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

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 SMA∆7 (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 was diluted in PBS/dH₂O and administered through IP injection using a 30-gauge needle (0.2 mg/kg dose). Control animals received equal volumes of vehicle alone. SMA∆7 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.

BAY 55-9837 was purchased from Tocris Bioscience. p38 inhibitor SB239063 was purchased from Sigma. The antibodies used in this study were SMN/Smn (BD Transduction Laboratories), Actin (Abcam), Tubulin (Abcam), Phospho-p38 (Cell signalling) and Total p38 (Cell signalling).

Human neuron-committed teratocarcinoma (NT2), mouse motor neuron derived (MN-1) cells and SMA type I patient fibroblasts were maintained in standard conditions (37°C in a 5% CO₂ humidified atmosphere) in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 1% antibiotics (100 units/ml penicillin-streptomycin) and 2 mM glutamate.

NT2 or MN-1 cells were seeded in 6 well plates (5 × 10⁵cells/well) and treated 24 h later with BAY 55-9837 (0.25 μM) for 24h. For time course experiment, NT2 cells were seeded in 6 well plates (5 × 10⁵cells/well) and treated 24h later with BAY 55-9837 (0.25 μM) for up to 24h. For p38 inhibitor treatment, NT2 were seeded in 6 well plates (5 × 10⁵cells/well) and pre-treated with p38 inhibitor SB239063 for 2 h followed by BAY 55-9837 treatment (0.25 μM) for 24 h.

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.

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.

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.

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[12‐16]. This class of mRNA 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].

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 file1: Figure S1a & b) and spinal cord samples (Additional file1: Figure S1c & d) following BAY 55-9837 treatment with the greatest induction (~2 fold), seen at 0.2 mg/kg dose in CD-1 mice.

We next explored the impact of BAY 55-9837-induced SMN upregulation in a severe mouse model of the disease (SMA∆7 mouse; mSmn-/-;hSMN2+/+, hSMNΔ7+/+[18]). SMA∆7 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). In keeping with these results, VPAC2 receptors are expressed in CNS as well as in peripheral tissues[19‐25]. 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].

We next examined the effect of BAY 55-9837 treatment on SMA∆7 mouse disease phenotype. The SMA∆7 mice are significantly underweight and have reduced motor activity compared to heterozygous and WT littermates. SMA∆7 mice were given twice daily BAY 55-9837 or vehicle IP injections starting at P1; their weight and motor function were assessed daily. SMA∆7 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 SMA∆7 mice (Figure 4a & b).

We also examined the impact of BAY 55-9837 on survival in two different severe SMA mouse models (SMA∆7[18] and Taiw/Jax-SMA{Cross between Taiwanese mice (Smn1 ^tm1Hung Tg(SMN2)2Hung/J; stock# 005058) and heterozygous Smn knock-out mice (Smn^+/-)[11]}. Significant extension of survival was observed in both mouse models upon treatment with BAY 55-9837 (median survival of 19.5 d for SMA∆7 mice from 14d and 12 days from 8d for Taiw/Jax SMA mice) as compared with vehicle-treated (Figure 4c & d). To account for the treatment effect variability between various laboratories and mouse models, the ratio of median survival of treated to non treated animals was used to assess drug response on survival. With BAY 55-9837 we have achieved a ratio of 1.39 (19.5d/14d for SMA∆7) & 1.5 (12d/8d for Taiw/Jax-SMA) for the two different models. These numbers compare favourably with the other small compounds previously used for SMA treatment such as TSA (1.2; 19d/16d albeit P5 TSA initiation in SMA∆7)[10], SAHA (1.3; 12.9d/9.9d in Taiw/Jax-SMA)[11], celecoxib (1.38; 18d/13d)[7] and Prolactin (1.5; 21d/14d)[27].

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)[28‐36]. In this regard the widespread presence of the VPAC2 receptor augurs well for this pathway as a therapeutic SMA target[25].