Acta Neuropathol (2013) 125:413–423

DOI 10.1007/s00401-013-1088-7

ORIGINAL PAPER

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 Mori • Sven Lammich • Ian R. A. Mackenzie • Ignasi Forné • Sonja Zilow • Hans Kretzschmar •

Dieter Edbauer • Jonathan Janssens • Gernot Kleinberger • Marc Cruts • Jochen Herms •
Manuela Neumann • Christine Van Broeckhoven • Thomas Arzberger • Christian Haass

Received: 28 November 2012 / Revised: 22 January 2013 / Accepted: 22 January 2013 / Published online: 5 February 2013
Ó Springer-Verlag Berlin Heidelberg 2013

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

Neurodegeneration
TDP-43
Electronic supplementary material The online version of this
article (doi:10.1007/s00401-013-1088-7) contains supplementary
material, which is available to authorized users.

K. Mori
S. Lammich
S. Zilow
G. Kleinberger
C. Haass J. Janssens
G. Kleinberger
M. Cruts
C. Van Broeckhoven
Adolf-Butenandt-Institute, Biochemistry, Neurodegenerative Brain Diseases Group, Department
Ludwig-Maximilians-University, Schillerstr. 44, of Molecular Genetics, VIB, Universiteitsplein 1,
80336 Munich, Germany 2610 Antwerp, Belgium

I. R. A. Mackenzie J. Janssens
G. Kleinberger
M. Cruts
C. Van Broeckhoven

Department of Pathology and Laboratory Medicine, Laboratory of Neurogenetics, Institute Born-Bunge, University
University of British Columbia, Vancouver, Canada of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium

I. Forné J. Herms
C. Haass

Adolf-Butenandt-Institute, Protein Analysis Unit, Munich Cluster for Systems Neurology (SyNergy),
Ludwig-Maximilians-University, Munich, Germany
Schillerstr. 44, 80336 Munich, Germany
M. Neumann
H. Kretzschmar
T. Arzberger DZNE, German Center for Neurodegenerative Diseases,
Center for Neuropathology and Prion Research, Tübingen, Germany
Ludwig-Maximilians-University, Munich, Germany
M. Neumann
D. Edbauer
G. Kleinberger
J. Herms
C. Haass (&) Department of Neuropathology, University of Tübingen,
DZNE, German Center for Neurodegenerative Diseases, Tübingen, Germany
Munich, Germany
e-mail: [email protected]

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Introduction that trapping of essential RNA-binding proteins may be

involved in the disease. On the other hand, the finding of
Neurodegenerative disorders are generally characterized by decreased expression of C9orf72 mRNA and decreased
disease-signifying protein deposits. Moreover, in a number transcriptional activity of the C9orf72 promoter on inter-
of neurodegenerative diseases mutations causing geneti- mediate (7–24 repeats) alleles [8, 14, 39] implies a loss of
cally inherited variants of the disease were associated with function as a disease-causing mechanism. These scenarios
the genes encoding the protein deposits, their precursors or are not mutually exclusive and may even occur in parallel.
their modulating enzymes. Functional analysis of these We aimed to identify proteins capable to bind to the
genetic variants fundamentally helped to understand dis- GGGGCC repeat and to verify their pathological relevance
ease-associated mechanisms of Alzheimer’s disease (AD) by investigating their distribution in cases with C9orf72
and Parkinson’s disease [13, 16]. Similarly, research into hexanucleotide expansion mutations.
amyotrophic lateral sclerosis (ALS) and frontotemporal
lobar degeneration (FTLD) was dramatically accelerated
by the identification of the RNA/DNA-binding protein Materials and experimental procedures
TDP-43 (Tar DNA-binding protein of 43 kDa) as an
abundant deposited protein [2, 25] and by the discovery DNA synthesis and plasmid construction
that mutations in TARDBP cause familial variants of both
diseases [4, 36]. These findings also helped to develop the 120 base single-stranded DNA containing GGGGCC or
concept that ALS and FTLD are multisystem disorders AAAACC hexanucleotide repeats with restriction enzyme
with overlapping clinical and pathological characteristics sites (NheI or HindIII) were synthesized. 100 lM of
and similar functional and genetic causes [27, 33]. Besides complementary DNA strands were annealed in the pres-
TDP-43 and the long known SOD1 (super oxide dismutase ence of 10 % GC-RICH solution (Roche) and GC-RICH
1) gene [30], a number of other ALS and/or FTLD-related PCR Reaction buffer (Roche) and cloned into pcDNA3.1
genes/risk factors were discovered including FUS (fused in (?) vector (invitrogen). Plasmids containing 7, 17 and 23
sarcoma) [20, 40], OPTN (optineurin) [23], Ataxin 2 [11], repeats of GGGGCC and 17 repeat of AAAACC were
Chmp2B [35], VCP (valosin containing protein) [41], obtained. The sequence of all constructs was verified.
TMEM106B [38], GRN (progranulin) [3, 7], PFN (profilin)
[43] and the C9orf72 gene [8, 14, 29]. Pathological repeat In vitro transcription of RNA probes
expansions in C9orf72 have been found in about 40 % of
familial ALS patients and 20 % of familial FTLD [27], pcDNA3.1-GGGGCC923 and pcDNA3.1-AAAACC917
demonstrating that C9orf72 is the most common genetic constructs were linearized with HindIII and used as tem-
cause for these incurable disorders. Thus, C9orf72 may plates for RNA synthesis. In vitro RNA transcription was
provide a key not only for the understanding of ALS/FTLD performed with T7 Ribomax Express Large Scale RNA
causing mechanisms, but also for the identification of drug Production System (Promega) as described by the manu-
targets and the design of therapeutic strategies. Indepen- facturer supplemented with 40 U of RNase inhibitor
dent evidence originally obtained by three different (RiboLock, Thermo Scientific). To achieve equal levels of
research groups [8, 14, 29] and subsequently confirmed by biotinylation between these probes, different concentra-
numerous other groups demonstrated that a large expansion tions of biotin-14-CTP (2 mM for GGGGCC probe and
of a hexanucleotide repeat (GGGGCC) in the first intron of 0.29 mM for AAAACC probe) were added in each reac-
C9orf72 segregates with the disease. In healthy humans the tion. Following DNase treatment, biotinylated RNA
repeat length is apparently below 26, whereas for ALS/ products were purified with phenol/chloroform. Biotinyla-
FTLD patients a repeat length from 60 to 1,600 was tion efficacy of RNA probes was evaluated using the
determined [8, 14, 29, 39]. Repeat expansions in non- BrightStar BioDetect kit (Ambion). For competition
coding regulatory regions of genes can cause a disease experiments non-biotinylated GGGGCC repeat RNA was
principally by two different mechanisms. Due to the in vitro RNA transcribed using the MEGA script kit
immense length of the repeat expansion transcription and/ (Ambion) as described by the manufacturer. Expected
or splicing may be affected leading to haploinsufficiency lengths of repeat RNA probes and competitor were con-
[39]. On the other hand, RNA toxicity caused by seques- firmed with formaldehyde gel electrophoresis.
tration of RNA-binding proteins may also be causative
[28]. Currently, evidence for both possibilities exists. The Cell culture and preparation of nuclear extracts
observation of nuclear RNA foci in patients with
GGGGCC hexanucleotide repeat expansions [8], a finding HEK293 cells were grown with DMEM supplemented with
which however is still controversially discussed, suggests 10 % FCS and penicillin/streptomycin. Nuclear extract was

