Targeted Depletion of TDP-43 Expression in the Spinal Cord
Motor Neurons Leads to the Development of Amyotrophic
Lateral Sclerosis-like Phenotypes in Mice*□
Lien-Szu Wu‡§, Wei-Cheng Cheng‡, and C.-K. James Shen‡§1
their diseased spinal cord motor neurons.
Results: Mice with targeted depletion of TDP-43 expression in the spinal cord motor neurons developed a range of ALS-like
Conclusion: TDP-43 is essential for the survival and functioning of mammalian spinal cord motor neurons.
Significance: Loss of TDP-43 function could be one major cause for neurodegeneration in ALS with TDP-43 proteinopathies.
ALS, or amyotrophic lateral sclerosis, is a progressive and
fatal motor neuron disease with no effective medicine. Impor-
tantly, the majority of the ALS cases are with TDP-43 pro-
teinopathies characterized with TDP-43-positive, ubiquitin-
mismetabolism of TDP-43 in the pathogenesis of ALS with
TDP-43 proteinopathies is unclear. Using the conditional
mouse gene targeting approach, we show that mice with inacti-
vation of the Tardbp gene in the spinal cord motor neurons
(HB9:Cre-Tardbplx/?) exhibit progressive and male-dominant
development of ALS-related phenotypes including kyphosis,
motor dysfunctions, muscle weakness/atrophy, motor neuron
loss, and astrocytosis in the spinal cord. Significantly, ubiquiti-
nated proteins accumulate in the TDP-43-depleted motor neu-
rons of the spinal cords of HB9:Cre–Tardbplx/?mice with the
ALS phenotypes. This study not only establishes an important
role of TDP-43 in the long term survival and functioning of the
loss of TDP-43 function could be one major cause for neurode-
generation in ALS with TDP-43 proteinopathies.
Amyotrophic lateral sclerosis (ALS)2(also known as Lou
Gehrig’s disease and motor neuron disease) is a progressive,
primary motor cortex, corticospinal tracts, brainstem, and spi-
nal cord, leading to paralysis of voluntary muscles (1–3). Cur-
prevalence is 4–6 per 100,000 of the total population, with a
ALS are sporadic but ?10% of patients have a familial history
progressive manifestations of dysfunction of the lower motor
toms (3). Age and gender are documented sALS risk factors (5)
with a male-to-female ratio of 3:2 among patients. Among the
oxide dismutase (SOD1) gene have long been thought to cause
the antioxidant function of the SOD1 enzyme (6). Other
genes with mutations associated with the fALS include alsin
(ALS2), senataxin (ALS4), vesicle-associated membrane
protein (VAPB, ALS8), Angiogenin, and the p150 subunit of
dynactin (DCTN1) (3). More than 30 mutations in the TDP-
43-coding region of Tardbp have also been identified in ALS
patients with or without apparent family history, correspond-
ing to ?4% of fALS and less than 1% of sALS (7). Most patients
with the TDP-43 mutation(s) develop a classical ALS pheno-
type without cognitive deficit suggesting an important role of
TDP-43 in the development of ALS (7–9).
tously expressed nuclear protein encoded by one of the mRNA
isoforms from the highly conserved Tardbp gene (11). It is a
RNA-binding protein involved in transcriptional repression,
pre-mRNA splicing, and translation (12, 13). TDP-43 has also
been identified as the major pathological signature protein of
the intracellular inclusions typical for disease cells of a range of
neurodegenerative diseases, including the frontotemporal
lobar degeneration with ubiquitin-positive, Tau- and ?-sy-
nuclein-negative inclusions (FTLD-U) and ALS (13–17).
TDP-43 molecules in the diseased cells of the patient brains or
spinal cords are characterized by abnormal ubiquitination,
hyperphosphorylation, and partial cleavage to generate ?25-
kDa and 35-kDa C-terminal fragment(s). Furthermore,
TDP-43 is partially or completely cleared from the nuclei of
either neuronal or glial cells containing the TDP-43(?) and
ubiquitin(?) aggregates/inclusions, or UBIs, in the cytoplasm
Science Council and the Academia Sinica (AS), Taipei Taiwan.
