Characterization of Dlk1 knockout animals

The Dlk1 knockout animals did not show obvious defects besides higher body weight due to increased body fat. The defects in eye or rip cage development depicted by Moon and colleagues have not been observed so far (Moon et al. 2002). Dlk1^-/- animals were born in the expected Medelian ration. Also, no increased early postnatal

mortality or reduced birth weight was found in Dlk1^-/- animals compared to their wild type littermates. These differences might be caused by differing genetic backgrounds of the mouse lines or defects carried over from the embryonic stem cells used to generate these mice.

5.6.2.1 The clasping phenotype in Dlk1 knockout mice

Fig. 5.23 The clasping phenotype in Dlk1 knockout mice Dlk1 wild type animal in comparison to a knockout mouse showing strong a clasping behavior (A, B).

Clasping behavior in Dlk1 knockout and wild type animals before (unloaded) and after (loaded) running exercise (C). Clasping score for fore and hindlimb before running exercise (D). A summary of clasping scores before and after running exercise (E).

Dlk1 knockout animals exhibited a neurological impairment that resulted in a pathological fore and hind limb clasping behavior when the animals were subjected to tail suspension (Fig. 5.23 A & B). This phenotype was typically observed several seconds after suspension. To examine this phenotype, the mice response towards tail suspension was monitored and scored according to Methods 4.1.3.3. None of the wild type animals showed clasping in any of the trials (Fig. 5.23 C). The phenotype affected the forelimbs significantly stronger than the hind limbs (Fig. 5.23 D), which differs from other transgenic mouse models for neuromuscular diseases where it is more prominent in the hindlimbs (Mangiarini et al. 1996, Guy et al. 2001, Hansson et al. 2003, Bruestle et al. 2009). In order to test how increased neuromuscular workload is affecting the phenotype the animals were tested for clasping before and directly after being subjected to a short high intensity running exercise. After the running exercise the average clasping score increased by app. 35% to 2.166 points while wild type animals still did not show clasping (Fig. 5.23 C). These results indicate that neuromuscular stress enhances the pathological clasping phenotype possibly due to energy depletion or oxidative stress in the muscles or the associated MNs of Dlk1 knockout mice.

5.6.2.2 Running endurance test

The specification of FMNTs is directly linked to the fine tuned regulation of skeletal muscle outputs. Thus, to generally test neuromuscular performance of Dlk1^-/- animals, they were subjected to two forced treadmill running endurance paradigms. Running endurance assays are generally applied to test the integrity of the neuromuscular systems and voluntary running distance was used as a diagnostic marker for the disease progression in mouse models of neuromuscular diseases. Two different protocols were applied in this assay in order to test the performance of different motor units. The medium intensity protocol tested the ability to run at a gradually increasing speed on a slight slope of 10° while the high intensity protocol used the same running speeds but in combination with a 20° incline.

Fig. 5.24 Running endurance of Dlk1 knock out and wild-type mice Dlk1 knockout and wildtype animals were subjected to a treadmill running protocol at 10°

(A) and 20° (B) incline. The test was performed with six Dlk1 wild-type control mice and ten Dlk1^-/- experimental animals.

In this assays the running endurance of Dlk1^-/- mice was reduced compared to wild-type animals in the medium and high intensity running protocol. Although there were high individual differences observed between the experimental animals the results were significant (p<0.05) in this assay. In the medium intensity running paradigm the Dlk1 knockout mice traveled a distance of 300.1 m (SD +/-54.31 m) while the wild type controls reached a distance of 400.5 m (SD +/-103.15 m). These results were verified by the high intensity running test in which the Dlk1 knockout mice traveled a distance 196.6 m (SD +/-31.8 m) compared to 288.1 m (SD +/-99.5 m) for the wild-type controls. However, due to the systemic loss of the protein to this point it could not be determined if the observed phenotype was caused by a neuronal impairment, a muscle defect or the higher body weight of the Dlk1^-/- animals.

5.6.2.3 Grip strength assay

Forelimb grip strength assays allow testing the neuromuscular performance of rodents. This is based on the grasping reflex that makes the animals holding on to a handlebar upon being pulled backwards. Grip strength is also routinely used as an indicator for neuromuscular disease progression (Henriques et al. 2010). The grip strength of ten male Dlk1-/- and six male Dlk1 +/+ control mice was examined at three subsequent days. Each animal was tested five times per day and the highest value was taken.

The Dlk1 knockout animals revealed significantly lower absolute and relative grip strength compared to wild type mice. The absolute grip strength decreased from 164 g (SD +/-6.84 g) in wild-type animals to 138 g (SD +/-4.64 g) in knockout mice. A similar result was obtained for the relative grip strength, which was reduced from 6.35g/g body weight (SD +/-0.32 g) to 5.14 g/g body weight (SD +/-0.47 g). The individual variances observed in this experiment were lower than that observed in running endurance test. However, the measured force values slightyly increased over the three experimental days suggesting a possible familiarization of the animals towards the experimental setup. However, as stated for the impairment in running endurance observed in these animals also this phenotype might be caused due to a systemic effect reducing general neuromuscular performance. Thus, to obtain a more direct measurement of the effects of Dlk1 towards neuromuscular performance, it will

be necessary to repeat the experiments with animals selectively deficient of Dlk1 in MNs as soon as they become available.

Fig. 5.25 Grip strength measurement of Dlk1 knockout and wild-type mice Ten four month old knockout and six age matched wild-type control animals were tested.

Error bars are indicating standard deviation. The absolute grip strength (A) as well as the grip strength normalized to body weight (B) was determined.