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prepared as previously described [9]. Protein concentration Proteome Software Inc., Portland, OR; Peptide Thresholds:
was determined by the BCA method. 90.0 % minimum, Protein Threshold: 99.0 % minimum
and 2 peptides minimum).
siRNA-mediated knockdown of hnRNP A3
Western blotting
Cells were plated without antibiotics at a density of
600,000 cells per 6 wells. Transfection of ON-TARGET Samples were separated on 10 % Tris–glycine gel and
plus SMARTpool Human HNRNPA3 or ON-TARGETplus transferred on PVDF membranes. After blocking for 1 h
Non-targeting pool siRNA (Thermo SCIENTIFIC) was with 0.2 % I-Block (Applied Biosystems) in PBS, the
performed with Lipofectamine RNAiMax. Three days after membrane was incubated with indicated antibody over-
transfection cell lysates were harvested. night. The antibody signal was detected with HRP-
conjugated secondary antibodies (Promega) using the ECL
Purification of hexanucleotide-repeat-binding proteins reagents (GE healthcare) and exposed to X-ray films
(SuperRX, Fujifilm).
A total of 0.6 mg of HEK293 cell nuclear extract was
diluted in 4 ml of protein-binding buffer [10 % glycerol, Immunohistochemistry
10 mM HEPES, 50 mM KCl, 1 U/ml RNase inhibitor,
0.15 lg/ml yeast tRNA (10109495001, Roche), 1 mM Immunohistochemistry was performed on 3-lm-thick par-
EDTA, 1 mM DTT and 0.5 % Triton X100 in DEPC affin sections. Following antigen retrieval by microwaving
water]. The diluted extract was then precleared with hep- in 0.1 M citrate buffer pH 6 and blocking of endogenous
arin-agarose (H6508, Sigma) and streptavidin-agarose peroxidase with 5 % H2O2 in methanol, sections were
(15942-050, Invitrogen). The precleared nuclear extracts settled in PBS with 0.02 % Brij35 (Sigma-Aldrich). After
were incubated with streptavidin lMACS-microbeads blocking with 2 % FBS in PBS for 5 min, the respective
(MACS molecular) and 150 pmol of biotinylated repeat primary antibody was applied for 1.5 h at RT or overnight
RNA in the presence of 50 mM KCl for 1 h. For compe- at 4 °C. When the first antibody was derived from goat or
tition experiments, nuclear extracts were incubated for 1 h rat, rabbit-anti-goat antibody (DAKO) or rabbit anti-rat
with 7.5 nmol (50-fold excess) of non-biotinylated IgG (PARIS-biotech) was used as secondary antibody for
GGGGCC competitor RNA before addition of the biotin- 1 h. After rinsing with 0.02 % Brij35 in PBS, antibody
ylated probe. The reaction mixture was loaded on a binding was detected and enhanced by DCS Super Vision 2
lMACS column and subsequently washed three times with HRP-Polymer-Kit (DCS Innovative Diagnostik-Systeme,
protein-binding buffer. GGGGCC-repeat binding proteins Hamburg, Germany) or NovoLink DS Polymer Detection
were sequentially eluted with increasing concentrations of Systems (Leica) using DAB as chromogen. Counterstain-
NaCl. Each eluate was TCA precipitated and subjected to ing for cellular structures was performed with haemalum.
SDS-PAGE. Gels were stained with Silver Quest Silver Microscopic images were obtained using a BX50 micro-
staining kit (Invitrogen) according to manufacturer’s pro- scope and Cell-D software (Olympus).
tocol and prepared for mass spectrometry.
Immunofluorescence
Protein identification and quantification by LC–MS
For double immunofluorescence, the secondary antibodies
Gel lanes of interest were excised into 25 pieces per lane conjugated to Alexa Fluor 555 or Alexa Fluor 488-conju-
and transferred into a 96-well plate and digested as gated antibodies (anti-rabbit, anti-mouse, and anti-rat) were
described before with minor modifications [32, 42]. For the used with TO-PRO-3 (Invitrogen) for nuclear counter-
LC–MS analysis, the peptides were injected in an Ultimate staining. To reduce autofluorescence, slides were incubated
3000 HPLC system (LC Packings) and the effluent was with 0.3 % Sudan Black in 70 % ethanol. Confocal images
directly electrosprayed into a LTQ-Orbitrap mass spec- were obtained with an inverted laser scanning confocal
trometer (Thermo) as described elsewhere [31]. Proteins microscope (Zeiss LSM510 or LSM710) with a 409 or
were identified using Mascot (Matrix Science, London, 639/1.4 oil immersion lens, using a pinhole diameter of 1
UK; version Mascot) against SwissProt_2011.02 database Airy unit. If necessary for printing, brightness and contrast
for human proteins (Fragment Tolerance: 0.80 Da, Fixed of each image were linearly enhanced using LSM software
Modification for carbamidomethyl cysteine, Variable (Zeiss). Co-localization of inclusions on these images was
Modification for methionine oxidation, Max Missed manually determined using ImageJ software with ‘‘Cell
Cleavage:2). Proteins were quantified using the ‘‘Quanti- Counter’’ plug-in. Six merged images per section per case
tative Value’’ from Scaffold (version Scaffold_3.4.9, were analyzed.

