only. The experiment in Fig. 2 also demonstrates that the effect of bAP25 – 35 is not reversible, since replacement of the peptide by Tyrode’sr2 mM Ca causes immediate influx of calcium.
3.1.2. b AP1 – 4 2 Exposure of hNT cells to an unfiltered suspension of bAP1 – 42 in Tyrode’sr2 mM Ca, aggregated for 24 h at 378C, causes a highly significant influx of Ca2q ŽFig. 1.. The effect with this peptide also shows a strong concentration dependence Ždata not shown..
3.2. NMDA and non-NMDA ligand-gated channels Fig. 3 indicates that 0.5 mM or 1 mM magnesium chloride in the presence of bAP25 – 35 totally blocks the peptide-induced influx of calcium. Influx caused by bAP25 – 35 was also abolished by CNQX at 22 mM or DNQX at 8 mM; both are known to block specifically
Fig. 5. Decoy peptide DP3 blocks bAP25 – 35 . Each column represents the mean wCa2q x int of 7–10 cells " S.D. measured as described in Section 2. DMSO stock solutions of DP3 had not been filtered. Each group of three columns represents the same set of cells with measurements after application of DP3 q bAP25 – 35 in Tyrode’sr2 mM Ca at ; 5 min Ž5., 20 min Ž20. and after application of 20 mM EGTAq DP3 q bAP25 – 35 in Tyrode’sr2 mM Ca within ; 2 min ŽE., respectively. DP3 Žnon-filtered DMSO stock. q bAP25 – 35 in Tyrode’sr2 mM Ca at a molar ratio of 0.25:1Ž5 mM 20 mM. is the only set that shows a significant increase in wCa2q x int as compared to 20 mM DP3 by itself and 20 mM bAP25 – 35 by itself. DP3 q bAP25 – 35 at molar ratios of 0.5:1 Ž10 mM q 20 mM., 1:1Ž20 mM q 20 mM. and 2:1 Ž40 mM q 20 mM. show a decrease in wCa2q x int . The significance of the means from the mean of the related bAP25 – 35 measurements Ž5 or 20 min after exposure. is: ) P - 0.05; ) ) P - 0.01; ) ) ) P - 0.001.
B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
45
Fig. 6. Effect of adding decoy peptide DP3 to aggregated bAP25 – 35 . Each column represents the mean wCa2q x int of 9–13 cells " S.D. and separate aggregation conditions, measured as described in Section 2. bAP25 – 35 and DP3 are both at 20 mM. The first column shows bAP25 – 35 after aggregation for 2 h at RT. In the second column, bAP25 – 35 is allowed to aggregate for 2 h before DP3 is added. In the third column, DP3 is diluted into Tyrode’s 2 mM Ca, allowed to stand for 1 h, and then bAP25 – 35 is added. The significance of the difference of the mean of the third column relative to bAP25 – 35 measurements is ) ) P - 0.01.
non-NMDA ligand-gated channels ŽFig. 4.. On the other hand, DL-AP5, a specific blocker of NMDA ligand-gated channels, did not decrease at all the bAP25 – 35 peptide-induced influx of calcium at 50 mM concentration ŽFig. 4., or at 200 mM Žnot shown.. 3.3. Selection and design of decoy peptides When we began the present experiments, we interpreted our patch-clamp studies w16x of calcium influx by aggregated bAP25 – 35 as being due to the formation of an ionophore, by analogy with Arispe et al. w1,2x who used artificial membranes. Since Durell et al. w6x have proposed a model for b-amyloid ionophore formation that has a series of glycines ‘lining’ the putative ion conductance channel, we included glycine in our original collection of peptides. However, the presence of glycine would make a peptide susceptible to proteolytic digestion. The most recently synthesized decoy peptide DP8 replaces glycines with D-alanines; all other amino acids are D-amino acids. We note that the decoy peptides so far selected Žsee Table 2. tend to begin with a cluster of b-branched amino acid. b-Sheet-forming potential of the selected decoy pep-
tides is indicated in Table 2. 1 Peptides were synthesized either with a –COOH terminus or as the amide. The presence or absence of a C-terminal negative charge can affect the ability of the peptide to interfere with calcium influx caused by bAP25 – 35 Žsee Fig. 8; DP4 and DP5.. The conformational rules that apply to L-amino acid peptides apparently also apply to peptides containing D-amino acids ŽFasman, personal communication.. We have also synthesized and tested a second generation peptide ŽDP8, Table 2., in which Glycine4 of decoy peptide DP3 is replaced with Arginine, and C-terminal spacer glycines are replaced by D-alanine. Without affecting b-sheet forming potential w4x, this change introduces a charged side chain into the middle of the sequence. We hope to affect the function of a b-sheet structure that might form when DP8 and bAP25 – 35 are allowed to aggregate together. Indeed, this is the result ŽFig. 7.. Interestingly, the charged side chains — R in DP8 and K
1
Assessment of b-sheet-forming ability is based on the Chou–Fasman rules, as used in the software ‘Peptide Companion’ w4x.
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B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
in bAP25 – 35 — are adjacent to the regions with highest Žanti-parallel. b-sheet-forming potential ŽTable 2. in the two sequences. 3.4. Effect of decoy peptides on the calcium influx caused by b-amyloid peptides Our first experiments with decoy peptides used unfiltered DMSO stocks of peptides and DP3 at 20 mM showed potential for blocking bAP25 – 35 aggregation ŽFig. 5.. However, when prepared from DMSO stock solution that had ‘seeds’ removed by filtration before dilution, decoy peptide DP3 by itself at 20 mM in Tyrode’sr2 mM Ca produces a much smaller increase of wCa2q x int ŽFig. 7.. Interestingly, when decoy peptide DP3 from unfiltered stocks and b-amyloid peptide were mixed at molar ratios of 0.5:1 or greater, we observed a marked decrease in the ability of bAP25 – 35 to induce influx of calcium ŽFig. 5.. This inhibition became more marked as the proportion of decoy peptide increased. At a molar ratio of 0.25:1, we did not observe a decrease in calcium influx but instead a considerable increase. We have at present no explanation for this observation. Subsequent experiments used filtered stocks.
Addition of decoy peptide DP3 from filtered stock after bAP25 – 35 has aggregated does not reduce the induction of calcium influx by the b-amyloid peptide ŽFig. 6.. Apparently, the inhibitory effect of this decoy peptide is not due to competition at the cell surface site between aggregated bAP25 – 35 and possibly aggregated DP3. When DP3 is present from the beginning of the experiment, the effect of bAP25 – 35 on wCa2q x int is greatly reduced. This suggests that DP3 interferes with the bAP25 – 35 aggregation itself, resulting in a modified fibril that is inactive in our system. Since the selected decoy peptides have by their method of selection considerable, or even strong, b-sheet forming potential, they might also allow calcium influx to occur when they themselves are applied to cells. This is true for DP1, DP3, DP6 and DP7 ŽFig. 7., though these are not as effective as bAP25 – 35 at equimolar concentration Ž20 mM.. It is important to note that DP4, DP5 and DP8 by themselves do not cause calcium influx ŽFig. 7.. When added at the beginning to bAP25 – 35 , DP1, DP3, DP4, DP6, DP7 and DP8 either reduce or abolish the rise in internal calcium due to bAP25 – 35 ŽFig. 5, Fig. 7, Fig. 8.. Only DP5 augments the calcium influx caused by bAP25 – 35 . The reason for this behavior is not yet known. When decoy peptide DP8 from filtered DMSO stocks
Fig. 7. Effectiveness of decoy peptides. Each column represents the mean wCa2q xint of 5–21 cells " S.D. measured as described in Fig. 1 and Section 2. bAP25 – 35 and all decoy peptides Žexcept for DP1 which is water soluble. were prepared as DMSO stock solutions at ; 50 mM, spin-filtered through a Millipore filter ) 30 000 Mr Žcat. no. UFC3LTK00.; aliquots were stored at y408C. Decoy peptides DP1, and DP3–DP8 were diluted in Tyrode’sr2 mM Ca or mixed with bAP25 – 35rTyrode’sr2 mM Ca at a ratio of 1:1 Ž20 mM q 20 mM.. The significance of the difference of certain means from the mean of bAP25 – 35 treatment is expressed as: ) P - 0.05; ) ) P - 0.01; ) ) ) ) P - 0.0001.
