THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 273, No. 42, Issue of October 16, pp. 27640 –27644, 1998 Printed in U.S.A.
Amyloid b-Peptide Possesses a Transforming Growth Factor-b Activity* (Received for publication, July 23, 1998)
Shuan Shian Huang‡, Franklin W. Huang‡§, Jan Xu¶, Shawei Chen¶, Chung Y. Hsu¶, and Jung San Huang‡i From the ‡Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104 and ¶Center for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110
Amyloid b-peptide (Ab)1 of 39 – 42 amino acid residues comprises the major proteinaceous component of amyloid deposits in the brains of patients with Alzheimer’s disease (1– 6). The
deposition of Ab aggregates (fibrils) is believed to be an early and critical event in the pathogenesis of Alzheimer’s disease. The mechanisms by which Ab aggregates exert their detrimental effects are not well understood, but may involve effects through interactions with specific cell-surface receptors or binding proteins. Several receptors and binding proteins have been reported to interact with Ab, but none appears to be able to discriminate Ab monomers from Ab aggregates (7–9). Recently, we have identified a putative TGF-b active-site motif (WSXD) in TGF-b isoforms (TGF-b1 and TGF-b2) (10). Synthetic peptides containing this motif in the middle of the peptide exhibit TGF-b antagonist activity. Multiple conjugation of these peptides to carrier proteins not only enhances TGF-b antagonist activity but also confers partial TGF-b agonist activity (10). Since Ab contains a motif (FAED) that is similar to the putative TGF-b active-site motif (WSXD) and since Ab aggregates would provide multivalencies with many copies of the putative active-site motif (11), we hypothesize that the Ab monomer and Ab aggregates may function as TGF-b antagonist and partial TGF-b agonist, analogous to previously described monovalent and multivalent TGF-b peptide antagonist/partial agonist, respectively (10). To test this hypothesis, we investigated the TGF-b antagonist/agonist activity of the Ab-(1– 40) monomer and Ab-(1– 40)-bovine serum albumin conjugate (Ab-(1– 40)-BSA) which contains ;5–10 Ab-(1– 40) peptides per molecule of protein and mimics Ab aggregates in multivalencies (11). In this communication, we demonstrate that Ab-(1– 40) monomers inhibited 125I-labeled TGF-b1 binding to cell-surface TGF-b receptors in mink lung epithelial cells (Mv1Lu cells). We also show that Ab-(1– 40)-BSA exhibited a potent cytotoxicity toward bovine cerebral endothelial (BCE) cells and rat post-mitotic differentiated hippocampal neuronal cells (H19-7 cells), and strongly inhibited 125I-TGF-b binding to cell-surface TGF-b receptors and TGF-b-induced expression of plasminogen activator inhibitor 1 (PAI-1). Ab-(1– 40)-BSA but not Ab-(1– 40) monomers inhibited cellular proliferation of Mv1Lu cells. EXPERIMENTAL PROCEDURES
* This work was supported by the National Institutes of Health Grant CA38808. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Summer research student from Harvard University. i To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8135; Fax: 314-577-8156; E-mail:
[email protected]. 1 The abbreviations used are: Ab, amyloid b-peptide; TGF, transforming growth factor; BSA, bovine serum albumin; BCE, bovine cerebral endothelial; PAI, plasminogen activator inhibitor; FAD, familial Alzheimer’s disease; H19-7, rat post-mitotic differentiated hippocampal neuronal cells; TbR, TGF-b receptor; Mv1Lu, mink lung epithelial.
