Separating the Wheat from the Chaff: The Use of Upstream Regulator Analysis to Identify True Differential Expression of Single Genes within Transcriptomic Datasets.

The development of DNA microarray and RNA-sequencing technology has led to an explosion in the generation of transcriptomic differential expression data under a wide range of biologic systems including those recapitulating the monogenic muscular dystrophies. Data generation has increased exponentially due in large part to new platforms, improved cost-effectiveness, and processing speed. However, reproducibility and thus reliability of data remain a central issue, particularly when resource constraints limit experiments to single replicates. This was observed firsthand in a recent rare disease drug repurposing project involving RNA-seq-based transcriptomic profiling of primary cerebrocortical cultures incubated with clinic-ready blood-brain penetrant drugs. Given the low validation rates obtained for single differential expression genes, alternative approaches to identify with greater confidence genes that were truly differentially expressed in our dataset were explored. Here we outline a method for differential expression data analysis in the context of drug repurposing for rare diseases that incorporates the statistical rigour of the multigene analysis to bring greater predictive power in assessing individual gene modulation. Ingenuity Pathway Analysis upstream regulator analysis was applied to the differentially expressed genes from the Care4Rare Neuron Drug Screen transcriptomic database to identify three distinct signaling networks each perturbed by a different drug and involving a central upstream modulating protein: levothyroxine (DIO3), hydroxyurea (FOXM1), dexamethasone (PPARD). Differential expression of upstream regulator network related genes was next assessed in in vitro and in vivo systems by qPCR, revealing 5× and 10× increases in validation rates, respectively, when compared with our previous experience with individual genes in the dataset not associated with a network. The Ingenuity Pathway Analysis based gene prioritization may increase the predictive value of drug-gene interactions, especially in the context of assessing single-gene modulation in single-replicate experiments.

Celecoxib increases SMN and survival in a severe spinal muscular atrophy mouse model via p38 pathway activation.

The loss of functional Survival Motor Neuron (SMN) protein due to mutations or deletion in the SMN1 gene causes autosomal recessive neurodegenerative spinal muscle atrophy (SMA). A potential treatment strategy for SMA is to upregulate the amount of SMN protein originating from the highly homologous SMN2 gene, compensating in part for the absence of the functional SMN1 gene. We have previously shown that in vitro activation of the p38 pathway stabilizes and increases SMN mRNA levels leading to increased SMN protein levels. In this report, we explore the impact of the p38 activating, FDA-approved, blood brain barrier permeating compound celecoxib on SMN levels in vitro and in a mouse model of SMA. We demonstrate a significant induction of SMN protein levels in human and mouse neuronal cells upon treatment with celecoxib. We show that activation of the p38 pathway by low doses celecoxib increases SMN protein in a HuR protein-dependent manner. Furthermore, celecoxib treatment induces SMN expression in brain and spinal cord samples of wild-type mice in vivo. Critically, celecoxib treatment increased SMN levels, improved motor function and enhanced survival in a severe SMA mouse model. Our results identify low dose celecoxib as a potential new member of the SMA therapeutic armamentarium.

p38 Mitogen-activated protein kinase stabilizes SMN mRNA through RNA binding protein HuR.

Spinal muscle atrophy (SMA) is an autosomal recessive neurodegenerative disease which is characterized by the loss of alpha motor neurons resulting in progressive muscle atrophy. Reduced amount of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene is the cause of SMA. A potential treatment strategy for SMA is to upregulate levels of SMN protein originating from the SMN2 gene compensating in part for the absence of functional SMN1 gene. Although there exists a sizeable literature on SMN2 inducing compounds, there is comparatively less known about the signaling pathways which modulate SMN levels. Here, we report a significant induction in SMN mRNA and protein following p38 activation by Anisomycin. We demonstrate that Anisomycin activation of p38 causes a rapid cytoplasmic accumulation of HuR, a RNA binding protein which binds to and stabilizes the AU-rich element within the SMN transcript. The stabilization of SMN mRNA, rather than transcriptional induction results in an increase in SMN protein. Our demonstration of SMN protein regulation through the p38 pathway and the role of HuR in this modulation may help in the identification and characterization of p38 pathway activators as potential therapeutic compounds for the treatment of SMA.

High-throughput kinome-RNAi screen identifies protein kinase R activator (PACT) as a novel genetic modifier of CUG foci integrity in myotonic dystrophy type 1 (DM1).

Myotonic Dystrophy Type 1 (DM1) is the most common form of adult muscular dystrophy (~1:8000). In DM1, expansion of CTG trinucleotide repeats in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene results in DMPK mRNA hairpin structures which aggregate as insoluble ribonuclear foci and sequester several RNA-binding proteins. The resulting sequestration and misregulation of important splicing factors, such as muscleblind-like 1 (MBNL1), causes the aberrant expression of fetal transcripts for several genes that contribute to the disease phenotype. Previous work has shown that antisense oligonucleotide-mediated disaggregation of the intranuclear foci has the potential to reverse downstream anomalies. To explore whether the nuclear foci are, to some extent, controlled by cell signalling pathways, we have performed a screen using a small interfering RNA (siRNA) library targeting 518 protein kinases to look at kinomic modulation of foci integrity. RNA foci were visualized by in situ hybridization of a fluorescent-tagged (CAG)10 probe directed towards the expanded DMPK mRNA and the cross-sectional area and number of foci per nuclei were recorded. From our screen, we have identified PACT (protein kinase R (PKR) activator) as a novel modulator of foci integrity and have shown that PACT knockdown can both increase MBNL1 protein levels; however, these changes are not suffcient for significant correction of downstream spliceopathies.

Anisomycin Activates Utrophin Upregulation Through a p38 Signaling Pathway.

Duchenne muscular dystrophy is a recessive X-linked disease characterized by progressive muscle wasting; cardiac or respiratory failure causes death in most patients by the third decade.  The disease is caused by mutations in the dystrophin gene that lead to a loss of functional dystrophin protein. Although there are currently few treatments for Duchenne muscular dystrophy, previous reports have shown that upregulating the dystrophin paralog utrophin in Duchenne muscular dystrophy mouse models is a promising therapeutic strategy. We conducted in silico mining of the Connectivity Map database for utrophin-inducing agents, identifying the p38-activating antibiotic anisomycin. Treatments of C2C12, undifferentiated murine myoblasts, and mdx primary myoblasts with anisomycin conferred increases in utrophin protein levels through p38 pathway activation.  Anisomycin also induced utrophin protein levels in the diaphragm of mdx mice.  Our study shows that repositioning small molecules such as anisomycin may prove to have Duchenne muscular dystrophy clinical utility.

Current and emerging treatment options for spinal muscular atrophy.

Spinal muscular atrophy is one of the most common inherited neuromuscular conditions; our understanding of the genetic pathology and translational research coming from this insight has made significant progress over the past decade. This short review provides the background of the disease along with the bench to bedside progress of some promising treatment options to develop better understanding of the present state of the disease.

Sodium Channel Inhibitors Reduce DMPK mRNA and Protein.

Myotonic dystrophy type 1 (DM1) is caused by an expanded trinucleotide (CTG)n tract in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene. This results in the aggregation of an expanded mRNA forming toxic intranuclear foci which sequester splicing factors. We believe down-regulation of DMPK mRNA represents a potential, and as yet unexplored, DM1 therapeutic avenue. Consequently, a computational screen for agents which down-regulate DMPK mRNA was undertaken, unexpectedly identifying the sodium channel blockers mexiletine, prilocaine, procainamide, and sparteine as effective suppressors of DMPK mRNA. Analysis of DMPK mRNA in C2C12 myoblasts following treatment with these agents revealed a reduction in the mRNA levels. In vivo analysis of CD1 mice also showed DMPK mRNA and protein down-regulation. The role of DMPK mRNA suppression in the documented efficacy of this class of compounds in DM1 is worthy of further investigation.

Human Growth Hormone Increases SMN Expression and Survival in Severe Spinal Muscular Atrophy Mouse Model.

BACKGROUND: Autosomal recessive spinal muscle atrophy (SMA) is characterized by the loss of α motor neurons resulting in progressive muscle loss and respiratory failure. SMA is one of the most common inherited causes of infant death with a carrier frequency of 1 in 50 and a calculated prevalence of about 1 in 11,000 live births in the US. The low amount of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene causes SMA. OBJECTIVE: A potential treatment strategy for SMA is to upregulate levels of SMN protein originating from the paralog SMN2 gene compensating in part for the absence of the SMN1 gene. Our group has previously shown that activation of the STAT5 pathway by lactation hormone prolactin (PRL) increased SMN levels, improved motor function and enhanced survival in a severe SMA mouse model. Given that human growth hormone (HGH) is also known to activate the STAT5 signalling pathway and is already used extensively in clinical settings, we thus elected to assess its impact on SMN levels. METHODS AND RESULTS: Administration of HGH in NT2 cells activated STAT5 pathway which resulted into significant induction in SMN protein levels. Furthermore, systemic administration of HGH to transgenic SMA mice induced SMN protein levels in the brain and spinal cord samples. Critically, HGH treatment improved disease phenotype and increased survival in two severe SMA mouse models. CONCLUSIONS: Our results confirm earlier work suggesting STAT5 pathway activators as potential therapeutic compounds for the treatment of SMA and identify HGH as one such promising agent.

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.

Prolactin increases SMN expression and survival in a mouse model of severe spinal muscular atrophy via the STAT5 pathway.

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease that is characterized by the loss of motor neurons, resulting in progressive muscle atrophy. It is caused by the loss of functional survival motor neuron (SMN) protein due to mutations or deletion in the SMN1 gene. A potential treatment strategy for SMA is to upregulate levels of SMN protein. Several agents that activate STAT5 in human and mouse cell lines enhance SMN expression from the SMN2 gene and can compensate, at least in part, for the loss of production of a functional protein from SMN1. Here, we have shown that prolactin (PRL) increases SMN levels via activation of the STAT5 pathway. PRL increased SMN mRNA and protein levels in cultured human and mouse neuronal cells. Administration of STAT5-specific siRNA blocked the effects of PRL, indicating that the PRL-induced transcriptional upregulation of the SMN-encoding gene was mediated by activation of STAT5. Furthermore, systemic administration of PRL to WT mice induced SMN expression in the brain and spinal cord. Critically, PRL treatment increased SMN levels, improved motor function, and enhanced survival in a mouse model of severe SMA. Our results confirm earlier work suggesting STAT5 pathway activators as potential therapeutic compounds for the treatment of SMA and identify PRL as one such promising agent.