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47
Fig. 8. Decoy peptide DP8 blocks bAP25 – 35 . Each column represents the mean wCa2q x int of 9–31 cells " S.D. measured as described in Section 2 at varying molar ratios of DP8 to bAP25 – 35 — 0.2:1 Ž5 mM q 20 mM., 1:1 Ž10 mM q 10 mM., 2:1 Ž20 mM q 11 mM.. The significance of the difference of certain means from the mean of bAP25 – 35 treatment is expressed as ) ) ) ) P - 0.0001.
Fig. 9. Decoy peptide DP8 blocks bAP1 – 42 . Each column represents the mean wCa2q x int of 9–10 cells, measured as described in Section 2. The first column shows a significant increase in wCa2q x int after 10 min application of 20 mM aggregated bAP1 – 42 Žwater soluble stock solution, incubated at 378C for 48 h.. Columns 2 and 3 show wCa2q xint when hNT cells are challenged with 20 mM DP8 q 20 mM bAP1 – 42 after co-incubation at 378C for 24 h measured after 10 min and 60 min, respectively. The significance of the difference of certain means from the mean of bAP1 – 42 treatment is expressed as: )) P - 0.01; ) ) ) ) P - 0.0001.
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B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
was added to hNT cells Ž n s 74 cells., there was no or very little effect on calcium influx ŽFig. 7, Fig. 8.. It is interesting that this decoy peptide when mixed with bAP25 – 35 is able to reduce the effect of the b-amyloid peptide completely at molar ratios of 1:1 and 2:1 ŽFig. 8. and also with bAP1 – 42 at a molar ratio of 1:1 ŽFig. 9..
4. Discussion The formation of the pathological b-amyloid peptides in Alzheimer’s disease is not well understood. The precursor protein is a very large transmembrane protein whose normal turnover degradation would cleave the presumptive b-amyloid peptide in the middle, thus making it inactive as a neurotoxic agent. How this is avoided in AD is only gradually becoming clear. In addition, the future Cterminus of b-amyloid peptides is buried in the middle of the lipid membrane. While there are theories as to its cleavage, there is no complete explanation as yet. At least three b-amyloid peptides, bAP1 – 40 , bAP1 – 42 , and bAP25 – 35 , are strongly neurotoxic when applied to cultured cells w20x. While bAP1 – 40 and bAP1 – 42 are the most prominent components of senile plaques, it is not clear whether our model peptide, bAP25 – 35 , occurs in the brains of AD individuals. It is considered by many researchers that the development of AD is paralleled by, or perhaps caused by, a great increase in the relative amount of bAP1 – 42 produced. Our early experiments were carried out with bAP25 – 35 , but currently we use bAP1 – 42 . It is worth noting that the b-amyloid peptides are homologous to the tachykinin neuropeptides, as indicated in the box below. Substance P can protect brain tissue against the neurotoxicity of the b-amyloid peptides w11x, as do substance P and the tachykinin phaesalaemin in cell culture w20x.
The bAP1 – 43 peptide, and related shorter peptides, are cytotoxic towards cultured neuronal cells at mM concentrations, but are neurotrophic at nM concentrations w20x. Others have observed that the peptide is cytotoxic also in
vivo w11x. Variability in results from different laboratories can perhaps be ascribed to variability in speed or degree of aggregation of particular b-amyloid peptides in aqueous solution before and during application. It has been suggested that long term cytotoxicity towards cells in culture resides in insoluble aggregates w5,10,12–14,20x. The early molecular mechanism of this cytotoxicity is not well known, perhaps because most of the reported experiments examine chronic cytotoxic effects only after 24–48 h of exposure. Yankner et al. w13x have reported that only the aggregated FIBRILLAR form of bAP1 – 40w42x is neurotoxic. Our early experiments with bAP25 – 35 show that this peptide also forms very long uniform fibrils or ribbons 8–10 nm in diameter, when diluted out of DMSO solution into aqueous Tyrode’sr2 mM Ca Žnegative staining, EM; J.R.Butler and V.M. Ingram, unpublished.. The width of the fibril corresponds approximately to the length of the undecapeptide. It seems likely that the fibril assembles at right angles to the peptide chain, as might be expected. We do not know the nature of the interaction between such a well-formed bsheet fibril and a putative receptor — the calcium permeant channel itself or some intermediate receptor. The aggregated bAP25 – 35 does react positively with Congo red, indicating the expected b-sheet structure ŽA. Hiniker and V.M. Ingram, unpublished.. Our earlier patch-clamp experiments had shown that scrambled bAP25 – 35 was completely inactive. Furthermore, filtrates from suspensions of bAP25 – 35 had no activity w16x. In recent experiments we find that aggregated bAP1 – 42 also causes pronounced influx of calcium. This b-amyloid peptide occurs in Alzheimer brains and is a major component of the senile plaques. The ability of b-amyloid peptides such as bAP1 – 40 to form cation-selective ionophores has been postulated as a mechanism for cytotoxicity w1,2x. However, these experiments were carried out with artificial membranes. While in actual cells the ionophore mechanism might be an important factor, there are at least two other possible mechanisms: direct or indirect interaction between the b-amyloid peptides and existing ion channels, or penetration of the peptides into the cell with consequent release of calcium from internal stores. We can eliminate the last mechanism because our experiments show convincingly that the rise in wCa2q x int is entirely due to influx from the external medium. Clearly, in our experiments the effect of aggregated bAP25 – 35 on hNT cells is a large influx of Ca2q, as measured by the ratiometric fura2 method. Since this is completely blocked by CNQX and DNQX, but not at all by DL-AP5, we deduce w9x that the aggregated peptide interacts with a non-NMDA receptor cation channel that admits Ca2q. The effect is also completely blocked by Mg 2q. This is an accepted blocker of NMDA channels, but Mg 2q is also reported by some w7x as a blocker of non-NMDA receptors. hNT cells are reported to contain both non-NMDA and NMDA channels w21x. In addition,
B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
