Hironobu Naiki

In β₂-microglobulin-related (Aβ₂M) amyloidosis, a serious complication in patients on long-term dialysis, partial unfolding of β₂-microglobulin (β₂-m) is believed to be prerequisite to its assembly into Aβ₂M amyloid fibrils. Many kinds of amyloid-associated molecules, (e.g., apolipoprotein E (apoE), glycosaminoglycans (GAGs), proteoglycans (PGs)) may contribute to the development of Aβ₂M amyloidosis. In 1990s, the formation of Aβ₂M amyloid fibrils in vitro was first observed at low pH (2.0–3.0). Very recently, low concentrations of 2,2,2-trifluoroethanol (TFE) and the sub-micellar concentration of sodium dodecyl sulfate, a model for anionic phospholipids, have been reported to cause the extension of Aβ₂M amyloid fibrils at a neutral pH, inducing partial unfolding of β₂-m and stabilization of the fibrils. Moreover, apoE, GAGs, and PGs were found to stabilize Aβ₂M amyloid fibrils at a neutral pH, forming a stable complex with the fibrils. Some GAGs, especially heparin, enhanced the fibril extension in the presence of TFE at a neutral pH. Some PGs, especially biglycan also induced the polymerization of acid-denatured β₂-m. These findings are consistent with the hypothesis that in vivo, specific molecules that affect the conformation and stability of β₂-m and amyloid fibrils will have significant effects on the deposition of Aβ₂M amyloid fibrils.

β₂-Microglobulin–related (Aβ2M) amyloidosis is a frequent and serious complication in patients on long-term dialysis, and β₂-microglobulin is a major structural component of Aβ2M amyloid fibrils. Several biologic molecules inhibiting the depolymerization of Aβ2M amyloid fibrils at a neutral pH were found recently. The effect of trifluoroethanol and glycosaminoglycans (GAG) on the extension of the fibrils at a neutral pH was investigated with the use of fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy. Trifluoroethanol at concentrations of up to 20% (vol/vol) caused fibril extension of heparin-stabilized seeds, inducing a subtle change in the tertiary structure of β₂-microglobulin and stabilizing the fibrils at a neutral pH. This extension reaction followed a first-order kinetic model. In addition, some GAG, especially heparin, dose-dependently enhanced the fibril extension. These results suggest that some GAG, especially heparin, may bind to the fibrils and enhance their deposition in vivo. Thus, the experimental system described here should be useful to search for the factors that accelerate Aβ2M amyloid deposition in vivo. In addition, the interference of the binding of GAG to Aβ2M amyloid fibrils may be an attractive therapeutic modality. E-mail: naiki@fmsrsa.fukui-med.ac.jp

We compared the mechanisms of apolipoprotein E- (apoE-) and antioxidant- (AO-) mediated inhibition of β-amyloid fibril (fAβ) formation in vitro, based on a nucleation-dependent polymerization model using fluorescence spectroscopy with thioflavin T. We first applied a kinetic plot to transform a sigmoidal time-course curve of fAβ formation from freshly prepared amyloid β-peptides (Aβ) into a straight line. Mathematical treatment of this plot demonstrated that the above-described sigmoidal curve is a logistic curve and provided us with a kinetic parameter t_1/2, the time when the rate of fAβ formation is maximum. t_1/2 of β-amyloids (Aβ) (1−42) and (1−40) were 18.7 ± 1.7 min and 6.3 ± 0.2 h, respectively (mean ± SD, n = 3) and were independent of the initial Aβ concentration examined. Although apoE extended t_1/2 of both Aβs in a dose-dependent manner, AO did not. On the other hand, the final amount of fAβ formed was decreased by both apoE and AO dose-dependently. We then analyzed the effect of apoE and AO on the extension reaction of fAβ, based on a first-order kinetic model. Although apoE extended the time to proceed to equilibrium in a dose-dependent manner, AO did not. On the other hand, both apoE and AO dose-dependently decreased the final amount of fAβ formed. These results indicate that apoE and AO inhibit fAβ formation in vitro by different mechanisms and suggest the existence of multiple pharmacological targets for the prevention of fAβ formation.

Recently, many research groups have examined the effect of apolipoprotein E (apoE) on β-amyloid fibril (βAf) formation in vitro. However, their data were somewhat controversial and no exact kinetic assessment of the role of apoE has thus far been available. We examined the effect of human apoE on βAf formation in vitro, starting with various concentrations of freshly prepared β-amyloid(1−40) (β1−40) and using fluorescence spectroscopy with thioflavine T. When 50 μM of β1−40 was incubated with a 1:1000 to 1:100 molar ratio of apoE, a dose-dependent inhibitory effect of apoE was observed. Both the nucleation and extension phases of βAf formation in vitro were inhibited by apoE. On the other hand, when 300 μM of β1−40 was incubated with a 1:100 molar ratio of apoE, the inhibitory effect of apoE was completely abolished. We then focused our study on the kinetics of the inhibitory effect of apoE on the extension phase of βAf formation in vitro, utilizing the recently established first-order kinetic model of βAf extension in vitro [Naiki, H., & Nakakuki, K. (1996) Lab. Invest. 74, 374−383]. The mathematical treatment of the data suggests that apoE inhibits the extension of βAf in vitro, by making a complex with β1−40, thus eliminating free β1−40 from the reaction mixture. The equilibrium association constant with β1−40 was practically the same among the three major recombinant apoE isoforms. These results indicate that the effects of apoE on βAf formation in vitro is differential and could settle some of the controversy about β-amyloid-apoE interaction in vitro.