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. 2010 Aug 11;30(32):10851-9.
doi: 10.1523/JNEUROSCI.1630-10.2010.

Wild-type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits, and early mortality in transgenic mice

Ya-Fei Xu  1 , Tania F Gendron, Yong-Jie Zhang, Wen-Lang Lin, Simon D'Alton, Hong Sheng, Monica Castanedes Casey, Jimei Tong, Joshua Knight, Xin Yu, Rosa Rademakers, Kevin Boylan, Mike Hutton, Eileen McGowan, Dennis W Dickson, Jada Lewis, Leonard Petrucelli
Affiliations

Wild-type human TDP-43 expression causes TDP-43 phosphorylation, mitochondrial aggregation, motor deficits, and early mortality in transgenic mice

Ya-Fei Xu et al. J Neurosci. .

Abstract

Transactivation response DNA-binding protein 43 (TDP-43) is a principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). Mutations in TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, yet multiple neurodegenerative diseases exhibit TDP-43 pathology without known TARDBP mutations. While TDP-43 has been ascribed a number of roles in normal biology, including mRNA splicing and transcription regulation, elucidating disease mechanisms associated with this protein is hindered by the lack of models to dissect such functions. We have generated transgenic (TDP-43PrP) mice expressing full-length human TDP-43 (hTDP-43) driven by the mouse prion promoter to provide a tool to analyze the role of wild-type hTDP-43 in the brain and spinal cord. Expression of hTDP-43 caused a dose-dependent downregulation of mouse TDP-43 RNA and protein. Moderate overexpression of hTDP-43 resulted in TDP-43 truncation, increased cytoplasmic and nuclear ubiquitin levels, and intranuclear and cytoplasmic aggregates that were immunopositive for phosphorylated TDP-43. Of note, abnormal juxtanuclear aggregates of mitochondria were observed, accompanied by enhanced levels of Fis1 and phosphorylated DLP1, key components of the mitochondrial fission machinery. Conversely, a marked reduction in mitofusin 1 expression, which plays an essential role in mitochondrial fusion, was observed in TDP-43PrP mice. Finally, TDP-43PrP mice showed reactive gliosis, axonal and myelin degeneration, gait abnormalities, and early lethality. This TDP-43 transgenic line provides a valuable tool for identifying potential roles of wild-type TDP-43 within the CNS and for studying TDP-43-associated neurotoxicity.

