FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1993593920> ?p ?o ?g. }

• W1993593920 endingPage "38722" @default.
• W1993593920 startingPage "38715" @default.
• W1993593920 abstract "Transforming growth factor-β (TGF-β) receptor-mediated signaling has been proposed to mediate both the beneficial and deleterious roles for this cytokine in amyloid-β protein (Aβ) function. In order to assess receptor dependence of these events, we used PC12 cell cultures, which are devoid of TGF-β receptors. Surprisingly, TGF-β potentiated the neurotoxic effects of the 40-residue Aβ peptide, Aβ-(1–40), in this model suggesting that there may be a direct, receptor-independent interaction between TGF-β and Aβ-(1–40). Surface plasmon resonance confirmed that TGF-β binds with high affinity directly to Aβ-(1–40) and electron microscopy revealed that TGF-β enhances Aβ-(1–40) oligomerization. Immunohistochemical examination of mouse brain revealed that hippocampal CA1 and dentate gyrus, two regions classically associated with Aβ-mediated pathology, lack TGF-β Type I receptor immunoreactivity, thus indicating that TGF-β receptor-mediated signaling would not be favored in these regions. Our observations not only provide for a unique, receptor-independent mechanism of action for TGF-β, but also help to reconcile the literature interpreting the role of TGF-β in Aβ function. These data support a critical etiological role for this mechanism in neuropathological amyloidoses. Transforming growth factor-β (TGF-β) receptor-mediated signaling has been proposed to mediate both the beneficial and deleterious roles for this cytokine in amyloid-β protein (Aβ) function. In order to assess receptor dependence of these events, we used PC12 cell cultures, which are devoid of TGF-β receptors. Surprisingly, TGF-β potentiated the neurotoxic effects of the 40-residue Aβ peptide, Aβ-(1–40), in this model suggesting that there may be a direct, receptor-independent interaction between TGF-β and Aβ-(1–40). Surface plasmon resonance confirmed that TGF-β binds with high affinity directly to Aβ-(1–40) and electron microscopy revealed that TGF-β enhances Aβ-(1–40) oligomerization. Immunohistochemical examination of mouse brain revealed that hippocampal CA1 and dentate gyrus, two regions classically associated with Aβ-mediated pathology, lack TGF-β Type I receptor immunoreactivity, thus indicating that TGF-β receptor-mediated signaling would not be favored in these regions. Our observations not only provide for a unique, receptor-independent mechanism of action for TGF-β, but also help to reconcile the literature interpreting the role of TGF-β in Aβ function. These data support a critical etiological role for this mechanism in neuropathological amyloidoses. The 39–43-mer amyloid-β (Aβ) 1The abbreviations used are: Aβ, amyloid-β; TGF, transforming growth factor; MTT, 4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium.1The abbreviations used are: Aβ, amyloid-β; TGF, transforming growth factor; MTT, 4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium. peptide is derived from the membrane-bound amyloid precursor protein (APP) as an aberrant cleavage product (1Selkoe D.J. Trends Cell Biol. 1998; 8: 447-453Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar). Transgenic APP mouse models exhibit age-related extracellular amyloid deposits (plaques) and neurodegeneration as well as cerebral amyloid angiopathy (CAA) comparable to that found in human Alzheimer's disease (AD) brain (2Sturchler-Pierrat C. Abramowski D. Duke M. Wiederhold K.H. Mistl C. Rothacher S. Ledermann B. Burki K. Frey P. Paganetti P.A. Waridel C. Calhoun M.E. Jucker M. Probst A. Staufenbiel M. Sommer B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13287-13292Crossref PubMed Scopus (1248) Google Scholar, 3Calhoun M.E. Burgermeister P. Phinney A.L. Stalder M. Tolnay M. Wiederhold K.H. Abramowski D. Sturchler-Pierrat C. Sommer B. Staufenbiel M. Jucker M. Proc. Natl, Acad, Sci. U. S. A. 1999; 96: 14088-14093Crossref PubMed Scopus (351) Google Scholar). These same models respond to both active and passive immunization against Aβ as evidenced by the reduction in levels of Aβ, the prevention and/or clearance of amyloid plaques, and the improvement in cognitive behavior (4Schenk D. Nat. Rev. Neurosci. 