we know from our earlier patch-clamp experiments w16x that the inward calcium current caused by bAP25 – 35 is not due to an opening of voltage-gated calcium channels, because the current is not blocked by cadmium chloride. Our findings make an immediate connection with the neurobiology of memory formation, since NMDA w18x and non-NMDA AMPA channels w3x are said to be involved, and since interference with memory is one of the early hallmarks of AD. A non-NMDA channel that is constantly open cannot respond appropriately to normal stimuli. Moreover, there is now evidence w19x that fibrillar bAP1 – 42 interacts with the RAGE receptor, which directly or indirectly causes changes in the functioning of the cell. We postulate that the well-known cell-type specificity of neuronal loss in Alzheimer’s is due to the uneven distribution of RAGE and of non-NMDA receptors. Since these observations involve the obligatory role of b-amyloid peptide aggregates, they open the way to experiments that seek to delay or inactivate the formation of bAP25 – 35 aggregates. It is expected that compounds capable of doing this will alleviate neurotoxicity and the harmful cell death of Alzheimer’s disease. It is also possible to think of ways to modulate the calcium-passing ability of affected non-NMDA channels in order to develop a second therapeutic strategy. This notion leads one to propose that cation channel blockers of the non-NMDA kind, such as CNQX, DNQX, NBQX, CBPD, etc., are potentially useful in treating Alzheimer’s disease. It is likely that ultimately the successful control of the disease process in AD will require multiple simultaneous therapies. Selection of decoy peptides from a combinatorial peptide library produced eight hexapeptides with a tri-glycine spacer and with varying degrees of b-sheet-forming potential. We chose the chemically synthesized combinatorial library over the phage-display library because we wanted to obtain peptides that were composed of D-amino acids which would be resistant to the proteolytic attack expected when drugs are given by mouth or by injection. We believe that the stereochemical parameters that are involved when b-sheets are formed from peptides would be the same for peptides wholly formed from D-amino acids, as they are for L-amino acid peptides. The mechanism of interaction between decoy peptide and b-amyloid peptide is not yet clear, but then the mechanism of aggregation of the b-amyloid peptides themselves is not clear. For example, bAP25 – 35 aggregates rapidly and the active aggregate can be removed by filtration soon after the stock DMSO solution is diluted into an aqueous buffer. On the other hand, bAP1 – 42 , when diluted from an aqueous stock solution into neutral buffer immediately has aggregates ) 30 000 Mr , yet is not active on hNT cells; activity requires incubation at 378C for G 24 h. We postulate that aggregation of bAP1 – 42 occurs in at least two stages; only the aggregate formed in the last stage causes calcium influx. Certain decoy peptides prevent the activity of bAP25 – 35 only when the decoy peptide
49
is already present when stock solution of b-amyloid peptide bAP25 – 35 is diluted ŽFig. 6.. Similarly, the decoy peptide DP8 totally prevents activity by bAP1 – 42 when added at the beginning of incubation at 378C ŽFig. 9.. We believe that b-amyloid peptide and decoy peptide co-aggregate to form a new fibril that cannot induce calcium influx. The experiment of Fig. 6 shows that aggregates of pure b-amyloid peptide bAP25 – 35 remain active in causing Ca2q-influx in the presence of subsequently added decoy peptide DP3, unlike aggregates formed when the same decoy peptide is present from the beginning. Preliminary experiments ŽJ. Butler, A. Hiniker and V.M. Ingram, unpublished data. suggest that the decoy peptide DP8 can delay somewhat the time course of b-amyloid aggregation, as measured by light scattering or complex formation with Congo red. We have identified so far six decoy peptides, DP1, DP3, DP4, DP6, DP7 and DP8, that abolish in mM concentration the influx of calcium into hNT cells caused by the action of the b-amyloid peptide bAP25 – 35 . Two of these, DP3 and DP8, have been examined in some detail. DP8 also abolishes calcium influx caused by bAP1 – 42 . We are currently studying the other decoy peptides. The preliminary results with bAP1 – 42 are encouraging because bAP1 – 42 occurs naturally in the human Alzheimer brain. Since we believe that the calcium influx into the human hNT cells caused by this b-amyloid peptide and its homologs is the molecular cause of the massive loss of neurons seen in Alzheimer’s disease, these six decoy peptides are candidate compounds to be developed as therapeutic agents to reduce the neuronal cell loss, or at least to delay its progress. The candidate decoy peptides will have to be evaluated for possible toxicity, and additional completely non-toxic peptides will need to be developed. We hope that the decoy peptides will be able to cross the blood–brain barrier because they are very small and have a high proportion of hydrophobic side chains. Since they are selected by their sequence, i.e. by their ability to complex with a b-amyloid peptide, they might have enough specificity not to interact with other proteins and not to cause side effects.
5. Note added in review Since these experiments were completed, an interesting report from Soto et al. w17x has come to our notice. The authors find that peptides homologous to bAP25 – 35 and with a ‘similar degree of hydrophobicity’ but low b-sheetforming potential can partially inhibit A b fibril formation and partially disaggregate preformed fibrils in vitro. These inhibitory peptides were synthesized from D-amino and contained a few proline residues. A b aggregation was mostly measured by interaction with the b-sheet-binding fluorescent dye Thioflavin-T. Their inhibitor peptides were only active at high Ž10 = . molar excess and were at most
50
B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
50% effective. The authors did not investigate possible effects of aggregated A b on cells or on the wCa2q x int of cells.
Acknowledgements Helpful discussions with our colleagues J. Butler, C.-H. Chen, A. Hiniker, and R. Rabindran are gladly acknowledged. N. Rafizadeh helped to edit the manuscript. The research was supported by the Baum Fund for Alzheimer Research, by the John and Dorothy Wilson Fund, by the Johanna and Kurt Immerwahr Fund for Alzheimer Research, and by MIT’s Undergraduate Research Opportunities Program.
References w1x N. Arispe, H.B. Pollard, E. Rojas, Giant multilevel cation channels formed by Alzheimer disease amyloid b-protein wA b P-Ž1–40.x in bilayer membranes, Proc. Natl. Acad. Sci. USA 90 Ž1993. 10573– 10577. w2x N. Arispe, E. Rojas, H.B. Pollard, Alzheimer disease amyloid b protein forms calcium channels in bilayer membranes: Blockade by tromethamine and aluminum, Proc. Natl. Acad. Sci. USA 90 Ž1993. 567–571. w3x A. Barria, D. Muller, V. Derkach, L.C. Griffith, T.R. Scoderling, Regulatory phosphorylation of AMPA-type glutamate receptors by CAM-KII during long-term potentiation, Science 276 Ž1997. 2042– 2045. w4x P.Y. Chou, G.D. Fasman, Prediction of the secondary structure of proteins from their amino acid sequence, Adv. Enzymol. Relat. Areas Mol. Biol. 47 Ž1978. 45–148. w5x C.W. Cotman, C.J. Pike, A. Copani, Beta-amyloid neurotoxicity: a discussion of in vitro findings, Neurobiol. Aging 13 Ž1992. 587–590. w6x S.R. Durell, H.R. Guy, N. Arispe, E. Rojas, H.B. Pollard, Theoretical models of the ion channel structure of amyloid b-protein, Biophys. J. 67 Ž1994. 2137–2145. w7x D.R. Giovannucci, E.L. Struenkel, Glutamate receptor agonists modulate wCa2q x i in isolated rat melanotropes, Neuroendocrinology 62 Ž1995. 111–122.
