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hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations

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Abstract

Genetic analysis revealed the hexanucleotide repeat expansion GGGGCC within the regulatory region of the gene C9orf72 as the most common cause of familial amyotrophic lateral sclerosis and the second most common cause of frontotemporal lobar degeneration. Since repeat expansions might cause RNA toxicity via sequestration of RNA-binding proteins, we searched for proteins capable of binding to GGGGCC repeats. In vitro-transcribed biotinylated RNA containing hexanucleotide GGGGCC or, as control, AAAACC repeats were incubated with nuclear protein extracts. Using stringent filtering protocols 20 RNA-binding proteins with a variety of different functions in RNA metabolism, translation and transport were identified. A subset of these proteins was further investigated by immunohistochemistry in human autopsy brains. This revealed that hnRNP A3 formed neuronal cytoplasmic and intranuclear inclusions in the hippocampus of patients with C9orf72 repeat extensions. Confocal microcopy showed that these inclusions belong to the group of the so far enigmatic p62-positive/TDP-43 negative inclusions characteristically seen in autopsy cases of diseased C9orf72 repeat expansion carriers. Thus, we have identified one protein component of these pathognomonic inclusions.

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Acknowledgments

We thank Iryna Pigur for expert technical assistance, Axel Imhof, Harald Steiner, Akio Fukumori, Richard Page, and Eva Bentmann for providing tools and technologies and Dorothee Dormann for critically reading the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (SFB-596), the Competence Network for Neurodegenerative Diseases (KNDD) of the Bundesministerium für Bildung und Forschung (BMBF) to C.H. and the Consortium of Centers of Excellence in Neurodegenerative Brain Diseases (CoEN) to C.H., M.C., D.E., and C.V.B. K.M. was supported by a postdoctoral fellowship from the Alexander von Humboldt Foundation. D.E. was supported by the Helmholtz Young Investigator Program HZ-NG-607. The Agency for Innovation by Science and Technology provides a PhD fellowship to J.J. The authors acknowledge the Antwerp biobank of the Institute Born-Bunge for the brain samples as well as the neurologists S. Engelborghs and P.P. De Deyn and neuropathologist J.J. Martin for the clinical and pathological diagnoses. The Antwerp site is supported for the genetic research of neurodegenerative brain diseases by the Belgian Science Policy Office Interuniversity Attraction Poles program, the Foundation for Alzheimer Research (SAO/FRA), the Medical Foundation Queen Elisabeth, the Flemish Government Methusalem excellence program, the research Foundation Flanders (FWO) and the Special Research Fund of the University of Antwerp, Belgium.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Schillerstr. 44, 80336, Munich, Germany

    Kohji Mori, Sven Lammich, Sonja Zilow, Gernot Kleinberger & Christian Haass

  2. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada

    Ian R. A. Mackenzie

  3. Adolf-Butenandt-Institute, Protein Analysis Unit, Ludwig-Maximilians-University, Schillerstr. 44, 80336, Munich, Germany

    Ignasi Forné

  4. Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany

    Hans Kretzschmar & Thomas Arzberger

  5. DZNE, German Center for Neurodegenerative Diseases, Munich, Germany

    Dieter Edbauer, Gernot Kleinberger, Jochen Herms & Christian Haass

  6. Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, Universiteitsplein 1, 2610, Antwerp, Belgium

    Jonathan Janssens, Gernot Kleinberger, Marc Cruts & Christine Van Broeckhoven

  7. Laboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, Universiteitsplein 1, 2610, Antwerp, Belgium

    Jonathan Janssens, Gernot Kleinberger, Marc Cruts & Christine Van Broeckhoven

  8. Munich Cluster for Systems Neurology (SyNergy), Munich, Germany

    Jochen Herms & Christian Haass

  9. DZNE, German Center for Neurodegenerative Diseases, Tübingen, Germany

    Manuela Neumann

  10. Department of Neuropathology, University of Tübingen, Tübingen, Germany

    Manuela Neumann

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Correspondence to Christian Haass.

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Mori, K., Lammich, S., Mackenzie, I.R.A. et al. hnRNP A3 binds to GGGGCC repeats and is a constituent of p62-positive/TDP43-negative inclusions in the hippocampus of patients with C9orf72 mutations. Acta Neuropathol 125, 413–423 (2013). https://doi.org/10.1007/s00401-013-1088-7

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