2002; 3: 824-828Crossref PubMed Scopus (413) Google Scholar). However, an effective preparation free of significant side effects in humans is still awaited. Indeed, clinical trials involving Aβ vaccination have been discontinued following the development of inflammation in patients brains (5Haass C. Nat. Med. 2002; 8: 1195-1196Crossref PubMed Scopus (20) Google Scholar). Cerebral microhemmorhaging has also been observed in similarly immunized mice (6Pfeifer M. Boncristiano S. Bondolfi L. Stalder A. Deller T. Staufenbiel M. Mathews P.M. Jucker M. Science. 2002; 298: 1379Crossref PubMed Scopus (456) Google Scholar). Although it has been suggested that antibodies capable of recognizing other Aβ epitopes or conformations should be screened (6Pfeifer M. Boncristiano S. Bondolfi L. Stalder A. Deller T. Staufenbiel M. Mathews P.M. Jucker M. Science. 2002; 298: 1379Crossref PubMed Scopus (456) Google Scholar), perhaps a closer examination of modulators of Aβ fibrillogenesis may reveal a target better suited for immunotherapy. Among the modulators proposed to date, which include apolipoprotein E, cholesterol, and α2-macroglobulin, we were particularly interested in the cytokine transforming growth factor-β (TGF-β). The TGF-β1 isoform was recently implicated as a co-factor for amyloid deposition with the observation that cerebrovascular amyloid deposits, which are strikingly similar to those seen in patients with AD and CAA, and the ensuing neuropathological development are accelerated in bigenic mice overexpressing both TGF-β1 and human APP (hAPP), relative to hAPP transgenic mice controls (7Wyss-Coray T. Masliah E. Mallory M. McConlogue L. Johnson-Wood K. Lin C. Mucke L. Nature. 1997; 389: 603-606Crossref PubMed Scopus (360) Google Scholar). These authors suggested that cerebrovascular amyloid deposition might reflect TGF-β receptor-mediated induction of extracellular matrix deposition. Aβ binding proteins within these extracellular matrix components could enhance the formation and/or stability of Aβ fibrils (8Gupta-Bansal R. Frederickson R.C. Brunden K.R. J. Biol. Chem. 1995; 270: 18666-18671Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 9Castillo G.M. Ngo C. Cummings J Wight T.N. Snow A.D. J. Neurochem. 1997; 69: 2452-2465Crossref PubMed Scopus (224) Google Scholar). A link between TGF-β and Aβ deposition was already being considered following the localization of TGF-β immunoreactivity to senile plaques and neurofibrillary tangle-bearing neurons in AD patient brain (7Wyss-Coray T. Masliah E. Mallory M. McConlogue L. Johnson-Wood K. Lin C. Mucke L. Nature. 1997; 389: 603-606Crossref PubMed Scopus (360) Google Scholar, 10Flanders K.C. Ren R.F. Lippa C.F. Prog. Neurobiol. 1998; 54: 71-85Crossref PubMed Scopus (335) Google Scholar). TGF-βs had also been shown to enhance the formation of amyloid deposits in rats when co-injected intracerebroventricularly with Aβ-(1–40) (11Frautschy S.A. Yang F. Calderon L. Cole G.M. Neurobiol. Aging. 1996; 17: 311-321Crossref PubMed Scopus (124) Google Scholar) and to increase the number of Aβ plaque-like deposits in hippocampal slice culture in a subfield-selective manner (12Harris-White M.E. Chu T. Balverde Z. Sigel J.J. Flanders K.C. Frautschy S.A. J. Neurosci. 1998; 18: 10366-10374Crossref PubMed Google Scholar). Finally, APP production and Aβ-(1–40)/Aβ-(1–42) processing were promoted by TGF-β1 in transgenic mice (13Lesne S. Docagne F. Gabriel C. Liot G. Lahiri D.K. Buee L. Plawinski L. Delacourte A. MacKenzie E.T. Buisson A. Vivien D. J. Biol. Chem. 2003; 278: 18408-18418Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In contrast to its role as a risk factor, TGF-β1 facilitates Aβ clearance and plaque burden reduction following activation of parenchymal glial cells in TGF-β1/hAPP bigenic mouse brain (14Wyss-Coray T. Lin C. Yan F. Yu G.Q. Rohde M. McConlogue L. Masliah E. Mucke L. Nat. Med. 2001; 7: 612-618Crossref PubMed Scopus (510) Google Scholar). TGF-βs have also been shown to protect neuronal cell cultures against Aβ-mediated insult (15Prehn J.H. Bindokas V.P. Jordan J. Galindo M.F. Ghadge G.D. Roos R.P. Boise L.H. Thompson C.B. Krajewski S. Reed J.C. Miller R.J. Mol. Pharmacol. 