Loss of murine TDP-43 disrupts motor function and plays an essential role in embryogenesis
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- Kraemer, B.C., Schuck, T., Wheeler, J.M. et al. Acta Neuropathol (2010) 119: 409. doi:10.1007/s00401-010-0659-0
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Abnormal TDP-43 aggregation is a prominent feature in the neuropathology of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. Mutations in TARDBP, the gene encoding TDP-43, cause some cases of ALS. The normal function of TDP-43 remains incompletely understood. To better understand TDP-43 biology, we generated mutant mice carrying a genetrap disruption of Tardbp. Mice homozygous for loss of TDP-43 are not viable. TDP-43 deficient embryos die about day 7.5 of embryonic development thereby demonstrating that TDP-43 protein is essential for normal prenatal development and survival. However, heterozygous Tardbp mutant mice exhibit signs of motor disturbance and muscle weakness. Compared with wild type control littermates, Tardbp+/− animals have significantly decreased forelimb grip strength and display deficits in a standard inverted grid test despite no evidence of pathologic changes in motor neurons. Thus, TDP-43 is essential for viability, and mild reduction in TDP-43 function is sufficient to cause motor deficits without degeneration of motor neurons.
Abnormal TDP-43 protein inclusions are part of the neuropathology of amyotrophic lateral sclerosis (ALS) , Alzheimer’s disease (AD) [2, 30], frontotemporal lobar dementia-ubiquitin type (FTLD-U) , and other neurodegenerative diseases. In these disorders, collectively known as TDP-43 proteinopathies, aggregated ubiquinated TDP-43 is observed in neurons and in glial cells. Recently, a consensus conference recommended that FTLD-U now be referred to as FTLD-TDP . In ALS, TDP-43 deposits occur in the spinal cords of almost all patients, with the notable exception of ALS caused by SOD1 mutations [26, 32, 43]. Some rare cases of familial ALS are caused by mutations in TARDBP, the gene encoding TDP-43 [16, 21, 38, 45, 51]. Thus, abnormal TDP-43 is not merely a marker for ALS, but is causal in some cases. In FTLD-TDP, TDP-43 deposits occur in cases caused by mutations in either progranulin (PGRN) or in vasolin containing protein (VCP). In AD, approximately 20–50% of cases have TDP-43 deposits [2, 27]. Thus, TDP-43 inclusions are clearly implicated in the pathogenesis of multiple neurodegenerative disorders.
TDP-43, a ubiquitously expressed nuclear protein, was originally identified as a 414 amino acid protein that binds the TAR DNA motif of the human immunodeficiency virus type 1 (HIV-1) (for recent review of TDP-43 function see ). The DNA binding activity of TDP-43 regulates HIV-1 gene expression in vivo . TDP-43 also acts as a transcriptional regulator of the mouse spermatogenesis protein SP-10 encoding gene . Other work showed TDP-43 binds RNA and controls RNA splicing of transcripts from the cystic fibrosis transmembrane conductance regulator (CFTR) , survival of motor neuron (SMN) , and apolipoprotein A-II (APOA2)  genes. TDP-43 also binds a sequence in the 3′ UTR region of neurofilament light chain (NEFL) mRNA and potentially affects the cytoplasmic stability of this message . TDP-43 has both nuclear export and import signals and shuttles between the nucleus and the cytoplasm, although in normal cells its distribution is primarily nuclear . In addition to these trafficking sequences, TDP-43 has two RNA-binding motifs  followed by a glycine-rich region near the carboxyl terminus. Cross-species comparisons of TDP-43 proteins from human, Drosophila, and C. elegans show a high degree of amino acid sequence conservation with the greatest identity found in the RNA recognition motifs (RRMs). Indeed, this sequence conservation translates into functional conservation as proteins from man, worm, and fly all share equivalent RNA and DNA recognition specificities . Therefore, TDP-43 is a multifunctional nuclear protein involved in several different cellular processes, but its functions are not yet fully understood.
In TDP-43 proteinopathies, aggregates of this protein occur in the nucleus, the cytoplasm, or in neurites (for recent reviews of TDP-43 pathology see [15, 23]). In some cells where cytoplasmic inclusions are seen, the nucleus is cleared of TDP-43 [20, 32]. These observations led to the hypothesis that compromised neuronal function may be caused by the depletion of available TDP-43 protein due to sequestration into aggregated protein inclusions. This hypothesis is viable if TDP-43 is indeed required for neuronal function. To test this hypothesis, we generated mice lacking TDP-43 and found that homozygous loss of TDP-43 protein is embryonic lethal while heterozygous loss of TDP-43 causes defects in motor function.