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Human tissue expanded repeats, because the repetitive sequence will

likely form a similar secondary structure and RNA-binding
All cases provided by the Neurobiobank Munich, Ludwig- proteins typically bind to rather short sequences motifs or
Maximilians-University Munich and the University of structures. In fact a four-repeat GGGGCC repeat RNA is
British Columbia were collected and distributed according sufficient to form a complex G-quadruplex structure [12].
to the guidelines of the local ethical committee. Autopsy Moreover, repeats of intermediate length, such as the 23
material of a control individual was provided by the repeats used here, may be a risk for developing the disease
Antwerp Bio Bank at the Institute Born-Bunge, University [15, 39]. Biotinylated in vitro-transcribed RNA probes
of Antwerp, Antwerp, Belgium. Brain autopsy was per- were incubated with nuclear extracts from HEK 293 cells
formed on the basis of informed consent. in the absence or presence of a 50-fold excess of non-
biotinylated competitor RNA containing the GGGGCC
repeat (Fig. 1a). The competitor RNA prevented binding as
Results indicated by strongly enhanced flow through (Fig. 1a,
lane 4). Using increasing salt concentrations for the elution
Identification of GGGGCC hexanucleotide- of bound proteins, we observed differential protein-binding
repeat-binding proteins affinity to the GGGGCC/AAAACC repeats upon elution
with 500 mM NaCl (Fig. 1a, lanes 17 and 18). Proteins
To identify proteins, which could be bound by GGGGCC eluting with 500 mM NaCl from the GGGGCC or the
hexanucleotide repeats, we performed pull down assays AAAACC repeats were subjected to LC–MS/MS. This
using in vitro-transcribed biotinylated RNAs containing allowed the identification of 235 proteins in three replicates
either 23 GGGGCC or 17 AAAACC repeats as control. We (Suppl. Tab. 1). 188 proteins were identified at least twice
choose this repeat length to circumvent the still unsolved in the three pull down experiments. Of these 188 proteins,
problems of cloning and transcribing the extremely long binding of 127 proteins could be competed with an excess
hexanucleotide repeats observed in patients with C9orf72 of non-biotinylated GGGGCC probes. 72 proteins showed
mutations. We expect such shorter repeats to have a similar at least a twofold stronger binding to GGGGCC than to
ability and specificity for selective protein binding like AAAACC. All proteins with an abundance higher than 20

b
Input (4%)

FT Elution by NaCl (mM)

a
Input (4%)

