Expte: PI 5480/22 RGEP 13038

PLS3 has previously been proven to be a fully protective modifier in unaffected SMN1-

deleted siblings of discordant SMA families (Oprea et al., 2008). Therefore, it was the main aim of the present study to investigate, whether PLS3 overexpression is also able to rescue the phenotype of an SMA mouse model.

PLS3-overexpressing mice were produced by targeting a V5-tagged version of the human PLS3 gene (PLS3V5) controlled by the CMV enhancer/chicken β-actin (CAG) fusion

promoter into the Rosa26 locus. Since a LoxP sites flanked stop-cassette was present between promoter and transgene (Figure 8), PLS3V5 expression could be driven either ubiquitously or tissue specifically. We induced a motor neuron specific expression by breeding PLS3V5-floxed mice with the Cre-expressing line Hb9-Cre (Arber et al., 1999).

For transgenesis, the hybrid ES cell line V6.5 (SV129 and C57BL/6N) was used in this study (Eggan et al., 2002). V6.5 ES cells offer the big advantage of being insensitive and robust in handling. Furthermore, favorable germline transmission rates have been reported for those cells by various laboratories. Altogether, two rounds of ES cell transgenesis (chapter 5.2) and injection into blastocysts were performed using V6.5 ES cells before a single PLS3V5fl_st/wt transgenic female could be identified by genotyping PCR. Nevertheless, to study a possible modifying function of PLS3V5, a clean genetic background was required. Therefore, resulting PLS3V5fl_st/wt founder animals had to be backcrossed for more than 7 generations to reach a statistical genetic purity of 100 % prior to further analysis.

While constantly backcrossing PLS3V5fl_st/wt miceonto a clean C57BL/6N background, the same line was bred with the ubiquitously Cre-expressing line CMV-Cre (Schwenk et al., 1995) in order to permanently delete the stop cassette between CAG promoter and transgene. Importantly, the Cre transgene is located on the X-chromosome in CMV-Cre mice. In animals of the genotype PLS3V5fl_st/wt;CMV-CreX_tg/Y the transgene was expected to be expressed ubiquitously in every cell type (chapter 5.3.3). However, using a PCR allowing to detect the presence or absence of the stop cassette, it was found that the stop cassette was present in ~50 % of PLS3V5fl_st/wt;CMV-CreX_tg/Y mice despite the presence of Cre, likely due to insufficient penetrance of the Cre enzyme (Figure 20, A). In line with this, the presence of the stop cassette in PLS3V5fl_st/wt;CMV-CreX_tg/Y mice was correlated with an absence of PLS3V5 expression on protein level (Figure 20, B). Importantly, this finding proved that the stop cassette between CAG promoter and PLS3V5 transgene successfully prevents uncontrolled transgene expression. This observation was of particular importance with respect to motor neuron specific overexpression of the PLS3V5 transgene.

Cre expression has been shown to act toxic on cells in a dose dependent manner in

possible (Silver and Livingston, 2001). Moreover, Cre recombinase has been shown to induce proliferation disturbances in Drosophila melanogaster, resulting in phenotypic aberrations (Heidmann and Lehner, 2001). Finally, mice expressing Cre at high levels in spermatids display chromosome scrambling after meiosis II, causing complete male sterility (Schmidt et al., 2000). Since Cre was not necessary to activate PLS3V5 expression once the stop cassette had been deleted in the germline and to avoid unspecific side effects of Cre,

PLS3V5 was outcrossed by breeding PLS3V5fl_st/wt;CMV-CreX_tg/Y mice with C57BL/6N wt animals (chapter 5.3.3).

This way, heterozygous PLS3V5tg/wt animals of the so termed PLS3V5-ubi line (Table 13) were established, invariably carrying the stop-cassette-deleted allele in every cell and tissue. Notably, PLS3V5tg/wt animals displayed completely normal behavior, motoric ability, weight gain and fertility and were indistinguishable by their wt littermates. This suggested that

PLS3V5 overexpression, at least when driven by the CAG promoter, is not per se toxic to

cells. Additionally, the absence of toxic effects of PLS3V5 transgene expression allowed to model the situation in unaffected SMA siblings best possible, namely by ubiquitously overexpressing PLS3V5 in the murine SMA background.