1996; 49: 319-328PubMed Google Scholar, 16Ren R.F. Flanders K.C. Brain Res. 1996; 732: 16-24Crossref PubMed Scopus (61) Google Scholar, 17Ren R.F. Hawver D.B. Kim R.S. Flanders K.C. Mol. Brain Res. 1997; 48: 315-322Crossref PubMed Scopus (38) Google Scholar) as a direct consequence of TGF-β Type II receptor activation (17Ren R.F. Hawver D.B. Kim R.S. Flanders K.C. Mol. Brain Res. 1997; 48: 315-322Crossref PubMed Scopus (38) Google Scholar). These apparently opposing actions of TGF-β in the brain illustrate the multifunctionality of this cytokine and the dependence of its effects on the specific cellular context in which it is expressed. Additional investigation is needed to characterize the cellular and molecular processes that underlie the effects of TGF-β during Aβ-mediated pathology. To date, the actions of TGF-β have centered on receptor-mediated events. In this report, we demonstrate a direct interaction between TGF-β and Aβ that promotes Aβ fibrillogenesis and neurotoxicity. Our data support a role for a unique, receptor-independent mode of action for TGF-β and define a new molecular point of intervention for inhibiting Aβ fibrillogenesis. TGF-β Receptor Competition Assays—Mink lung epithelial Mv1Lu cells (ATCC: CCL-64) were seeded at 2 × 105 cells/ml in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 h at 37 °C (5% CO2). Cell surface receptors were cross-link-labeled with 200 pm125I-TGF-β1 (Perkin Elmer Life Science Products) in the presence of increasing concentrations (1–50 μm) of Aβ-(1–40), Aβ-(25–35), and Aβ-(12–28) (BIOSOURCE, Camarillo, CA). Bis(sulfosuccinimidyl) suberate (Pierce) was used as the cross-linking reagent, and the receptors were analyzed by 4–11% gradient SDS-PAGE and autoradiography. Signal intensities were quantitated using the ImageQuaNT system (Molecular Dynamics, Sunnyvale, CA). Circular Dichroism Spectroscopy (CD)—Aβ-(1–40) was prepared by dissolution into 50 mm phosphate buffer (pH 7.0) to yield a final concentration of 1 mg/ml. Samples were incubated at ambient temperature (22 °C) without stirring and CD measurements (at 22 °C) were performed periodically using a 0.1 cm pathlength quartz cell (Hellma, Forest Hills, NY) and an Aviv Model 62A DS spectropolarimeter (Aviv Associates, Lakewood, NJ). The scan rate was 1 nm/sec at a bandwidth of 1 nm. Three independent sets of experiments, each comprised of triplicate scans performed from 250–198 nm, were done. The buffer spectrum was subtracted from the scans and the resulting functions were smoothed. Data could not be acquired at wavelengths lower than 198 nm due to saturation of the photomultiplier. However, this range is sufficient to accurately evaluate the secondary structure state of the samples (18Kirkitadze M.D. Condron M.M. Teplow D.B. J. Mol. Biol. 2001; 312: 1103-1119Crossref PubMed Scopus (602) Google Scholar). Protein concentrations were determined a posteriori by quantitative amino acid analysis, thus enabling accurate calculation of molar ellipticities (Θ). Neurotoxicity Assay—Rat pheochromocytoma PC12 cells (ATCC: CRL-1721) were cultured on rat tail collagen-coated plates. Both PC12 cells and human neuroblastoma SH-SY5Y cells (ATCC: CRL-2266) were maintained in Dulbecco's modified Eagle's medium containing 10% horse serum (PC12) and 5% fetal bovine serum (PC12 & SH-SY5Y) at 37 °C (5% CO2). Cells (5 × 103/well) were differentiated to a neuronal phenotype using 100 ng/ml of NGF. The toxicity of Aβ-(1–40) was determined by treatment with 50 μm Aβ-(1–40) either alone or in combination with 1 nm TGF-βs (R&D Systems) for 72 h. The reverse peptide, Aβ-(40–1), was used as a peptide control. Mitochondrial function, as an index of cell viability, was monitored using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) dye conversion assay. MTT (20 μl of a 5 mg/ml solution in sterile phosphate-buffered saline) was added to the cells and allowed to incubate for 3 h at 37 °C. The reaction was terminated by addition of 20% SDS in water/N,N-dimethylformamide (1:1), pH 4.7. Optical density was measured at 590 nm. Surface Plasmon Resonance—Between 1200 and 1600 resonance units of Aβ peptides [e.g. Aβ-(1–40), Aβ-(25–35), and Aβ-(12–28)] were immobilized using the standard amine-coupling procedure onto CM5 sensor chips which were docked in a Surface Plasmon Resonance-based biosensor (BIAcore™; Biosensor AB, Uppsala, Sweden). Briefly, an injection of 50 μl of NHS/EDC was followed by manual injection at a flow rate of 5 μl/min of freshly prepared Aβ peptide