300 500 1000

Sample buffer

FT Wash Elution by NaCl Repeat-probe- A G G A G G A GG A G G

(mM)
100 200 300 500 1000 2000 Competitor - - - + - - + - - + - - +
lane 1 2 3 4 5 6 7 8 9 10 11 12 13
Repeat-probe - AG G AG G A GG A GG A G G AG G A GG A GG 50
Competitor - - - + - - + - - + - - + - - + - - + - - + - - + hnRNP A3
lane 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 36
hnRNP A2/B1
36
250
148 98 SFPQ
98 98 ILF3
kDa 64
64 64 NONO
kDa 50
50
64 hnRNP L

36 64 IL2BP1
50 ILF-2
22
64 FUS
50 hnRNP F

Fig. 1 Identification of GGGGCC hexanucleotide repeat-specific protein identification by LC–MS/MS. b Western blot analysis
binding proteins. a Representative silver-stained gels showing confirming GGGGCC-repeat-specific binding of selected proteins.
proteins pulled down by the respective repeat containing RNAs. Aliquots of proteins eluted at different salt concentrations were
HEK293 nuclear extracts were incubated with indicated RNA probes subjected to electrophoresis, and Western blotting was performed
with (?) or without (-) 50-fold excess of non-biotinylated RNA using the indicated antibodies. All proteins show GGGGCC-repeat-
competitor. RNA–protein interaction was weakened with increasing specific binding at high NaCl concentrations (compare lane 8 and 9).
concentrations of NaCl. In the presence of GGGGCC competitor Note that binding was completely blocked by a 50-fold excess of non-
(G?), RNA–protein binding was inhibited (lanes 10, 13, 16, 19, 22, labeled GGGGCC (?) (lanes 7, 10). hnRNP F, which is not one of the
25), and proteins in the flow through fraction (lane 4) were increased. 20 GGGGCC-repeat-specific binding proteins, is used as negative
Boxed lanes in 500 mM NaCl elution fractions were excised for control. A AAAACC repeats, FT flow through, G GGGGCC repeats

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in the GGGGCC fraction were finally selected. These derived from an autopsy case (case EM1) with genetically
stringent selection criteria resulted in 20 top candidate confirmed C9orf72 repeat expansion. Hippocampus was
proteins, of which most were known RNA interacting selected as a representative region, in which the character-
factors such as heterogenous ribonucleoproteins (hnRNPs), istic inclusion body pathology of cases with C9orf72
splicing factors and mRNA-binding proteins (Table 1). A mutations is seen consisting of TDP-43-positive neuronal
selection of these 20 proteins for which specific antibodies cytoplasmic inclusions (NCI) (Fig. 2a), and p62-positive/
were commercially available (Suppl. Tab. 2) was then TDP-43-negative dot-like NCIs, dot-like neuronal intranu-
confirmed by Western blotting of the elution fractions. All clear inclusions (NII) (Fig. 2b) and star-like NCIs (Fig. 2c)
antibodies tested including antibodies to hnRNP A3, [1]. Among the antibodies tested (Suppl. Tab. 2; Suppl.
hnRNP A2B1, SFPQ, ILF3, NONO, hnRNP L, IL2BP1, Fig. 1) only the antibody against hnRNP A3 [Suppl. Tab. 2
ILF-2, and FUS revealed strong and selective binding to (ab1)] visualized specific inclusion pathology in the C9orf72
the GGGGCC repeat in the 500 mM NaCl fraction. These cases (Fig. 2d). This antibody recognized a 40 kDa protein
signals were completely blocked by 50-fold excess of the in cerebellar lysates of C9orf72 carriers and controls (Suppl.
non-labeled GGGGCC probe (Fig. 1b, lanes 8–10). Fig. 2a). siRNA-mediated knockdown of hnRNP A3 mes-
senger RNA results in a significant reduction of hnRNP A3
Identification of hnRNP A3-positive inclusions protein in cultured cells (Suppl. Fig. 2b) again confirming
in C9orf72 mutation cases the specificity of the hnRNP A3 antibody. To investigate if
these hnRNP A3-positive inclusions are characteristic for
To investigate a potential pathological involvement of these C9orf72 GGGGCC repeat expansion carriers, we stained a
proteins, we performed immunohistochemical screenings series of hippocampal sections from 13 cases with C9orf72
using antibodies against the identified GGGGCC RNA mutations, 7 FTLD-TDP cases without C9orf72 mutation, 2
repeat binding proteins on hippocampal paraffin sections ALS-TDP cases without C9orf72 mutation, 1 case with