w8x H. Han, P.H. Weinreb, T. Peter, J. Lansbury, The core Alzheimer’s peptide NAC forms amyloid fibrils which seed and are seeded by b-amyloid: is NAC a common trigger or target in neurodegenerative disease?, Chem. Biol. 2 Ž1995. 163–169. w9x B. Hille, Ionic Channels of Excitable Membranes, 2nd Edition, Sinauer Associates, 1992, ch. 6. w10x K.S. Kosik, The Alzheimer’s disease sphinx: a riddle with plaques and tangles, J. Cell. Biol. 127 Ž1994. 1501–1504. w11x N.W. Kowall, M.F. Beal, J. Busciglio, L.K. Duffy, B.A. Yankner, An in vivo model for the neurodegenerative effects of b amyloid and protection by substance P, Proc. Natl. Acad. Sci. USA 88 Ž1991. 7247–7251. w12x K.S. Lam, S.E. Salmon, E.M. Hersh, V.J. Hruby, W.M. Kazmierski, R.J. Knapp, A new type of synthetic peptide library for identifying ligand-binding activity, Nature 354 Ž1991. 82–84. w13x A. Lorenzo, B.A. Yanker, Beta-amyloid neurotoxicity requires fibril formation and is inhibited by congo red, Proc. Natl. Acad. Sci. USA 91 Ž1994. 12243–12247. w14x C.J. Pike, A.J. Walencewicz, C.G. Glabe, C.W. Cotman, In vitro aging of beta-amyloid protein causes peptide aggregation and neurotoxicity, Brain Res. 563 Ž1991. 311–314. w15x S.J. Pleasure, N.M. Lee, NTera 2 cells: A human cell line which displays characteristics of human committed neuronal progenitor cell, J. Neurosci. Res. 35 Ž1993. 585–602. w16x K.L. Sanderson, L. Butler, V.M. Ingram, Aggregates of a b-amyloid peptide are required to induce calcium currents in neuron-like human teratocarcinoma cells: relation to Alzheimer’s disease, Brain Res. 744 Ž1997. 7–14. w17x C. Soto, M.S. Kindy, M. Baumann, B. Frangione, Inhibition of Alzheimer’s amyloidosis by peptides that prevent b-sheet conformation, Biochem. Biophys. Res. Commun. 226 Ž1996. 672–680. w18x J.Z. Tsien, D.F. Chen, D. Gerber, C. Tom, E.H. Mercer, D.J. Anderson, M. Mayford, E.R. Kandel, S. Tonegawa, Subregion- and cell type-restricted gene knockout in mouse brain, Cell 87 Ž1996. 1317–1326. w19x S.D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern, A.M. Schmidt, RAGE and amyloid-b peptide neurotoxicity in Alzheimer’s disease, Nature 382 Ž1996. 685–691. w20x B.A. Yankner, L.K. Duffy, D.A. Kirschner, Neurotrophic and neurotoxic effects of amyloid b protein: reversal by tachykinin neuropeptides, Science 250 Ž1990. 279–282. w21x D.P. Younkin, C.-M. Tang, M. Hardy, U.R. Reddy, Q.-Y. Shi, S.J. Pleasure, V.M.Y. Lee, D. Pleasure, Inducible expression of neuronal glutamate receptor channels in the NT2 human cell line, Proc. Natl. Acad. Sci. USA 90 Ž1993. 2174–2178.
Research report
Mechanism and prevention of neurotoxicity caused by b-amyloid peptides: relation to Alzheimer’s disease Barbara J. Blanchard, Genevieve Konopka, Margaret Russell, Vernon M. Ingram
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Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Accepted 29 July 1997
Abstract In Alzheimer’s disease, neurotoxic b-amyloid peptides cause a deleterious influx of calcium ions into neurons. This increase in wCa2q x int is expected to trigger intracellular events that eventually cause cell dysfunction and cell death. We find that the aggregated b-amyloid peptide bAP25 – 35 opens irreversibly a Ca2q-carrying channel, as does aggregated bAP1 – 42 . The opening of this channel is unaffected by DL-AP5, but it is blocked by Mg 2q, CNQX and DNQX, suggesting a non-NMDA channel. External calcium enters and cytosolic calcium levels rise several-fold, as measured by fura-2 ratiometric analysis. Our findings illustrate a very early molecular event in the neurotoxicity of Alzheimer’s disease. To combat the neurotoxic effect of aggregated b-amyloid peptides, we have devised a series of very short antagonistic peptides. Using a combinatorial library of hexapeptides made from D-amino acids, we have selected peptides by their ability to complex with the tagged b-amyloid peptide bAP25 – 35. Certain of these so-called ‘decoy peptides’, as well as some modified decoy peptides, are able to abolish the calcium influx caused by aggregated, probably fibrillar, b-amyloid peptides bAP25 – 35 and bAP1 – 42 . q 1997 Elsevier Science B.V. Keywords: b-Amyloid peptide; b-Amyloid fibril; Calcium ion channel; Calcium homeostasis; Calcium ion concentration; Decoy peptide; hNT neuron; Non-NMDA channel
1. Introduction The development of Alzheimer’s disease ŽAD. is an age-related process involving the dysfunction and eventual death of certain types of neurons in particular regions of the brain. It is important to realize that the differences between the slow neurodegenerative changes associated with ‘normal’ aging and the much more rapid changes in AD are driven by additional factors, genetic or environmental.Thus, there may be several preconditions leading to AD. However, the neuropathology of AD is remarkably similar in all cases. Apparently, different root causes lead to the same or very similar molecular scenarios. It has been proposed w5,10x that the molecular basis of Alzheimer pathogenesis is the appearance in the brain of abnormally high concentrations of the neurotoxic bamyloid peptides that are themselves abnormal degradation products of the large transmembrane precursor protein, APP. Neurotoxicity occurs through the ability of aggregates of these peptides to interact with sites on neurons
)
Corresponding author.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 0 0 3 - 2
that carry the appropriate receptors. The result of this interaction is an influx of external calcium into these neurons that is too large for the cell to control by sequestration or by pumping out. Neurons of older subjects are especially prone to this attack, because they suffer from an age-related deficiency in mitochondrial function and ATP production, both needed to sequester unwanted calcium. There can be many end effects of the increased calcium: activation of protein kinases Že.g. ERKs., consequent hyperphosphorylation of t proteins and formation of NFTs, activation of immediate early gene products, possible induction of apoptosis, etc. Certainly the normal response of the affected neurons to stimuli will be much altered. The early findings of Yankner’s group in 1990 w20x indicated that the b-amyloid peptides bAP25 – 35 and bAP1 – 42 were neurotoxic towards cultured hippocampal neurons in micromolar concentration. To investigate the immediate molecular mechanism of the neurotoxicity, we exposed cultured human hNT neuronal cells to the aggregated b-amyloid peptide bAP25 – 35 as a simple model system. In each experiment the peptide was diluted from a DMSO stock solution into the aqueous external solution and thereby aggregated. We also used aggregated bAP1 – 42 ,
B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
which gave similar results. These treatments cause a large persistent influx of Ca2q from outside of the cell through channels that are described in this paper. It is known that the b-amyloid peptides are neurotoxic when they are in aggregated Žfibrillar. form w13x and that an amorphous precipitate is not toxic to cultured cells. We undertook the present study in order to interfere with b-sheet aggregation that is involved in fibril formation by selecting and designing ‘decoy peptides’ ŽDPs.. These have themselves sufficient b-sheet forming potential to associate with the multimer-forming b-amyloid peptide, and either block the usual aggregation or incorporate into the multimer so as to make it inactive. We describe a number of peptides that have these properties. The successful DP should be resistant to proteolytic digestion, if it is to be useful in therapy. Therefore, we chose to use peptides composed of D-amino acids.
2. Materials and methods 2.1. Experimental treatment hNT cells were from Stratagene. DL-AP5 ŽDL-2-amino5-phosphonovaleric acid . , C N Q X Ž 6-cyano-7nitroquinoxaline-2,3-dione . and DNQX Ž 6,7-dinitroquinoxaline-2,3Ž1 H,4 H .-dione. were from Sigma. Fura-2-AM was from Molecular Probes. bAP25 – 35 was synthesized by the Biopolymers Laboratory at MIT and purified by HPLC. bAP1 – 42 was purchased from Quality Control Biochemicals. hNT cells are human post-mitotic NT2-N neurons, derived by retinoic acid treatment of a teratocarcinoma cell line w15x. They have good neuronal morphology and possess NMDA and non-NMDA receptors w21x and voltagegated Ca-channels w16x. hNT cells were plated 4–20 days before use on poly-D-lysine-coated acid-washed glass coverslips. Only single phase-bright cells, connected by extensive neurite extensions, were used. For wCa2q x int estimation, the coverslips were placed in a coverslip holder ŽMedical Systems Corp... We find bAP25 – 35 to be insoluble in millimolar concentrations in aqueous media at neutral pH. Stock solutions of bAP25 – 35 in DMSO at 20–100 mM tend to aggregate during frozen storage, as well as at room temperature, due to the presence of seed aggregates. We filter our DMSO stock solutions immediately w8x by spin-filtering with Ultrafree-MC filters of low-binding regenerated cellulose with cut-off at 30 000 Mr ŽMillipore cat. no. UFC3LTK00, Bedford, MA.. Concentration of the filtrate is determined by amino acid analysis w8x. Aliquots can be stored for weeks at y408C. To treat cells, the DMSO stock solution of the peptide is diluted into the aqueous Tyrode’s solution, with or without calcium, shortly before use. Final DMSO concentrations are kept near 0.1%.