Materials and methods
Strains and cells
The BayGenomics ES cell line RB030 was obtained from the mutant mouse regional resource center (MMRRC, University of California-Davis). Targeting of the Tardbp gene by the gene trapping vector pGT1lxf was reconfirmed by sequencing of Tardbp-specific RT-PCR products. Also the genetrap insertion site was confirmed by DNA sequencing of the Tardbp genomic region flanking the genetrap. C57BL/6 mice were obtained from Jackson Labs. The TardbpGt(RB030)Byg chimeric mice were generated in the Murine targeted genomics laboratory at the MMRRC from the RB030 ES cells described above. The TardbpGt(RB030)Byg heterozygous mice were backcrossed to C57BL/6 mice five times.
Mice were genotyped using DNA prepared from tail clips for live mice and whole embryo or liver tissue of killed animals. The Tardbp gene was genotyped in a duplexed PCR using primers: TDP intron 2-23129S (AACTGCGCTAGCCCAAGTCTTGAGT), TDP-intron2 23511A (CCCACCTTCTATTTCCTGCCTCAGC), and pGT_A_501(CCATCCACTACTCAGTGCAGTGCAGT) yielding a 400-bp product for the wild-type Tardbp allele and a 635-bp product for TardbpGt(RB030)Byg. Single-cell PCR (reviewed in ) was conducted using Hotstar PCR reagents (Qiagen) as recommended by the manufacturer to genotype 3.5 day old embryos. Early post-implantation embryos cannot be reliably genotyped as separating maternal tissue from the embryo is very difficult; therefore, genotypes were inferred from Tardbp expression of day 5.5–9.5 embryos. For day 12.5 embryos, embryonic tissue was dissected away from maternal tissue, subjected to DNA extraction using Qiagen DNAeasy reagents, and genotyped using the same scheme as above using Hotstar PCR reagents (Qiagen).
Adult Tardbp+/− or Tardbp+/+ mice were euthanized and brains dissected. Whole brain was homogenized in Trizol reagent according to manufacturer’s recommendations (Invitrogen) for purification of RNA. Poly(A)+ RNA was selected using poly(A) purist MAG reagents as recommended by the manufacturer (Ambion). Four micrograms of poly(A)+ RNA was glyoxylated, resolved, transferred, and analyzed as previously described  using a TDP-43 exon 1 specific and beta-geo specific 32P radiolabeled probe.
Adult Tardbp+/− or Tardbp+/+ mice were euthanized and internal organs dissected. Tissue samples were homogenized in homogenization buffer (0.1 M Tris, 0.05 M NaCl, 0.001 M EDTA, pH 7.4) by sonication. Protein concentration was measured by Bradford assay as recommended (Biorad). Protein concentrations were normalized and resolved on a precast 4–15% gradient SDS PAGE gel (Biorad), and transferred to PVDF membrane (Biorad). Immunoblots were probed with polyclonal pan-TDP-43 antibody (ProteinTec Group 1:1,500), N-terminal TDP-43 (1:1,000), C-terminal TDP-43 antibody (1:1,000) , and lacZ (beta-geo1:1,000.) specific antibody (DSHB).
Grip strength and endurance measurements
Subjects were male and female Tardbp+/− heterozygous mice (n = 18) and control Tardbp+/+ littermates (n = 22), ranging in age from 342 to 840 days at the time of testing. The two groups had well-matched age distributions, with a mean age of 536 and 581 days for control and Tardbp+/− groups, respectively. The grip strength of the animals was tested in two ways: the inverted grid test and specific forelimb strength measurements. The inverted grid test was performed by placing each animal onto a metal grid which was held 12 in. above a padded surface and slowly inverted. The amount of time each animal gripped the grid was measured, up to a maximum of 2 min. Each animal was given three trials, with a rest period of 30 min between trials. Forelimb strength was measured using a digital force gage (Extech Instruments) fitted with a wire grid for animals to grip. Each animal was allowed to grip the grid with forepaws only and was gently pulled by the tail to measure maximum grip strength. Data represent the average of three trials for each animal. Data for both experiments were initially analyzed by ANOVA using sex and genotype as grouping factors, with effects of α ≤ 0.05 considered significant. No interactions with sex were identified, and thus the sexes were collapsed into a single group for subsequent analyses. Linear regression analysis was used to examine potential correlations between grip strength measurements and age or body weight.