SThis article contains supplemental Figs. S1–S4.
1An Academia Sinica Investigator Awardee. To whom correspondence
kang, Taipei, Taiwan 115, Republic of China. Tel.: 886-2-27824188; Fax:
886-2-26518055; E-mail: email@example.com.
2The abbreviations used are: ALS, amyotrophic lateral sclerosis; ChAT, cho-
line acetyltransferase; GFAP, glial fibrillary acidic protein.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 33, pp. 27335–27344, August 10, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
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Several mouse models have been established for ALS disease
including the strains of rodents that are transgenic with SOD1,
ALS2 knock-out mice, and mice with genetically engineered
genes coding for the neurofilament subunits (reviewed in Refs.
19 and 20). Among these, the mutant human SOD1 (hSOD1)
transgenic mouse model is currently the most widely used
because it shares several clinical phenotypes with the ALS
patients. The first symptom of the hSOD1 mice is a fine “jitter-
ing/tremor” in one or more of the limbs, which appears at ?90
to 100 days of age (21, 22). At later stages, the cytopathological
features of the hSOD1 transgenic mice include motor neuron
including Lewy body-like hyaline inclusions/astrocyte hyline
inclusions, and vacuole formation (23).
Overexpression of TDP-43 in transgenic rodents could also
lead to development of motor neuron disease-like symptoms.
related pathology, and neuronal loss (24–27). The lifespans of
cytotoxicity of the overexpressed TDP-43 as well as the rela-
tively lower motor neuron specificity of the promoters, e.g.
Thy1, prion, etc., used to express the transgenes (24, 26–28).
Finally, the appearance of cells with cytoplasmic TDP-43(?)
UBIs and TDP-43-depleted nuclei at later stages of pathogene-
sis of the TDP-43 transgenic mice (25, 26) suggest that the dis-
ease phenotypes in the TDP-43 transgenic mice may result in
part from loss of function of TDP-43. However, the pathologi-
of toxicity from overexpression of the exogenous TDP-43.
Thus, the relative contributions of loss of function and gain
of cytotoxicity to the neurodegeneration in FTLD-U and ALS
with TDP-43(?) UBIs are not clear (reviewed in Refs. 16 and
29–31). Also, regardless of its currently known biochemical
and structural properties, the physiological functions of
viously, we have shown, by gene targeting approaches in mice,
that TDP-43 is important for mouse early embryonic develop-
ment (32). As described in the following, we have taken advan-
tage of the Tardbplxmouse line from that study and generated
mice with spinal cord motor neuron-specific knock-out of
ical phenotypes in striking similarity to ALS.
Generation of HB9 Promotor-driven Conditional Tardbp Gene
Knock-out Mice—The Tardbp allele of the C57BL/6j mice was
knocked out specifically in the postmitotic motor neurons in
the spinal cord by crossing mice carrying the Tardbp conditional
driven by the HB9-promoter (HB9:Cre)(see “Results” for more
details). The viabilities and weights of the mice were monitored
regularly. Genotyping of the mice was performed by PCR of
genomic DNAs from the tail biopsies in comparison to the nail
the mice were suspended by pulling their tails. For the rotarod
in motivation and motor learning. Mice were first trained for
rod rotating constantly at 2.5 rpm, and the speed was gradually
when the mice fell from the rod or when they gripped the rod
and started to rotate with it.
Tissue Preparation and Immunostaining Analysis—The
mice were sacrificed under deep anesthesia, and perfused tran-
scardially with 4% paraformaldehyde in PBS (pH 7.4). The spi-
nal cords were dissected and the lumber segments (L3–L6)
were identified using the ribs and vertebrae as the guide. The
segments were processed for cryoprotection, and 100 serial
FIGURE 1. Targeted disruption of the mouse Tardbp gene in spinal cord motor neurons. Exons 2 (E2) and 3 (E3) of TDP-43 were replaced with a ”floxed“
the two exons were removed by HB9 promoter-driven Cre recombinase from the HB9:Cre mice. The genotypes of mice carrying the different alleles were
validated by PCR of their genomic DNAs using primers (a, b, and c) shown in the figure.
