VPAC2 receptor agonist BAY 55-9837 increases SMN protein levels and moderates disease phenotype in severe spinal muscular atrophy mouse models

Background Spinal Muscular Atrophy (SMA) is one of the most common inherited causes of infant death and is caused by the loss of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene. One of the treatment strategies for SMA is to induce the expression of the protein from the homologous SMN2 gene, a rescuing paralog for SMA. Methods and results Here we demonstrate the promise of pharmacological modulation of SMN2 gene by BAY 55-9837, an agonist of the vasoactive intestinal peptide receptor 2 (VPAC2), a member of G protein coupled receptor family. Treatment with BAY 55-9837 lead to induction of SMN protein levels via activation of MAPK14 or p38 pathway in vitro. Importantly, BAY 55-9837 also ameliorated disease phenotype in severe SMA mouse models. Conclusion Our findings suggest the VPAC2 pathway is a potential SMA therapeutic target.


Background
Spinal muscular atrophy (SMA), is an untreatable recessive neuromuscular disorder; with an incidence of 1:11000, it is a leading genetic cause of pediatric death [1]. The loss of lower motor neurons from the ventral horn of spinal cord is the major pathological feature of the disease and results in generalized weakness, progressive muscle loss and respiratory failure [2]. SMA is caused by the pathologic reduction in survival of motor neuron (SMN) protein levels due to deletions and mutations in SMN1 gene [3]. Although the complete loss of SMN protein is embryonically lethal, the presence of the paralogous SMN2, a result of a recent duplication event, which produces a limited full length SMN mRNA (~10%) precludes this outcome in humans [4,5]. Thus all SMA patients have 2 or more copies of SMN2 gene which in part compensates for the loss of SMN1 gene. The inverse correlation between the severity of the disease phenotype and copy number of SMN2, both confirms the gene's disease modifying function and has made the induction of SMN2 a common SMA therapeutic goal. In this regard we have showed the post transcriptional stabilization of SMN mRNA through activation of p38 pathway leads increased SMN levels [6]. We have recently reported that the activation of the p38 pathway through celecoxib upregulates SMN protein levels and can ameliorate disease phenotype in SMA mouse model [7]. In this regard, Vasoactive intestinal peptide receptor 2 (VPAC2), a member of G protein coupled receptor family when activated has been reported to activate p38 pathway in vivo [8,9].
We thus decided to assess the blood brain barrier (BBB) penetrant VPAC2 receptor agonist BAY 55-9837 for its potential SMA therapeutic utility. We show here that BAY 55-9837 conferred an increase in SMN protein levels via p38 activation in human neuronal cells. Importantly, we show that treatment with BAY 55-9837 also increases brain and spinal cord SMN protein levels as well as improving disease phenotype and survival in a severe SMA mouse model. Our results provide further evidence that p38 MAPK pathway activators act as potential therapeutic compounds for the treatment of SMA and identify the VPAC pathway as one means of achieving such activation.

Animals
All protocols were approved by Animal Care and Veterinary Services (ACVS) and Ethics board of University of Ottawa. All experiments were carried out in accordance with the Canadian Institute of Health Research (CIHR) Guidebook and ACVS legislation. CD-1 mice were obtained from Charles River Laboratory. The original breeding pair of heterozygous SMAΔ7 (mSmn+/-, hSMN2+/+, hSMNΔ7+/+; stock# 005025), Taiwanese mice (Smn1 tm1Hung Tg(SMN2)2Hung/J; stock# 005058) and heterozygous Smn knock-out mice (Smn +/− ) on the FVB background were provided by the Jackson Laboratory. The animals were maintained in an air-conditioned ventilated animal facility. Survival, righting time and weight were monitored daily as described by Aviva et al [10].
BAY 55-9837 administration BAY 55-9837 was diluted in PBS/dH 2 O and administered through IP injection using a 30-gauge needle (0.2 mg/kg dose). Control animals received equal volumes of vehicle alone. SMAΔ7 and Taiw/Jax SMA mice were genotyped at P0 and BAY 55-9837 treatment was started from P1. Animals were sacrificed within twenty four hours of the final dose.