Table 1 List of 20 selected proteins specifically binding to the GGGGCC repeat

Identified proteins Accession Average quantitative value GC/AC ratio

AC GC Competition

1 Heterogeneous nuclear ribonucleoproteins A2/B1 ROA2_HUMAN 110.3 369.8 1.7 3.4

2 Splicing factor, proline- and glutamine-rich SFPQ_HUMAN 51.7 130.5 0.1 2.5
3 Splicing factor 3B subunit 3 SF3B3_HUMAN 51.1 118.8 6.3 2.3
4 ELAV-like protein 1; (Hu-antigen R) ELAV1_HUMAN 9.6 117.1 0.0 12.1
5 Interleukin enhancer-binding factor 3 ILF3_HUMAN 24.7 87.4 0.0 3.5
6 Non-POU domain-containing octamer-binding protein NONO_HUMAN 13.4 80.4 0.0 6.0
7 Heterogeneous nuclear ribonucleoprotein R HNRPR_HUMAN 28.8 74.1 0.0 2.6
8 Heterogeneous nuclear ribonucleoprotein A3 ROA3_HUMAN 23.8 70.6 0.0 3.0
9 Heterogeneous nuclear ribonucleoprotein L HNRPL_HUMAN 21.7 57.2 0.0 2.6
10 Scaffold attachment factor B1 SAFB1_HUMAN 27.5 55.0 0.0 2.0
11 Insulin-like growth factor 2 mRNA-binding protein 1 (IMP1) IF2B1_HUMAN 19.5 47.9 0.6 2.5
12 Scaffold attachment factor B2 SAFB2_HUMAN 20.0 45.0 0.0 2.3
13 Heterogeneous nuclear ribonucleoprotein A1 ROA1_HUMAN 20.8 44.9 0.1 2.2
14 Double-stranded RNA-specific adenosine deaminase (ADAR1) DSRAD_HUMAN 16.5 42.3 0.3 2.6
15 Putative pre-mRNA-splicing factor ATP-dependent RNA DHX15_HUMAN 13.0 31.0 1.0 2.4
16 Interleukin enhancer-binding factor 2 ILF2_HUMAN 8.3 25.3 0.0 3.0
17 Putative ATP-dependent RNA helicase DHX30 DHX30_HUMAN 6.3 23.5 0.0 3.8
18 Heterogeneous nuclear ribonucleoprotein K HNRPK_HUMAN 10.7 23.2 0.0 2.2
19 Nucleolar RNA helicase 2 DDX21_HUMAN 7.5 23.0 0.7 3.1
20 RNA-binding protein FUS FUS_HUMAN 7.0 20.0 0.0 2.9

20 proteins were selected as specific GGGGCC-repeat binding proteins based on the criteria described in the result section and in the legend of
supplementary Table 1. The quantitative value reflects the relative protein amount estimated from the intensity of LC–MS/MS signal derived from the
500 mM elution fraction using Scaffold software. In the presence of non-biotinylated GGGGCC repeat competitor (competition) binding of all proteins is
efficiently suppressed. Furthermore, these proteins show at least 2 times more binding to the GGGGCC RNA repeat (GC) compared to AAAACC RNA
repeat (AC). Quantitative values listed here are the averages of three independent experiments. Standard errors of these average quantitative values are
shown in supplementary Table 1
Positive results are described in bold

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a pTDP b p62 c p62

C9 EM1
C9 EM1

C9 EM1
GL GL CA3

d A3 e A3 f A3

Control EM16
C9 EM1

C9 EM3
GL GL GL

g A3 h A3 i A3

C9 EM10
C9 EM10

C9 EM8

GL GL CA4

j A3 k pTDP l A3
FTLD-TDP EM20

FTLD-TDP EM20
C9 EM7

CA4 GL GL

Fig. 2 Immunohistochemical detection of phosphorylated TDP-43 dot-like NCIs (red arrowheads) and NIIs (red arrows) are detectable
(pTDP), p62, and hnRNP A3 in hippocampi of cases with C9orf72 with varying frequency confirming the findings in case EM1 (d). i In
hexanucleotide expansions, a FTLD-TDP case without C9orf72 CA4 of a case with expanded C9orf72 repeats (case EM10), a star-
mutation, and a control case. a–c C9orf72 mutation case EM1 with like hnRNP A3-positive NCI is seen (green arrowhead), whose size
characteristic large pTDP-43-positive (red arrowheads in a) [24], and shape is similar to the p62-positive NCIs characteristically found
small dot-like p62-positive NCIs (red arrowheads in b), small dot- in CA regions of cases with expanded C9orf72 repeats (green
like p62-positive NIIs (red arrows in b) in GL and large star-like p62- arrowheads in c). j In CA4 of a C9orf72 mutation case (case EM7) a
positive NCIs in CA3 (green arrowheads in c). d In GL of C9orf72 hnRNP A3-positive dystrophic neurite can be detected. k, l In a
mutation case EM1 small dot-like hnRNP A3-positive NCIs (red FTLD-TDP case without C9orf72 repeat expansion (case EM20),
arrowheads) and NIIs (red arrow) are seen whose shapes are similar there are numerous often ring-shaped pTDP-positive NCIs in GL (k),
to those of p62-positive inclusions shown in b. e In GL of non- but no hnRNP A3-positive NCIs or NIIs (l). Counterstains for
diseased control case EM16, there is a strong nuclear and a weak visualizing cellular structures were done with haemalum. CA3 cornu
cytoplasmic immunoreactivity for hnRNP A3, but there are no ammonis region 3, CA4 cornu ammonis region 4, GL granular layer
hnRNP A3-positive NCIs or NIIs. f–h In GL of other cases with of the dentate gyrus, NCI neuronal cytoplasmic inclusion, NII
expanded C9orf72 repeats (EM3, EM10, EM8) hnRNP A3-positive neuronal intranuclear inclusion. Scale bar 20 lm

