Analysis of the modifying influence of Plastin 3 (PLS3) onSpinal Muscular Atrophy (SMA) by generation of transgenic mouse models

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by the loss of α-motor neurons in the ventral horn of the spinal cord. Depending on the severity, the clinical spectrum of SMA ranges from early infant death to normal adult life with only mild muscle weakness. To date, no cure is available. SMA is caused by the homozygous loss of the survival motor neuron gene 1 (SMN1). Besides SMN1, another nearly identical copy of the gene is present in the human genome, thus called SMN2. In the SMN2 gene, a C to T transition in exon 7 leads to the disruption of an exonic splicing enhancer, resulting in alternative splicing of SMN2 pre-mRNA and skipping of exon 7 in 90 % of total transcript. However, about 10 % of full length (FL) transcripts are still being produced by SMN2. Through gene duplication events, SMN2 is present in varying copy numbers in the human population, ranging from the total absence to a maximum of 4 SMN2 copies per allele. Since every copy produces about 10 % FL transcript, SMA severity is inversely correlated with SMN2 copy number. For a long time, SMN2 was the only known modifying gene and has therefore been target for the development of therapeutic strategies. The observation of SMN1-deleted patients with either extremely weakened or even absent symptoms in the presence of only a small number of SMN2 copies has in the past been associated with the existence of SMA-modifying genes. Finally, in 2008 the Actin-bundling protein Plastin 3 (PLS3) has been identified as a protective modifying gene showing high expression in asymptomatic homozygously SMN1- deleted siblings of discordant SMA families in our group (Oprea et al., 2008). Cell culture as well as in vivo experiments in a zebrafish SMA model revealed that PLS3 overexpression rescues the axonal outgrowth phenotype. The goal of the present study was to address the question whether PLS3 overexpression is able to rescue the phenotype in an existing SMA mouse model. Furthermore, it was asked which effect PLS3 overexpression has on the development of neuromuscular junctions (NMJ) as well as muscle development and function. Using the Cre/loxP system, transgenic mice were generated expressing a V5-tagged version of human PLS3 (PLS3V5) either ubiquitously or motor neuron specifically (Hb9 promoter). In a next step, PLS3V5 transgenic mice were crossed onto the SMA background using the Hung mouse model. All mice were crossed congenic onto clean C57BL/6N background to exclude any background modifying effects. Transgenic PLS3V5 mice on wildtype (wt) as well as on SMA background were histologically (Motor neurons, NMJs, muscle) and functionally (Motoric tests, weight, survival) analyzed in detail. Ubiquitous as well as motor neuron specific PLS3V5 expression was proven by qRT-PCR, Western blot analysis and immunohistochemistry. PLS3V5 transgenic mice on wt as well as on SMA background showed remarkable influence of PLS3 overexpression on motor neuron and NMJ phenotype: The motor neuron and Acetylcholine receptor (AChR) cluster size was highly increased as compared to wt or SMA littermates. Time course measurements of presynaptic sprouting during the process of axonal pruning revealed highly improved axonal connectivity. These findings raised the question whether the increase of AChR cluster size in transgenic animals is an effect of pre- or postsynaptic PLS3V5 overexpression. To address this issue, motor neuron specific PLS3V5 overexpression was analyzed in wt mice. Strikingly, motor neuron specific overexpression of PLS3V5 was sufficient to increase AChR cluster and muscle fiber size, further strengthening the positive impact of neuronal PLS3V5 expression on neurotransmission. Moreover, electrophysiological analyzes (NMJ quantal content measurements, Motor Unit Number Estimate (MUNE)) will be performed in the future and correlated with the recent findings. Despite this positive impact on axon biology, PLS3 overexpression neither increased survival nor improved motoric ability in the severe type I-like SMA mouse model used in this study. Therefore, it was hypothesized that a certain amount of SMN is required – similar to asymptomatic humans, who own at least 3 SMN2 copies – to rescue the SMA phenotype. To test this hypothesis, the PLS3V5 transgene is currently crossed onto a milder SMA background. Additionally, PLS3V5 was up to now only heterozygously overexpressed in SMA mice. Since also higher PLS3V5 levels might be necessary to finally improve SMA symptoms, PLS3V5 will be homozygously expressed in the SMA background in the future.