mTh1 driven expression of hTDP-43 results in typical ALS/FTLD neuropathological symptoms
mTh1 driven expression of hTDP-43 results in typical ALS/FTLD neuropathological symptoms
Barbara Scherz 0 1
Roland Rabl 0 1
Stefanie Flunkert 0 1
Siegfried Rohler 1
Joerg Neddens 0 1
Nicole Taub 0 1
Magdalena Temmel 0 1
Ute Panzenboeck 1
Vera Niederkofler 0 1
Robert Zimmermann 1
Birgit Hutter-Paier 0 1
0 QPS Austria GmbH , Grambach , Austria , 2 Karl-Franzens University, Institute of Molecular Biosciences , Graz , Austria , 3 Medical University Graz, Institute of Pathophysiology and Immunology , Graz , Austria
1 Editor: Emanuele Buratti, International Centre for Genetic Engineering and Biotechnology , ITALY
Transgenic mouse models are indispensable tools to mimic human diseases and analyze the effectiveness of related new drugs. For a long time amyotrophic lateral sclerosis (ALS) research depended on only a few mouse models that exhibit a very strong and early phenotype, e.g. SOD1 mice, resulting in a short treatment time window. By now, several models are available that need to be characterized to highlight characteristics of each model. Here we further characterized the mThy1-hTDP-43 transgenic mouse model TAR6/6 that overexpresses wild type human TARDBP, also called TDP-43, under control of the neuronal Thy-1 promoter presented by Wils and colleagues, 2010, by using biochemical, histological and behavioral readouts. Our results show that TAR6/6 mice exhibit a strong TDP-43 expression in the hippocampus, spinal cord, hypothalamus and medulla oblongata. Apart from prominent protein expression in the nucleus, TDP-43 protein was found at lower levels in the cytosol of transgenic mice. Additionally, we detected insoluble TDP-43 in the cortex, motoneuron loss, and increased neuroinflammation in the central nervous system of TAR6/6 animals. Behavioral analyses revealed early motor deficits in the clasping- and wire suspension test as well as decreased anxiety in the elevated plus maze. Further motor tests showed differences at later time points compared to non-transgenic littermates, thus allowing the observation of onset and severity of such deficits. Together, TAR6/6 mice are a valuable tool to test new ALS/FTLD drugs that target TDP-43 expression and insolubility, neuroinflammation, motoneuron loss or other TDP-43 related downstream signaling pathways since these mice exhibit a later pathology as previously used ALS/FTLD mouse models.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The authors greatly thank the whole
research team of QPS Austria GmbH for their
technical support. This work was supported by the
Austrian FFG: Forschungspartnerschaften,
Industrienahe Dissertationen: #847249 as well as
QPS Austria GmbH. The Austrian FFG provided
support in the form of part of the salary of BS, but
did not have any additional role in the study design,
The transactive response DNA-binding protein 43 (TARDBP; TDP-43) has been identified to
play a crucial role in the development of neurodegenerative diseases, especially in amyotrophic
lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions
data collection and analysis, decision to publish, or
preparation of the manuscript. QPS Austria GmbH
provided support in the form of part or full salaries
of BS, RR, SF, JN, NT, MT, VN, BHP. As QPS
employees, these authors had main roles in the
study design, data collection and analysis, decision
to publish, and preparation of the manuscript.
Competing interests: BS, RR, SF, JN, NT, MT, VN,
BHP are employees of QPS Austria GmbH. This
does not alter our adherence to PLOS ONE policies
on sharing data and materials.
Abbreviations: AAALAC, Association for
Assessment and Accreditation of Laboratory
Animal Care; ALS, Amyotrophic lateral sclerosis;
ANOVA, Analysis of variance; C or cyt, cytoplasmic;
CD11b, Cluster of Differentiation 11b; ChAT,
Choline acetyltransferase; CTF, C-terminal
fragment; DAPI, 4',6-Diamidin-2-phenylindol;
FTLD-U/ FTLD-TDP, frontotemporal lobar
degeneration with ubiquitin-positive inclusions;
Glycerinaldehyd-3-phosphatDehydrogenase; GFAP, Glial fibrillary acidic protein;
IR, immunoreactive; N or nuc, nuclear; ntg,
nontransgenic; SOD1, Superoxide dismutase 1; TAR6/
6, homozygous transgenic mice; TAR6,
hemizygous transgenic mice; TARDBP, transactive
response DNA-binding protein 43; TDP-43,
transactive response DNA-binding protein 43.
(FTLD-U). TDP-43 protein has been recognized to be the main component of ubiquitinated
inclusions in human ALS and FTLD samples [
] and FTLD-U was since renamed FTLD-TDP.
Physiological TDP-43 is ubiquitously expressed and mainly localized in the nucleus. The
protein is synthesized in the cytoplasm but shuttles between the nucleus and cytoplasm [3±5].
TDP-43 can bind DNA as well as RNA [
] and seems to be critical for fat metabolism and
embryonic stem cell survival . Pathological TDP-43 is highly ubiquitinated [
], and mislocated to the cytoplasm [
]. Truncated isoforms of TDP-43 have
been found in the telencephalon of ALS patients suggesting that TDP-43 is prone to be cleaved.
In addition, disease-related TDP-43 is relatively insoluble, a feature that might be caused by
]. Insolubility of TDP-43 might furthermore support aggregation of
the protein in the cytoplasm, suggesting not only a loss of function but also a gain of toxic
function (discussed in [
]). Currently, 47 missense and one truncating TARDBP gene
mutations have been reported (for review see [
]), most of them located in the glycine-rich domain
causing predominantly ALS and only rarely FTLD (summarized in [
]). The distribution
pattern of TDP-43 inclusions can be distinguished between ALS and FTLD-TDP patients. ALS
patients exhibit strong TDP-43 pathology in the motor cortex, spinal cord and basal ganglia,
while FTLD-TDP patients show TDP-43 expression throughout the central nervous system
(CNS), except for the occipital cortex and cerebellum [
]. In ALS, TDP-43 histopathology is
characterized by cytoplasmic inclusions of skein-like or compact granular shape and at the
same time a complete absence of TDP-43 in the nucleus [
Since 2009, a whole series of genetically altered TDP-43 mouse models were generated (for
review see [
]). These models express wild type or mutated TDP-43 under the control of
different promoters, such as the prion or Thy-1 promoter, constitutionally or conditionally
[15±20]. A major drawback of most of these models is that some barely show any phenotype
[16,18] and others have such a strong and early phenotype resulting in severely shortened
survival times [15±18], so that neither type of these models is suitable for treatment studies
designed to characterize new compounds against ALS/FTLD. In 2010, Wils and colleagues
generated two transgenic ALS mouse models, homozygous TAR4/4 and TAR6/6. Both
founder lines express wild type human TDP-43 (hTDP-43) under control of the neuron
specific Thy-1.2 promoter . While the TAR4/4 model presents a very strong and early
phenotype with an average survival time of 25 days, the TAR6/6 model develops a later phenotype
onset and an average survival time of 6.5 months . TAR6/6 mice were shown to have 1.2
fold increased hTDP-43 expression levels compared to murine TDP-43, an abnormal hind
limb reflex at two months, and disturbed motor behavior at approximately four months of age.
