Phenylalanine assembly into toxic fibrils suggests amyloid etiology in phenylketonuria

Journal name:
Nature Chemical Biology
Volume:
8,
Pages:
701–706
Year published:
DOI:
doi:10.1038/nchembio.1002
Received
Accepted
Published online

Abstract

Phenylketonuria (PKU) is characterized by phenylalanine accumulation and progressive mental retardation caused by an unknown mechanism. We demonstrate that at pathological concentrations, phenylalanine self-assembles into fibrils with amyloid-like morphology and well-ordered electron diffraction. These assemblies are specifically recognized by antibodies, show cytotoxicity that can be neutralized by the antibodies and are present in the hippocampus of model mice and in parietal cortex brain tissue from individuals with PKU. This is, to our knowledge, the first demonstration that a single amino acid can form amyloid-like deposits, suggesting a new amyloidosis-like etiology for PKU.

At a glance

Figures

  1. The single aromatic amino acid, phenylalanine, self-assembles into supramolecular fibrillar structures.
    Figure 1: The single aromatic amino acid, phenylalanine, self-assembles into supramolecular fibrillar structures.

    (a) TEM images of elongated phenylalanine fibrils. Scale bar is 1 μm. (b) SEM images of phenylalanine fibrils in human serum. Scale bar is 20 μm. (c) Microscopic examination under polarized light following Congo red staining of phenylalanine fibrils. Scale bar is 500 μm. (d) Confocal microscopy image of fibrils dyed with ThT. Scale bar is 10 μm. (e) Representative snapshot of the filamentous structure obtained by molecular dynamics simulations started with 27 monodisperse phenylalanine molecules (cyan) at high pH in the presence of counterions (yellow spheres). The tight packing of the aromatic rings is emphasized by their van der Waals envelope (gray surface). (f) Distribution of distances between pairs of atoms in different phenylalanine molecules in the aggregates obtained by molecular dynamics simulations. The distances between the center of mass of all pairs of the 27 phenylalanines were used for these histograms, and quantitatively similar histograms were obtained using distances between atoms instead of those between center of masses. The very similar distributions at 280 K (black) and 310 K (red) show that the ordered aggregates of phenylalanine are essentially the same in this temperature range and that the simulations have reached convergence.

  2. Specific antibodies against phenylalanine fibrils and the toxic effect and interaction of phenylalanine fibrillar structures with cell cultures.
    Figure 2: Specific antibodies against phenylalanine fibrils and the toxic effect and interaction of phenylalanine fibrillar structures with cell cultures.

    (a) Cell viability was determined using the MTT assay. The PC12 cell line was maintained in the presence of phenylalanine fibrils (black bars) or the control amino acid, alanine (gray bars). *P < 0.05. (b) TEM micrographs of phenylalanine fibrils, visualized using antibodies that were specifically bound to phenylalanine fibrils and then were marked with a secondary antibody conjugated to 18-nm gold particles. Scale bar is 2 μm. (c) Cell viability was determined using the MTT assay, CHO cell cultures were maintained in the presence of an increasing amount of phenylalanine fibrils (black bars) or immunoprecipitated (IP) solutions of phenylalanine depleted of fibrils (gray bars). As control, phenylalanine fibrils were also incubated with preimmune serum (white bars). *P < 0.05; **P < 0.001. Results for a and c are presented as mean ± s.e.m. (d) CHO cell cultures were incubated with phenylalanine fibrils, fixed, and then incubated with anti-Phe and stained using Alexa 488–conjugated antibody (green, marked with arrows). The cell membrane was marked with phalloidin (red). As controls, CHO cell cultures were incubated with phenylalanine fibrils then fixed and incubated with preimmune serum or without primary antibodies. Scale bars are 10 μm.

  3. Dot-blot analysis and histological staining indicates the presence of phenylalanine fibrils in model mice and PKU patient brain tissues.
    Figure 3: Dot-blot analysis and histological staining indicates the presence of phenylalanine fibrils in model mice and PKU patient brain tissues.

    (a) Dot-blot analysis of phenylalanine fibrils. Column 1, serum of homozygous mouse (Pahenu2) strongly bound the phenylalanine fibrils. Column 2, serum of heterozygous mouse (Pahenu2) did not bind the phenylalanine fibrils. Column 3, serum of wild-type mouse did not bind phenylalanine fibrils. (b) Histological staining of homozygous Pahenu2 mouse brain. The 20-μm-thick brain slices were stained with rabbit anti–Phe fibril antibodies and Congo red and then were examined using fluorescent microscopy. The detected amyloid-like plaques showed colocalization of the fluorescent signal obtained from Congo red and antibody staining. (c) The brain of an individual with PKU was stained with anti–Phe fibril serum or with preimmune serum, examined using light microscopy and co-stained with Congo red. Phenylalanine-positive depositions were found in the parietal cortex. Scale bars are 100 μm.

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Author information

Affiliations

  1. Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

    • Lihi Adler-Abramovich,
    • Lilach Vaks,
    • Ohad Carny &
    • Ehud Gazit
  2. Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.

    • Dorit Trudler &
    • Dan Frenkel
  3. Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.

    • Dorit Trudler &
    • Dan Frenkel
  4. Department of Biochemistry, University of Zurich, Zurich, Switzerland.

    • Andrea Magno &
    • Amedeo Caflisch

Contributions

L.A.-A., O.C. and E.G. conceived and designed the experiments. L.A.-A. and L.V. planned and performed the experiments. D.T., L.V. and L.A.-A. designed and performed the mouse and human histology experiments. D.F. designed and coordinated the mice and human histology experiments. A.M. designed and performed the molecular dynamics simulations. A.C. designed and coordinated the molecular dynamics simulations. L.A.-A. and E.G. wrote the paper. All authors discussed the results and commented on the manuscript.

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The authors declare no competing financial interests.

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