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Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis

Authors

  • Masato Hasegawa PhD,

    Corresponding author
    1. Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, Kamikitazawa, Setagaya-ku, Tokyo
    • Departments of Molecular Neurobiology and Psychogeriatrics, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585
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  • Tetsuaki Arai MD, PhD,

    Corresponding author
    1. Department of Psychogeriatrics, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, Kamikitazawa, Setagaya-ku, Tokyo
    • Departments of Molecular Neurobiology and Psychogeriatrics, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585
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  • Takashi Nonaka PhD,

    1. Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, Kamikitazawa, Setagaya-ku, Tokyo
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  • Fuyuki Kametani PhD,

    1. Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, Kamikitazawa, Setagaya-ku, Tokyo
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  • Mari Yoshida MD, PhD,

    1. Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Yazako, Nagakute-cho, Aichi-gun, Aichi, Japan
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  • Yoshio Hashizume MD, PhD,

    1. Department of Neuropathology, Institute for Medical Science of Aging, Aichi Medical University, Yazako, Nagakute-cho, Aichi-gun, Aichi, Japan
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  • Thomas G. Beach MD, PhD,

    1. Sun Health Research Institute, Sun City, AZ
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  • Emanuele Buratti PhD,

    1. International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
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  • Francisco Baralle MD, PhD,

    1. International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
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  • Mitsuya Morita MD, PhD,

    1. Department of Neurology, Jichi Medical University, Shimotsuke-shi, Tochigi, Japan
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  • Imaharu Nakano MD, PhD,

    1. Department of Neurology, Jichi Medical University, Shimotsuke-shi, Tochigi, Japan
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  • Tatsuro Oda MD, PhD,

    1. Department of Neuropsychiatry, National Shimofusa Mental Hospital, Chiba, Japan
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  • Kuniaki Tsuchiya MD, PhD,

    1. Department of Laboratory Medicine and Pathology, Tokyo Metropolitan Matsuzawa Hospital, Setagaya-ku, Tokyo, Japan
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  • Haruhiko Akiyama MD, PhD

    1. Department of Psychogeriatrics, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, Kamikitazawa, Setagaya-ku, Tokyo
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Abstract

Objective

TAR DNA-binding protein of 43kDa (TDP-43) is deposited as cytoplasmic and intranuclear inclusions in brains of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Previous studies reported that abnormal phosphorylation takes place in deposited TDP-43. The aim of this study was to identify the phosphorylation sites and responsible kinases, and to clarify the pathological significance of phosphorylation of TDP-43.

Methods

We generated multiple antibodies specific to phosphorylated TDP-43 by immunizing phosphopeptides of TDP-43, and analyzed FTLD-U and ALS brains by immunohistochemistry, immunoelectron microscopy, and immunoblots. In addition, we performed investigations aimed at identifying the responsible kinases, and we assessed the effects of phosphorylation on TDP-43 oligomerization and fibrillization.

Results

We identified multiple phosphorylation sites in carboxyl-terminal regions of deposited TDP-43. Phosphorylation-specific antibodies stained more inclusions than antibodies to ubiquitin and, unlike existing commercially available anti–TDP-43 antibodies, did not stain normal nuclei. Ultrastructurally, these antibodies labeled abnormal fibers of 15nm diameter and on immunoblots recognized hyperphosphorylated TDP-43 at 45kDa, with additional 18 to 26kDa fragments in sarkosyl-insoluble fractions from FTLD-U and ALS brains. The phosphorylated epitopes were generated by casein kinase-1 and -2, and phosphorylation led to increased oligomerization and fibrillization of TDP-43.

Interpretation

These results suggest that phosphorylated TDP-43 is a major component of the inclusions, and that abnormal phosphorylation of TDP-43 is a critical step in the pathogenesis of FTLD-U and ALS. Phosphorylation-specific antibodies will be powerful tools for the investigation of these disorders. Ann Neurol 2008

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