Additionally, histological analyses revealed neuronal loss and neuroinflammation in the motor
cortex as well as in the spinal cord of 6 months old TAR6/6 mice. These results already indicate
that TAR6/6 mice exhibit an ALS/FTLD like phenotype with a moderate progression rate. To
be able to use TAR6/6 mice for ALS/FTLD compound tests, we validated TDP-43 expression
in the spinal cord and further characterized this model for histopathological and biochemical
features and motor deficits for the first time at presymptomatic, symptomatic and late disease
stages. Our results verify and extend the results by Wils and colleagues and emphasize the
strength of TAR6/6 mice as a valid ALS/FTLD model and its suitability to test new compounds
against these devastating, incurable diseases.
Materials & methods
TAR6 mice and corresponding littermates were provided by `Neurodegenerative Brain
Diseases Group', Department of Molecular Genetics, VIB, Antwerp, Belgium . TAR6/6
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transgenic mice express human wild type TDP-43 under the control of the murine Thy-1.2
promoter, mThy1-hTDP-43 mice . Homozygous mice (TAR6/6) were bred by mating
hemizygous mice (TAR6). Non-transgenic (ntg) littermates served as control for all
experiments. Equal numbers of male and female TAR6/6, TAR6 and ntg mice were used and kept in
individually ventilated cages under a 12 hour light/dark cycle at the QPS Austria, AAALAC
accredited and pathogen free animal facility. Animals were provided with standard rodent
chow (Altromin™, Germany) normal tap water ad libitum and a 50 mm square piece of pressed
cotton (nestlets) as nesting material. Room temperature and humidity were kept at
approximately 24ÊC and 40±70%, respectively. Before starting the experiments animals were
grouphoused, but since mice had to be single-housed for the first behavioral test, all animals were
kept single housed from that time on to prevent fighting between male groups caused by
regrouping. Single housing is shown to influence several aspects of the mouse phenotype like
body weight, activity and anxiety but also striatal changes in dopamine and serotonin turnover
], therefore male and female mice were single housed after the nest building test to
keep housing conditions equal between sexes. Since all animals were housed equally, possible
alterations should be similar between genotypes. Mice of four cohorts were analyzed: The first
cohort was analyzed for behavioral deficits at the age of 1.5 months and afterwards analyzed by
biochemical and histological methods. The second cohort was analyzed longitudinally from
the age of 3 to 4.75 months. The third and fourth cohorts were only analyzed by biochemical
and histological methods at the age of 3 and 6 months, respectively. Actual animal numbers
are given in the figure legends. Animal studies conformed to the Austrian guidelines for the
care and use of laboratory animals and were approved by the Styrian government, Austria
(FA10A-78Jo-69-2010, ABT13-78Jo168-2015). All efforts were made to minimize suffering.
All behavioral tests were performed during the early phase of the light cycle. Animals were
allocated to the corresponding experimental group according to their genotype. The following
behavioral tests were performed in week 6 (cohort I) or repeatedly from week 12, 14±17
(cohort II) in the following order: nest building, clasping, wire suspension, Rota Rod.
Additionally, animals were tested in the elevated plus maze and fear conditioning test in week 18
(cohort II). Hemizygous mice were analyzed at the age of 1.5, 9, or 12 months.
The nest building test was performed as previously described [
To analyze animals for the hind limb reflex, mice were lifted on the base of their tail and
clasping was scored. Healthy mice spread their hind limbs but with increasing pathology mice
press their hind limbs against each other. The hind limb position was scored as follows: Score
3: mice with normal hind limb position. Score 2: mice with intermediate hind limb position.
Score 1: mice with closed hind limb position .
The wire suspension test and Rota Rod test were conducted as previously described [
The elevated plus maze test analyzes the anxiety of mice. The animal was placed in the
center of a four-arm-maze from which it can choose any of the four narrow arms. Two arms are
well lit and open. The two alternating arms are enclosed with walls and dimmed. Healthy
rodents will prefer the closed arms but will venture out into the open arms. The time, animals
spent in the different arms was measured by automated video-tracking software (Videomot,
All mice were sedated by standard inhalation anesthesia (isofluorane). Once animals were
deeply anesthetized, mice were transcardially perfused with physiological (0.9%) saline.
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Thereafter, spinal cords, including vertebrae, and brains were rapidly removed and brains
were hemisected. The spinal cords of half of the animals and the right hemispheres of all
animals were fixed by immersion at 4ÊC in freshly prepared 4% formaldehyde in phosphate buffer
(pH 7.4) for 2 or 24 hours, respectively. After cryo-conservation in a 15% sucrose/phosphate
buffered saline (PBS) solution, right hemispheres were shock frozen in dry-ice cooled liquid
isopentane. Spinal cords and the right hemispheres were than stored at -80ÊC until used for
histological analyses. The spinal cords were further dissected from vertebrae, embedded in
paraffin and stored at room temperature.
The spinal cords of half of the animals and the left hemispheres of all animals that were
used as whole or dissected into cortex, hippocampus and midbrain, were shock frozen on dry
ice and stored at -80ÊC until used for biochemical analyses.
Homogenization of frozen tissue samples
The frozen, unfixed tissue samples [brain (left hemisphere), hippocampus and spinal cord]
were weighed and homogenized for 20 seconds using the highest level of the Tissue Ruptor
(Qiagen, Germany) in 5 volumes per weight RIPA buffer (50 mM Tris-HCl, pH 7.4, 1 mM
EDTA, 150 mM NaCl, 1% NP-40 or 0.1% Triton-X, 2% SDS, 1 μM NaF, 0.2 mM sodium
deoxycholate, 80 μM glycerophosphate 1x protease inhibitor (Calbiochem, Germany), 1x
phosphatase inhibitor (Sigma, USA).
Samples were further analyzed by Western blotting using antibodies against
human-TDP43 (1:1,000, Abnova, Germany, H00023435-M01), total TDP-43 (1:5,000, Proteintech, USA
10782-2-AP), or β-tubulin isotype III (1:5,000, Sigma-Aldrich, USA, T8660). Protein levels of
tTDP-43, hTDP-43 and û-tubulin were densitometrically quantified using Image J. For
densitometric quantification, protein signals of tTDP-43, hTDP-43 and truncated TDP-43 were
analyzed by Image J.