A subset of the animals that were tested for grip strength as described above were also tested for gait abnormalities (n = 11 Tardbp+/−; n = 18 controls). Animals were trained to walk down a narrow alley with a white paper floor and a darkened goal box at the opposite end. After they had successfully demonstrated the ability to walk straight down the alley into the goal box, the hindpaws of each animal were dipped in nontoxic paint to produce a record of their footprints on the paper. The distance between sequential footprints on the same side was measured, to obtain the average stride length. The distance between sequential footprints on opposite sides was measured, to obtain the average hindbase width.
Histology and immunohistochemistry
Mouse uteri/embryos on days E5.5, 6.5, 7.5, 8.5, and 12.5 of gestational age were fixed overnight in 10% neutral buffered formalin, paraffin embedded and serially sectioned at 6 μm. Further, brains and spinal cords from eight Tardbp+/− mice and seven normal littermate controls were similarly fixed, embedded, and sectioned. Study mice for histology were as follows: Tardbp+/− mice were 13, 13, 18, 24, 24, 24, 27, and 28 months of age while normal control animals were 13, 16, 18, 24, 24, 25, and 28 months of age. Mice used for histology and IHC had been tested for grip strength, endurance, and gait analysis prior to killing for histology. Sections from the uteri/embryos, brains and spinal cords at all time points were stained with hematoxylin and eosin (H&E) and immunohistochemistry (IHC) was performed on E7.5, 8.5, and 12.5 uteri/embryos as described . Briefly, after antigen retrieval with citrate buffer (Vector Antigen Unmasking Solution H3300) using the Biogenex EZ-Retriever with microwave treatment for 15 min at 99°C, sections were stained with an anti-TDP-43 polyclonal antibody (1:2,000; ProteinTec Rabbit anti-TDP-43, Catalog# PTG10782) followed by a light hematoxylin counterstain. For the brain, spinal cord and muscle of adult Tardbp+/− and wild-type mice, additional antibodies to synaptic, cytoskeletal, and disease proteins were used including: phospho-TDP43 at 1:1,000 with the same microwave antigen retrieval protocol used for the polyclonal TDP43 antibody ; synaptophysin (Abcam cat # 8049 mouse monoclonal) at 1:200; MAP 2 1:2,000 (rabbit polyclonal in house antibody); GFAP (Dako cat #Z0334 rabbit polyclonal) at 1:10,000; NFL (in house rabbit polyclonal) at 1:2,000; alpha synuclein (in house SYN 303 mouse monoclonal) at 1:4,000 with 5 min antigen retrieval in 88% formic acid and phosphorylated tau (AT8 Innogenetics cat #90206 mouse monoclonal) at 1:2,000 as described in previous publications [14, 31, 52].
Deletion of mouse Tardbp
To generate mice with a targeted disruption of Tardbp, ES cell line RB030 was injected into blastocysts subsequently implanted into pseudopregnant female mice. Twenty chimeric pups resulted, as determined by coat color assessment ranging from 5 to 80% chimerism. Among these were two male mice exhibiting 80% chimeric coat color, which were mated to C57BL/6 females. Germline transmission was achieved from one of the highly chimeric males resulting in heterozygous TardbpGt(RB030)Byg progeny. For purposes of simplicity, we refer to the TardbpGt(RB030)Byg allele as Tardbp–, and the wild-type Tardbp allele as Tardbp+.
Tardbp+/− heterozygous mice are viable
To confirm that Tardbp+/− mice do indeed contain a targeted allele, we examined the Tardbp locus by southern blotting with TDP-43 and genetrap-specific probes (Fig. S1). Findings from southern blotting are consistent with a single genetrap insertion within the Tardbp locus in the genome of Tardbp+/− mice.
We also find other low-molecular-weight non-predicted TDP-43 isoforms in tissues such as kidney and liver. Although the size and origin of these products are not known, we speculate they may arise from alternative splicing and/or proteolytic processing of the TDP-43 mRNA and protein, respectively. The mouse Tardbp gene produces as many as 11 different transcripts encoding a variety of TDP-43 isoforms , which may account for the lower molecular weight bands seen here in different tissues. Alternatively, proteolytic processing of full-length normal TDP-43 could occur in these tissues. Furthermore, the mouse genome contains at least one Tardbp-like pseudogene predicted to encode a truncated version of TDP-43. We also observe higher molecular weight bands corresponding to the Tardbp exon 2/beta-geo fusion protein in Tardbp+/− mice, but not wild-type animals. To confirm that the higher molecular weight product does indeed contain the beta-geo genetrap encoded product we probed a duplicate immunoblot with lacZ specific antibody and observed the same high-molecular-weight products only in Tardbp+/− animals (Fig. 3b).