diluted to 25 μg/ml in 10 mm acetic acid (pH 4.0). The remaining active sites on the surface were blocked by injection of 50 μl ethanolamine (pH 8.0). Binding curves were obtained by injecting fresh solutions of TGF-β1 or TGF-β2 (12.5–150 nm) in Hepes-buffered saline (pH 7.4) over the test surfaces. The binding curves from mock surfaces (no Aβ peptide immobilized) were subtracted from the corresponding experimental curves. Morphological Characterization of Aβ-(1–40) Fibrils—Aβ-(1–40) was incubated at a concentration of 1 mg/ml (∼235 μm) in 50 mm phosphate buffer (pH 7.0) either alone or in combination with 5 μg/ml (∼200 nm) of either TGF-β1 or TGF-β2 for 48 h at room temperature without agitation. A small amount of specimen was placed onto 200-mesh Formvar-coated grids, blotted, and then air-dried. The specimen was negatively stained with 1% (w/v) potassium phosphotungstic acid (pH 7.0) prior to examination with a JEM-2000FX electron microscope (JEOL, Ltd., Tokyo, Japan) using an accelerating voltage of 80 kV. Immunolocalization of TGF-β Receptors in Mouse Hippocampus— Mice (C3H/C57Bl; Charles River Laboratories) were terminated by cervical dislocation, and the brains were quickly removed, frozen in liquid nitrogen and kept at -80 °C until use. Animal maintenance and manipulation was performed according to the recommendations of our ethical committees (Biotechnology Research Institute-NRC). Affinity-purified polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were used for localization of TGF-β RI (V-22, 2 μg/ml), RII (C-16, 1 μg/ml), and RIII (C-20, 2 μg/ml) receptors. Antibody specificity was confirmed using biosensor and Western blot analyses. Serial sagittal sections (8-μm thick) were incubated with the primary antibody in phosphate-buffered saline/1% bovine serum albumin in a moist chamber overnight at 4 °C. The specificity of the immunoreactions was tested by exclusion of the primary antibody as well as by competition, i.e. preblocking the primary antibody with the immunizing peptide (2–5 μm). Following several rinses in phosphate-buffered saline, the sections were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody. Antigen expression was visualized (0.3% diaminobenzidine, 0.025% H2O2, 2–10 min) by light microscopy. Cell soma were counterstained with methyl green. This series of experiments was repeated 4–6 times using tissue obtained from both male and female mice ranging in age from 7 to 10 months. Statistics—Data were analyzed by one- or two-way analysis of variance using p < 0.05 as the criterion for significance. Post-hoc analysis relied on Dunnet's Multiple Comparisons Test. Aβ-(1–40) Diminishes 125I-TGF-β1 Labeling of Cell Surface Receptors—Three high affinity cell surface receptors for TGF-β have been identified, i.e. the type I, II, and III receptors (RI, RII, and RIII, respectively; Ref. 19Massagué J. Nat. Rev. Mol. Cell. Biol. 2000; 1: 169-178Crossref PubMed Scopus (1649) Google Scholar). One approach to investigating the mechanism of action of TGF-β on Aβ-mediated effects is to determine the effect of freshly dissolved Aβ on TGF-β binding to these three receptors. We analyzed this using 125I-TGF-β1 binding on Mv1Lu cells which express relatively high levels of these receptors. Analysis of variance of all treatment groups revealed significant reductions in binding of 125I-TGF-β1 to all three types of receptors, i.e. RI (F(10,69) = 3.853, p = 0.0004), RII (F(10,56) = 2.653, p = 0.0101) and RIII (F(10,69)= 2.824, p = 0.0054). Post-hoc analysis indicated that this effect occurred, in a dose-dependent manner, with the physiologically relevant peptide Aβ-(1–40), but not with the Aβ-(12–28) and Aβ-(25–35) fragments (Fig. 1). Aβ-(1–40)-mediated Neurotoxicity Is Potentiated by TGF-β Isoforms in a Receptor-independent Manner—To examine the receptor-dependence of TGF-β on Aβ-(1–40)-mediated neurotoxicity, we chose to use cells that do not express TGF-β receptors (i.e. PC12 cells) and comparing these to cells that express all three TGF-β receptor types (i.e. SH-SY5Y) (Ref. 20Massagué J. Cheifetz S. Boyd F.T. Andres J.L. Ann. N. Y. Acad. Sci. 1990; 593: 59-72Crossref PubMed Scopus (206) Google Scholar and confirmed in our laboratory). Using PC12 cells eliminates the possibility of any event being obscured by TGF-β RII receptor-mediated neuroprotection (10Flanders K.C. Ren R.F. Lippa C.F. Prog. Neurobiol. 