Neuronal production of transthyretin in human and murine Alzheimer's disease: is it protective?

Abstract

Transthyretin (TTR), a systemic amyloid precursor in the human TTR amyloidoses, interacts with β-amyloid (Aβ) in vitro, inhibits Aβ fibril formation, and suppresses the Alzheimer's disease (AD) phenotype in APP23 mice bearing a human APP gene containing the Swedish autosomal dominant AD mutation. In the present study, we show that TTR is a neuronal product upregulated in AD. Immunohistochemical analysis reveals that, in contrast to brains from non-demented age-matched individuals and control mice, the majority of hippocampal neurons from human AD and all those from the APP23 mouse brains contain TTR. Quantitative PCR for TTR mRNA and Western blot analysis show that primary neurons from APP23 mice transcribe TTR mRNA, and the cells synthesize and secrete TTR protein. TTR mRNA abundance is greatly increased in cultured cortical and hippocampal embryonic neurons and cortical lysates from adult APP23 mice. Antibodies specific for TTR and Aβ pulled down TTR/Aβ complexes from cerebral cortical extracts of APP23 mice and some human AD patients but not from control brains. In complementary tissue culture experiments, recombinant human TTR suppressed the cytotoxicity of soluble Aβ aggregates added to mouse neurons and differentiated human SH-SY5Y neuroblastoma cells. The findings that production of Aβ, its precursor, or its related peptides induces neuronal TTR transcription and synthesis and the presence of Aβ/TTR complexes in vivo suggest that increased TTR production coupled with interaction between TTR and Aβ and/or its related peptides may play a role in natural resistance to human AD.

Figure 1.

TTR expression in primary cultured embryonic cortical and hippocampal neurons. Choroid plexus-free neuron cultures were established from C57BL/6 [WT (B6)], APP23, mttr^−/−, hTTR⁺, APP23/mttr^−/−, APP23/hTTR⁺ strains. A–C, Immunostaining of APP23 neurons with anti-TTR and anti-Aβ antibodies. A, TTR staining (Dako; green). B, Aβ or related peptide staining (6E10; red). C, Merge of A and B. D, TTR-positive human hepatoblastoma HepG2 cells. E, TTR-negative human cardiac myocyte AC16 cells. F, Second antibody alone with HepG2 cells. G, H, TTR staining in neurons cultured from WT (B6) (G) and APP23 (H) embryos shows increased TTR signal in APP23 cells. I, Embryonic cortical and hippocampal neurons stained with the neuronal marker MAP2 (green) and counterstained by Hoechst 33342 (blue). Scale bars, 10 μm.

Figure 2.

TTR is expressed and upregulated in APP23 embryonic cortical and hippocampal neurons. A, TTR mRNAs were detected by RT-PCR. TTR mRNA was detected in WT (B6), APP23, and hTTR⁺ neurons collected by FACS for MAP2 positivity, and mttr^−/− served as a negative control. B, Quantitation of ttr mRNA in cultured primary neurons. Relative expression was calculated based on qPCR using ΔΔCt method. Error bars indicate SD. *p < 0.05. C, TTR immunoprecipitation of cultured neurons. TTR protein was immunoprecipitated from neuronal lysates with protein A/G plus agarose beads and anti-TTR antibody. Western blots of the immunoprecipitates analyzed by SDS-PAGE were developed with an anti-TTR antibody. D, Immunodetection of TTR in medium of neuronal cultures. Neurons were incubated in B27 free Neurobasal medium overnight. TTR released by the neurons was immunoprecipitated from the medium and processed as in C.

Figure 3.

Brain extracts of APP23 mice transgenic for hTTR have decreased Aβ peptide. Lysates of cortical and hippocampal tissue from APP23/hTTR⁺/mttr^−/− and APP23 (mttr^+/+) mice (>1 year) were centrifuged at 10,000 × g for 10 min at 4°C. Protein in supernatant (S) and resuspended pellet (P) were quantified by Bradford assay. Identical amounts of total protein/lane were analyzed in a 15% Tris-Tricine SDS-PAGE. The electrophoresed proteins were transferred onto a PVDF membrane, and the Aβ species were detected with 6E10 antibody.

Figure 4.

Coimmunoprecipitation of in vivo Aβ and TTR complexes. A, Mouse brains. Cortices and hippocampi of WT (B6), hTTR⁺/mttr^−/−, APP23/hTTR⁺/mttr^−/−, APP23, and APP23/mttr^−/− mice (>1 year) were dissected free of choroid plexus and homogenized in lysis buffer with protease inhibitors, precleared with protein A/G plus agarose beads, and then incubated with anti-TTR antibody cross-linked to protein A magnetic beads overnight at 4°C. Eluted complexes were analyzed in 15% Tris-Tricine SDS-PAGE, and Aβ was detected by Western blot (6E10 antibody). B, Human brains. Human brain homogenates were processed as in A. P indicates brain from AD patient; Ctr indicates age-matched control brain.

Figure 5.

Recombinant human TTR suppresses Aβ cytotoxicity and ROS induction. A, Aβ_1–40 soluble aggregates are more cytotoxic than resuspended amyloid fibrils. Cell viability was measured by resazurin reduction assay. B, hTTR (2.5 μm) suppressed the cytotoxicity induced by Aβ_1–40 (10 μm) aggregates on WT (B6), mttr^−/−, hTTR⁺, and APP23 neurons. C, hTTR (5 μm) reduces Aβ_1–42 oligomer (20 μm) cytotoxicity for cultured WT (B6) neurons. D, hTTR suppressed ROS formation in neurons treated with Aβ_1–42 oligomers. WT (B6) neurons were treated with Aβ_1–42 oligomers with or without TTR, and the fluorescence intensity of oxidized DHE was measured. E, hTTR prevents Aβ_1–42 oligomer cytotoxicity for differentiated SH-SY5Y human neuroblastoma cells. F, Adding hTTR to Aβ_1–40 fibrilization process suppressed Aβ cytotoxicity on WT (B6) neurons. Aβ_1–40 solutions with ([Aβ+TTR]ag) or without (Aβ_1–40) TTR, or TTR alone (TTRagctl) was subjected fibril formation condition. Solutions were diluted to final concentration of 10 μm Aβ and 2.5 μm TTR on WT (B6) neurons. Ten micromolar Aβ with 2.5 μm hTTR (Aβ+TTR) or 2.5 μm hTTR (hTTR) were also incubated with cells as control. Cell viability was measured as above. *p < 0.05 and **p < 0.01. Error bars indicate SD.