Lewy body disease (LBD), 1 case with AD, 1 case with a cytoplasmic hnRNP A3 expression (Fig. 2e; Suppl. Fig 3a).
combination of AD and LBD, 1 case with Pick’s disease, and In contrast to that, a significant reduction of intranuclear
2 cases with Huntington’s disease (HD). To determine the hnRNP A3 staining expression was observed in 10 out of 13
physiological distribution pattern of hnRNP A3, five control C9orf72 mutation cases (Fig. 2d, f–i). In all C9orf72
cases without neurodegenerative alterations were added. All mutation cases dot-like hnRNP A3-positive NCIs consis-
cases investigated are listed in Table 2. In most (4 out of 5) tently occurred in the granular layer of the dentate gyrus
control cases hippocampal neurons and some glial cells (Fig. 2d, f–h; Table 2). However, hnRNP A3-positive
showed a moderate to intense nuclear and a weak dot-like inclusions were less frequently observed than

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Table 2 Cases investigated with source, neuropathological diagnosis, C9orf72 genotyping and results on immunhistochemical hnRNPA3 stains
Case Source Neuropathological FTLD-TDP GGGGCC hnRNP A3 expression
diagnosis type repeat expansion
DG CA CBL
NCI NII NCI NII DN NCI NII

EM1 LMU FTLD/ALS-TDP B ? ? ? ? – ? – –

EM2 LMU FTLD/ALS-TDP B ? ? ? ? – ? – –
EM3 LMU FTLD/ALS-TDP B ? ? ? ? – ? ? ?
EM4 VA FTLD-TDP B ? ? ? ? ? ? – –
EM5 VA FTLD/ALS-TDP B ? ? – – – ? – –
EM6 VA FTLD-TDP A?B ? ? ? ? – ? ? –
EM7 VA FTLD-TDP A?B ? ? ? ? – ? ? –
EM8 VA FTLD/ALS-TDP B ? ? – – – – – –
EM9 VA FTLD-TDP A?B ? ? ? ? ? ? ? –
EM10 VA FTLD-TDP B ? ? ? ? – ? ? ?
EM11 VA FTLD-TDP B ? ? ? – – – – –
EM12 VA FTLD-TDP B ? ? – ? – – ? –
EM13 VA FTLD/ALS-TDP B ? ? – – ? – – –
EM14 LMU Control – – – – – – – – –
EM15 LMU Control – – – – – – – – –
EM16 VA Control – – – – – – – nd nd
EM17 VA Control – – – – – – – nd nd
EM18 VIB Control – – – – – – – – –
EM19 LMU FTLD-TDP B – – – – – ? – –
EM20 LMU FTLD-TDP B – – – – – ? – –
EM21 VA FTLD-TDP B – – – – – – nd nd
EM22 VA FTLD-TDP A – – – – – – nd nd
EM23 VA FTLD-TDP C – – – – – ? nd nd
EM24 VA FTLD-TDP B – – – – – – nd nd
EM25 VA FTLD-TDP A – – – – – – nd nd
EM26 LMU ALS-TDP B – – – – – – – –
EM27 LMU ALS-TDP – – – – – – – – –
EM28 VA LBD – – – – – – – nd nd
EM29 VA AD/LBD – – – – – – – nd nd
EM30 VA AD – – – – – – – nd nd
EM31 VA Pick – – – – – – – nd nd
EM32 LMU HD – nd – – – – – nd nd
EM33 LMU HD – nd – – – – – nd nd
FTLD-TDP subtyping is based on Mackenzie et al. [22]
AD Alzheimer’s disease, ALS amyotrophic lateral sclerosis, CA cornu ammonis, CBL cerebellum, DG dentate gyrus, DN dystrophic neuritis,
FTLD frontotemporal lobar degeneration, HD Huntington’s disease, LBD Lewy body disease, LMU Ludwig-Maximilians-University, Munich;
NCI neuronal cytoplasmic inclusion, NII neuronal intranuclear inclusion, nd not determined, VA Vancouver General Hospital, Vancouver; VIB
Antwerp VIB Department of Molecular Genetics, Antwerp

p62-positive dot-like inclusions (compare Fig. 2b and 2d). immunopositive dystrophic neurites (DN) were found in 9
In addition, a subset of star-like p62-positive/TDP-43- out of 13 C9orf72 mutation cases (Fig. 2j; Table 2). No
negative NCIs known to be pathognomonic for cases with hnRNP A3-positive NCIs or NIIs were detectable in FTLD-
C9orf72 hexanucleotide repeat expansions [1] were immu- TDP and ALS-TDP cases without GGGGCC repeat
nopositive for hnRNP A3 (arrowheads in Fig. 2c, i). 10 out expansions (Fig. 2k, l; Suppl. Fig. 3b), AD, LBD, Pick or
of 13 C9orf72 mutation cases also showed hnRNP A3- HD cases (Suppl. Fig. 3c–g; Table 2). However, few
positive NIIs (Fig. 2d, f–g; Table 2). Additionally, hnRNP A3-positive DNs were also found in 3 out of 7

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FTLD-TDP cases without C9orf72 hexanucleotide case EM1 in Fig. 3h). No hnRNP A3 positive NCIs or
expansions, but not in cases with other diseases NIIs were detectable in the cerebella of 3 control, 2
(Table 2). No hnRNP A3-positive NCIs, NIIs or DNs FTLD-TDP and 2 ALS-TDP cases (Fig. 3i–l; Table 2). In
were seen in any control case (Fig. 2e; Suppl. Fig. 3a; C9orf72 mutation cases co-localization experiments
Table 2). These findings were confirmed by another anti- revealed that 16.5–27.6 % of the p62 inclusions in the
hnRNP A3 antibody (A3 ab2), which also visualized granular layer of the dentate gyrus were also immuno-
neuronal inclusions in a case with C9orf72 hexanucleo- positive for hnRNP A3 (Fig 4a, d), but only 3.2 % of
tide expansions (Suppl. Fig. 3h) but not in a control case phosphorylated TDP-43 inclusions were also immuno-
(Suppl. Fig. 3i). In the granular layer of cerebellum dot- positive for hnRNP A3 (Fig 4b, data not shown). In
like neuronal hnRNP A3 aggregates were found in 6 out cerebellum of three C9orf72 cases with the most frequent
of the 13 C9orf72 cases (positive and negative examples cerebellar hnRNP A3 inclusions, 2–17 % of p62 inclu-
are shown in Fig. 3a–g; Table 2), while p62-positive sions also contained aggregated hnRNP A3 proteins
inclusions were consistently observed in all cases (e.g., (Fig. 4c, d).

a b A3 c A3
C9 EM3

C9 EM10
C9 EM10

CBL CBL CBL

d A3 e A3 f A3
C9 EM9

C9 EM8
C9 EM6

CBL CBL CBL

g A3 h p62 i A3
Control EM18
C9 EM1

C9 EM1

CBL CBL CBL

j A3 k A3 l A3
FTLD-TDP EM20

ALS-TDP EM27
Control EM15

CBL CBL CBL

Fig. 3 Immunohistochemical detection of hnRNP A3 in the cerebel- case EM1 no hnRNP A3-positive NCIs are seen (g), while p62-
lar granular layer of cases with C9orf72 hexanucleotide expansions, a positive dot-like NCIs (red arrowheads) are present (h). Strong
control case, a FTLD-TDP, and a ALS-TDP case without C9orf72 nuclear staining of hnRNP A3 in control case EM18 (i). No hnRNP
mutation. a–e In C9orf72 mutation cases EM3, EM10, EM6 and EM9 A3-positive NCIs and a variable degree of nuclear staining is
hnRNP A3-positive neuronal cytoplasmic inclusions (NCIs red observed in the cerebellar granular layer of control case EM15 (j),
arrowheads), neuronal intranuclear inclusions (red arrows), and a FTLD-TDP case EM20 (k), and ALS-TDP case EM27 (l). Counter-
reduced nuclear staining are observed in varying frequency. In stains for visualizing cellular structures were done with haemalum.
C9orf72 mutation case EM8, there is a strong nuclear staining of CBL cerebellum. Scale bar 20 lm
hnRNP A3 but no hnRNP A3-positive NCIs (f). In C9orf72 mutation

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Acta Neuropathol (2013) 125:413–423 421

Discussion mRNA trafficking trans-acting factors in neurons [21].

Consistent with our findings in human non-diseased control
Taken together, we identified hnRNP A3 as a first component cases, hnRNP A3 is mainly expressed in cell nuclei of mouse
of the so far enigmatic TDP-43-negative/p62-positive NCIs brain and human cell lines and to a lesser extent in the
and NIIs in hippocampus, which are pathognomonic for cytosol [26]. However, in C9orf72 cases an apparent redis-
cases with C9orf72 repeat expansions [1]. hnRNP A3 is a tribution from the nucleus to the cytosol is observed, which
member of hnRNP A/B-type family of proteins (A1, A2/B1, may cause a loss of an essential nuclear function. Of note, we
A3, and A0) that contain two N-terminal RNA recognition also observed a similar redistribution in some cases with
motifs followed by a C-terminal glycine-rich auxiliary other neurodegenerative disorders. Therefore, further studies
domain [17]. The members of the hnRNP A/B family are are required to prove the selectivity of this observation.
known to shuttle between nucleus and cytoplasm. The Interestingly, nuclear clearance accompanied by a potential
hnRNP A/B-type family of proteins performs multiple loss of function seems to be a more general phenomenon, as
functions in alternative pre-mRNA splicing, nuclear import it is also observed for TDP-43 and to some extend for FUS as
and cytoplasmic trafficking of mRNA, mRNA stability and well [2, 20, 25, 40]. Furthermore, the major cause of ALS-
turnover, and translation [17]. Most studies have been per- FUS are mutations affecting a PY-nuclear localization sig-
formed with the homologs hnRNP A1 and A2/B1. However, naling, further supporting a loss of nuclear function as one
evidence exists that hnRNP A3 and A2/B1, but not A1 act as reason for the disease [10].