Probing duplicate blots with N-terminal and C-terminal specific TDP-43 antibodies  are consistent with these findings (Fig S2). The N-terminal antibody recognizes an epitope between amino acids 6 and 24 of human TDP-43, a region completely conserved between human and mouse. The C-terminal antibody recognizes an epitope between amino acids 394 and 414 of human TDP-43, a region completely conserved between human and mouse. The TDP-43 N-terminal specific antibody recognized the higher molecular weight bands, but the TDP-43 C-terminal specific antibody did not, in keeping with the structure of the predicted Tardbp exon 2/beta-geo fusion product (Fig. 1c).
Homozygous loss of Tardbp causes early embryonic lethality
Genotype analysis of mouse pups and embryos from Tardbp+/− intercrosses
Degeneration of TDP-43 deficient zygotes
Tardbp+/− heterozygous mice exhibit motor defects resulting from muscle weakness
Pathology of Tardbp+/− heterozygous mice
After extensive sampling of brain, spinal cord and muscle of Tardbp+/− heterozygous and wild-type mice, no unequivocal differences were seen between these mice by H&E staining or IHC (see Figs. S4, S5). Thus, while the behavioral testing revealed motor weakness, this is not reflected by evidence of neurodegeneration in the CNS or muscle atrophy in the tissues examined here suggesting subtle non-structural motor neuron deficiencies, metabolic changes, or other systemic abnormalities account for these motor impairments.
We generated mice with a targeted disruption of the Tardbp gene, which replaces the normal TDP-43 protein coding sequence with a beta-galactosidase/neomycin resistance fusion protein coding sequence. Mice homozygous for the Tardbp disruption do not survive to term and die in utero by day E9.5 of embryogenesis. Immunostaining of degenerating embryos shows a pronounced lack of detectable TDP-43 protein in nuclei of necrotic embryos. Because the lethality occurs early in embryogenesis at about E7.5, it is not possible to determine whether the defect is global or if failure to generate specific organ(s) is responsible for embryonic demise. The findings presented here demonstrate an absolute requirement for TDP-43 protein during early to mid embryogenesis. This is not surprising since TDP-43 participates in multiple cellular functions. For example, TDP-43 influences splicing of CFTR, SMN, and APOA2, and probably participates in splicing regulation of numerous other genes. Correct regulation of splicing during embryogenesis is critical, and in adults altered splicing of the tau mRNA causes FTLD-tau [11, 19]. Lack of TDP-43 may also cause early defects in transcriptional regulation of multiple genes including several cell proliferation factors . TDP-43 appears to be localized to a number of nuclear bodies that potentially are complexes important in transcription and splicing . The embryonic lethal phenotype observed demonstrates that TDP-43 is an essential protein and suggests that, at least in embryos, there is no functionally redundant protein equivalent to TDP-43.
In Tardbp+/− animals, we observed impaired motor function, expressed as decreased forelimb strength and performance deficits in the inverted grid test. Despite behavioral evidence of motor weakness, comparative examination of the CNS and muscle tissues from Tardbp+/− and wild type revealed no differences that could be ascribed to the Tardbp+/− genotype. The Tardbp+/− mice showed no evidence of neurodegeneration or muscle atrophy, and did not exhibit any gait abnormalities or any sign of limb clasping. This weakness occurs in aged mice, but not in young mice, indicating that the phenotype is associated with the aging process. This leaves us with two possible explanations. First, weakness could be a result of aging-related defects in motor neuron function without detectable pathologic or structural changes. Second, weakness could occur in the context of aging-related metabolic abnormalities. Future studies will distinguish between these possibilities to more fully account for the motor impairments in the Tardbp+/− mice. However, loss of a single copy of Tardbp does not change TDP-43 protein levels in any tissue examined, suggesting that an auto-regulatory mechanism controls TDP-43 protein levels. These findings are consistent with the suggestion that TDP-43 function is critical for cellular homeostasis . Furthermore, at least one TARDBP mutation (Y374X) in ALS is a sporadic mutation causing a premature stop codon in TARDBP . This is consistent with loss of TARDBP driving motor neuron disease in humans, although this is also consistent with TDP-43 truncation being pathological.