Two mid brain and 2 cortex samples of 4 ntg, 4 TAR6 and 4 TAR6/6 mice at an age of 3 months
were fractionated into cytoplasm and nucleus using NE-PER™ Nuclear and Cytoplasmic
Extraction Reagent (Thermo Scientific, USA #78833). Fractionation was performed according
to the manual. Samples were additionally analyzed by Western blotting using antibodies against
total TDP-43 (1:5,000, Proteintech, USA 10782-2-AP), human-TDP-43 (1:1,000, Abnova,
Germany, H00023435-M01), glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, 1:10,000,
Sigma-Aldrich, USA, G9545) as marker for cytoplasmic fraction and HDAC3 (1:250, Abcam,
UK, ab7050) as marker for nuclear fraction. Protein levels of tTDP-43, hTDP-43 and truncated
TDP-43 were densitometrically quantified using Image J.
Five frozen brain samples of 3 months old ntg or TAR6/6 mice were thawed on ice and
sonicated in 5x v/w RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5%
sodium deoxycholate, and 0.1% SDS, pH 8.0) containing protease and phosphatase inhibitor
cocktails (Sigma, USA). Afterwards, samples were centrifuged at 100,000 g for 35 min at 4ÊC
and supernatants collected as RIPA soluble fractions. The pellets were washed with RIPA
buffer and sonicated in 2x (v/w) urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, and 30 mM
Tris, pH 8.5). After centrifugation at 100,000 g for 35 min at 22ÊC the supernatants were taken
as RIPA insoluble (urea soluble) fractions. Samples were further analyzed by Western blotting
using antibodies against total TDP-43 (1:5,000, Proteintech, USA 10782-2-AP),
human-TDP43 (1:1,000, Abnova, Germany, H00023435-M01), or GAPDH (1:10,000, Sigma-Aldrich, USA,
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G9545) as loading control. Protein levels of tTDP-43, hTDP-43 and truncated TDP-43 were
densitometrically quantified using Image J.
Frozen brain hemispheres and paraffin-embedded spinal cords were cut sagittally or
transversely, respectively. Unless indicated otherwise, a systematic uniform random set of five
mounted 10 μm thick brain sections from five medio-lateral levels and three mounted 5 μm
thick spinal cord sections per animal were used for immunofluorescent labeling.
TDP-43 labeling. Sagittal sections were treated for 15 min with citrate buffer at 95ÊC and
a 1 mg/ml sodium borohydride/PBS solution and subsequently blocked with 10% horse serum
for one hour. Thereafter, sections were labeled with a human-TDP-43 antibody (1: 2,000;
Proteintech, USA, 60019-2-Ig, monoclonal). Binding of the primary antibody was visualized using
a highly cross-absorbed DyLight secondary antibody (DyLight650, 1:500, Jackson
ImmunoResearch, USA, 712-495-153).
Co-labeling of human and total TDP-43. Sagittal sections were treated for 15 min with
citrate buffer at 95ÊC and a 1 mg/ml sodium borohydride/PBS solution and subsequently
blocked with M.O.M. Blocking Reagent in 0.3% Triton X-100/PBS for one hour at room
temperature. Thereafter, sections were labeled with a human-TDP-43 antibody (1:1,500; Abnova,
Taiwan, H00023435-M01, monoclonal), total TDP-43 antibody (1:400; Proteintech, USA,
10782-2-AP, polyclonal) and NeuN antibody (1:2,000, Synaptic Systems, Germany, 266 004,
polyclonal) over night at room temperature. Binding of the primary antibody was visualized
using highly cross-absorbed Alexa Fluor and DyLight secondary antibodies (Alexa Fluor555;
1:500; Abcam, USA, ab150110; DyLight650, 1:500, Abcam, USA, ab96922; Alexa Fluor 488,
1:500, Jackson ImmunoResearch, USA, 706-545-148) for 1 hour at room temperature followed
by nuclear staining with 4',6-Diamidin-2-phenylindol (DAPI, PanReac AppliChem GmbH,
Germany, A1001) for 15 minutes.
GFAP and CD11b labeling. Sagittal sections were treated with a 1 mg/ml sodium
borohydride/PBS solution followed by 1% hydrogen peroxide in methanol. To block unspecific
labeling sections were treated with 10% donkey serum for 30 min. Further, sections were incubated
with a GFAP (1:500; Dako, USA, Z0334) and CD11b antibody (1:1,000, BioRad Laboratories,
USA, MCA711) for 1 h at room temperature. Binding of primary antibodies was visualized
using highly cross-absorbed DyLight secondary antibodies (DyLight488, 1:800, Jackson
ImmunoResearch, USA, 711-485-152; DyLight649, 1:500, Abcam, USA, ab102263) and a Cy3
secondary antibody (Cy3, 1:500, Jackson ImmunoResearch, USA, 115-165-166). Cell nuclei
were labeled by 4',6-Diamidin-2-phenylindol (DAPI) counterstaining (PanReac AppliChem
GmbH, Germany, A1001).
ChAT labeling. Cervical and lumbar spinal cord sections of 3 months old animals were
treated for 15 min with citrate buffer at 95ÊC and a 1 mg/ml sodium borohydride/PBS solution
and subsequently blocked with 10% horse serum for one hour. Thereafter sections were
labeled with a ChAT antibody (1:300, Millipore, USA, AB144P). Binding of the primary
antibody was visualized using a highly cross-absorbed DyLight secondary antibody (DyLight650,
1:500, Abcam, USA, ab96938). Cell nuclei were labeled by DAPI counterstaining (PanReac
AppliChem GmbH, Germany, A1001).
Grey-scale images for quantitative analysis were recorded with an AxioCam MRm camera
mounted on a Zeiss Axio Imager.Z1 epifluorescence microscope at 10x magnification.
Exposure time and additional setting details were kept constant within the same quantification.
ChAT+ cells were counted manually (overlapping of ChAT and DAPI staining). For evaluation
of TDP-43, GFAP and CD11b expression, regions of interest (ROI) were defined by individual
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delineation of the whole isocortex, spinal cord, hypothalamus and medulla oblongata. These
brain regions were chosen after manual evaluation of TDP-43 expression on sagittal sections.
Immunofluorescence signals were measured by adequate thresholding and morphological
filtering (size, shape) throughout the ROIs of all images using ImageProPlus Software (Version
6.2). Individual means were determined from three to five sections per animal. Group means
were calculated by averaging the individual means. Analyzed parameter were immunoreactive
area (IR) in percent of the ROI area and summarized object intensity in a.u./mm .
Data analysis was performed in GraphPad Prism™ 4.03 (GraphPad Software Inc., USA).
Graphs show group means and standard error of the mean (SEM). The significance level was
set at p < 0.05. Group means were compared using Student's t-test with Welch's correction or
a Two-way analysis of variance (ANOVA) with a subsequent post-hoc test. The utilized
statistical tests and exact sample numbers are mentioned in the figure legends.
hTDP-43 protein is highly expressed in the CNS of TAR6/6 mice
For quantification of total, truncated and human TDP-43 protein levels, Western blot analyses
of total brain, hippocampus and spinal cord tissue obtained from 1.5, 3 and 6 months old
TAR6/6 and non-transgenic (ntg) littermates were performed (Fig 1A, 1E and 1I).
Total TDP-43 levels (tTDP-43) were analyzed in ntg and TAR6/6 mice (Fig 1B, 1F and 1J).
tTDP-43 was approximately 1.5-to 2-fold overexpressed in brain and spinal cord samples and
2- to 3-fold increased in hippocampus homogenates of TAR6/6 mice. While tTDP-43 levels
did not significantly alter over age in the brain and hippocampus, levels were reduced in ntg
spinal cord samples at an age of 6 months. Furthermore, the 35 kDa C-terminally truncated
fragment (CTF-35) of TDP-43 was found in TAR6/6 samples and 20-fold increased in the
hippocampus, 10-fold in the spinal cord and 5-fold in whole brain samples (Fig 1C, 1G and 1K).
In addition to tTDP-43 and CTF-35, densitometric analyses of immunoreactive signals
revealed high hTDP-43 levels in the central nervous system already at the age of 1.5 months in
TAR6/6 mice (Fig 1D, 1H and 1L). hTDP-43 levels stayed unchanged over age in the whole
brain and hippocampus, while levels progressively decreased in the spinal cord of TAR6/6
mice (Fig 1L). As an exemplary analysis, hTDP-43 was also investigated in hippocampus of 1.5
and 3 months old hemizygous TAR6 mice (Fig 1H). An overexpression of hTDP-43 could be
detected in hemizygous TAR6 but it was not as strong as in homozygous TAR6/6 mice.
Interestingly, even after 2 hours of exposure, the 35 kDa CTF could not be detected by the hTDP-43
antibody (Fig 1A, 1E and 1I).
Furthermore, histological analysis of hTDP-43 expression levels in the hypothalamus,
medulla oblongata and spinal cord grey matter, including anterior and posterior horn areas,
revealed a high expression of hTDP-43 protein in TAR6, TAR6/6 and ntg mice already at the
age of 1.5 months (Fig 2A±2E). We observed even higher expression of hTDP-43 protein in 3
months old TAR6 and TAR6/6 mice compared to 1.5 months old animals. Further aging of
TAR6 and TAR6/6 animals caused a significant hTDP-43 decrease in both genotypes
compared to 3 months old animal. In general, TAR6/6 animals expressed significantly more
hTDP-43 than TAR6 as shown by an increased immunoreactive area (IR). Analysis of the
sum object intensity resulted in very similar results exempt in the hypothalamus of 6 months
old TAR6 and TAR6/6 mice, were the signal was strongly reduced compared to younger mice
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Fig 1. Densitometric analysis of tTDP-43, CTF-35 and hTDP-43 expression in ntg, TAR6 and TAR6/6 mice. (A) Brain homogenates, (E) hippocampal
and (I) spinal cord homogenates from ntg, TAR6 and TAR6/6 mice at the age of 1.5, 3 and 6 months were analyzed by Western blotting and probed with
the indicated antibodies (long exp. = long time ECL exposure for 30 min). One representative example of 3 is shown. Densitometric analysis of tTDP-43,
CTF-35 and hTDP-43 levels normalized to β-tubulin levels of brain (B- D), hippocampal (F-H) and spinal cord (J-L) homogenates. Two-way ANOVA
followed by Bonferroni`s post-hoc test. Mean+SEM. significances between genotypes, #significances between age groups. p<0.05, p<0.01, p<0.001.
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Fig 2. Quantification of hTDP-43 expression in ntg, TAR6 and TAR6/6 mice. Quantitative hTDP-43 expression in hypothalamus (A), medulla
oblongata (B) and spinal cord (C) analyzed by immunofluorescent labeling of CNS samples. (D, E) Representative overview image and
magnifications of hTDP-43 labeling of a brain section (olfactory bulb (1), cortex (2), hippocampus (3), thalamus (4), hypothalamus (5) and
medulla oblongata (6)) of 3 months old TAR6/6 (D) and ntg (E) mice. Scale bars: Overview images = 1000 μm; magnification images = 20 μm. (A,
B) 1.5 months: ntg: n = 4; TAR6: n = 10; TAR6/6: n = 5; 3 months: ntg: n = 5; TAR6: n = 8; TAR6/6: n = 5; 6 months: ntg: n = 3; TAR6: n = 3;
TAR6/6: n = 3. (C) n as in A, B exempt: 1.5 months: ntg: n = 3; TAR6: n = 11. (A-C) Two-way ANOVA followed by Bonferroni`s post-hoc test.
Mean+SEM. significances between genotypes, #significances between age groups. p<0.05, p<0.01, p<0.001.
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Cytoplasmic localization of hTDP-43 is increased in TAR6/6 mice
To investigate the cellular localization of TDP-43 protein, midbrain and cortex tissue of 3
months old ntg, TAR6 and TAR6/6 mice was separated into cytoplasmic and nuclear fractions
and analyzed by Western blot (Fig 3A±3D). tTDP-43 was predominantly localized in the
nucleus in all three genotypes (Fig 3A) and significantly enriched in TAR6 and TAR6/6
samples (Fig 3B). Moreover, after long-time ECL exposure of the tTDP-43 blot, CTFs could not
only be detected in the nuclear fraction of TAR6 and TAR6/6 mice but was also significantly
increased in the cytosolic fraction of TAR6/6 mice (Fig 3A (long exp.), C). In addition,
hTDP43 staining could confirm that overexpressed TDP-43 accumulates in the cytoplasm of TAR6/
6 mice and that nuclear CTFs are produced by hTDP-43 (Fig 3A and 3D).
Insoluble TDP-43 accumulates in TAR6/6 mice
Furthermore, it was analyzed whether overexpression of hTDP-43 enhances insolubility of
TDP-43. For this purpose, brain samples of 3 months old TAR6/6 and ntg mice were
separated into RIPA soluble and RIPA insoluble (UREA) fractions and analyzed by Western blot
using antibodies against tTDP-43 and hTDP-43 (Fig 3E±3H). In the RIPA soluble fraction, a
signal for total full-length TDP-43 in TAR6/6 and ntg samples (Fig 3F) and after long-time
ECL exposure a specific 35 kDa CTF in the TAR6/6 samples could be detected (Fig 3G).
Additionally, a signal for human full length TDP-43 could be detected in TAR6/6 but not in
ntg mice (Fig 3H). Furthermore, TAR6/6 mice showed high levels of tTDP-43 (Fig 3F) and
hTDP-43 (Fig 3H) in the insoluble UREA fraction. Interestingly, no differences in CTF levels
between TAR6/6 and ntg in the UREA fraction by using antibodies against tTDP-43 (Fig
3G) and hTDP-43 (data not shown) could be detected. This result suggests that fragment
bands of the UREA fraction shown by tTDP-43 labeling are non-specific and do not depend
on the genotype.
Co-localization of human and total TDP-43 by immunofluorescent labeling
In order to visualize the subcellular localization of human and total TDP-43 protein as
observed by cell fractionation and Western blotting, co-labeling of both proteins by
immunofluorescence was performed (Fig 4) in 3 months old ntg (Fig 4A) and TAR6/6 (Fig 4B) mice.
High magnification images of the cortex and hippocampus show high immunofluorescence of
human and total TDP-43 in the nucleus and only minor immunofluorescence in the cytoplasm
of TAR6/6 mice, consistent with the biochemical analysis. In the cortex, more cells were
positive for total TDP-43 than for human TDP-43. Human TDP-43 immunoreactivity was absent
from ntg brain tissue (Fig 4A). The histological results generally support the biochemical
findings on expression and subcellular localization of TDP-43 protein.
Neuroinflammation in TAR6/6 mice
Neuroinflammation has been described as one of the major hallmarks of neurodegenerative
diseases (for review see [
]) and also linked to ALS/FTLD [
]. For this reason, IR area of
glial fibrillary acidic protein (GFAP) as marker for reactive astrocytes was quantified over age.
Significantly increased levels in TAR6/6 and TAR6 mice compared to ntg animals could be
found in the cortex (Fig 5A), medulla oblongata (Fig 5B) and spinal cord (Fig 5C) at the age of
1.5 months as well as in the cortex of TAR6/6 mice at an age of 3 months (Fig 5A). Analysis of
GFAP at the age of 6 months revealed no significant differences between genotypes. TAR6
mice followed a similar, but weaker expression pattern as TAR6/6 (Fig 5A, 5B and 5C).
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Fig 3. Posttranslational changes in TAR6/6 mice. (A-D) Localization of tTDP-43, CTF-35 and hTDP-43 after
cellular fractionation of 3 months old TAR6/6 mice. (A) Cell fractions of midbrain and cortex samples of 3 months old
ntg, TAR6 and TAR6/6 mice were analyzed by Western blotting and probed with the indicated antibodies (GAPDH as
cytoplasmic and HDAC3 as nuclear marker; long exp. = long time ECL exposure for 30 min). One representative
example of 4 is shown (C: cytoplasm, N: nucleus). (B- D) Densitometric analysis of tTDP-43, CTF-35 and hTDP-43
levels in cell fractions (cyt: cytoplasm, nuc: nucleus). Nuclear tTDP-43 and CTF-35 levels of ntg and hTDP-43 of
TAR6/6 were set as 100%. (E-H) Soluble and insoluble TDP-43 protein levels in brains of 3 months old TAR6/6 mice.
Brain samples were separated into RIPA soluble (R) and RIPA insoluble / UREA fractions (U). Three representative
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examples of 5 are shown. Fractions were analyzed by Western blotting and probed with the indicated antibodies (long
exp. = long time ECL exposure for 30 min). Coomassie Blue staining was performed as loading control for UREA
fraction. (F-H) Densitometric analysis of tTDP-43, CTFs and hTDP-43 levels in RIPA and UREA fractions. For
tTDP43 and CTF evaluation, RIPA fraction of ntg was set as 100%, whereas for hTDP-43 analysis, RIPA sample of TAR6/6
was set as 100%. (B-D; F-H) Two-way ANOVA followed by Bonferroni`s post-hoc test. Mean+SEM. significances
between genotypes, #significances between fractions. p<0.05, p<0.01, p<0.001.
Fig 4. Immunofluorescent double-labeling of human and total TDP-43. Cortical and hippocampal tissue of three-months old ntg (A) and TAR6/6 (B) mice was
labeled with antibodies against human TDP-43 (green) and total TDP-43 (red). Tissue was additionally labeled with NeuN antibody (white) and DAPI (blue) to
visualize neuronal somata and nuclei, respectively. The merged images in the right column show human TDP-43, total TDP-43 and DAPI labeling. (C) The
preferred nuclear localization of both human TDP-43 and total TDP-43 is obvious when the outlines of NeuN-positive somata are projected on the TDP-43
channels. Scale bars: Overview images = 500 μm; magnification images = 20 μm; (C) = 20 μm.
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Labeling of cortical tissue with cluster of differentiation 11b (CD11b) as marker for activated
microglia revealed a slight increase at 3 months of age and a severe microglia activation in
6 months old TAR6/6 mice compared to ntg and TAR6 animals (Fig 5D). In the medulla
oblongata the same trend was detected (Fig 5E). Representative images of labelings are shown
in S2 Fig.
Neuronal loss in the spinal cord of TAR6/6 mice
Since ALS pathology is accompanied by loss of motoneurons in the spinal cord, the number of
choline acetyltransferase (ChAT) positive neurons was counted in cervical and lumbar spinal
cord regions of 3 months old ntg and TAR6/6 mice (Fig 6A±6D). In both regions ChAT
positive neurons were significantly decreased in the ventral horn indicating degeneration of
motoneurons in TAR6/6 mice.
TAR6/6 mice develop severe motor deficits
TAR6/6 mice were tested for hind limb reflexes at the age of 1.5 and 3 months. Our results
show that already at the age of 1.5 months TAR6/6 mice reached a significantly higher clasping
score compared to ntg animals (Fig 7A). This effect was also prominent at the age of 3 months.
Further analyses of TAR6/6 mice for motor abilities show that 1.5 months old TAR6/6 mice
presented severe neuromuscular deficits as revealed by the significantly decreased hanging
time in the wire suspension test (Fig 7B). Analysis of 3 months old animals in this test
confirmed the results obtained with young animals. Thus, TAR6/6 mice exhibit highly decreased
muscle strength. Additional analyses of TAR6/6 mice in the Rota Rod test over age showed a
strongly decreased latency to fall from the rod starting at the age of 3.5 months (Fig 7C). Even
though mice showed an early muscle weakness in the wire suspension test, the first deficits
in the Rota Rod were only detectable at the age of 3.5 months (Fig 7B vs. 7C). Next to these
explicit motor assays, TAR6/6 mice were analyzed in the nest building test. TAR6/6 mice
reached a significantly lower nesting score at the age of 3 months compared to ntg littermates
TAR6/6 mice exhibit reduced anxiety
To test TAR6/6 mice for anxiety, mice were assessed in the elevated plus maze test. 1.5 and 4.5
months old TAR6/6 mice spent significantly more time in the open arms of the elevated plus
maze compared to ntg littermates (Fig 7E) indicating reduced anxiety levels of TAR6/6 mice.
Decreased body weight of TAR6/6
To validate that motor deficits are not caused by obesity of TAR6/6 mice, the first cohort of
animals was weight once at 1.5 months of age and a second cohort from the age of 3.25 to 4.25
months. Analysis by Two-way ANOVA revealed a comparable body weight at young age but a
significantly decreased body weight of TAR6/6 mice at the age of 3.25 to 3.75 months. With
increasing age, this difference attenuates to comparable levels (Fig 7F).
Analysis of up to 12 months old hemizygous TAR6 mice in a similar test battery did not
reveal significant behavioral abnormalities compared to ntg littermates (S3 Fig).
In summary, we observed a strong hTDP-43 overexpression followed by ALS/FTLD
pathologies, such as enriched cytoplasmic and insoluble TDP-43, neuroinflammation as revealed by
increased astrogliosis and microgliosis, loss of motor neurons in the spinal cord as well as
pronounced motor abnormalities in homozygous TAR6/6 mice (Table 1).
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Fig 5. Quantification of GFAP and CD11b expression in the central nervous system of ntg, TAR6 and TAR6/6 mice. (A-C) GFAP expression
levels labeling reactive astrocytes in the cortex (A), medulla oblongata (B) and spinal cord (C); and CD11b expression levels labeling reactive
microglia in the cortex (D) and medulla oblongata (E) as IR area in percent. (A) 1.5 months: ntg: n = 4; TAR6: n = 9; TAR6/6: n = 5; 3 months: ntg:
n = 4; TAR6: n = 9; TAR6/6: n = 5; 6 months: ntg: n = 3; TAR6: n = 3; TAR6/6: n = 3. (D) n as in A except: 1.5 months: TAR6: n = 10; 3 months: ntg:
n = 5; TAR6: n = 8. (B, E) n as in D except: 3 months: TAR6/6: n = 7. C) n as in B except: 1.5 months: TAR6: n = 5; TAR6/6: n = 3; 3 months: ntg:
n = 3; TAR6: n = 7; TAR6/6: n = 4. (A-E) Two-way ANOVA followed by Bonferroni`s post-hoc test. Mean+SEM. significances between genotypes.
p<0.05, p<0.01, p<0.001.
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Fig 6. Quantification of neuron loss in the spinal cord of 3 months old TAR6/6 mice. (A, C) Number of ChAT+ neurons in the ventral horn of the cervical (A)
and lumbar (C) spinal cord grey matter. (B, D) Representative images of ChAT labeling in the cervical (B) and lumbar (D) spinal cord of 3 months old TAR6/6
mice compared to ntg animals. Nuclei of cells were stained by DAPI. (A) ntg: n = 4, TAR6/6: n = 3. (C) ntg: n = 3, TAR6/6: n = 3. Unpaired Student's t-test
followed by Welch's correction. Mean+SEM. p<0.05.
To represent a suitable new transgenic animal model for compound testing it is essential that
the model ensures a high construct and face validity. Since TDP-43 is well known to be a
major factor in the development of human ALS and the here presented TAR6/6 mouse model
overexpresses human wild type TDP-43 protein , the construct validity is well given. With
the present data, the face validity of the TAR6/6 model was analyzed in detail.
Biochemical analysis of hTDP-43 levels showed a strong expression in the total brain,
hippocampus and spinal cord of TAR6/6 mice and a slightly weaker expression in TAR6 mice.
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Fig 7. Motor impairments and reduced anxiety in TAR6/6 mice. (A) Clasping behavior scored from 1±3 of 1.5 and 3 months old TAR6/6 mice
compared to ntg littermates (score of 1: normal clasping). (B) Latency to fall in the wire suspension test in seconds of 1.5 and 3 months old TAR6/6
mice compared to ntg littermates. (C) Latency to fall from the rotating rod in the Rota Rod test of 1.5 to 4.25 months old TAR6/6 mice compared to
ntg littermates. (D) Nesting behavior scored from 1±5 of 3 months old TAR6/6 mice compared to ntg littermates (high score: perfect nest). (E) Time
in seconds of 1.5 and 4.5 months old TAR6/6 mice spent in the open arms of the elevated plus maze compared to ntg littermates. F: Body weight in
gram of 1.5 to 4.25 months old TAR6/6 mice compared to ntg mice of the same age. (A, B) 1.5 months: TAR6/6: n = 5, ntg: n = 7; 3 months: TAR6/6:
n = 13, ntg: n = 16. (C) 1.5 months: TAR6/6: n = 5, ntg: n = 7; 3.5 months: TAR6/6: n = 8, ntg: n = 7; 3.75 months: TAR6/6: n = 12, ntg: n = 16; 4
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months: TAR6/6: n = 10, ntg: n = 16; 4.25 months: TAR6/6: n = 9, ntg: n = 16. (D) TAR6/6: n = 16, ntg: n = 12. E: 1.5 months: TAR6/6: n = 6, ntg:
n = 4; 4.5 months: TAR6/6: n = 8, ntg: n = 16. (F) 1.5 months: TAR6/6: n = 5, ntg: n = 7; 3.25 months: TAR6/6: n = 7, ntg: n = 12; 3.5 months: TAR6/
6: n = 12, ntg: n = 18; 3.75 months: TAR6/6: n = 10, ntg: n = 18; 4.0 months: TAR6/6: n = 8, ntg: n = 18; 4.25 months: TAR6/6: n = 8, ntg: n = 18.
(A-C, E, F). Two-way ANOVA followed by Bonferroni`s post-hoc test. D: Mann Whitney U-test. A,B,E: Mean+SEM; C,D,F: Mean±SEM. p<0.05,
The expression pattern of hTDP-43 is comparable with evaluated tTDP-43 levels which are 1.5
to 2.5 fold increased depending on the tissue. The overexpression might seem to be rather
weak but Ayala and colleagues previously showed that endogenous TDP-43 levels can be
downregulated by binding of overexpressed TDP-43 in the 3'UTR region of the endogenous
]. We hypothesize that endogenous TDP-43 is widely depleted and the detected
tTDP-43 levels mainly consist of hTDP-43. In addition to increased tTDP-43 levels, the 35
kDa C-terminally truncated fragment (CTF-35) of TDP-43 that is clearly detectable in human
FTLD samples [
], could be found in TAR6/6 mice. This result is consistent with a previous
analysis of FTLD and ALS cases and therefore presenting the biochemical similarity of TAR6/
6 mice and human TDP-43 related diseases [
]. On the other hand, the 25 kDa fragment,
which has also been associated with FTLD [
], could not be detected in transgenic animals
assuming that the model mimics rather ALS than FTLD pathology where fragments are less
Overall histological analyses showed similar expression levels of TDP-43 in different brain
regions. We observed an increase of hTDP-43 in the hypothalamus, medulla oblongata and
spinal cord up to an age of 3 months, followed by a slight decrease at the age of 6 months
which was observed by Western blot in the spinal cord but not in brain and hippocampal
tissue. The decrease in TDP-43 expression at higher age might be caused by a loss of hTDP-43
expressing neurons. The reduced ChAT labeling in the spinal cord of TAR6/6 samples thus
indicates an increased motoneuron death which is accompanied by neuronal loss of motor
cortex layer V and anterior horn neurons detected by analyzing Cresyl violet staining as
described by Wils et al. . Furthermore, CD11b labeling of activated microglia is slightly
Results of cellular pathology, neuroinflammation, neuron loss as well as motor and anxiety tests in TAR6/6 mice compared to ntg mice at an age of 1.5, 3±4.5 and 6
months. N/A: not available. N.S.: not significant. ": increase, #: decrease.
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increased in the cortex at an age of 3 months and highly increased in 6 months old TAR6/6
mice supporting the assumption that neurons start to degenerate at 3 months. On the contrary,
GFAP labeling of activated astrocytes was strongly increased at an age of 1.5 months in the
cortex, medulla oblongata and spinal cord and decreased over age in TAR6/6 mice while only
slight activation of astroglia and microglia was observed in the cortex of 1.5 and 3 months old
TAR6 mice, respectively. In 2010, Wils et al. analyzed inflammation processes in the TAR6/6
mice by GFAP and Iba-1 staining. The group also revealed activation of microglia in 6 months
old animals but additionally activation of astrocytes by GFAP at the age of 6 months in the
motor cortex layer V and anterior horn of the spinal cord . The observed astrogliosis and
microgliosis in different brain regions of TAR6/6 mice provides evidence for a similar
neuroinflammatory pathology compared to another TDP-43 transgenic mouse [
], the SOD1
mouse model [
], human ALS cases [
] or human FTLD with mutated progranulin [
Neuroinflammation is thus a mutual neuropathological feature of ALS/FTLD occurring in the
most common types of genetically altered ALS models.
Although expression levels of hTDP-43 and inflammation processes in hemizygous TAR6
and homozygous TAR6/6 mice differ, our biochemical and histological data consistently
indicate that TDP-43 is strongly enriched in the nucleus whereas immunoreactivity is much lower
in the cytoplasm. Of note, we did not detect a shift of subcellular hTDP-43 from the nucleus to
the cytoplasm. On the contrary, the genotypes display significantly increased nuclear hTDP-43
although in human ALS/FTLD samples the absence of nuclear TDP-43 has been described and
correlated to crucial pathological features of ALS/FTLD [
]. Furthermore, 35 kDa CTFs can
be found in the nuclear fractions by staining for tTDP-43 and hTDP-43 and in the cytoplasmic
fraction by staining for tTDP-43. The 35 kDa CTF is known to be cleaved by caspase-3 [
cleavage site DETD89 [
] which has been shown to be activated in TAR6/6 mice by Wils
et al. . The fragment is thought to be involved in the formation of cytoplasmic inclusion
bodies as well as pulling the functional full-length TDP-43 out of the nucleus, which is then
additionally integrated in the cytosolic inclusion bodies . One interesting difference
between TAR6 and TAR6/6 mice is the presence of the 35 kDa fragment only in the cytoplasm
of TAR6/6 mice. This cytoplasmic CTF might cause the described pathogenic effects of
fulllength TDP-43 and contribute to the development of phenotypic symptoms of TAR6/6 mice.
In another experiment, the influence of overexpressed hTDP-43 on the solubility of
TDP43 was investigated. In 3 months old TAR6/6 mice, tTDP-43 as well as hTDP-43 was highly
enriched in the RIPA insoluble (UREA) fraction. Thus, insoluble TDP-43 was accumulated in
brains of TAR6/6 animals and mimics one of the well described hallmarks of human ALS/
FTLD pathology [
]. The 35 kDa CTF has been described to induce aggregation because of its
own insolubility [
] and is supposed to be concentrated in the insoluble UREA sample.
However, no differences in the pattern of the CTF bands between ntg and TAR6/6 mice in the
UREA fraction could be observed in the here presented study. In contrast, the 35 kDa CTF
was significantly increased in the soluble RIPA fraction. This unusual fractionation might
result in a pathology start caused by high hTDP-43 levels in the nucleus as well as the
cytoplasm at 3 months of age. As a result of toxic concentrations, full-length TDP-43 would be
processed and cleaved into CTFs which might still be soluble but already induce insolubility of
full-length TDP-43 and would become insoluble at a later age. To clarify this hypothesis,
experiments with older animals are needed in future.
To demonstrate a similarity of behavioral deficits between TAR6/6 mice and ALS patients,
several specific tests were performed. Mice not only presented altered reflexes and muscle
weakness but also strong deficits in motor coordination as shown with the Rota Rod test.
These results are comparable with muscle weakness, gait disturbance, coordination problems
as well as weakness in arm and leg muscles described in ALS patients (reviewed in [
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Besides these typical motor disturbances TAR6/6 mice exhibited decreased anxiety. A similar
phenotype could also be observed in a conditional TDP-43 transgenic mouse model [
for many published TDP-43 transgenic mouse models, the anxiety status is not described yet
]. It is thus not possible to evaluate, if this phenotype is typical in such models
although increased anxiety levels are common in ALS patients [
]. Nest building in
rodents is a multifaceted behavior conducted for thermoregulation and protection of offspring
and requires fine motor skills . Reduced nest building behavior can thus be indicative
for a general reduction of well-being. When observed in combination with motor deficits as
observed in TAR6/6 mice, deficits of fine motor skills of the fore paws and orofacial motor
abilities are most likely. Additionally, we could already show that TAR6/6 mice display severe
gnawing deficits as analyzed with the pasta gnawing test [
]. These data can be related to
dysphagia and other bulbar deficits that are well known in ALS/FTLD patients [45±47] and
support the results of the nest building test. The analysis of learning and memory deficits in
TAR6/6 mice would also be of great value, since many patients show such symptoms that even
correlate with hippocampal volume [
] and a shorter survival [
]. Many learning tests for
mice such as the Morris water maze or two choice swim test require good motor performance
since animals often have to navigate or even swim. Due to strong motor deficits of TAR6/6
mice the analysis of learning would require the use of a learning test that is not solely
dependent on motor performance, e.g. the poke hole test, radial arm maze or Barnes maze.