Western blot analysis
Cells were washed 2 times with 1 ml PBS (1X) and lysed in 150μl RIPA buffer containing 10 mg/ml each of aprotinin, PMSF and leupeptin (all from Sigma), 5 mM β-Glycerolphosphate, 50 mM NaF and 0.2 μM sodium orthovanadate for 30 min at 4°C, followed by centrifugation at 13 000 × g for 30 min; supernatants were then collected and kept frozen at -20°C. Tissue samples were homogenized in 0.5 ml RIPA (10 mg/ml each of aprotinin, PMSF and leupeptin) and then sonicated for 15 seconds. Total protein concentrations were determined by Bradford protein assay using a Bio-Rad protein assay kit. For western blot analysis, protein samples were separated by 11% SDS-PAGE. Proteins were subsequently transferred onto nitrocellulose membrane and incubated in blocking solution (PBS, 5% non-fat milk, 0.2% Tween-20) for 1 h at room temperature followed by overnight incubation with primary antibody at 4°C at the dilution prescribed by the manufacturer. Membranes were washed with PBS-T (PBS, and 0.2% Tween-20) 3 times followed by incubation with secondary antibody (anti-mouse or rabbit, Cell signalling) for 1 h at room temperature. Antibody complexes were visualized by autoradiography using the ECL Plus and ECL western blotting detection systems (GE Healthcare). Quantification was performed by scanning the autoradiographs and signal intensities were determined by densitometric analysis using the ImageJ program.

Primer sequences For genotyping
Genotyping was performed as previously described by Aviva et al [10] for SMAΔ7 mice using the following primers mSmn WT Forward: 5′-TCTGTGTTCGTGCGTGGTG ACTTT-3′.

Statistical methods
GraphPad Prism software package was used for the Kaplan-Meier survival analysis. The log-rank test was used and survival curves were considered significantly different at P < 0.05.
Data in figures (histograms, points on graphs) are mean values with the standard error mean (SEM) shown as error bars. The Student's two-tail t test was used to test for statistical differences between samples and were considered significantly different at P < 0.05.

Results and discussion
SMA is a frequently severe neurodegenerative disease which most frequently affects children; many of them do not survive beyond the first few years of life. Although there is no effective therapy for SMA, one translational approach is to induce the paralogous gene SMN2. This results in the production of more SMN protein, which can partially compensate for the loss of SMN1 gene and to moderate the disease phenotype.
BAY 55-9837 treatment upregulates SMN protein in vitro VPAC2 receptor activation has been reported to activate the p38 kinase pathway [8,9] which, in turn, we have shown to stabilize SMN transcript and increase SMN protein level [6]. In order to assess the potential of VPAC-2 receptor activation in the regulation of SMN gene expression; human NT2, mouse MN-1 cells and SMA I patient fibroblasts were treated with VPAC2 receptor agonist BAY 55-9837 (25 μM) for 24 h and subsequently harvested for western blot analysis. SMN protein levels were found to be increased by~2 fold in all cell lines upon treatment with BAY 55-9837 (Figure 1a-f). These results were encouraging in that the increase in SMN protein levels was observed in both neuronal cell lines and patient fibroblasts suggesting that the induction was not specific to a given cell line.

BAY 55-9837 conferred increase in SMN protein levels is mediated by p38 MAPK activation
The p38 MAPK pathway regulates a number of cellular process including post-transcriptional stabilization of a distinct class of mRNAs that contain AU rich elements (ARE) mapping to their 3′ UTRs [12][13][14][15][16]. This class of  includes that encoded by SMN2 [14]; we have previously reported that p38 MAPK increases SMN protein expression by virtue of the binding of HUR protein to SMN2 3′UTR [6]. The VPAC2 receptor agonist Ro 25-1553 has been previously shown to activate the p38 MAPK pathway [9]; we wished to confirm that BAY 55-9837 could elicit the same p38 activation and that this was underlying the observed SMN protein induction. NT2 cells were therefore treated with BAY 55-9837 and then harvested; western blot analysis at the indicated time intervals revealed within one hour an increase in the ratio of phosphorylated/ total p38 protein (up to 24 hrs after BAY 55-9837 treatment) consistent with p38 activation (Figure 2a-b). p38 MAPK activation was concurrent with the increase in SMN protein levels in NT2 cells (Figure 2a & c). To confirm the role of p38 in the observed SMN protein induction, NT2 cells were pre-treated with the p38 inhibiting agent SB-239063 [17] for 2 h prior to treatment with BAY 55-9837 for 24 h. Western blot analysis revealed that p38 inhibition effectively blocked the BAY 55-9837-mediated increase in SMN protein (Figure 2d & e). These results demonstrate that activation of p38 pathway presumable through binding of VPAC2 receptor agonist to its receptor confers the increase in SMN protein levels observed upon BAY 55-9837 treatment. This result is consistent with our previous observation of increased SMN protein levels conferred by the p38 MAPK activating small compounds anisomycin and celecoxib [6,7].

