Loss of murine TDP-43 disrupts motor function and plays an essential role in embryogenesis

Abstract

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.

Fig. 1

Genetrap targeting of the Tardbp gene. a The genetrap insertion site on mouse chromosome 4 is depicted. Vertical bars are exons, intervening lines represent introns. The position on chromo-some 4 is indicated. b The predicted mRNAs encoding TDP-43 and the truncated genetrap product are indicated. c The protein domain structure of normal TDP-43 and the TDP-43/beta-geo fusion protein are indicated

Fig. 2

Expression of Tardbp mRNA. Northern blot of brain mRNA purified from Tardbp^+/+ and Tardbp^+/− animals. The normal short and long 3′ UTR forms of the TDP-43 encoding message are indicated (7.7 and 2.7 kb products). The genetrap truncated mRNA is the intermediate 5.4 Kb band only present in Tardbp^+/− animals. The exon 1 targeted radio labeled probe is indicated (left panel). Genetrap specific probe binds beta-geo coding sequence (right panel)

Fig. 3

Expression of TDP-43 protein in various tissues. Immunoblotting of brain, heart, kidney, liver, lung, muscle, and spleen tissue protein extracts. Duplicate blots were probed with a pan-TDP-43 specific polyclonal antibodies and b beta-geo specific antibody. Arrow indicates the TDP-43(1–65)/beta-geo fusion protein. Arrowhead indicates the major 43 kDa brain isoform of TDP-43 protein. c beta-Actin specific antibody

Fig. 4

Degeneration of in utero embryos lacking TDP-43 protein. a H&E stained section of E7.5 embryo (arrow) undergoing necrosis; the surrounding tissue is uterine deciduas. b H&E stained section of a normal E7.5 embryo surrounded by uterine decidual tissue. c TDP immunohistochemistry demonstrating lack of TDP staining in the degenerating E7.5 embryo (arrow) surrounded by TDP positive uterine decidual tissue. d TDP immunohistochemistry normal E7.5 embryo (arrow) showing positive staining in the embryo as well as in the surrounding uterine decidual tissue

Fig. 5

Tardbp^+/− animals display deficits in grip strength relative to littermate controls. a Tardbp^+/− animals (n = 18) hung on an inverted wire grid for significantly less time than control animals (n = 22) and b also exhibit lower maximum forelimb grip strength. Individual scores are the average of three trials, and the error bars represent standard deviation (**p= 0.01; ***p= 0.0002) c A subset of these animals was also examined for differences in gait, as measured by average stride length and the width between hindpaws while walking. No significant difference between the genotypes was detected