Materials—Na125I (17 Ci/mg) and [methyl-3H]thymidine (67 Ci/ mmol) were purchased from ICN Radiochemicals (Irvine, CA). High molecular mass protein standards (myosin, 205 kDa; b-galactosidase, 116 kDa; phosphorylase, 97 kDa; bovine serum albumin, 66 kDa), Ab-(1– 40), Ab-(1–16), and Ab-(1–20) were obtained from Sigma. Ab-(1– 40), Ab-(25–35), and Ab-(12–28) were obtained from Bachem Bioscience, Inc. (King of Prussia, PA). Disuccinimidyl suberate was obtained from Pierce. TGF-b1 was purchased from Austral Biologicals (San Ramon, CA). b125-(41– 65), a synthetic pentacosapeptide with an amino acid sequence corresponding to the 41st to 65th amino acid residues of TGF-b1, was synthesized as described previously (10). Mv1Lu cells were grown in 10% fetal calf serum in Dulbecco’s modified Eagle’s medium. Preparation of BSA Conjugates of Ab-(1– 40) and Ab Fragments—
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Amyloid b-peptide (Ab) of 39 – 42 amino acid residues is a major constituent of Alzheimer’s disease neurite plaques. Ab aggregates (fibrils) are believed to be responsible for neuronal damage and dysfunction, as well as microglia and astrocyte activation in disease lesions by multiple mechanisms. Since Ab aggregates possess the multiple valencies of an FAED motif (20th to 23rd amino acid residues), which resembles the putative transforming growth factor-b (TGF-b) active site motif, we hypothesize that Ab monomers and Ab aggregates may function as TGF-b antagonists and partial agonists, analogous to previously described monovalent and multivalent TGF-b peptide antagonists and agonists (Huang, S. S., Liu, Q., Johnson, F. E., Konish, Y., and Huang, J. S. (1997) J. Biol. Chem. 272, 27155–27159). Here, we report that the Ab monomer, Ab-(1– 40) and its fragment, containing the motif inhibit radiolabeled TGF-b binding to cell-surface TGF-b receptors in mink lung epithelial cells (Mv1Lu cells). Ab-(1– 40)-bovine serum albumin conjugate (Ab-(1– 40)-BSA), a multivalent synthetic analogue of Ab aggregates, exhibited cytotoxicity toward bovine cerebral endothelial cells and rat postmitotic differentiated hippocampal neuronal cells (H19-7 cells) and inhibitory activities of radiolabeled TGF-b binding to TGF-b receptors and TGF-b-induced plasminogen activator inhibitor-1 expression, that were ;100 – 670 times more potent than those of Ab-(1– 40) monomers. At less than micromolar concentrations, Ab(1– 40)-BSA but not Ab-(1– 40) monomers inhibited proliferation of Mv1Lu cells. Since TGF-b is an organizer of responses to neurodegeneration and is also found in neurite plaques, the TGF-b antagonist and partial agonist activities of Ab monomers and aggregates may play an important role in the pathogenesis of the disease.
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FIG. 1. Amino acid sequences of TGF-b peptide antagonists (b125-(41– 65) and b225-(41– 65)), Ab-(1– 40), and Ab fragments. The amino acid residues underlined are the putative TGF-b active-site motifs. Identical amino acid residues are boxed with a solid line, whereas functionally homologous residues are boxed with a broken line. Dutch, Dutch-type Alzheimer’s disease.
RESULTS AND DISCUSSION
Ab and its fragments have been found to exert cytotoxic and trophic effects on cells in culture. Ab-(25–35) mimics the cyto-
toxic activity of Ab (5), whereas Ab-(1–28), Ab-(25–28), and Ab-(1– 40) exhibit acetylcholine release inhibitory activity (4). The sequence of VFF (residues 18 –20) has been implicated in mediating the amnestic activity of Ab fragments (17). These cytotoxic and trophic effects of Ab appear to be mediated by different domains, e.g. the sequences of residues 25–35, 25–28, and 18 –20 (4, 5, 17). We noted that, in addition to these domains, Ab possesses a motif (FAED, 20th to 23rd amino acid residues), which is similar to the WSXD putative TGF-b activesite motif. Known TGF-b peptide antagonists b125-(41– 65) and b225-(41– 65) that contain this motif are synthetic peptides with amino acid sequences corresponding to the 41st to 65th residues of TGF-b1 