41
2.2. Fura-2 measurements of internal calcium concentration hNT cells were loaded with 3 mM fura-2-AM at room temperature in Tyrode’s solution containing 2 mM calcium, as described by the supplier ŽMolecular Probes.. Cells were subsequently washed and allowed to recover at 378C for 30 min either in Tyrode’sr2 mM Ca solution alone or with DMSO Ž< 1%. equivalent to the dilution of DMSO stock solutions of the peptides. Loaded cells were examined at room temperature in 400 ml of control Tyrode’sr2 mM Ca, containing the appropriate concentration of DMSO, the vehicle. Typically, wCa2q x int was determined for 6–12 cells per coverslip first in the control buffer Tyrode’sr2 mM Ca, then again after exchanging the buffer for the test solution. Calcium measurements were taken on a Nikon Diaphot inverted microscope with a Fluor-40 objective using Photon Technology International ŽPTI. ratiometric technology ŽRM-M system. and PTI Felix software. The emission fluorescence at 510 nm was measured over an excitation range of 320–400 nm. The ratio of 510 nm emissions at 340 nm and at 380 nm was used to calculate wCa2q x int from a standard calcium concentration curve made with fura-2–Na salt and standard Ca2q solutions ŽMolecular Probes, cat. no. C3009.. Control values of wCa2q x int were usually between 10 and 100 nM. The effects of externally adding bAP25 – 35 , bAP1 – 42, or other reagents, to the cell culture were recorded during the first 15 min of exposure; however, we often noticed a small decrease in wCa2q x int after 30–45 min. The presence of the small concentration of DMSO made little difference. 2.3. Statistics Individual hNT cells Ž; 10. were examined on a glass coverslip. We recorded the change in wCa2q x int of each individual cell as it became exposed to one or more treatments with peptides and reagents. Statistical evaluation was therefore based on the behavior of individual cells and the significance of differences was calculated using paired Student’s t-test analysis, two populations, when the wCa2q x int themselves are reported. When percent changes of wCa2q x int are described, we used the individual Student’s t-test, two populations, to assess the significance of differences between means. The mean and its standard deviation are recorded in the figures; n s number of individual cells that responded to the treatment. 2.4. Selection of ‘decoy peptides’ The MIT Biopolymer Lab. ŽDirector Richard Cook. made a library w12x of some 43 000 individual sequences, each from the D-amino acids Ala, Ile, Val, Ser, Thr and from Gly, all of which are represented in b-amyloid
42
B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
peptides. The peptides are six amino acids long and originally did not have charged side chains. The peptides are attached by their C-terminus to the polystyrene bead via a linker of three Gly residues to facilitate interaction between a member of the peptide library and the dissolved FITC- or Dansyl-tagged bAP25 – 35 peptide. There are many copies of a unique peptide sequence per bead. We have used bAP25 – 35 tagged with a fluorescent label, FITC- or Dansyl-, FITC–GGGGSNKGAIIGLM–COOH and Dansyl–GGGGSNKGAIIGLM–COOH. There is a Gly3 linker separating the bAP-sequence from the fluorescent label. An aliquot Ž100 ml. of a 50 mM solution of FITC-Gly3bAP25 – 35 was added to a suspension of ; 35 mg of the resin-bound hexapeptide library in 0.5 ml PBS. The suspension was very briefly vortexed six times during the next 15 min at room temperature. The beads were washed three times in 1 ml of PBS, spread out in a dish, and examined under ultraviolet light. Out of thousands of
beads, only very few fluoresced brightly on their surface. Beads were transferred with fine forceps onto glass filters for sequencing at MIT’s Biopolymers Laboratory. Similarly, a 12 mM solution in PBS of DANSYL-Gly3bAP25 – 35 was used. Very few blue fluorescent beads were seen, and these were also sequenced.
3. Results 3.1. Aggregated [Ca 2 q ]i n t
b AP2 5 – 35
and
b AP1 – 4 2
increase
3.1.1. b AP2 5 – 35 Application of unfiltered solutionsrsuspensions of bAP25 – 35 to a group of hNT cells loaded with fura-2-AM causes a very marked increase in cytosolic calcium within a few minutes ŽFig. 1.. There is a strong trend of increas-
Fig. 1. An unfiltered suspension of bAP25 – 35 in Tyrode’sr2 mM Ca applied to hNT cells causes influx of Ca2q. A: hNT cells in Tyrode’sr2 mM Ca Žcolumn 1. have an average wCa2q x int of 80 nM. Exposure to bAP25 – 35 at 20 mM Žfrom unfiltered stock solution. causes a stable rise of wCa2q xint to approximately 200 nM Žcolumn 2., decreasing only slightly after 30 min Žcolumn 3.. Final DMSO concentration is at 0.04% in both conditions. Each column represents the mean wCa2q x int of 8 cells " the S.D. of the mean. The significance of the means from the mean of the control values is expressed as: )))) P - 0.0001. B: an unfiltered suspension of aggregated bAP1 – 42 in Tyrode’sr2 mM Ca applied to hNT cells causes influx of Ca2q. Control Žcolumn 1. hNT cells in control Tyrode’sr2 mM Ca have an average wCa2q x int of 50 nM. Exposure to unfiltered aggregated bAP1 – 42 Ž378C, 24 h.. at 20 mM causes a stable rise of wCa2q x int to 94 mM Žcolumn 2.; ) ) ) ) P - 0.0001. C: display of wCa2q xint in individual cells in A. Open squares, control; upward triangle, immediate bAP25 – 35 effect; inverted triangle, after 30 min. D: display of wCa2q x int in individual cells in B. Open squares, control; upward triangle, immediate bAP1 – 42 effect.
B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
43
Table 1 Effect of different concentrations of bAP25 – 35 on wCa2q x int in hNT cells w bAP25 – 35 x
% increase in wCa2q x int Žmean"S.D..
P
a
10 mM 20 mM 50 mM 112–114 mM 130 mM 172 mM 334 mM
170" 85 286"179 343"245 366"260 623"555 367"108 508"561
- 0.05 -10y9 -10y5 -10y5 - 0.05 -10y6 - 0.05
nb 9 70 22 16 11 8 9
hNT cells in Tyrode’sr2 mM Ca have an average wCa2q x int of 80 nM. Exposure to unfiltered dilutions of bAPbAP25 – 35 in Tyrode’sr2 mM Ca for ;10 min causes a stable rise of wCa2q xint . a Difference from Tyrode’sr2 mM Ca control; by paired Student’s t-test. b Number of responding cells 145r163s89%.
ing cytosolic calcium with increasing peptide concentration ŽTable 1.. The effect decreases only slightly during the first hour when peptide is present. It is not readily reversible for as long as 1 h after the peptide is removed from the external medium Ždata not shown; see also Fig. 2.. We often observed that hNT cells treated with the peptide will detach when a second change of external medium is attempted; there is no obvious change in the well-differentiated neuronal morphology of the treated cells before lift-off. The data reported here are derived from experiments in which the designated treatment immediately followed the control Tyrode’s solution. Within each experiment involving 6–12 cells on one coverslip, there is considerable variability from cell to cell in the magnitude
Fig. 2. The increased wCa2q x int is derived from the external medium only. Each column represents wCa2q xint "S.D. after the external medium was changed as indicated; ns6. The same cells were exposed to Tyrode’sr0 mM Ca plus 50 mM bAP25 – 35 Žfrom unfiltered stock., Tyrode’sr2 mM Ca without bAP25 – 35 , and Tyrode’sr2 mM Ca plus 50 mM bAP25 – 35 . Some measurements were repeated after 20 or 15 min, as indicated. The mean control value for cells in Tyrode’sr0 mM Ca was 44 nM. The results are shown"S.D. The significance of the means of the last 3 columns from the initial Tyrode’sr0 mM Ca condition was calculated by the paired Student’s t-test: ) ) ) P - 0.001; ) ) ) ) P - 0.0001.