Furthermore, ALS patients often show increased aggression [
] or impairments to set shifting
behavior from one response to another [
]. It would therefore also add value to this ALS
model to analyze TAR6/6 mice in corresponding behavioral tests.
TAR4/4 mice, which were in detail characterized by Wils and colleagues, have an average
survival of only 24 days while TAR6/6 mice live on average 6±7 months . This extended
life span strongly improves the usability of TAR6/6 mice for the analysis of new compounds
against ALS/FTLD symptoms and pathologies, since these mice present a reasonable time
window for a compound to be effective. A shorter life span could mask a drug effect while a longer
life span would not properly mimic the severe progression of human ALS symptoms.
Additionally, some of the here and previously described behavioral deficits of TAR6/6 mice exhibit
a delayed onset, such as the body weight, Rota Rod and pasta gnawing test [
], allowing a
treatment start before onset of these symptoms and thus for the analysis of preventive
In summary, our study confirms results obtained by Wils et al., such as expression of
hTDP-43 in the CNS, increased microgliosis and motor deficits evaluated by the Rota Rod
and clasping behavior . In addition, we show that TAR6/6 mice exhibit further prominent
ALS/FTLD pathologies, such as cytoplasmic and insoluble TDP-43, CTFs of TDP-43 and loss
of ChAT+ neurons in the spinal cord (Table 1). TAR6/6 mice thus represent not only
pathological TDP-43 expression but also disease-relevant posttranslational changes, substantiating the
face validity of this animal model. Combining the results of already published data and the
here presented results it can be assumed that TAR6/6 mice represent a valid ALS/FTLD
model, well-suitable for preclinical compound tests.
In summary, our results show that TAR6/6 mice are a valid ALS/FTLD model to mimic
prominent pathologies of these devastating neurodegenerative diseases. TAR6/6 mice are therefore
an adequate model to test new compounds against ALS/FTLD that target TDP-43
overexpression, fragmentation, insolubility, neuroinflammation, motoneuron loss or other TDP-43
downstream signaling pathways.
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S1 Fig. (A-C) Quantification of human TDP-43 expression in 1.5, 3 and 6 months old ntg,
TAR6 and TAR6/6 mice. Sum object intensity of hTDP-43 expression in hypothalamus (A),
medulla oblongata (B) and spinal cord (C) analyzed by immunofluorescent labeling of CNS
samples. (A, B) 1.5 months: ntg: n = 4; TAR6: n = 10; TAR6/6: n = 5; 3 months: ntg: n = 5;
TAR6: n = 8; TAR6/6: n = 5; 6 months: ntg: n = 3; TAR6: n = 3; TAR6/6: n = 3. (C) n as in A, B
exempt: 1.5 months: ntg: n = 3; TAR6: n = 11. (A-C) Two-way ANOVA followed by
Bonferroni`s post-hoc test. Mean+SEM. significances between genotypes, #significances between age
groups. p<0.05, p<0.01, p<0.001.
S2 Fig. GFAP and CD11b labelling in ntg and TAR6/6 mice. (A) Cortical, (B) medulla
oblongata, (C) spinal cord GFAP expression levels labelling reactive astrocytes and (D) CD11b
expression levels labeling reactive microglia. Representative images of 1.5 months old (A, B, C)
and 6 months old (D) animals. Cell nuclei are stained by DAPI.
S3 Fig. Motor performance and anxiety levels in hemizygous TAR6 mice compared to
non-transgenic littermates. (A) Clasping behavior scored from 1±3 of 1.5 months old TAR6
mice (score of 1: normal clasping). (B) Latency to fall in the wire suspension test in seconds of
1.5, 9 and 12 months old TAR6 mice. (C) Latency to fall from the rotating rod in the Rota Rod
test of 1.5 months old TAR6 mice. (D) Nesting behavior score of 1.5 months old TAR6 and
TAR6/6 mice (high score: perfect nest). (E) Time in seconds 1.5 months old TAR6 mice spent
in the open arms of the elevated plus maze. (F) Body weight in gram of 1.5, 9 and 12 months
old TAR6 mice. (A, C) TAR6: n = 11, ntg: n = 7; (E) TAR6: n = 11, ntg: n = 6. (D) TAR6: n = 9,
ntg: n = 4, TAR6/6: n = 4. (B, F) 1.5 months: TAR6: n = 11, ntg: n = 7; 9 months: TAR6: n = 15,
ntg: n = 12; 12 months: TAR6: n = 15, ntg: n = 13; Mean+SEM. A: One sample t-test; C-E:
unpaired t-test. B,F: Two-way ANOVA; A-F: non significant (p>0.05).
S1 Table. Raw data of all quantifications presented in the manuscript.
Conceptualization: Barbara Scherz, Roland Rabl, Stefanie Flunkert, Nicole Taub, Robert
Zimmermann, Birgit Hutter-Paier.
Data curation: Barbara Scherz, Roland Rabl, Joerg Neddens, Vera Niederkofler.
Formal analysis: Barbara Scherz, Roland Rabl, Siegfried Rohler, Joerg Neddens, Magdalena
Funding acquisition: Barbara Scherz, Stefanie Flunkert, Birgit Hutter-Paier.
Investigation: Roland Rabl, Siegfried Rohler, Nicole Taub, Vera Niederkofler.
Methodology: Barbara Scherz, Roland Rabl, Joerg Neddens, Nicole Taub, Magdalena
Project administration: Stefanie Flunkert, Birgit Hutter-Paier.
Resources: Birgit Hutter-Paier.
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Supervision: Stefanie Flunkert, Nicole Taub, Ute Panzenboeck, Vera Niederkofler, Robert
Zimmermann, Birgit Hutter-Paier.
Validation: Stefanie Flunkert, Magdalena Temmel.
Visualization: Barbara Scherz, Stefanie Flunkert, Joerg Neddens.
Writing ± original draft: Barbara Scherz, Stefanie Flunkert.
Writing ± review & editing: Barbara Scherz, Stefanie Flunkert, Siegfried Rohler, Joerg
Neddens, Nicole Taub, Magdalena Temmel, Ute Panzenboeck, Vera Niederkofler, Robert
Zimmermann, Birgit Hutter-Paier.
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