1998; 54: 71-85Crossref PubMed Scopus (335) Google Scholar, 17Ren R.F. Hawver D.B. Kim R.S. Flanders K.C. Mol. Brain Res. 1997; 48: 315-322Crossref PubMed Scopus (38) Google Scholar). We first confirmed the conformation state of freshly dissolved Aβ-(1–40) using circular dichroism. Aβ underwent a well-defined random coil → β-sheet transition (Fig. 2), as expected of a species undergoing a conformational transition associated with fibril formation (18Kirkitadze M.D. Condron M.M. Teplow D.B. J. Mol. Biol. 2001; 312: 1103-1119Crossref PubMed Scopus (602) Google Scholar, 21Walsh D.M. Hartley D.M. Kusumoto Y. Fezoui Y. Condron M.M. Lomakin A. Benedek G.B. Selkoe D.J. Teplow D.B. J. Biol. Chem. 1999; 274: 25945-25952Abstract Full Text Full Text PDF PubMed Scopus (982) Google Scholar). We used neurite retraction and mitochondrial conversion of the tetrazolium redox dye, MTT, as early indicators of cytotoxicity. TGF-β1 and TGF-β2 alone had no observable effect on either neurite retraction or MTT dye conversion in PC12 cells (F(2,29)= 0.3632, p = 0.6985, Fig. 3A), as expected of cells that do not express TGF-β receptors. Also as expected, Aβ-(1–40) treatment caused neurite retraction (Fig. 3A) and inhibition of MTT dye conversion (reduced by 25% relative to untreated PC12 cells, t 0.05,23 = 3.426, p = 0.0023, Fig. 3B). Both TGF-β1 and TGF-β2 potentiated the cytotoxic effect of Aβ-(1–40) as evidenced by further neurite retraction (Fig. 3A) and a further reduction of MTT dye conversion (to 50% of control, F(2,19) = 4.968, p = 0.0184, Fig. 3B). Aβ-(1–40) also induced a reduction in MTT dye conversion in SH-SY5Y cells (F(5,22)= 24.78, p = 0.0001, Fig. 3B), however, this effect was not exacerbated by TGF-βs which is expected of a cell model expressing all three TGF-β receptor types. In fact, a marginal reversal of the effect of Aβ-(1–40) was observed during treatment with TGF-βs. These combined data demonstrate that TGF-β receptor-mediated events supersede the receptor-independent events. The reverse peptide, Aβ-(40–1), did not exert any effect on MTT reduction either alone or in combination with TGF-βs (data not shown). The extremely low ratio of TGF-βs to Aβ-(1–40) used during these experiments, i.e. a 1 to 50,000 molar ratio, supports the idea that TGF-β may have a seeding effect and emphasizes the potential physiological relevance of these observations.Fig. 3TGF-βs enhance Aβ-(1–40)-mediated toxicity in NGF-differentiated PC12 cells. A, the extensive neuritic arborization present in control NGF-differentiated PC12 cell cultures is unaffected by treatment with either TGF-β1 or TGF-β2 (top) (original magnification: ×150). Neurite retraction (arrows) is readily apparent following treatment with Aβ-(1–40) (bottom) and is even more evident following co-treatment with the individual TGF-β isoforms. B, the viability of similarly treated PC12 cells was quantitated using MTT dye conversion. Absorbance values (n = 6–8 experiments done in triplicate) are expressed as mean ± S.E. percent control. Aβ-(1–40) reduced MTT dye conversion to ∼75% of control levels. TGF-βs alone had no effect on TGF-β receptor-null PC12 cell viability, but they were able to potentiate the toxic effect of Aβ-(1–40) (top). In contrast, TGF-βs did not promote the Aβ-(1–40)-mediated toxicity in TGF-β receptor-expressing SH-SY5Y cell culture (bottom). The reverse peptide, Aβ-(40–1), did not affect MTT reduction, either alone or in combination with TGF-βs (as these data do not add anything to the figure, they are not shown). **, p < 0.01 compared with control and *, p < 0.05 compared with Aβ-(1–40) alone.View Large Image Figure ViewerDownload Hi-res image Download (PPT) TGF-β Isoforms Bind Directly to Aβ-(1–40) with Low Nano-molar Affinities—We used a Surface Plasmon Resonance-based biosensor to test for a direct physical interaction between TGF-β isoforms and Aβ peptides. The biosensor would detect mass accumulation resulting from binding of the individual TGF-β isoforms to the covalently immobilized Aβ peptides as a change in the refractive index of the surface matrix and would generate a curve recorded in arbitrary resonance units (RUs). Both TGF-β1 and TGF-β2 were observed to bind significantly to freshly dissolved Aβ-(1–40), with the binding of TGF-β2 