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cross-sections were made of the lumbar spinal cords at a thick-
ness of 10 ?m. Every 12th section (12 sections total from each
animal) was stained with 1% cresyl violet (Sigma). Each section
was visualized with an Axioimage-Z1 light microscope at ?20
magnification, and the cells were counted manually by tracing
within an area demarcated by a horizontal line drawn through
matter to include layers 7–9. Initially, we identified the motor
neurons using the following criteria: 1) the presence of a large
nucleolus located within the nucleus surrounded by light blue-
motor neurons range in their soma areas from 100 to 250 ?m2,
whereas the soma areas of the larger ? motor neurons range
from 250 to 1100 ?m2.
In parallel sets of the sections, the motor neurons were also
analyzed by immunofluorescence staining with single or com-
bined use of several different antibodies: a goat antibody directed
against the choline acetyltransferase (ChAT; Chemicon), a rabbit
bit anti-Iba1 (Wako), a mouse anti-MAP2 (Sigma), a mouse anti-
(NeuN), and a mouse anti-human ERR? (PPMX). The sections
were preincubated in PBS solution containing 10% (v/v) normal
rabbit/mouse IgG (Jackson ImmunoResearch) was used for sec-
Immunoblotting Analysis—Total extracts of the spinal cords
were prepared by homogenization of the tissue in the RIPA
5 mM EDTA, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0) contain-
urea-soluble fractions of the proteins from the spinal cords
were obtained by centrifugation of the tissue homogenate at
15,000 ? g at 4 °C for 30 min. The supernatant was collected as
RIPAbufferandthensolublizedintheureabuffer(7 Murea,2 M
thiourea, 4% CHAPS, 30 mM Tris-HCl, pH 8.5) to give the
“urea-soluble” fraction. 4 ?g of RIPA extract or ?4 equivalent
volumes of urea extract per lane were separated on a 10% Tris
glycine SDS-PAGE gel. Immunoblotting analysis of the RIPA-
soluble and urea-soluble fractions of the spinal cord extracts
followed standard procedures with use of appropriate antibod-
ies. For comparing the protein levels of WT, Tardbplx/?mice,
was performed using Image J software (NIH). For TDP-43/
right panel as compared with a control littermate in the left panel. C, rotarod test. The indices (average time on the rotarod after normalization to the control,
weeks of age. The lower 4 panels are the contrast enhancement radiography of the skeletons. n ? 6 for each group.
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ChAT expression quantification, protein levels relative to wild
type (WT) mice was determined using TDP-43/ChAT immu-
noblots within the linear range of band intensity after normal-
ization to tubulin expression.
Generation of Mice with Conditional Knock-out of Tardbp
Expression in the Spinal Cord Motor Neurons—The Tardbp
cre recombination system. We first generated a conditional
allele of the Tardbp locus by flanking exons 2 and 3 with loxP
sites (Tardbplxallele), which was suitable for Cre-mediated
Tardbp-floxed gene inactivation (Fig. 1A; Ref. 32). We then
crossed the mice with motor neuron-specific Mnx1 (HB9):Cre
mice to generate the HB9:Cre-Tardbplx/?mice (Fig. 1).
selectively in motor neurons in the developing spinal cord
(E9.5) and is essential for differentiation of the postmitotic
motor neurons (33). Also, HB9:Cre mice express the cre gene
specifically in the spinal cord motor neurons and have been
used previously to manipulate gene expression in the motor
neurons (33). The motor neuron specificity of the HB9-driven
Cre expression have also been verified by crossing the HB9:Cre
mouse line to a ROSA26:LacZ reporter mice in several studies
(33–38). Examination of the GFP signals in HB9:GFP trans-
the motor neurons, but not in the sensory neurons (39).