and TGF-b2, respectively (Fig. 1) (10). Replacement of the tryptophan residue in the motif by a phenylalanine residue does not affect the antagonist activity of b125-(41– 65).2 Thus, the FAED in the Ab monomer may be a functional TGF-b active-site motif. To test this possibility, we determined the effects of Ab-(1– 40) monomers and Ab fragments possessing and lacking the FAED motif on 125I-TGF-b1 binding to cellsurface TGF-b receptors in Mv1Lu cells, a standard model system for investigating TGF-b receptor types and TGF-binduced cellular responses (12, 13). As shown in Fig. 2, Ab-(1– 40), and Ab-(12–28), both of which contain the motif, exhibited 125 I-TGF-b1 receptor binding inhibitory activities with IC50 of ;3 and ;30 mM, respectively. Ab-(25–35), Ab-(1–20), and Ab(1–16), all of which lack the motif, failed to show 125I-TGF-b1 binding inhibitory activity at any concentration up to 30 mM. These results indicate that Ab-(1– 40) possesses a functional TGF-b active-site motif. It has been reported that the aggregation of Ab monomers is not easily controlled in vitro, as it is strongly affected by Ab concentration, pH, ionic strength, and incubation time (18 –22). In order to produce a multivalent stable inhibitor, we prepared Ab-(1– 40)-bovine serum albumin conjugate (Ab-(1– 40)-BSA, Mr ;90,000 –100,000) containing ;5–10 Ab-(1– 40) per molecule of BSA according to the procedure of Huang, et al. (10). Unlike the rather unstable Ab-(1– 40) aggregates, Ab-(1– 40)BSA is stable (at 4 °C) for at least a few weeks and has a consistent valence due to the covalent nature of the attachment of Ab-(1– 40) to BSA. This conjugate is meant to mimic Ab-(1– 40) aggregates by possessing multiple valences of Ab-(1– 40) per molecule and cytotoxicity toward BCE cells and rat postmitotic differentiated H19-7 cells. As shown in Fig. 3, Ab-(1– 40)-BSA exhibited cytotoxicity toward BCE cells and H19-7 cells that was ;670 times more potent than that of the Ab-(1– 40) monomer (Fig. 3, A and B). Ab-(1– 40)-BSA at 75 nM was as potent as 50 mM Ab-(1– 40) in causing cell death of BCE cells and H19-7 cells as determined by the 3-(4,5-dimethyl thiazol2-yl)-2,5-diphenyl tetrazolium bromide assay (16) and trypan blue exclusion assay (14). Similar cytotoxic effects of Ab-(1– 40) 2
S. S. Huang, F. W. Huang, and J. S. Huang, unpublished results.
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About 0.25 mmol of Ab-(1– 40), Ab-(1–16), Ab-(12–28), or Ab-(25–35) dissolved in 167 ml of H2O were mixed with 133 ml of 0.1 M NaHCO3 and 150 ml of 0.1 M NaHCO3 containing 0.25 mg of BSA. After adjusting the pH to 7.8 – 8.0, 10 ml of 27 mM disuccinimidyl suberate (a bifunctional cross-linking agent) in Me2SO was added into the solution. After mixing at 4 °C for 16 h, 50 ml of 1 M ethanolamine were added, and the reaction mixture was mixed at room temperature for 2 h, then dialyzed (with dialysis tubing, Mr cutoff, 25,000) against 2 liters of 0.1 M NaHCO3 (the pH was adjusted at 8.0). The dialysates were changed four times. The BSA conjugates were kept at 4 °C prior to use and were determined to contain ;5–10 peptides per molecule of protein based on analyses of amino acid composition and SDS-polyacrylamide gel electrophoresis (10). Specific Binding of 125I-TGF-b1 to Mv1Lu Cells—125I-TGF-b1 was prepared by iodination of TGF-b1 with Na125I and chloramine T according to our published procedure (10, 12, 13). 125I-TGF-b1 binding to cell-surface TGF-b receptors was assayed by incubating Mv1Lu cells with 0.1 nM 125I-TGF-b1 in the presence of various concentrations of Ab-(1– 40), Ab fragments, and their BSA conjugates at 0 °C for 2.5 h. The specific binding of 125I-TGF-b1 to cell-surface TGF-b receptors was estimated as described previously (10, 12, 13). 