Amyloid β-Peptide Possesses a Transforming Growth Factor-β Activity*

Amyloid β-peptide (Aβ) of 39–42 amino acid residues is a major constituent of Alzheimer’s disease neurite plaques. Aβ 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 Aβ aggregates possess the multiple valencies of anFAED motif (20th to 23rd amino acid residues), which resembles the putative transforming growth factor-β (TGF-β) active site motif, we hypothesize that Aβ monomers and Aβ aggregates may function as TGF-β antagonists and partial agonists, analogous to previously described monovalent and multivalent TGF-β 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 Aβ monomer, Aβ-(1–40) and its fragment, containing the motif inhibit radiolabeled TGF-β binding to cell-surface TGF-β receptors in mink lung epithelial cells (Mv1Lu cells). Aβ-(1–40)-bovine serum albumin conjugate (Aβ-(1–40)-BSA), a multivalent synthetic analogue of Aβ aggregates, exhibited cytotoxicity toward bovine cerebral endothelial cells and rat post-mitotic differentiated hippocampal neuronal cells (H19-7 cells) and inhibitory activities of radiolabeled TGF-β binding to TGF-β receptors and TGF-β-induced plasminogen activator inhibitor-1 expression, that were ∼100–670 times more potent than those of Aβ-(1–40) monomers. At less than micromolar concentrations, Aβ-(1–40)-BSA but not Aβ-(1–40) monomers inhibited proliferation of Mv1Lu cells. Since TGF-β is an organizer of responses to neurodegeneration and is also found in neurite plaques, the TGF-β antagonist and partial agonist activities of Aβ monomers and aggregates may play an important role in the pathogenesis of the disease.

deposition of A␤ aggregates (fibrils) is believed to be an early and critical event in the pathogenesis of Alzheimer's disease. The mechanisms by which A␤ 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 A␤, but none appears to be able to discriminate A␤ monomers from A␤ aggregates (7)(8)(9).
Preparation of BSA Conjugates of A␤-(1-40) and A␤ Fragments-* 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. After mixing at 4°C for 16 h, 50 l of 1 M ethanolamine were added, and the reaction mixture was mixed at room temperature for 2 h, then dialyzed (with dialysis tubing, M r cutoff, 25,000) against 2 liters of 0.1 M NaHCO 3 (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 ϳ5-10 peptides per molecule of protein based on analyses of amino acid composition and SDS-polyacrylamide gel electrophoresis (10).

H]Thymidine Incorporation and Northern Blot Analysis-
The [methyl-3 H]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 g/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 g/ml streptomycin, 50 units/ml penicillin, and 200 g/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.
Dimerization is known to be required for TGF-␤ activity (23) and multivalent TGF-␤ peptide antagonists have been shown to exhibit partial TGF-␤ agonist activity as assayed by growth inhibition (10). We therefore examined the TGF-␤ agonist activity of A␤-(1-40)-BSA by measuring its inhibition of DNA synthesis using Mv1Lu cells. As shown in Fig. 5A (12,24). If the DNA synthesis inhibition by A␤-(1-40)-BSA is mediated by cell surface TGF-␤ receptors, R1B cells, which lack expression of the functional type I TGF-␤ receptor (12,24), should respond very little if any to A␤-(1-40)-BSA DNA synthesis inhibition. As shown in Table I, A␤-(1-40)-BSA did not significantly affect DNA synthesis of R1B cells. This result is consistent with the suggestion that the A␤-(1-40)-BSA exhibits TGF-␤ agonist activity in growth inhibition.
In summary, A␤-(1-40)-BSA is a stable multivalent analogue of naturally occurring A␤ aggregates seen in Alzheimer's disease lesions and is more potent than A␤-(1-40) as a TGF-␤ antagonist that blocks TGF-␤ binding to TGF-␤ receptors. The cytotoxicity of A␤-(1-40)-BSA toward BCE cells and H19-7 cells is ϳ670 times more potent than that of A␤-(1-40). Furthermore, A␤-(1-40)-BSA, which has multiple A␤-(1-40) peptides per BSA molecule, possesses partial TGF-␤ agonist activity (growth inhibition). These results suggest that A␤ monomers and A␤ aggregates may participate in the pathogenesis of neuronal death in Alzheimer's disease patients through their TGF-␤ antagonist and agonist activities. TGF-␤ has been shown to protect neurons from cell death (28 -32). Since TGF-␤ expression has been detected in Alzheimer's disease lesions (28,(33)(34)(35), we hypothesize that the TGF-␤ antagonist activity (TGF-␤ receptor binding inhibitory activity) of A␤-(1-40) monomers and aggregates may counteract this neuroprotective effect of TGF-␤. As both glial cells and monocytes have been shown to express TGF-␤ (35) and to respond to TGF-␤ stimulation (28), the partial TGF-␤ agonist activity (growth inhibition) of A␤ 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-␤ activesite motif (FAED) in A␤, 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-␤ activesite motif with particularly robust function on the basis of studies of various motifs in synthetic TGF-␤ peptide antagonists 2 (10). If the 2nd and 3rd amino acid residues in the motif are amino acids with small side chains (Gly, Ser, Cys, and Ala  residues) and noncharged amino acids, respectively, in the TGF-␤ peptide antagonist motif (WXXD), the potency of TGF-␤ antagonism is enhanced. Determining the TGF-␤ activities of FAD and Dutch-type A␤ mutant peptides would test the hypothesis that the TGF-␤ activities of these peptides are important in the mechanism of A␤ in the neuronal degeneration of Alzheimer's disease.