a p62 A3 b pTDP A3
C9 EM1

C9 EM1

TOPRO Merge TOPRO Merge

DG-GL DG-GL
%A3(+) inclusion/p62(+) inclusion

c p62 A3 d 30
DG-GL

20

10

0
C9 EM3

1 2 3 4 10 12
TOPRO Merge 20
15
CBL

10
5
0
3 10 12
CBL C9 Case (EM)
Fig. 4 Double immunofluorescence analysis of p62, phosphorylated (arrowheads). Double immunofluorescence for pTDP (green signal)
TDP-43 (pTDP), and hnRNP A3 in hippocampus and cerebellum of and hnRNP A3 (red signal, arrow) in b does not show co-localization
cases with C9orf72 hexanucleotide expansions. a In DG-GL of a of the cytoplasmic aggregates. c Partial co-localization of p62 and
C9orf72 mutation case EM1 double immunofluorescence for p62 hnRNP A3 the in granular layer of the cerebellum (arrows). Again, not
(green signal) and hnRNP A3 (red signal) reveals dot-like aggregates all p62 aggregates contain hnRNP A3 (arrowheads). d Percentage of
for both proteins. Merging both signals demonstrates that hnRNP A3 p62 inclusions that co-localize hnRNP A3 in DG-GL and CBL of
and p62 aggregates are co-localized in the cytoplasm of granular cells indicated C9orf72 cases. Nuclei were marked with TO-PRO-3. DG-GL
(arrow). Note that not all p62 aggregates contain hnRNP A3 granular layer of the dentate gyrus, CBL cerebellum. Scale bar 10 lm

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We found that hnRNP A3 binds to the GGGGCC repeats. 2. Arai T, Hasegawa M, Akiyama H et al (2006) TDP-43 is a
Moreover, since hnRNP A3 is known to act in mRNA export component of ubiquitin-positive tau-negative inclusions in fron-
totemporal lobar degeneration and amyotrophic lateral sclerosis.
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repeat expansions could initiate aberrant export of C9orf72 3. Baker M, Mackenzie IR, Pickering-Brown SM et al (2006)
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Acknowledgments We thank Iryna Pigur for expert technical import of FUS. EMBO J 31(22):4258–4275
assistance, Axel Imhof, Harald Steiner, Akio Fukumori, Richard 11. Elden AC, Kim HJ, Hart MP et al (2010) Ataxin-2 intermediate-
Page, and Eva Bentmann for providing tools and technologies and length polyglutamine expansions are associated with increased
Dorothee Dormann for critically reading the manuscript. This work risk for ALS. Nature 466(7310):1069–1075
was supported by the Deutsche Forschungsgemeinschaft (SFB-596), 12. Fratta P, Mizielinska S, Nicoll AJ et al (2012) C9orf72 hexanucleotide
the Competence Network for Neurodegenerative Diseases (KNDD) of repeat associated with amyotrophic lateral sclerosis and frontotem-
the Bundesministerium für Bildung und Forschung (BMBF) to C.H. poral dementia forms RNA G-quadruplexes. Sci Rep 2:1016
and the Consortium of Centers of Excellence in Neurodegenerative 13. Gasser T, Hardy J, Mizuno Y (2011) Milestones in PD genetics.
Brain Diseases (CoEN) to C.H., M.C., D.E., and C.V.B. K.M. was Mov Disord 26(6):1042–1048
supported by a postdoctoral fellowship from the Alexander von 14. Gijselinck I, Van Langenhove T, van der Zee J et al (2012) A
Humboldt Foundation. D.E. was supported by the Helmholtz Young C9orf72 promoter repeat expansion in a Flanders-Belgian cohort
Investigator Program HZ-NG-607. The Agency for Innovation by with disorders of the frontotemporal lobar degeneration-amyo-
Science and Technology provides a PhD fellowship to J.J. The trophic lateral sclerosis spectrum: a gene identification study.
authors acknowledge the Antwerp biobank of the Institute Born- Lancet Neurol 11(1):54–65
Bunge for the brain samples as well as the neurologists S. Enge- 15. Gomez-Tortosa E, Gallego J, Guerrero-Lopez R et al (2013)
lborghs and P.P. De Deyn and neuropathologist J.J. Martin for the C9ORF72 hexanucleotide expansions of 20–22 repeats are
clinical and pathological diagnoses. The Antwerp site is supported for associated with frontotemporal deterioration. Neurology. doi:
the genetic research of neurodegenerative brain diseases by the Bel- 10.1212/WNL.0b013e31827f08ea
gian Science Policy Office Interuniversity Attraction Poles program, 16. Haass C, Selkoe DJ (2007) Soluble protein oligomers in neuro-
the Foundation for Alzheimer Research (SAO/FRA), the Medical degeneration: lessons from the Alzheimer’s amyloid beta-
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excellence program, the research Foundation Flanders (FWO) and the 17. He Y, Smith R (2009) Nuclear functions of heterogeneous nuclear
Special Research Fund of the University of Antwerp, Belgium. ribonucleoproteins A/B. Cell Mol Life Sci 66(7):1239–1256
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Conflict of interest The authors declare that they have no conflict defective pre-mRNAs and intermediates. Mol Cell 12(6):1453–1465
of interest. 19. Keller BA, Volkening K, Droppelmann CA et al (2012) Co-
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