being greater than that of TGF-β1 (Fig. 4, A and B). The extent of binding of the TGF-β isoforms to Aβ-(12–28) was significantly lower than that observed with Aβ-(1–40), while no detectable binding was observed on surfaces to which Aβ-(25–35) was immobilized. Subsequent biosensor experiments were focused on the physiologically relevant Aβ-(1–40) peptide given the overall lack of interaction of the pharmacological fragments Aβ-(25–35) and Aβ-(12–28) with TGF-β isoforms in the present biosensor study. The specificity of the interaction between injected TGF-β2 and the immobilized Aβ-(1–40) was confirmed by co-injection of the TGF-β ligand specific antibody 3C7 (Celtrix Pharmaceuticals, Inc.) (Fig. 5), thus excluding the possibility that the binding to Aβ-(1–40) that we observed might be due to a protein contaminant in the commercial TGF-β preparation.Fig. 5Confirmation of the TGF-β-Aβ-(1–40) complex. Injection of TGF-β2 (100 nm) over the Aβ-(1–40) surface gave the characteristic binding curve. Co-injection of TGF-β2 with the TGF-β ligand specific antibody, 3C7, confirmed the presence of TGF-β2 within the binding complex. Injection of 3C7 alone did not recognize the immobilized Aβ-(1–40).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The binding of TGF-β isoforms to freshly dissolved Aβ-(1–40) was characterized in more detail using the biosensor by varying the concentration of TGF-β1 and TGF-β2. The curves (Fig. 6, A and D) clearly indicated that both TGF-β1 and TGF-β2 bound to Aβ-(1–40) in a concentration-dependent manner. Fitting of these binding data using nonlinear least squares analysis and numerical integration of the differential rate equations (22De Crescenzo G. Grothe S. Lortie R. Debanne M.T. O'Connor-McCourt M.D. Biochemistry. 2000; 39: 9466-9476Crossref PubMed Scopus (48) Google Scholar) demonstrated that the binding of TGF-β1 to Aβ-(1–40) could not be described well by a simple binding model (A+B → AB), as judged by the variance in the residuals between the calculated and experimental data (Fig. 6B). However, the data was well represented by a rearrangement model (A+B → AB → AB*) (Fig. 5, A and C) with an apparent K D of 60.5 ± 5.2 nm. The fitting of the rearrangement model suggests that the initial TGF-β1-Aβ-(1–40) complex undergoes a kinetically detectable rearrangement, perhaps a structural transition. This observation may point to a direct effect of TGF-β1 on Aβ-(1–40) fibril formation since Aβ-(1–40) is known to undergo a conformational transition from a predominantly random coil → β-sheet-rich form during fibrillogenesis. In the case of TGF-β2 (Fig. 6B), when the curves derived from all of the TGF-β2 concentrations were taken into account, neither the simple model (Fig. 6E) nor rearrangement model (data not shown) represented the data well. However, the lower concentration curves for TGF-β2 could be fit using a simple model, resulting in an apparent K D of 96.1 ± 17.9 nm (Fig. 6, D and F). When the 100 and 150 nm TGF-β2 curves were predicted using the constants derived from the fitting of the lower concentration curves, the experimental curves were found to have significantly higher plateau values than the predicted curves (Fig. 6D), illustrating the complexity of the TGF-β2-Aβ-(1–40) interaction. The greater than predicted RU values at higher TGF-β2 concentrations may result from a change in density of the biosensor matrix due to the formation of aggregates or fibrils on the surface. The interaction between TGF-βs and Aβ-(1–40) is not generalized to growth factors. Indeed, this was confirmed by the absence of binding between Aβ-(1–40) and 150 nm nerve growth factor (NGF; data not shown), which, along with TGF-βs, is a member of the cystine-knot-containing superfamily of growth factors (23Wiesmann C. de Vos A.M. Nat. Struct. Biol. 2000; 7: 440-442Crossref PubMed Scopus (15) Google Scholar). Subsequent binding of TGF-β2 to the same surface confirmed the presence of covalently immobilized Aβ-(1–40) (data not shown). TGF-β Isoforms Promote Aβ-(1–40) Fibril Formation in Vitro—Our biosensor studies confirmed that TGF-βs interact directly with Aβ-(1–40) and suggested a structural transition possibly affecting fibril growth. We used electron microscopy to examine the characteristics of Aβ-(1–40) fibrils formed in the absence and presence of