125 I-TGF-b1 Affinity Labeling of Cell-surface TGF-b Receptors in Mv1Lu Cells—The 125I-TGF-b1 affinity labeling of cell-surface TGF-b receptors in Mv1Lu cells was performed as described previously (10, 12, 13). After affinity labeling, the labeled TGF-b receptors were analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. [methyl-3H]Thymidine Incorporation and Northern Blot Analysis— The [methyl-3H]thymidine incorporation into cellular DNA and Northern blot analysis of PAI-1 and glyceraldehyde-3-phosphate dehydrogenase were performed as described previously (10, 13). The relative intensity of transcript on the autoradiogram was quantitated by a PhosphorImager. Cytotoxicity Assay Using BCE Cells and Rat Postmitotic Differentiated H19-7 Cells—BCE cells were prepared from bovine brain as described previously (14) and cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, heparin (0.5 mg/ml), and endothelial growth supplements (75 mg/ml). Rat hippocampal progenitor cells with neuronal lineage (H19-7 cells) were immortalized with a temperature-sensitive SV40 large T antigen provided by Drs. Eva M. Eves and Marsha R. Rosner, Ben May Institute for Cancer Research, University of Chicago (15). H19-7 cells were cultured at 33 °C in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 50 mg/ml streptomycin, 50 units/ml penicillin, and 200 mg/ml G418. H19-7 cells grown under this condition were defined as mitotic progenitor cells with neuronal lineage. To achieve a postmitotic differentiated state, N2 supplement and basic fibroblast growth factor (10 ng/ml) were added to the medium, and the temperature shifted to the nonpermissive range for a temperature-sensitive SV40 large T antigen (39 °C) for 24 h to allow differentiation (15) before cytotoxicity assay. BCE cells grown on 96-well cluster dishes were incubated with various concentrations of Ab-(1– 40) or Ab-(1– 40)-BSA in serum-free Dulbecco’s modified Eagle’s medium. After 48 h at 37 °C, the extent of cell death was determined by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (16). The postmitotic and differentiated H19-7 cells grown on 24-well cluster dishes were incubated with various concentrations of Ab-(1– 40) or Ab-(1– 40)-BSA. After 24 h, the cell survival was assessed by trypan blue exclusion assay (14).
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FIG. 3. Cytotoxic effects of Ab-(1– 40) and Ab-(1– 40)-BSA in BCE cells and H19-7 cells. A, BCE cells were exposed to Ab-(1– 40) or Ab-(1– 40)-BSA at concentrations indicated for 48 h. The extent of cell survival (mean 6 S.D.) was determined in quadruplicate cell cultures by the MTT assay. The asterisk denotes a p value of ,0.05. The data are representative of two experiments that gave comparable results. B, H19-7 cells were exposed to Ab-(1– 40) or Ab-(1– 40)-BSA at concentrations indicated for 24 h. The extent of cell survival (mean 6 S.D.) was determined in quadruplicate cell cultures by the trypan blue exclusion assay. The asterisk denotes a p value of ,0.05. The data are representative of two experiments that gave comparable results.
and Ab-(1– 40)-BSA in BCE cells and H19-7 cells were also noted using lactate dehydrogenase assay (14) and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, respectively (data not shown). To determine the TGF-b antagonist activity of Ab-(1– 40)BSA, we examined the 125I-TGF-b1 receptor binding inhibitory
FIG. 4. Effects of the BSA conjugates of Ab-(1– 40) and its fragments on 125I-TGF-b1 binding (A) and 125I-TGF-b1-affinity labeling (B) in Mv1Lu cells. A, cells were incubated with various concentrations of the BSA conjugates of Ab-(1– 40), Ab-(12–28), Ab-(25–35), or Ab-(1–16) at 0 °C for 25 h. The specific binding of 125I-TGF-b1 without the BSA conjugates was taken as 0% inhibition. The error bars are means 6 S.D. of triplicate cell cultures. The data are representative of five experiments that gave comparable results. B, after 125I-TGF-b1 binding, in the absence (control) and presence of 10 mM b125(41– 65), 0.1 mM Ab-(1– 40)-BSA, or 5 mM Ab-(1– 40), the 125I-TGF-b1 affinity labeling of cell-surface TGF-b receptors was performed. The 125I-TGF-b1 affinity-labeled receptors were analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography. The brackets indicate the locations of the types I, II, III, and V TGF-b receptors (TbR-I, TbR-II, TbR-III, and TbR-V).