Fig. 3. Mg 2q blocks the influx of Ca2q caused by bAP25 – 35 . Each column represents wCa2q x int "S.D. for separate experiments in which hNT cells were first exposed to the control solution of Tyrode’sr2 mM Ca. The external solution was then replaced by a test solution containing bAP25 – 35 Ž ; 20 mM. and also MgCl 2 at either 0.5 mM Ž ns18. or 1 mM Ž ns 5., in Tyrode’sr2 mM Ca, as indicated.
of the effect on cytosolic calcium, as expressed by the standard deviation of the mean displayed in the graphs and the table. To determine the origin of the increased internal calcium, cells were exposed to the usual concentrations of peptide in an external Tyrode’s solution that had no calcium ŽFig. 2.. There was no increase in internal calcium, even after 15 min. In fact, there was a small decrease in
Fig. 4. Response of bAP25 – 35-induced calcium influx to NMDA and non-NMDA channel blockers. hNT cells were first exposed to the control solution of Tyrode’sr2 mM Ca. The external solution was then replaced by a test solution containing bAP25 – 35 Ž21 mM. and also DL-AP5 at 50 mM Ž ns10 cells., or CNQX at 22 mM Ž ns14 cells., or DNQX at 8 mM Ž ns6 cells., as indicated. As an exception, included among the CNQX-treated cells were four cells that had first been exposed to bAP25 – 35 Ž21 mM. and had shown the expected 270% increase in wCa2q x int before becoming blocked by CNQX. The results are shown" S.D. The significance of the mean of the DL-AP5 column from the initial Tyrode’sr2 mM Ca condition was calculated by the paired Student’s t-test: ) ) ) ) P - 0.0001.
B.J. Blanchard et al.r Brain Research 776 (1997) 40–50
44 Table 2 D-Amino acid decoy peptides C-Terminus –COOH
–CONH 2
FITC-bAP-Selected I.A.A.G.I.T.G.G.G T.V.I.G.T.I.G.G.G T.G.I.I.A.S.G.G.G
DP1 DP2 DP4
DP3 DP5
Dansyl-bAP-selected T.T.I.V.S.T.G.G.G A.G.V.I.S.I.G.G.G
DP6 DP7
Decoy peptide derived from DP3 T.V.I.Rq.T.I.A.A.A DP8
bAP25 – 35 G.S.N.Kq.G.A.I.I.G.L.M b-Sheet-forming potentials of regions of peptide, wsx s relative b-sheetforming potential, using the Chou–Fasman algorithm: Ts wsx barely above 1; T s wsx )1; Ts wsx 41.
wCa2q x int at 15 min which could indicate the outflow of calcium into the zero calcium external solution. Restoring external calcium to 2 mM, even in the absence of peptide,
at once caused wCa2q x int to rise substantially to almost 200% of control. This was increased slightly by further addition of external peptide. Evidently, the calcium increase comes from external sources only. The experiment in Fig. 2 also demonstrates that the effect of bAP25 – 35 is not reversible, since replacement of the peptide by Tyrode’sr2 mM Ca causes immediate influx of calcium.
3.1.2. b AP1 – 4 2 Exposure of hNT cells to an unfiltered suspension of bAP1 – 42 in Tyrode’sr2 mM Ca, aggregated for 24 h at 378C, causes a highly significant influx of Ca2q ŽFig. 1.. The effect with this peptide also shows a strong concentration dependence Ždata not shown..
3.2. NMDA and non-NMDA ligand-gated channels Fig. 3 indicates that 0.5 mM or 1 mM magnesium chloride in the presence of bAP25 – 35 totally blocks the peptide-induced influx of calcium. Influx caused by bAP25 – 35 was also abolished by CNQX at 22 mM or DNQX at 8 mM; both are known to block specifically
Fig. 5. Decoy peptide DP3 blocks bAP25 – 35 . Each column represents the mean wCa2q x int of 7–10 cells " S.D. measured as described in Section 2. DMSO stock solutions of DP3 had not been filtered. Each group of three columns represents the same set of cells with measurements after application of DP3 q bAP25 – 35 in Tyrode’sr2 mM Ca at ; 5 min Ž5., 20 min Ž20. and after application of 20 mM EGTAq DP3 q bAP25 – 35 in Tyrode’sr2 mM Ca within ; 2 min ŽE., respectively. DP3 Žnon-filtered DMSO stock. q bAP25 – 35 in Tyrode’sr2 mM Ca at a molar ratio of 0.25:1Ž5 mM 20 mM. is the only set that shows a significant increase in wCa2q x int as compared to 20 mM DP3 by itself and 20 mM bAP25 – 35 by itself. DP3 q bAP25 – 35 at molar ratios of 0.5:1 Ž10 mM q 20 mM., 1:1Ž20 mM q 20 mM. and 2:1 Ž40 mM q 20 mM. show a decrease in wCa2q x int . The significance of the means from the mean of the related bAP25 – 35 measurements Ž5 or 20 min after exposure. is: ) P - 0.05; ) ) P - 0.01; ) ) ) P - 0.001.
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Fig. 6. Effect of adding decoy peptide DP3 to aggregated bAP25 – 35 . Each column represents the mean wCa2q x int of 9–13 cells " S.D. and separate aggregation conditions, measured as described in Section 2. bAP25 – 35 and DP3 are both at 20 mM. The first column shows bAP25 – 35 after aggregation for 2 h at RT. In the second column, bAP25 – 35 is allowed to aggregate for 2 h before DP3 is added. In the third column, DP3 is diluted into Tyrode’s 2 mM Ca, allowed to stand for 1 h, and then bAP25 – 35 is added. The significance of the difference of the mean of the third column relative to bAP25 – 35 measurements is ) ) P - 0.01.
non-NMDA ligand-gated channels ŽFig. 4.. On the other hand, DL-AP5, a specific blocker of NMDA ligand-gated channels, did not decrease at all the bAP25 – 35 peptide-induced influx of calcium at 50 mM concentration ŽFig. 4., or at 200 mM Žnot shown.. 3.3. Selection and design of decoy peptides When we began the present experiments, we interpreted our patch-clamp studies w16x of calcium influx by aggregated bAP25 – 35 as being due to the formation of an ionophore, by analogy with Arispe et al. w1,2x who used artificial membranes. Since Durell et al. w6x have proposed a model for b-amyloid ionophore formation that has a series of glycines ‘lining’ the putative ion conductance channel, we included glycine in our original collection of peptides. However, the presence of glycine would make a peptide susceptible to proteolytic digestion. The most recently synthesized decoy peptide DP8 replaces glycines with D-alanines; all other amino acids are D-amino acids. We note that the decoy peptides so far selected Žsee Table 2. tend to begin with a cluster of b-branched amino acid. b-Sheet-forming potential of the selected decoy pep-
tides is indicated in Table 2. 1 Peptides were synthesized either with a –COOH terminus or as the amide. The presence or absence of a C-terminal negative charge can affect the ability of the peptide to interfere with calcium influx caused by bAP25 – 35 Žsee Fig. 8; DP4 and DP5.. The conformational rules that apply to L-amino acid peptides apparently also apply to peptides containing D-amino acids ŽFasman, personal communication.. We have also synthesized and tested a second generation peptide ŽDP8, Table 2., in which Glycine4 of decoy peptide DP3 is replaced with Arginine, and C-terminal spacer glycines are replaced by D-alanine. Without affecting b-sheet forming potential w4x, this change introduces a charged side chain into the middle of the sequence. We hope to affect the function of a b-sheet structure that might form when DP8 and bAP25 – 35 are allowed to aggregate together. Indeed, this is the result ŽFig. 7.. Interestingly, the charged side chains — R in DP8 and K
1
Assessment of b-sheet-forming ability is based on the Chou–Fasman rules, as used in the software ‘Peptide Companion’ w4x.