TGF-βs. Lower magnification electron microscopy of Aβ-(1–40) alone showed occasional short strands having a gross morphology characteristic of protofibrils (21Walsh D.M. Hartley D.M. Kusumoto Y. Fezoui Y. Condron M.M. Lomakin A. Benedek G.B. Selkoe D.J. Teplow D.B. J. Biol. Chem. 1999; 274: 25945-25952Abstract Full Text Full Text PDF PubMed Scopus (982) Google Scholar) (Fig. 7a). In contrast, protofibrils were more abundant and formed network-like assemblies in the Aβ-(1–40) + TGF-β1 (Fig. 7b) and Aβ-(1–40) + TGF-β2 (Fig. 7c) samples. No detectable structures were discerned with TGF-β1 or TGF-β2 alone (data not shown). The low ratio of TGF-βs to Aβ-(1–40) used during this experiment, i.e. a1–1000 molar ratio, together with our biosensor data indicating a structural transition in the TGF-β-Aβ-(1–40) complex, suggest that TGF-β may enhance fibrillogenesis by generating a conformationally altered form of Aβ-(1–40) with seeding ability. Higher magnification revealed that the Aβ-(1–40) protofibrils were 3–4 nm in width and were composed of a tight helical structure with a periodicity of 2–3 nm based on the coiling of 1 nm wide filaments (Fig. 7, d–f). At this magnification, the tight helical nature of the protofibrils was more evident for Aβ-(1–40) + TGF-β2 than for Aβ-(1–40) + TGF-β1, although further magnification of the Aβ-(1–40) + TGF-β1 sample (Fig. 7e, inset) confirmed the presence of loose helical structures in this sample. This magnification also revealed numerous flexible filaments 1 nm in width in the “interstrand” spaces (data not shown). Interestingly, the morphology of the 3-nm wide helical Aβ-(1–40) protofibrils that predominate in the presence of TGF-β1 and, particularly, TGF-β2 resembles that of in situ amyloid protofibrils obtained using advanced sample preparation methods such as cryofixation and freeze substitution (24Inoue S. Int. Rev. Cytol. 2001; 210: 121-161Crossref PubMed Scopus (20) Google Scholar). Mouse Hippocampal Field CA1 and Dentate Gyrus Lack TGF-β RI Receptors—The hippocampus is a structure particularly vulnerable during amyloid pathology. The increases in amyloid plaque burden that occur following co-treatment of hippocampal slices with Aβ and TGF-βs (12Harris-White M.E. Chu T. Balverde Z. Sigel J.J. Flanders K.C. Frautschy S.A. J. Neurosci. 1998; 18: 10366-10374Crossref PubMed Google Scholar) and in TGF-β1/hAPP bigenic mice (7Wyss-Coray T. Masliah E. Mallory M. McConlogue L. Johnson-Wood K. Lin C. Mucke L. Nature. 1997; 389: 603-606Crossref PubMed Scopus (360) Google Scholar, 14Wyss-Coray T. Lin C. Yan F. Yu G.Q. Rohde M. McConlogue L. Masliah E. Mucke L. Nat. Med. 2001; 7: 612-618Crossref PubMed Scopus (510) Google Scholar) were found to be subfield-specific, indicating the context-dependent nature of TGF-β action. Our observations indicate that TGF-β enhances Aβ-(1–40)-mediated neurotoxicity in a receptor-independent manner. We therefore examined the expression of all three TGF-β receptors within the hippocampal formation to determine if the vulnerability of particular subfields correlates with the pattern of TGF-β receptor expression. Although no TGF-β RI receptor expression was detected in field CA1 and dentate gyrus (Fig. 8A), expression was detectable, albeit weak, in the stratum pyramidale of fields CA2-CA3, while much stronger staining was found in the stratum lucidum of fields CA2-CA3 (Fig. 8C) through to the hilus of the dentate gyrus. TGF-β RII receptors were expressed throughout the hippocampal formation particularly in the stratum pyramidale of fields CA1-CA3 and the strata moleculare and granulosum of the dentate gyrus, with sparse staining also observed in t" @default.
• W1993593920 created "2016-06-24" @default.
• W1993593920 creator A5000908405 @default.
• W1993593920 creator A5010835197 @default.
• W1993593920 creator A5017314590 @default.
• W1993593920 creator A5017797419 @default.
• W1993593920 creator A5051792352 @default.
• W1993593920 creator A5054824818 @default.
• W1993593920 creator A5075549153 @default.
• W1993593920 creator A5085382860 @default.
• W1993593920 date "2003-10-01" @default.
• W1993593920 modified "2023-10-18" @default.
• W1993593920 title "A Direct Interaction between Transforming Growth Factor (TGF)-βs and Amyloid-β Protein Affects Fibrillogenesis in a TGF-βReceptor-independent Manner" @default.