activity of Ab-(1– 40)-BSA in Mv1Lu cells. As shown in Fig. 4A, Ab-(1– 40)-BSA inhibited 125I-TGF-b1 binding to cell-surface TGF-b receptors with an IC50 of ;30 nM. This is 100-fold more potent than Ab-(1– 40) monomers. The BSA conjugate of Ab(12–28), which contains the FAED motif, showed somewhat weaker 125I-TGF-b1 binding inhibitory activity (IC50 ; 0.3 mM). The BSA conjugates of the Ab-(1–16) and Ab-(25–35) fragments, which lack the motif, did not inhibit 125I-TGF-b1 binding. In control experiments, BSA subjected to conjugation conditions without peptide also had no inhibitory activity (10). 125 I-TGF-b1 affinity labeling analysis revealed that Ab-(1– 40)BSA conjugate almost completely inhibited 125I-TGF-b1 binding to type I, type II, type III, and type V TGF-b receptors (TbR -I, TbR-II, TbR-III, and TbR-V) at 0.1 mM, whereas Ab-(1– 40) only partially inhibited 125I-TGF-b1 binding to its receptors at
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FIG. 2. Effects of Ab-(1– 40) and Ab fragments on 125I-TGF-b1 binding in Mv1Lu cells. Cells were incubated with 0.1 nM 125I-TGF-b1 and various concentrations of Ab-(1– 40), Ab-(12–28), Ab-(25–35), Ab(1–20), and Ab-(1–16) at 0 °C for 2.5 h. The specific binding of 125ITGF-b without Ab-(1– 40) and Ab fragments was taken as 0% inhibition (5,429 6 780 cpm/well). The error bars are means 6 S.D. of triplicate cell cultures. The data are representative of seven experiments that gave comparable results.
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TABLE I Effect of Ab-(1– 40)-BSA and Ab-(1– 40) on DNA synthesis of type I TGF-b receptor-defective and wild-type mink lung epithelial cells (R1B and Mv1Lu cells) [methyl-3H]Thymidine incorporationa R1B cells
Mv1Lu cells cpm/well
Control 10.35 mM Ab-(1–40)-BSA 110 mM Ab-(1–40)
4890 6 240 4520 6 480 5019 6 320
23,940 6 1,520 15,082 6 929 24,120 6 2,001
a Cells were incubated with 0.35 mM Ab-(1– 40)-BSA or 10 mM Ab-(1– 40) at 37 °C for 16 h. DNA synthesis was determined by measuring [methyl-3H]thymidine incorporation into cellular DNA. The assay was carried out in triplicate cell cultures.
5 mM (Fig. 4B). These results indicate that the multiple valencies enhance the 125I-TGF-b1 binding-inhibitory activity of Ab-(1– 40). Dimerization is known to be required for TGF-b activity (23) and multivalent TGF-b peptide antagonists have been shown to exhibit partial TGF-b agonist activity as assayed by growth inhibition (10). We therefore examined the TGF-b agonist activity of Ab-(1– 40)-BSA by measuring its inhibition of DNA synthesis using Mv1Lu cells. As shown in Fig. 5A, 0.35 mM of multivalent Ab-(1– 40)-BSA produced ;35% inhibition of [methyl-3H]thymidine incorporation into DNA of Mv1Lu cells. Neither Ab-(1– 40) monomers (0.35 mM) nor BSA conjugated in the absence of peptide (at any concentration up to 10 mM) affected DNA synthesis in this system. The DNA synthesis inhibition induced by 0.35 mM Ab-(1– 40)-BSA was blocked in the presence of 10 mM b125-(41– 65), a specific TGF-b peptide antagonist (data not shown). These results suggest that multiple valencies of Ab-(1– 40) confer TGF-b agonist activity, i.e. inhibit cellular proliferation as measured by DNA synthesis. To support this suggestion, we determined the effect of Ab-(1– 40)BSA on DNA synthesis of type I TGF-b receptor-defective mutant and wild-type mink lung epithelial cells (R1B and Mv1Lu
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FIG. 5. Effects of BSA conjugates of Ab-(1– 40) and Ab fragments on DNA synthesis (A) and TGF-b1-induced PAI-1 expression (B) in Mv1Lu cells. A, cells were incubated with various concentrations of Ab-(1– 40) and BSA conjugates of Ab-(1– 40), Ab-(12–28), Ab-(25–35), and Ab-(1–20) at 37 °C for 16 h. DNA synthesis was determined by measuring [methyl-3H]thymidine incorporation into cellular DNA. The [methyl-3H]thymidine incorporations in the presence and absence of 10 pM TGF-b1 were taken as 100 and 0% inhibition (2,242 6 679 and 25,493 6 1,200 cpm/well, respectively). The error bars are means 6 S.D. of triplicate cell cultures. The data are representative of six experiments which gave comparable results. B, cells were treated with 0.5 pM TGF-b1 in the presence of various concentrations of Ab-(1– 40)-BSA at 37 °C for 2 h. PAI-1 expression was determined by Northern blot analysis. The expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as a control. The relative intensities of transcripts on the autoradiograms were estimated by a PhosphorImager.
