Supplementary Legends

hnRNP A3 binds to GGGGCC repeats and is a constituent of

p62 positive/TDP43 negative inclusions in the hippocampus of patients with C9orf72 mutations

Kohji Mori1, Sven Lammich1, Ian R.A. Mackenzie2, Ignasi Forné3, Sonja Zilow1, Hans Kretzschmar4, Dieter Edbauer5, Jonathan Janssens6,7, Gernot Kleinberger1,5,6,7, Marc Cruts6,7, Jochen Herms5,8, Manuela Neumann9,10, Christine Van Broeckhoven6,7, Thomas Arzberger4, Christian Haass1,5,8*

1Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University, Munich, Germany; 2Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada; 3Adolf-Butenandt-Institute, Protein Analysis Unit, Ludwig-Maximilians-University, Munich, Germany; 4Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany; 5DZNE - German Center for Neurodegenerative Diseases, Munich, Germany; 6Neurodegenerative Brain Diseases Group, Department of Molecular Genetics, VIB, Universiteitsplein 1, B-2610 Antwerp, Belgium; 7Laboratory of Neurogenetics, Institute Born-Bunge, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium; 8Munich Cluster for Systems Neurology (SyNergy), Munich, Germany; 9DZNE - German Center for Neurodegenerative Diseases, Tübingen, Germany; 10Department of Neuropathology, University of Tübingen, Tübingen, Germany

*To whom correspondence should be addressed: Christian Haass, Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-University and DZNE - German Center for Neurodegenerative Diseases, Schillerstr. 44, 80336 Munich, Germany, Tel.: +49 89 218075471; Fax: +49 89 218075415;

Supplementary figures and tables

Supplementary Figure 1. Immunohistochemical distribution pattern of GGGGCC repeat binding proteins in the granular cell layer of the dentate gyrus in C9orf72 mutation case EM1 and non-diseased control case EM18

In contrast to hnRNP A3 (Fig. 2) hnRNP A2/B1, SFPQ, SF3B3, ELAVL-1, and ILF-3 do not form any apparent intracellular aggregates in the C9orf72 mutation case. In the control cases there is a nuclear expression of all five proteins and cytoplasmic expression of hnRNP A2/B1, SF3B3 and ELAVL-1. Except for hnRNP A2/B1, whose expression seems to be reduced in the C9orf72 mutation case, there is no obvious difference in localization and staining intensity of the GGGGCC binding proteins between C9orf72 cases and controls. Scale bar=20μm.

Supplementary Figure 2. Characterization of the hnRNP A3 antibody using human cerebellar lysate and genetic knockdown experiment

a. Western blot analysis of cerebellar lysates derived from control cases EM14 and EM15 and C9orf72 cases EM1 and EM2 using the anti hnRNP A3 antibody.

b. siRNA mediated knockdown of hnRNP A3 in HEK293 cells proves the specificity of the hnRNP A3 antibody. The same membrane was re-probed with anti beta-actin antibody (clone AC-74 Sigma) as loading control.

Supplementary Figure 3. Immunohistochemical detection of hnRNP A3 in the hippocampal granular layer of control cases and several cases with different neurodegenerative diseases with or without C9orf72 hexanucleotide expansions

a-g. There are no cytoplasmic, intranuclear or neuritic aggregates in control case EM17, ALS-TDP case EM26, HD case EM32, Pick’s disease case EM31, AD case EM30, AD/LBD case EM29 or LBD case EM28. hnRNP A3 is seen in the nuclei and the cytoplasm with varying intensities. Note that Pick bodies (arrowheads in d) do not contain hnRNP A3. A second hnRNP A3 antibody (A3 ab2) visualizes neuronal cytoplasmic inclusions in C9orf72 mutation case (EM1) (h), but not in a control case (EM18) (i). AD Alzheimer’s disease, ALS Amyotrophic Lateral Sclerosis, GL granular layer of dentate gyrus, HD Huntington’s disease, LBD Lewy body disease. Scale bar=20μm.

Supplementary Table 1. List of all proteins bound to both RNA probes used. A total of 235 proteins were identified. To select specific GGGGCC repeat binding proteins, four selection criteria were applied. (1) Proteins labeled dark gray indicate proteins identified in only one of three independent experiments (N° 189-235). These proteins were not further investigated. (2) Proteins labeled gray (N° 128-188) were also excluded because competition with non-biotinylated GGGGCC RNA was not efficient (GC/Competition ratio < 2.0). (3) Proteins labeled light gray (N° 73-127) were excluded due to less selective binding to GGGGCC as compared to AAAACC (GC/AC ratio < 2.0). (4) A cut-off of GGGGCC Quantitative Value higher than 20 was applied. This resulted in a selection of 20 proteins highlighted with bold characters (N° 1-20). AVQ average quantitative value, AC AAAACC, GC GGGGCC, SE standard error.

Supplementary Table 2

List of antibodies used in this study. IHC immunohistochemistry, IF immunofluorescence, MW microwave. *no antibody available for immunohistochemistry, - not performed, x antibody did not function.

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