• W1993593920 cites W1579764797 @default.
• W1993593920 cites W1586876258 @default.
• W1993593920 cites W1605023308 @default.
• W1993593920 cites W1617304195 @default.
• W1993593920 cites W1636022999 @default.
• W1993593920 cites W1966180801 @default.
• W1993593920 cites W1969165181 @default.
• W1993593920 cites W1972104173 @default.
• W1993593920 cites W1972535964 @default.
• W1993593920 cites W1972567838 @default.
• W1993593920 cites W1978161256 @default.
• W1993593920 cites W1983670592 @default.
• W1993593920 cites W1990044150 @default.
• W1993593920 cites W1995344309 @default.
• W1993593920 cites W1995650022 @default.
• W1993593920 cites W2002520289 @default.
• W1993593920 cites W2009414873 @default.
• W1993593920 cites W2016401484 @default.
• W1993593920 cites W2020842716 @default.
• W1993593920 cites W2029923284 @default.
• W1993593920 cites W2039397725 @default.
• W1993593920 cites W2041192235 @default.
• W1993593920 cites W2050181337 @default.
• W1993593920 cites W2061786230 @default.
• W1993593920 cites W2067875575 @default.
• W1993593920 cites W2070488302 @default.
• W1993593920 cites W2073516240 @default.
• W1993593920 cites W2084348234 @default.
• W1993593920 cites W2089040344 @default.
• W1993593920 cites W2102460037 @default.
• W1993593920 cites W2103745407 @default.
• W1993593920 cites W2111952057 @default.
• W1993593920 cites W2118569739 @default.
• W1993593920 cites W2129459566 @default.
• W1993593920 cites W2146610745 @default.
• W1993593920 cites W2346002398 @default.
• W1993593920 cites W2397016425 @default.
• W1993593920 cites W2734291336 @default.
• W1993593920 cites W4296154147 @default.
• W1993593920 cites W58149416 @default.
• W1993593920 doi "https://doi.org/10.1074/jbc.m304080200" @default.
• W1993593920 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12867422" @default.
• W1993593920 hasPublicationYear "2003" @default.
• W1993593920 type Work @default.
• W1993593920 sameAs 1993593920 @default.
• W1993593920 citedByCount "22" @default.
• W1993593920 countsByYear W19935939202013 @default.
• W1993593920 countsByYear W19935939202014 @default.
• W1993593920 countsByYear W19935939202015 @default.
• W1993593920 countsByYear W19935939202018 @default.
• W1993593920 countsByYear W19935939202020 @default.
• W1993593920 countsByYear W19935939202021 @default.
• W1993593920 countsByYear W19935939202022 @default.
• W1993593920 crossrefType "journal-article" @default.
• W1993593920 hasAuthorship W1993593920A5000908405 @default.
• W1993593920 hasAuthorship W1993593920A5010835197 @default.
• W1993593920 hasAuthorship W1993593920A5017314590 @default.
• W1993593920 hasAuthorship W1993593920A5017797419 @default.
• W1993593920 hasAuthorship W1993593920A5051792352 @default.
• W1993593920 hasAuthorship W1993593920A5054824818 @default.
• W1993593920 hasAuthorship W1993593920A5075549153 @default.
• W1993593920 hasAuthorship W1993593920A5085382860 @default.
• W1993593920 hasBestOaLocation W19935939201 @default.
• W1993593920 hasConcept C118131993 @default.
• W1993593920 hasConcept C12554922 @default.
• W1993593920 hasConcept C125555471 @default.
• W1993593920 hasConcept C126322002 @default.
• W1993593920 hasConcept C134018914 @default.
• W1993593920 hasConcept C147259501 @default.
• W1993593920 hasConcept C170493617 @default.
• W1993593920 hasConcept C185592680 @default.
• W1993593920 hasConcept C27523624 @default.
• W1993593920 hasConcept C2775960820 @default.
• W1993593920 hasConcept C2778229498 @default.
• W1993593920 hasConcept C55493867 @default.
• W1993593920 hasConcept C71924100 @default.
• W1993593920 hasConcept C86803240 @default.
• W1993593920 hasConcept C95444343 @default.
• W1993593920 hasConceptScore W1993593920C118131993 @default.
• W1993593920 hasConceptScore W1993593920C12554922 @default.
• W1993593920 hasConceptScore W1993593920C125555471 @default.
• W1993593920 hasConceptScore W1993593920C126322002 @default.
• W1993593920 hasConceptScore W1993593920C134018914 @default.
• W1993593920 hasConceptScore W1993593920C147259501 @default.
• W1993593920 hasConceptScore W1993593920C170493617 @default.

• next