PLS3V5tg/wt animals on C57BL/6N wt background were further analyzed for PLS3V5 expression on mRNA and protein level. By comparing total plastin 3 mRNA levels (here defined as the sum of PLS3V5 + endogenous Pls3) between wt and PLS3V5tg/wt mice, it was shown that plastin 3 is significantly overexpressed in all examined tissues affected by SMA, including brain, spinal cord and muscle (Figure 22, A). However, total plastin 3 mRNA levels were only moderately increased in brain, spinal cord and muscle to 3.6, 3.4 and 21.5 fold of endogenous level, respectively. In other tissue types, e.g. heart or blood, plastin 3 levels were more significantly increased, with 76.2 and 206 fold overexpression. Notice that the fold-expression reflects the difference compared to the endogenous level. Since there is usually no Pls3 expression in blood, a 200 fold increase is easy to be achieved. On the other hand, endogenous Pls3 is highly expressed in spinal cord or muscle, which means that the total plastin 3 level observed in spinal cord or muscle of transgenic mice might still exceed the level in blood. To ultimately compare plastin 3 overexpression levels between different tissue types, an absolute quantification would be necessary.

On protein level, PLS3V5 was neither detectable in brain, nor in spinal cord of PLS3V5tg/wt mice using a PLS3 antibody recognizing both, endogenous Pls3 as well as PLS3V5 (Figure 24, A, B). In this regard, it was only possible to obtain the endogenous Pls3 protein band and only in combination with V5 antibody, also PLS3V5 was detectable. Together, these findings indicated that PLS3V5 is present in brain and spinal cord of PLS3V5tg/wt animals, however, in lower concentrations as was indicated on mRNA level. This finding gets further support from experiments with liver proteins, where total plastin 3 mRNA amount was 7.8 fold increased

compared to endogenous level (Figure 22, A). Concomitantly with an increased PLS3V5 expression on mRNA level, PLS3V5 protein was indeed detectable by using the PLS3 antibody (Figure 24, C`). Nevertheless, as assessed by densitometric analysis, the expression of total plastin 3 protein in liver of PLS3V5tg/wt mice was only ~2 fold increased compared to wt level. Therefore, it is likely that also in brain, spinal cord and muscle the real

plastin 3 upregulation is far below the levels indicated by mRNA expression analysis. These

findings suggest that PLS3V5 mRNA is not likewise converted into PLS3V5 protein, indicating translational regulation of the transgene or other possible negative feedback mechanisms on its own expression. In this context it might be possible that 5` and 3` sequence elements of the targeting vector might account for lower translation rates. Furthermore, the V5-tag of the human PLS3 transgene might negatively impact on protein folding or stability and might thus induce degradation via the ubiquitin-proteasome system.

Additionally, PLS3V5 was successfully activated specifically in motor neurons by crossing

PLS3V5fl_st/wt mice with the motor neuron specific Cre line Hb9-Cre. Via qRT-PCR and Western blotting it was tried to detect PLS3V5 expression on mRNA and protein level in spinal cord of PLS3V5fl_st/wt;Hb9-Cretg/wt mice. Unexpectedly, PLS3V5 expression was detectable neither on mRNA, nor on protein level (data not shown). It is known that only a small percentage, in the range of 3-5 % of the total cell population, of spinal cord consists of motor neurons (Arce et al., 1999, Wiese et al., 2010). Therefore, it must be considered that in spinal cord lysate PLS3V5 mRNA and protein also hold only a very small percentage of total transcripts and proteins. Since no expression differences were detectable between

PLS3V5fl_st/wt;Hb9-Cretg/wt animals and PLS3V5fl_st/wt as well as Hb9-Cretg/wt controls, it was concluded that qRT-PCR and Western blotting methods are not sensitive enough to detect such low levels of PLS3V5 mRNA and protein, respectively. A similar observation has been made in the context of motor neuron specific deletion of the splicing factor Sfrs10 in

Sfrs10fl/fl;Hb9-Cretg/wt mice by our group (Mende et al., 2010). Finally, motor neuron specific expression of PLS3V5 was proven via immunohistochemical stainings using a V5 antibody on spinal cord sections of PLS3V5fl_st/wt;Hb9-Cretg/wt mice.

6.2 The genetic background influences the phenotypic severity in Hung SMA