Lipocalin-type prostaglandin D synthase/beta-trace is a major amyloid beta-chaperone in human cerebrospinal fluid - PubMed (original) (raw)

Comparative Study

. 2007 Apr 10;104(15):6412-7.

doi: 10.1073/pnas.0701585104. Epub 2007 Apr 2.

Tadato Ban, Kosuke Aritake, Zhi-Li Huang, Wei-Min Qu, Issay Okazaki, Ikuko Mohri, Shigeo Murayama, Keiichi Ozono, Masako Taniike, Yuji Goto, Yoshihiro Urade

Affiliations

Comparative Study

Lipocalin-type prostaglandin D synthase/beta-trace is a major amyloid beta-chaperone in human cerebrospinal fluid

Takahisa Kanekiyo et al. Proc Natl Acad Sci U S A. 2007.

Abstract

The conformational change in amyloid beta (Abeta) peptide from its monomeric form to aggregates is crucial in the pathogenesis of Alzheimer's disease (AD). In the healthy brain, some unidentified chaperones appear to prevent the aggregation of Abeta. Here we reported that lipocalin-type prostaglandin D synthase (L-PGDS)/beta-trace, the most abundant cerebrospinal fluid (CSF) protein produced in the brain, was localized in amyloid plaques in both AD patients and AD-model Tg2576 mice. Surface plasmon resonance analysis revealed that L-PGDS/beta-trace tightly bound to Abeta monomers and fibrils with high affinity (K(D) = 18-50 nM) and that L-PGDS/beta-trace recognized residues 25-28 in Abeta, which is the key region for its conformational change to a beta-sheet structure. The results of a thioflavin T fluorescence assay to monitor Abeta aggregation disclosed that L-PGDS/beta-trace inhibited the spontaneous aggregation of Abeta (1-40) and Abeta (1-42) within its physiological range (1-5 microM) in CSF. L-PGDS/beta-trace also prevented the seed-dependent aggregation of 50 microM Abeta with K(i) of 0.75 microM. Moreover, the inhibitory activity toward Abeta (1-40) aggregation in human CSF was decreased by 60% when L-PGDS/beta-trace was removed from the CSF by immunoaffinity chromatography. The deposition of Abeta after intraventricular infusion of Abeta (1-42) was 3.5-fold higher in L-PGDS-deficient mice and reduced to 23% in L-PGDS-overexpressing mice as compared with their wild-type levels. These data indicate that L-PGDS/beta-trace is a major endogenous Abeta-chaperone in the brain and suggest that the disturbance of this function may be involved in the onset and progression of AD. Our findings may provide a diagnostic and therapeutic approach for AD.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

L-PGDS/β-trace immunostaining of amyloid plaques in Tg2576 mice and AD patients. (A–F) Amyloid plaques in the cerebral cortex of Tg2576 mice (A, arrowheads; B and C, asterisk) were immunopositive with anti-mouse L-PGDS/β-trace antibody (A and B), but not with preabsorbed antibody (C). Double immunofluorescence staining of Aβ (D) and L-PGDS/β-trace (E) showed that they were colocalized (F, merged image). (G and H) In the frontal cortex of a 70-year-old AD patient, amyloid plaques (G, arrowheads; H, asterisk) were immunostained by anti-human L-PGDS/β-trace polyclonal antibody. (Scale bars: A and G, 200 μm; B–F and H, 20 μm.)

Fig. 2.

Fig. 2.

SPR analysis of binding between L-PGDS/β-trace and Aβ. (A) Binding of L-PGDS/β-trace to 2 ng of immobilized Aβ (1–40). (B) Binding of Aβ (1–40) to 8 ng of immobilized Aβ L-PGDS/β-trace. Filled and open arrows show the starting points for sample injection and washing with buffer, respectively. Human serum albumin did not bind to Aβ (1–40) in the same concentration range.

Fig. 3.

Fig. 3.