cells) (12, 24). If the DNA synthesis inhibition by Ab-(1– 40)BSA is mediated by cell surface TGF-b receptors, R1B cells, which lack expression of the functional type I TGF-b receptor (12, 24), should respond very little if any to Ab-(1– 40)-BSA DNA synthesis inhibition. As shown in Table I, Ab-(1– 40)-BSA did not significantly affect DNA synthesis of R1B cells. This result is consistent with the suggestion that the Ab-(1– 40)BSA exhibits TGF-b agonist activity in growth inhibition. The transcriptional expression of PAI-1 is known to be stimulated by TGF-b1 (25–27). To determine whether Ab-(1– 40)BSA interferes with this effect, Mv1Lu cells were treated with 0.5 pM TGF-b1 plus various concentrations of Ab-(1– 40)-BSA for 2 h at 37 °C. Northern blot analysis was then performed. As shown in Fig. 5B, Ab-(1– 40)-BSA did not stimulate the expression of PAI-1 (lane 2 versus lane 1). However, Ab-(1– 40)-BSA diminished the PAI-1 expression stimulated by 0.5 pM TGF-b in a dose-dependent manner (lanes 4 – 6). This suggests that Ab(1– 40)-BSA can bind to TGF-b receptors and function as an antagonist for TGF-b as assayed by transcriptional activation. In summary, Ab-(1– 40)-BSA is a stable multivalent analogue of naturally occurring Ab aggregates seen in Alzheimer’s disease lesions and is more potent than Ab-(1– 40) as a TGF-b antagonist that blocks TGF-b binding to TGF-b receptors. The cytotoxicity of Ab-(1– 40)-BSA toward BCE cells and H19-7 cells is ;670 times more potent than that of Ab-(1– 40). Furthermore, Ab-(1– 40)-BSA, which has multiple Ab-(1– 40) peptides per BSA molecule, possesses partial TGF-b agonist activity (growth inhibition). These results suggest that Ab monomers and Ab aggregates may participate in the pathogenesis of neuronal death in Alzheimer’s disease patients through their TGF-b antagonist and agonist activities. TGF-b has been shown to protect neurons from cell death (28 –32). Since TGF-b expression has been detected in Alzheimer’s disease lesions (28, 33–35), we hypothesize that the TGF-b antagonist activity (TGF-b receptor binding inhibitory activity) of Ab-(1– 40) monomers and aggregates may counteract this neuroprotective effect of TGF-b. As both glial cells and monocytes have been shown to express TGF-b (35) and to respond to TGF-b stimulation (28), the partial TGF-b agonist activity (growth inhibition) of Ab aggregates may also play an important role in the chemotaxis and activation of astrocytes and microglia that are associated with Alzheimer’s disease. The familial Alzheimer’s disease (FAD) (36) and Dutch-type Alzheimer’s disease (37) patients may provide some clues to the structure/function relationship of the putative TGF-b activesite motif (FAED) in Ab, since these patients have mutations within this motif (Fig. 1). The mutations in both FAD (A692G) and Dutch-type (E693Q) patients may provide a TGF-b activesite motif with particularly robust function on the basis of studies of various motifs in synthetic TGF-b peptide antagonists2 (10). If the 2nd and 3rd amino acid residues in the motif are amino acids with small side chains (Gly, Ser, Cys, and Ala
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residues) and noncharged amino acids, respectively, in the TGF-b peptide antagonist motif (WXXD), the potency of TGF-b antagonism is enhanced. Determining the TGF-b activities of FAD and Dutch-type Ab mutant peptides would test the hypothesis that the TGF-b activities of these peptides are important in the mechanism of Ab in the neuronal degeneration of Alzheimer’s disease. Acknowledgments—We thank Drs. William S. Sly, Frank E. Johnson, and Uthay Ezekiel for critical comments and review of the manuscript and John McAlpin for preparing the manuscript. REFERENCES
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Amyloid β-Peptide Possesses a Transforming Growth Factor-β Activity Shuan Shian Huang, Franklin W. Huang, Jan Xu, Shawei Chen, Chung Y. Hsu and Jung San Huang J. Biol. Chem. 1998, 273:27640-27644. doi: 10.1074/jbc.273.42.27640
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