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Figures

Figure 1.
Figure 1.
TDP-43PrP mice expressing hTDP-43 in the brain and spinal cord display reduced brain and body weight and abnormal escape response. A, B, Immunohistochemistry shows hTDP-43 distributed throughout the gray matter of the spinal cord (A) and brain (B) in homozygous TDP-43PrP mice. C, Western blots of brain lysates from NT, as well as hemizygous and homozygous TDP-43PrP mice using antibodies that detect either both mTDP-43 and hTDP-43 (total TDP-43) or hTDP-43 only. GAPDH immunoreactivity was included to ensure equal loading. D, Compared to NT and hemizygous mice, homozygous TDP-43PrP mice had significant deficits in body weight. By 28 d, the average body weight of homozygous TDP-43PrP mice was approximately half that of controls. Data shown are the means ± SEM of 8 mice per group; *p < 0.05, ***p < 0.001, as assessed by two-way ANOVA followed by Bonferroni post hoc test. E, At 1 month, brain weight of homozygous TDP-43PrP mice was significantly lower than that of age-matched NT and hemizygous mice. Data shown are the means of 7–14 mice per group. ***p < 0.001, as assessed by one-way ANOVA. F, G, Upon tail elevation, homozygous mice (G) held their hindlimbs close to their body and failed to show proper escape extension, while NT mice (F) showed normal escape response by splaying their hindlimbs. H, Immunoblot of spinal cord lysates from NT, hemizygous and homozygous TDP-43PrP mice using an antibody that detects total TDP-43. Note that TDP-43 fragments (∼35 kDa and 25 kDa) in spinal cord comigrate with C-terminal fragments generated by staurosporine (STP)-induced caspase activation in human neuroglioma cells expressing hTDP-43. hemi, Hemizygous; homo, homozygous.
Figure 2.
Figure 2.
Neuropathology in TDP-43PrP mice. A, B, Immunostaining in spinal cord sections of a 1-month old NT and symptomatic homozygous TDP-43PrP mice using an antibody to hTDP-43 shows hTDP-43 in nuclei of TDP-43PrP mice (B, B′), with occasional cytoplasmic staining (arrows). hTDP-43 was not observed in NT mice (A, A′). C–F, IHC analysis of spinal cord (C–E) or cortical (F) neurons using an antibody for the detection of TDP-43 phosphorylated at serines 403/404 and hematoxylin (C) or eosin (D–F) counterstain. Shown in C are nuclear bodies immunoreactive for pTDP-43 within a spinal cord motor neuron, while cytoplasmic pTDP-43-immunoreactive inclusions are shown in D–F. Scale bars: A, B, 100 μm; A′, B′, 50 μm; C–F, 10 μm.
Figure 3.
Figure 3.
Neuropathology in TDP-43PrP mice. A, F, Hematoxylin and eosin staining in spinal cord sections of 1-month-old NT and symptomatic homozygous TDP-43PrP mice. Eosinophilic aggregates in spinal cord motor neurons from TDP-43PrP mice (F, arrows and inset) are not observed in NT mice (A). B, G, IHC analysis of spinal cord neurons in TDP-43PrP mice (G) and NT mice (B) using an antibody for the detection of TDP-43 phosphorylated at serines 403/404 and eosin counterstain. C, H, Abnormal ubiquitin immunoreactivity was present in the cytoplasm and nucleus of neurons in TDP-43PrP mice (H) but not in NT mice (C). Enhanced IBA-1 (I, D) and GFAP (J, E) immunoreactivity indicative of activated microglia and reactive astrogliosis, respectively, were observed in TDP-43PrP (I, J), but not NT mice (D, E). Scale bars: A, C–F, H–J, 50 μm; A, F, insets, 25 μm; B, G, 10 μm.
Figure 4.
Figure 4.
Axonal degeneration and myelin degeneration in TDP-43PrP mice. Silver staining with a method specific for neurodegeneration of neurites and neuronal cell bodies revealed argyrophilic degenerating neurites and neurons in spinal cord of symptomatic TDP-43PrP mice (B) compared to NT controls (A). Toluidine blue stains show myelin vacuolization, with myelin ovoids (arrows and inset) in anterolateral funiculi of spinal cords of symptomatic TDP-43PrP mice (C, D) but not in NT mice (E, F). Scale bars: A, B, 20 μm; C, E, 40 μm; D, F, 20 μm.
Figure 5.
Figure 5.
Ultrastructural evidence of abnormal aggregation of mitochondria in homozygous TDP-43PrP mice. The upper left inset of A depicts a low-power image of a motor neuron in the anterior horn of a 1-month old symptomatic homozygous TDP-43PrP mouse containing a cytoplasmic aggregate (arrow) and a peripherally located nucleus (N; scale bar, 5 μm). Enlargement of the aggregate (panel A, proper) reveals clustered mitochondria (scale bar, 2 μm). A large, abnormal mitochondrion with disorganized inner cristae (arrow) is shown in the right upper inset. Also observed are mitochondria with paucity of cristae and vacuoles within the mitochondrial matrix (arrowheads; scale bar, 0.2 μm). B, The accumulation of mitochondria of various shapes and sizes and of small and large vesicles is observed in a swollen dendrite (scale bar, 1 μm). C, Abnormally shaped (arrowheads) and degenerated (arrow) mitochondria, as well as autophagic vacuoles (*) are present within a swollen axon of a spinal cord neuron (scale bar, 0.25 μm). D, An axon with a vacuolated myelin sheath (*), containing degenerating mitochondria (arrowheads), many vesicles/vacuoles, and tightly packed neurofilaments (arrow) is shown (scale bar, 2 μm). E, Immunoblot analysis of mitofusin 1 (MFN1), Fis1, DLP1, and Ser616-phosphorylated DLP1 expression in brain lysates of nontransgenic, hemizygous, and homozygous TDP-43PrP mice. Densitometric analysis of Western blots is shown. While total DLP1 levels did not change, phosphorylation of DLP1 at Ser616 was significantly increased in homozygous TDP-43PrP mice compared to nontransgenic mice. Similarly, expression of Fis1, another component of the fission machinery was significantly upregulated in TDP-43PrP mice. In contrast, mitofusin 1 (MFN1) expression was significantly decreased in TDP-43PrP mice. *p < 0.05 compared to levels in nontransgenic mice, as assessed by one-way ANOVA (n = 3). hemi, Hemizygous; homo, homozygous. F, J, Following IHC against the mitochondrial marker, COX-IV, spinal cord sections were counterstained with eosin (G, K). Notice the COX-IV-positive aggregates, which are also eosinophilic, in TDP-43PrP mice (J, K) but NT mice (F, G). Likewise, COX-IV-positive aggregates (L) stained blue following staining with toluidine blue (M) in TDP-43PrP mice, supporting the presence of high phospholipid levels associated with mitochondria. No similar staining was observed in NT mice (H, I). Scale bar, 24 μm.
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