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in bAP25 – 35 — are adjacent to the regions with highest Žanti-parallel. b-sheet-forming potential ŽTable 2. in the two sequences. 3.4. Effect of decoy peptides on the calcium influx caused by b-amyloid peptides Our first experiments with decoy peptides used unfiltered DMSO stocks of peptides and DP3 at 20 mM showed potential for blocking bAP25 – 35 aggregation ŽFig. 5.. However, when prepared from DMSO stock solution that had ‘seeds’ removed by filtration before dilution, decoy peptide DP3 by itself at 20 mM in Tyrode’sr2 mM Ca produces a much smaller increase of wCa2q x int ŽFig. 7.. Interestingly, when decoy peptide DP3 from unfiltered stocks and b-amyloid peptide were mixed at molar ratios of 0.5:1 or greater, we observed a marked decrease in the ability of bAP25 – 35 to induce influx of calcium ŽFig. 5.. This inhibition became more marked as the proportion of decoy peptide increased. At a molar ratio of 0.25:1, we did not observe a decrease in calcium influx but instead a considerable increase. We have at present no explanation for this observation. Subsequent experiments used filtered stocks.
Addition of decoy peptide DP3 from filtered stock after bAP25 – 35 has aggregated does not reduce the induction of calcium influx by the b-amyloid peptide ŽFig. 6.. Apparently, the inhibitory effect of this decoy peptide is not due to competition at the cell surface site between aggregated bAP25 – 35 and possibly aggregated DP3. When DP3 is present from the beginning of the experiment, the effect of bAP25 – 35 on wCa2q x int is greatly reduced. This suggests that DP3 interferes with the bAP25 – 35 aggregation itself, resulting in a modified fibril that is inactive in our system. Since the selected decoy peptides have by their method of selection considerable, or even strong, b-sheet forming potential, they might also allow calcium influx to occur when they themselves are applied to cells. This is true for DP1, DP3, DP6 and DP7 ŽFig. 7., though these are not as effective as bAP25 – 35 at equimolar concentration Ž20 mM.. It is important to note that DP4, DP5 and DP8 by themselves do not cause calcium influx ŽFig. 7.. When added at the beginning to bAP25 – 35 , DP1, DP3, DP4, DP6, DP7 and DP8 either reduce or abolish the rise in internal calcium due to bAP25 – 35 ŽFig. 5, Fig. 7, Fig. 8.. Only DP5 augments the calcium influx caused by bAP25 – 35 . The reason for this behavior is not yet known. When decoy peptide DP8 from filtered DMSO stocks
Fig. 7. Effectiveness of decoy peptides. Each column represents the mean wCa2q xint of 5–21 cells " S.D. measured as described in Fig. 1 and Section 2. bAP25 – 35 and all decoy peptides Žexcept for DP1 which is water soluble. were prepared as DMSO stock solutions at ; 50 mM, spin-filtered through a Millipore filter ) 30 000 Mr Žcat. no. UFC3LTK00.; aliquots were stored at y408C. Decoy peptides DP1, and DP3–DP8 were diluted in Tyrode’sr2 mM Ca or mixed with bAP25 – 35rTyrode’sr2 mM Ca at a ratio of 1:1 Ž20 mM q 20 mM.. The significance of the difference of certain means from the mean of bAP25 – 35 treatment is expressed as: ) P - 0.05; ) ) P - 0.01; ) ) ) ) P - 0.0001.
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Fig. 8. Decoy peptide DP8 blocks bAP25 – 35 . Each column represents the mean wCa2q x int of 9–31 cells " S.D. measured as described in Section 2 at varying molar ratios of DP8 to bAP25 – 35 — 0.2:1 Ž5 mM q 20 mM., 1:1 Ž10 mM q 10 mM., 2:1 Ž20 mM q 11 mM.. The significance of the difference of certain means from the mean of bAP25 – 35 treatment is expressed as ) ) ) ) P - 0.0001.
Fig. 9. Decoy peptide DP8 blocks bAP1 – 42 . Each column represents the mean wCa2q x int of 9–10 cells, measured as described in Section 2. The first column shows a significant increase in wCa2q x int after 10 min application of 20 mM aggregated bAP1 – 42 Žwater soluble stock solution, incubated at 378C for 48 h.. Columns 2 and 3 show wCa2q xint when hNT cells are challenged with 20 mM DP8 q 20 mM bAP1 – 42 after co-incubation at 378C for 24 h measured after 10 min and 60 min, respectively. The significance of the difference of certain means from the mean of bAP1 – 42 treatment is expressed as: )) P - 0.01; ) ) ) ) P - 0.0001.
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was added to hNT cells Ž n s 74 cells., there was no or very little effect on calcium influx ŽFig. 7, Fig. 8.. It is interesting that this decoy peptide when mixed with bAP25 – 35 is able to reduce the effect of the b-amyloid peptide completely at molar ratios of 1:1 and 2:1 ŽFig. 8. and also with bAP1 – 42 at a molar ratio of 1:1 ŽFig. 9..
4. Discussion The formation of the pathological b-amyloid peptides in Alzheimer’s disease is not well understood. The precursor protein is a very large transmembrane protein whose normal turnover degradation would cleave the presumptive b-amyloid peptide in the middle, thus making it inactive as a neurotoxic agent. How this is avoided in AD is only gradually becoming clear. In addition, the future Cterminus of b-amyloid peptides is buried in the middle of the lipid membrane. While there are theories as to its cleavage, there is no complete explanation as yet. At least three b-amyloid peptides, bAP1 – 40 , bAP1 – 42 , and bAP25 – 35 , are strongly neurotoxic when applied to cultured cells w20x. While bAP1 – 40 and bAP1 – 42 are the most prominent components of senile plaques, it is not clear whether our model peptide, bAP25 – 35 , occurs in the brains of AD individuals. It is considered by many researchers that the development of AD is paralleled by, or perhaps caused by, a great increase in the relative amount of bAP1 – 42 produced. Our early experiments were carried out with bAP25 – 35 , but currently we use bAP1 – 42 . It is worth noting that the b-amyloid peptides are homologous to the tachykinin neuropeptides, as indicated in the box below. Substance P can protect brain tissue against the neurotoxicity of the b-amyloid peptides w11x, as do substance P and the tachykinin phaesalaemin in cell culture w20x.