BAY 55-9837 treatment upregulates SMN protein levels in vivo
In order confirm that BAY 55-9837-mediated SMN protein induction extends to the in vivo setting, a dose finding study was initiated. CD-1 mice were given daily intraperitoneal (IP) BAY 55-9837 injections for 5 days 0.02, 0.2 and 2 mg/kg and brain and spinal cord samples then isolated for western blot analysis. Increased SMN protein levels were observed both in brain (Additional file 1: Figure S1a   We next explored the impact of BAY 55-9837-induced SMN upregulation in a severe mouse model of the disease (SMAΔ7 mouse; mSmn-/-;hSMN2+/+, hSMNΔ7+/+ [18]). SMAΔ7 mice were given 0.2 mg/kg BAY 55-9837 IP injections twice daily from P1 until P6. Mice were euthanized 24 hours after their last treatment and brain, spinal cord, muscle and heart samples then harvested for western blot analysis. Mice treated with BAY 55-9837 demonstrated an approximate doubling in SMN2-derived full length SMN protein levels in all tissues except brain where an approximate quadrupling of SMN protein was observed when compared with vehicle treated animals Figure 3 BAY 55-9837 upregulates SMN protein in SMA mouse model. SMAΔ7 mice were treated daily with saline or BAY 55-9837 (200 μg/kg) from P1 for 6 days, then sacrificed at P7. Brain, spinal cord,skeletal muscle and heart tissues were harvested for western blot analysis. Representative western blots showing effect of BAY 55-9837 on SMN protein in brain (a), spinal cord (c), muscle (e) and heart (g) samples of SMAΔ7 mice treated with Saline (control, lane 1, 2 & 3) or BAY 55-9837 (treatment lane 1, 2 & 3 respectively) (each lane represents individual animal; all lanes were run on the same gel but were non-contiguous). Densitometric quantification of SMN relative to Actin/Tubulin [mean + SEM (bars)] is shown for brain (b; n=11), spinal cord (d; n=6), muscle (f; n=11) and heart (h; n=11) samples. *P < 0.05; **P < 0.01; t-test.
( Figure 3). In keeping with these results, VPAC2 receptors are expressed in CNS as well as in peripheral tissues [19][20][21][22][23][24][25]. The most modest (although still significant) induction of SMN protein was seen in muscle tissues compared to saline treated SMA mice, a possible result of the comparatively low amount of p38 transcript in SMA I muscle compared with normal muscle [26].
BAY 55-9837 treatment improves disease phenotype in SMA mice model We next examined the effect of BAY 55-9837 treatment on SMAΔ7 mouse disease phenotype. The SMAΔ7 mice are significantly underweight and have reduced motor activity compared to heterozygous and WT littermates. SMAΔ7 mice were given twice daily BAY 55-9837 or vehicle IP injections starting at P1; their weight and motor function were assessed daily. SMAΔ7 mice treated with BAY 55-9837 showed significant improvement in weight gain and motor function (as assessed by righting time), as compared to vehicle-treated SMAΔ7 mice (Figure 4a & b).
SMA is primarily considered as a motor neuron disease and consequently treatment strategies focus on drugs which can cross the blood brain barrier (BBB) to target tissues within central nervous system (CNS). However several recent studies challenge this notion and suggest that SMN has function above and beyond motor neurons and reclassify SMA as a multi-system disorder (including cardiovascular, peripheral necrosis, pancreatic and liver defects) [28][29][30][31][32][33][34][35][36]. In this regard the widespread presence of the VPAC2 receptor augurs well for this pathway as a therapeutic SMA target [25].

Conclusion
Re-purposing drugs for distinct disease indications is becoming a more common practice given the approximately 7000 orphan genetic disorders that are estimated to exist. This approach is both cost-effective as well as shortening the path to treatment for significant (and currently untreatable) disorders such as SMA. In the current study, BAY 55-9837 initially developed for the treatment of diabetes [37,38] has been used as a p38 activating compound for the treatment of murine SMA. Our results demonstrate that VPAC2 receptor agonist BAY 55-9837 increases SMN protein levels and attenuates disease progression in two distinct severe SMA mouse models ( Figure 5) providing a proof of concept and support for other VPAC2 agonists/p38 activating compounds to be tested as effective SMA therapies. Although the literature on the safety profile of BAY 55-9837 is divided (e.g. 38 and 39), in our experiments we did not observe any adverse effects. Nevertheless further work to obtain comprehensive safety profile for BAY 55-9837 will be beneficial [39]. This study provides a good supportive evidence as well as functional insight how p38 pathway can be targeted for its potential application towards development of therapeutics for SMA.

Additional file
Additional file 1: Figure S1. BAY 55-9837 upregulates Smn protein in wild type mice. 4 weeks old CD-1 wild type mice were treated daily with