On the other hand, it should be emphasized here that HB9
not be completely ruled out. For instance, expression of HB9:Cre
has also been observed in cells other than the ventral motor neu-
rons, which could be due to HB9 promoter activity in the visceral
have shown that Cre is expressed in motor neurons as well as in
some ventral interneurons at the embryonic stage (35, 36). In any
inated TDP-43 expression mainly in ChAT(?) motor neurons in
Development of ALS-like Morphological and Behavioral Phe-
notypes in HB9:Cre-Tardbplx/?Mice—Unlike the EIIa:Cre-
Tardbplx/lxmice we generated previously (32), which were
lethal at the peri-implantation stage, HB9:Cre–Tardbplx/?
FIGURE 3. Motor neuron loss in the spinal cords of 20-week-old HB9:Cre–Tardbplx/?mice. A, immunofluorescence co-staining of ChAT and TDP-43
showing that the number of ChAT-positive motor neurons (MN) in the ventral horn of the lumbar spinal cord of 20-week-old HB9:Cre–Tardbplx/?mice (lower
the age of 20 weeks (lower two panels) in comparison to a control littermate (upper two panels). The area of one ventral horn of each section is magnified for
(gray bars, by 38%) and the ? motor neurons (white bars, by 59%) in the mutant mice. n ? 4 for each group. *, p ? 0.05; **, p ? 0.01.
27338 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER33•AUGUST10,2012
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that TDP-43 was not essential for normal development of spi-
of HB9:Cre–Tardbplx/?mice was only slightly lower than the
controls at early births, the difference became more prominent
afterward (Fig. 2A). The body weight was shown previously to
be a simple and reliable measure for the disease “onset” and
progression in the ALS mouse model with transgenic expres-
sion of the mutant hSOD1 (G93A) (40), with the inflection
point of the weight curve providing a simple definition of the
genic mice, HB9:Cre–Tardbplx/?mice also showed a peak of
weight gain during 90 to 100 days (?13 weeks, Fig. 2A). Soon
after that, the mice started to show significant weight loss (Fig.
2A), abnormal hind limb clasping (Fig. 2B), and deficiency in
rotarod test (Fig. 2C). It should be noted here that throughout
ison to the HB9:Cre-Tardbplx/?mice. Like in the study of EIIa:
Cre-Tardbplx/?mice (32), the levels of TDP-43 expression in
the spinal cords of Tardbplx/?and the wild-type mice, either
10- or 20-week-old, were similar (supplemental Fig. S1A).
Although all of the above clinical signs were similar between
the HB9:Cre–Tardbplx/?mice and the mutant hSOD1 trans-
genic mice, the disease progression after the symptom onset
was somewhat slower in HB9:Cre–Tardbplx/?mice. Signifi-
cantly, HB9:Cre–Tardbplx/?mice exhibited a kyphosis pheno-
type beginning at 20 weeks of age and it became severe at 24
weeks, as exemplified in Fig. 2D. The kyphosis likely resulted
observed in ALS patients (41) as well as in the mutant hSOD1
and soleus muscles of the mice (supplemental Fig. S2). Finally,
similar to ALS (3), the development of ALS-like phenotypes in
HB9:Cre–Tardbplx/?mice were also male dominant, with a
male/female ratio of 3:1. The average lifespan of the HB9:Cre–
Loss of Motor Neurons and Enhancement of Astrocytosis in
as a neurodegenerative disorder characterized by progressive
in the primary motor cortex, brainstem, and spinal cord. Pro-
their corresponding motor neurons degenerated, then led to
weakening of the affected muscles. We reasoned that the
restricted population of the spinal cord motor neurons in HB9:
Cre-Tardbplx/?mice might be affected by the depletion of
TDP-43. Indeed, immunofluorescence staining and histology
analysis of the spinal cords showed a decrease of ChAT-positive
ing ChAT-positive motor neurons of HB9:Cre–Tardbplx/?mice,
in the confocal images (Fig. 3A and supplemental Fig. S3) and
statistically presented in the histogram of Fig. 3A.
As shown in supplemental Fig. S1B, the difference between the
FIGURE 4. Immunofluorescence co-staining analysis of the lumbar spinal cord motor neurons of 10-week-old and 20-week-old mice with use of
marginally, by 11%, in comparison to the control (black bars of the upper histogram; n ? 4 for each group; *, p ? 0.05).
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