The bAP1 – 43 peptide, and related shorter peptides, are cytotoxic towards cultured neuronal cells at mM concentrations, but are neurotrophic at nM concentrations w20x. Others have observed that the peptide is cytotoxic also in
vivo w11x. Variability in results from different laboratories can perhaps be ascribed to variability in speed or degree of aggregation of particular b-amyloid peptides in aqueous solution before and during application. It has been suggested that long term cytotoxicity towards cells in culture resides in insoluble aggregates w5,10,12–14,20x. The early molecular mechanism of this cytotoxicity is not well known, perhaps because most of the reported experiments examine chronic cytotoxic effects only after 24–48 h of exposure. Yankner et al. w13x have reported that only the aggregated FIBRILLAR form of bAP1 – 40w42x is neurotoxic. Our early experiments with bAP25 – 35 show that this peptide also forms very long uniform fibrils or ribbons 8–10 nm in diameter, when diluted out of DMSO solution into aqueous Tyrode’sr2 mM Ca Žnegative staining, EM; J.R.Butler and V.M. Ingram, unpublished.. The width of the fibril corresponds approximately to the length of the undecapeptide. It seems likely that the fibril assembles at right angles to the peptide chain, as might be expected. We do not know the nature of the interaction between such a well-formed bsheet fibril and a putative receptor — the calcium permeant channel itself or some intermediate receptor. The aggregated bAP25 – 35 does react positively with Congo red, indicating the expected b-sheet structure ŽA. Hiniker and V.M. Ingram, unpublished.. Our earlier patch-clamp experiments had shown that scrambled bAP25 – 35 was completely inactive. Furthermore, filtrates from suspensions of bAP25 – 35 had no activity w16x. In recent experiments we find that aggregated bAP1 – 42 also causes pronounced influx of calcium. This b-amyloid peptide occurs in Alzheimer brains and is a major component of the senile plaques. The ability of b-amyloid peptides such as bAP1 – 40 to form cation-selective ionophores has been postulated as a mechanism for cytotoxicity w1,2x. However, these experiments were carried out with artificial membranes. While in actual cells the ionophore mechanism might be an important factor, there are at least two other possible mechanisms: direct or indirect interaction between the b-amyloid peptides and existing ion channels, or penetration of the peptides into the cell with consequent release of calcium from internal stores. We can eliminate the last mechanism because our experiments show convincingly that the rise in wCa2q x int is entirely due to influx from the external medium. Clearly, in our experiments the effect of aggregated bAP25 – 35 on hNT cells is a large influx of Ca2q, as measured by the ratiometric fura2 method. Since this is completely blocked by CNQX and DNQX, but not at all by DL-AP5, we deduce w9x that the aggregated peptide interacts with a non-NMDA receptor cation channel that admits Ca2q. The effect is also completely blocked by Mg 2q. This is an accepted blocker of NMDA channels, but Mg 2q is also reported by some w7x as a blocker of non-NMDA receptors. hNT cells are reported to contain both non-NMDA and NMDA channels w21x. In addition,
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we know from our earlier patch-clamp experiments w16x that the inward calcium current caused by bAP25 – 35 is not due to an opening of voltage-gated calcium channels, because the current is not blocked by cadmium chloride. Our findings make an immediate connection with the neurobiology of memory formation, since NMDA w18x and non-NMDA AMPA channels w3x are said to be involved, and since interference with memory is one of the early hallmarks of AD. A non-NMDA channel that is constantly open cannot respond appropriately to normal stimuli. Moreover, there is now evidence w19x that fibrillar bAP1 – 42 interacts with the RAGE receptor, which directly or indirectly causes changes in the functioning of the cell. We postulate that the well-known cell-type specificity of neuronal loss in Alzheimer’s is due to the uneven distribution of RAGE and of non-NMDA receptors. Since these observations involve the obligatory role of b-amyloid peptide aggregates, they open the way to experiments that seek to delay or inactivate the formation of bAP25 – 35 aggregates. It is expected that compounds capable of doing this will alleviate neurotoxicity and the harmful cell death of Alzheimer’s disease. It is also possible to think of ways to modulate the calcium-passing ability of affected non-NMDA channels in order to develop a second therapeutic strategy. This notion leads one to propose that cation channel blockers of the non-NMDA kind, such as CNQX, DNQX, NBQX, CBPD, etc., are potentially useful in treating Alzheimer’s disease. It is likely that ultimately the successful control of the disease process in AD will require multiple simultaneous therapies. Selection of decoy peptides from a combinatorial peptide library produced eight hexapeptides with a tri-glycine spacer and with varying degrees of b-sheet-forming potential. We chose the chemically synthesized combinatorial library over the phage-display library because we wanted to obtain peptides that were composed of D-amino acids which would be resistant to the proteolytic attack expected when drugs are given by mouth or by injection. We believe that the stereochemical parameters that are involved when b-sheets are formed from peptides would be the same for peptides wholly formed from D-amino acids, as they are for L-amino acid peptides. The mechanism of interaction between decoy peptide and b-amyloid peptide is not yet clear, but then the mechanism of aggregation of the b-amyloid peptides themselves is not clear. For example, bAP25 – 35 aggregates rapidly and the active aggregate can be removed by filtration soon after the stock DMSO solution is diluted into an aqueous buffer. On the other hand, bAP1 – 42 , when diluted from an aqueous stock solution into neutral buffer immediately has aggregates ) 30 000 Mr , yet is not active on hNT cells; activity requires incubation at 378C for G 24 h. We postulate that aggregation of bAP1 – 42 occurs in at least two stages; only the aggregate formed in the last stage causes calcium influx. Certain decoy peptides prevent the activity of bAP25 – 35 only when the decoy peptide
49
is already present when stock solution of b-amyloid peptide bAP25 – 35 is diluted ŽFig. 6.. Similarly, the decoy peptide DP8 totally prevents activity by bAP1 – 42 when added at the beginning of incubation at 378C ŽFig. 9.. We believe that b-amyloid peptide and decoy peptide co-aggregate to form a new fibril that cannot induce calcium influx. The experiment of Fig. 6 shows that aggregates of pure b-amyloid peptide bAP25 – 35 remain active in causing Ca2q-influx in the presence of subsequently added decoy peptide DP3, unlike aggregates formed when the same decoy peptide is present from the beginning. Preliminary experiments ŽJ. Butler, A. Hiniker and V.M. Ingram, unpublished data. suggest that the decoy peptide DP8 can delay somewhat the time course of b-amyloid aggregation, as measured by light scattering or complex formation with Congo red. We have identified so far six decoy peptides, DP1, DP3, DP4, DP6, DP7 and DP8, that abolish in mM concentration the influx of calcium into hNT cells caused by the action of the b-amyloid peptide bAP25 – 35 . Two of these, DP3 and DP8, have been examined in some detail. DP8 also abolishes calcium influx caused by bAP1 – 42 . We are currently studying the other decoy peptides. The preliminary results with bAP1 – 42 are encouraging because bAP1 – 42 occurs naturally in the human Alzheimer brain. Since we believe that the calcium influx into the human hNT cells caused by this b-amyloid peptide and its homologs is the molecular cause of the massive loss of neurons seen in Alzheimer’s disease, these six decoy peptides are candidate compounds to be developed as therapeutic agents to reduce the neuronal cell loss, or at least to delay its progress. The candidate decoy peptides will have to be evaluated for possible toxicity, and additional completely non-toxic peptides will need to be developed. We hope that the decoy peptides will be able to cross the blood–brain barrier because they are very small and have a high proportion of hydrophobic side chains. Since they are selected by their sequence, i.e. by their ability to complex with a b-amyloid peptide, they might have enough specificity not to interact with other proteins and not to cause side effects.
5. Note added in review Since these experiments were completed, an interesting report from Soto et al. w17x has come to our notice. The authors find that peptides homologous to bAP25 – 35 and with a ‘similar degree of hydrophobicity’ but low b-sheetforming potential can partially inhibit A b fibril formation and partially disaggregate preformed fibrils in vitro. These inhibitory peptides were synthesized from D-amino and contained a few proline residues. A b aggregation was mostly measured by interaction with the b-sheet-binding fluorescent dye Thioflavin-T. Their inhibitor peptides were only active at high Ž10 = . molar excess and were at most
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50% effective. The authors did not investigate possible effects of aggregated A b on cells or on the wCa2q x int of cells.
Acknowledgements Helpful discussions with our colleagues J. Butler, C.-H. Chen, A. Hiniker, and R. Rabindran are gladly acknowledged. N. Rafizadeh helped to edit the manuscript. The research was supported by the Baum Fund for Alzheimer Research, by the John and Dorothy Wilson Fund, by the Johanna and Kurt Immerwahr Fund for Alzheimer Research, and by MIT’s Undergraduate Research Opportunities Program.
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