Despite the finding that Tardbp+/− animals exhibit approximately wild-type TDP-43 protein levels, we found clear evidence of muscle weakness in these animals. The inverted grid test is a standard test of motor function which has been previously used to characterize a variety of mouse models of neurodegeneration [10, 17, 36]. In particular, the inverted grid test was a sensitive measure of disease progression in the SOD1 mouse model of ALS . This test is a relatively complex measure of the strength of multiple muscle groups, as well as motor coordination and motivation. Thus, we also performed grip strength measurements using a force meter as a more specific test of forelimb strength. Similar tests have been used to characterize motor defects in other models [17, 39]. Our results indicate that loss of function of a single Tardbp allele in mice is sufficient to cause significant defects in adult motor function.
Including this study, three independent knockout mouse models examining loss of function in Tardbp, the gene encoding TDP-43, have been reported [35, 50]. All three models consistently show an early embryonic phenotype in Tardbp−/− mice arising from an early failure in cell division around the time of blastocyst implantation. In all three models the protein levels of heterozygous Tardbp+/− mice are nearly normal suggesting an auto-regulatory mechanism controls TDP-43 protein levels. Preliminary characterization of Tardbp+/− mice from two groups revealed no overt motor phenotypes [35, 50]; however, our analysis of aged animals revealed significant motor dysfunction present in Tardbp+/− mice without obvious neurodegenerative changes. The discrepancy in motor phenotypes among these three models is most likely due to differences in the age of animals examined. It will be interesting to observe what phenotypes materialize in the other two lines of Tardbp+/− animals as they age. Findings from Drosophila suggest TDP-43 is dispensable for viability in the fly [13, 25], which differs from the results presented for mouse Tardbp here and elsewhere [35, 50]. Fly TDP-43 appears to participate in motor neuron function, suggesting that loss of dTDP-43 can drive motor dysfunction, consistent with our observations in Tardbp+/− mice.
TARDBP mutations causing ALS establish that alterations in TDP-43 can cause neurodegeneration [6, 12, 16, 21, 22, 24, 38, 44, 45, 51]. These mutations result in the same type of TDP-43 aggregation seen in ALS cases without TARDBP mutations. The mechanism by which these mutations cause disease could be partial loss of function. However, as shown here (Fig. 3) in mice, loss of one Tardbp allele alters protein levels little in most tissues, presumably due to increased TDP-43 expression from the intact copy of Tardbp. However, even this subtle perturbation in TDP-43 activity is sufficient to cause detectable motor dysfunction in the absence of neurodegeneration. Therefore, a loss-of-function model for ALS mutations is consistent with the experimental data. In TDP-43 proteinopathies, TDP-43 aggregation may result in a more profound reduction in functional TDP-43 levels that cannot be compensated for by increased Tardbp expression. In some neurons where cytoplasmic aggregates are observed, the nuclei are devoid of TDP-43 detectable by immunohistochemical methods. Nuclear aggregates could act as a sink for TDP-43, leaving no free protein for essential functions. Such a complete loss of TDP-43 may result in death of the cell, a hypothesis supported by the embryonic lethality we observe in our Tardbp−/− embryos. Under this hypothesis, in the case of ALS mutations, both the mutant and the normal TDP-43 would aggregate and be removed from the soluble pool available for normal function.
The cause of TDP-43 aggregation is unknown. In other proteinopathies, mutations can cause increased aggregation rates for the mutated protein (e.g. tau [18, 29]). However, there is no direct evidence that ALS TARDBP mutations change the solubility of TDP-43. Once TDP-43 is depleted, any number of mechanisms could result in motor neuron dysfunction and/or cell death given the number of cellular processes that involve TDP-43. Clearly, further studies are needed, especially the generation of conditional Tardbp knockout mice to address these and other important aspects of the normal biology of TDP-43 and the pathobiology of TDP-43 in neurodegenerative proteinopathies.
This work was supported by NIH grant AG17586 to GDS, VMYL, and JQT. This work was also supported by grants to BCK from the Office of Research and Development Medical Research Service, Department of Veterans Affairs and NIH (NS064131). This work utilized facilities at the VA Puget Sound Health Care System, Seattle, Washington. We thank Eddie Lee for outstanding assistance examining neuropathology of Tardbp+/− animals and Elaine Loomis, Leojean Anderson, and Harmony Danner for excellent technical assistance. The lacZ monoclonal antibody was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa.