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. 2012 Aug 10;287(33):27335-44.
doi: 10.1074/jbc.M112.359000. Epub 2012 Jun 20.

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  1 , Wei-Cheng Cheng, C-K James Shen
Affiliations

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 et al. J Biol Chem. .

Abstract

ALS, or amyotrophic lateral sclerosis, is a progressive and fatal motor neuron disease with no effective medicine. Importantly, the majority of the ALS cases are with TDP-43 proteinopathies characterized with TDP-43-positive, ubiquitin-positive inclusions (UBIs) in the cytosol. However, the role of the 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 inactivation of the Tardbp gene in the spinal cord motor neurons (HB9:Cre-Tardbp(lx/-)) 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, ubiquitinated proteins accumulate in the TDP-43-depleted motor neurons of the spinal cords of HB9:Cre-Tardbp(lx/-) 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 mammalian spinal cord motor neurons, but also establishes that loss of TDP-43 function could be one major cause for neurodegeneration in ALS with TDP-43 proteinopathies.

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Figures

FIGURE 1.
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“ fragment containing exons 2 and 3 followed by a frt-flanking neo cassette. The neo cassette was then removed by germline expression of flp recombinase and 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.
FIGURE 2.
FIGURE 2.
ALS-like phenotypes of HB9:Cre–Tardbplx/− mice. A, top, time scale of the progression of ALS-like phenotypes of the HB9:Cre–Tardbplx/− mice, as exemplied in A–D. Below, time course of the body weight changes (weights at different ages, mean ± S.E.). The mean body weight of HB9:Cre–Tardbplx/− mice is statistically different from that of the Tardbplx/− mice after 8 weeks (*, p < 0.05; ***, p < 0.005). The disease onset of the HB9:Cre–Tardbplx/− mice is defined as the start of the weight loss at around 13 weeks. B, hindlimb clasping test. Abnormal clasping of a 20-week-old HB9:Cre–Tardbplx/− mouse is exemplied in the 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, mean ± S.E.) are plotted against the ages of the mice. D, kyphosis phenotype of the mutant mouse (right panels) in comparison to the control (left panels) at 20 weeks of age. The lower 4 panels are the contrast enhancement radiography of the skeletons. n = 6 for each group.
FIGURE 3.
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 two rows of panels) was reduced in comparison to the control (upper two rows). The 2nd and 4th rows are magnified photos of the boxed areas in the 1st and 3rd rows of photos, respectively. The scale bars are 50 μm. The quantitative comparison is shown by the bar histogram below the photographs. Note the depletion of TDP-43 in most, although not all, of the ChAT(+) cells, or motor neurons. Also, ∼62% of the ChAT(+) cells of the mutant mice are without TDP-43 expression. ***, p < 0.05. B, top, photomicrographs showing the cresyl violet (Nissl)-stained sections through the lumbar spinal cord from a HB9:Cre–Tardbplx/− mouse at 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 better visualization. The scale bar is 50 μm. Below, the quantitative analysis is presented by the bar histogram. Note the reduction of both the α motor neurons (gray bars, by 38%) and the γ motor neurons (white bars, by 59%) in the mutant mice. n = 4 for each group. *, p < 0.05; **, p < 0.01.
FIGURE 4.
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 anti-ChAT (white), anti-NeuN (green), DAPI (blue), and anti-TDP-43 (red). The photos are exemplified in the left 4 rows of panels. The scale bars are 50 μm. Right, the quantitative analysis is presented by the 2 bar histograms. Note the reduction of both the α (NeuN(+)/ChAT(+)) motor neurons (gray bars, by 46%) and the γ (NeuN(−)/ChAT(+)) motor neurons (white bars, by 25%) in the 20-week-old mutant mice (lower histogram, n = 4 for each groups; *, p < 0.05; ***, p < 0.005). On the other hand, the number of the total motor neurons (MN) in the lumbar spinal cord of the 10-week-old HB9:Cre–Tardbplx/− mice was reduced only marginally, by 11%, in comparison to the control (black bars of the upper histogram; n = 4 for each group; *, p < 0.05).
FIGURE 5.
FIGURE 5.
Reactive astrocytosis in the spinal cords of HB9:Cre–Tardbplx/− mice. Multiple sections of the ventral spinal cords from 20-week-old HB9:Cre–Tardbplx/− mice and control littermates were immunofluorescence stained with anti-GFAP and anti-MAP2, a marker of neurons. The patterns of one representative section each from the two groups of mice are shown. DAPI stainings indicate the locations of the nuclei. Note the enhanced level of anti-GFAP fluorescence in the mutant mice (lower panels) when compared with the control mice (upper panels). The scale bars are 50 μm. Below, quantitative analysis showing an ∼2-fold increase of the area occupied by the GFAP-positive cells within a specific AOI (area of interest) in the HB9:Cre–Tardbplx/− mice. Data are presented as mean ± S.E. **, p < 0.001, n = 4 for each group.
FIGURE 6.
FIGURE 6.
Accumulation of ubiquitinated proteins in the spinal cord motor neurons of HB9:Cre–Tardbplx/− mice. A, the lumbar sections of the spinal cords of 20-week-old control (upper 2 rows of panels) and HB9:Cre–Tardbplx/− (lower 2 rows of panels) mice were immunofluorescence co-stained with anti-ubiquitin (green), anti-TDP-43 (red), and anti-ChAT (white). DAPI (blue) stainings indicate the locations of the nuclei. The patterns of one representative section of the control mice and one representative section of HB9:Cre–Tardbplx/− are shown. The boxes mark the regions of the ventral horn magnified for better visualization of the staining signals. n = 4 for each group. Note that increased anti-ubiquitin signals were present mostly in the cytosol of ChAT(+)/TDP-43(−) cells of the HB9:Cre–Tardbplx/− mice, as exemplified by the two ChAT(+) cells in the lower left corners of the bottom row of panels. Almost all of the ChAT(+)/TDP(+) spinal cord cells of the control mice exhibited low ubiquitin signals, as exemplified by the ChAT(+) cell in the 2nd row from the top. The scale bars are 25 μm. The quantitative comparison is shown by the bar histograms below the photographs. Note that most, although not all, of the TDP-43(−)/ChAT(+) motor neurons of the HB9:Cre-Tardbplx/− mice are Ub(+), and vice versa. Furthermore, separate analysis of the immunofluorescence staining data showed that whereas ∼6% of the total cell population in the spinal cord of the HB9:Cre–Tardbplx/− mice were Ub(+), only 0.4% of the control Tardbplx/− spinal cord cells were Ub(+). B, representative immunoblots of polyubiquitinated proteins in fractionated spinal cord extracts. The total protein extracts were isolated from the spinal cords of Tardbplx/− control and HB9:Cre–Tardbplx/− mice, fractionated into the RIPA- and urea-soluble fractions, and analyzed by immunoblotting with use of anti-ubiquitin (top) and anti-actin (bottom). Note that the increase of high molecular weight ubiquitinated protein species (***) are in the urea-soluble fraction of the HB9:Cre–Tardbplx/− sample in comparison to the control sample. The arrows point to the two nonspecific bands on the anti-ubiquitin blots.
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