Inhibition of Aβ aggregation by L-PGDS/β-trace. (A and B) Spontaneous aggregation of 50 μM Aβ (1–40) (A) and Aβ (1–42) (B) in the absence (black) or presence of L-PGDS/β-trace (orange, 1 μM; green, 5 μM). (C and D) Observations of Aβ (1–40) seed-dependent aggregation by fluorescence abscence (black) or microscopy (C) and AFM (D) in the absence (Left) or presence (Right) of L-PGDS/β-trace. (Scale bars: C, 10 μm; D, 1 μm.) (E) CD spectra of 50 μM Aβ (1–40) before (black) and after incubation for 2 h with Aβ seed (10 μg/ml) in the absence (blue) or presence of 5 μM L-PGDS/β-trace (red). (F Upper) Inhibition of seed-dependent fibrillogenesis of 50 μM Aβ (1–40) by incubation for 1 h with L-PGDS/β-trace purified from human CSF (closed circles), recombinant human L-PGDS (open circles), heat-denatured L-PGDS/β-trace (closed triangles), or recombinant inactive Cys65Ala mutant of human L-PGDS (open triangles). (F Lower) Hill plot of the data obtained for L-PGDS/β-trace purified from human CSF.

Fig. 4.

Fig. 4.

Inhibitory effect of L-PGDS/β-trace in human CSF on Aβ aggregation. (A) SDS/PAGE and Western blot analysis revealed that L-PGDS/β-trace is a major protein in human CSF with a molecular mass of 26 kDa and is almost completely removed from the CSF by passage through a mouse monoclonal anti-L-PGDS antibody-conjugated column. (B) Representative time course of the spontaneous Aβ aggregation in the absence (black) or presence of 50% human CSF (red) or 50% L-PGDS/β-trace-free CSF (blue). (C) Inhibition of Aβ aggregation in the presence of 50% CSF or the L-PGDS/β-trace-free CSF. The percentage inhibition of Aβ aggregation was calculated by using the formula [1− (_F_a/_F_b)] × 100, where _F_b and _F_a are the ThT fluorescence intensities of 50 μM Aβ (1–40) incubated for 96 h or in the presence of 50% CSF or L-PGDS/β-trace-free CSF, respectively. Data are expressed as the mean ± SEM of three independent experiments. ∗∗, P < 0.01 vs. CSF (Student's t test).

Fig. 5.

Fig. 5.

Inhibition of Aβ deposition by L-PGDS/β-trace in vivo. (A–H) Aβ deposition in the brain of WT mice (C57BL/6; A and E), L-PGDS−/− mice (C57BL/6; B and F), WT mice (FVB; C and G), and L-PGDS-Tg mice (FVB; D and H). (Scale bars: A–D, 1 mm; E–H, 200 μm.) (I–L) Congo-red staining of Aβ deposition in WT (C57BL/6; I and K) and L-PGDS−/− mice (C57BL/6; J and L) brain as described above. (Scale bar: I and J, 1 mm; K and L, 200 μm.) (M and N) The Aβ deposition was quantified by binding of [125I]-streptavidin to tissue sections prepared from the brain of WT and L-PGDS−/− mice (C57BL/6; M) and of WT and L-PGDS-Tg mice (FVB; N), which sections had been reacted with biotin-labeled Aβ (1–42). Data are expressed as the mean ± SEM (n = 3–4). Significant difference was based on Student's t test; ∗, P < 0.05.

Fig. 6.

Fig. 6.

Proposed schema for inhibition of Aβ aggregation by L-PGDS/β-trace. Soluble Aβ monomers change their conformation from a random-coil dominant structure to the β-sheet-rich structure and then aggregate to insoluble fibrils. L-PGDS/β-trace can prevent Aβ aggregation by inhibiting the transformation of Aβ monomers to β-sheet-rich structure and the sequential seed-dependent Aβ fibrillogenesis.

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