Utility of survival motor neuron ELISA for spinal muscular atrophy clinical and preclinical analyses

Objectives

Genetic defects leading to the reduction of the survival motor neuron protein (SMN) are a causal factor for Spinal Muscular Atrophy (SMA). While there are a number of therapies under evaluation as potential treatments for SMA, there is a critical lack of a biomarker method for assessing efficacy of therapeutic interventions, particularly those targeting upregulation of SMN protein levels. Towards this end we have engaged in developing an immunoassay capable of accurately measuring SMN protein levels in blood, specifically in peripheral blood mononuclear cells (PBMCs), as a tool for validating SMN protein as a biomarker in SMA.

Methods

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed and validated for measuring SMN protein in human PBMCs and other cell lysates. Protocols for detection and extraction of SMN from transgenic SMA mouse tissues were also developed.

Results

The assay sensitivity for human SMN is 50 pg/mL. Initial analysis reveals that PBMCs yield enough SMN to analyze from blood volumes of less than 1 mL, and SMA Type I patients'' PBMCs show ∼90% reduction of SMN protein compared to normal adults. The ELISA can reliably quantify SMN protein in human and mouse PBMCs and muscle, as well as brain, and spinal cord from a mouse model of severe SMA.

Conclusions

This SMN ELISA assay enables the reliable, quantitative and rapid measurement of SMN in healthy human and SMA patient PBMCs, muscle and fibroblasts. SMN was also detected in several tissues in a mouse model of SMA, as well as in wildtype mouse tissues. This SMN ELISA has general translational applicability to both preclinical and clinical research efforts.     

A novel cell immunoassay to measure survival of motor neurons protein in blood cells

Background  

The motor neuron degenerative disease spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality and is caused by mutations in the survival of motor neurons (SMN) gene that reduce the expression levels of the SMN protein. A major goal of current therapeutic approaches is to increase SMN levels in SMA patients. The purpose of this study was to develop a reliable assay to measure SMN protein levels from peripheral blood samples.     

The E3 ubiquitin ligase mind bomb 1 ubiquitinates and promotes the degradation of survival of motor neuron protein

Spinal muscular atrophy is an inherited motor neuron disease that results from a deficiency of the survival of motor neuron (SMN) protein. SMN is ubiquitinated and degraded through the ubiquitin proteasome system (UPS). We have previously shown that proteasome inhibition increases SMN protein levels, improves motor function, and reduces spinal cord, muscle, and neuromuscular junction pathology of spinal muscular atrophy (SMA) mice. Specific targets in the UPS may be more efficacious and less toxic. In this study, we show that the E3 ubiquitin ligase, mind bomb 1 (Mib1), interacts with and ubiquitinates SMN and facilitates its degradation. Knocking down Mib1 levels increases SMN protein levels in cultured cells. Also, knocking down the Mib1 orthologue improves neuromuscular function in Caenorhabditis elegans deficient in SMN. These findings demonstrate that Mib1 ubiquitinates and catalyzes the degradation of SMN, and thus represents a novel therapeutic target for SMA.     

Spinal Muscular Atrophy and the Antiapoptotic Role of Survival of Motor Neuron (SMN) Protein

Spinal muscular atrophy (SMA) is a devastating and often fatal neurodegenerative disease that affects spinal motor neurons and leads to progressive muscle wasting and paralysis. The survival of motor neuron (SMN) gene is mutated or deleted in most forms of SMA, which results in a critical reduction in SMN protein. Motor neurons appear particularly vulnerable to reduced SMN protein levels. Therefore, understanding the functional role of SMN in protecting motor neurons from degeneration is an essential prerequisite for the design of effective therapies for SMA. To this end, there is increasing evidence indicating a key regulatory antiapoptotic role for the SMN protein that is important in motor neuron survival. The aim of this review is to highlight key findings that support an antiapoptotic role for SMN in modulating cell survival and raise possibilities for new therapeutic approaches.     

Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is currently incurable. SMA is caused by decreased levels of the survival motor neuron protein (SMN), as a result of loss or mutation of SMN1. Although the SMN1 homolog SMN2 also produces some SMN protein, it does not fully compensate for the loss or dysfunction of SMN1. Salbutamol, a β2-adrenergic receptor agonist and well-known bronchodilator used in asthma patients, has recently been shown to ameliorate symptoms in SMA patients. However, the precise mechanism of salbutamol action is unclear. We treated SMA fibroblast cells lacking SMN1 and HeLa cells with salbutamol and analyzed SMN2 mRNA and SMN protein levels in SMA fibroblasts, and changes in SMN protein ubiquitination in HeLa cells. Salbutamol increased SMN protein levels in a dose-dependent manner in SMA fibroblast cells lacking SMN1, though no significant changes in SMN2 mRNA levels were observed. Notably, the salbutamol-induced increase in SMN was blocked by a protein kinase A (PKA) inhibitor and deubiquitinase inhibitor, respectively. Co-immunoprecipitation assay using HeLa cells showed that ubiquitinated SMN levels decreased in the presence of salbutamol, suggesting that salbutamol inhibited ubiquitination. The results of this study suggest that salbutamol may increase SMN protein levels in SMA by inhibiting ubiquitin-mediated SMN degradation via activating β2-adrenergic receptor-PKA pathways.     

The contribution of mouse models to understanding the pathogenesis of spinal muscular atrophy

Spinal muscular atrophy (SMA), which is caused by inactivating mutations in the survival motor neuron 1 (SMN1) gene, is characterized by loss of lower motor neurons in the spinal cord. The gene encoding SMN is very highly conserved in evolution, allowing the disease to be modeled in a range of species. The similarities in anatomy and physiology to the human neuromuscular system, coupled with the ease of genetic manipulation, make the mouse the most suitable model for exploring the basic pathogenesis of motor neuron loss and for testing potential treatments. Therapies that increase SMN levels, either through direct viral delivery or by enhancing full-length SMN protein expression from the SMN1 paralog, SMN2, are approaching the translational stage of development. It is therefore timely to consider the role of mouse models in addressing aspects of disease pathogenesis that are most relevant to SMA therapy. Here, we review evidence suggesting that the apparent selective vulnerability of motor neurons to SMN deficiency is relative rather than absolute, signifying that therapies will need to be delivered systemically. We also consider evidence from mouse models suggesting that SMN has its predominant action on the neuromuscular system in early postnatal life, during a discrete phase of development. Data from these experiments suggest that the timing of therapy to increase SMN levels might be crucial. The extent to which SMN is required for the maintenance of motor neurons in later life and whether augmenting its levels could treat degenerative motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), requires further exploration.     

Increased susceptibility of spinal muscular atrophy fibroblasts to camptothecin is p53-independent

Background  

Deletion or mutation(s) of the survival motor neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA). The SMN protein is known to play a role in RNA metabolism, neurite outgrowth, and cell survival. Yet, it remains unclear how SMN deficiency causes selective motor neuron death and muscle atrophy seen in SMA. Previously, we have shown that skin fibroblasts from SMA patients are more sensitive to the DNA topoisomerase I inhibitor camptothecin, supporting a role for SMN in cell survival. Here, we examine the potential mechanism of camptothecin sensitivity in SMA fibroblasts.     

Motor defects in a Drosophila model for spinal muscular atrophy result from SMN depletion during early neurogenesis

Spinal muscular atrophy (SMA) is the most common autosomal recessive neurodegenerative disease, and is characterised by spinal motor neuron loss, impaired motor function and, often, premature death. Mutations and deletions in the widely expressed survival motor neuron 1 (SMN1) gene cause SMA; however, the mechanisms underlying the selectivity of motor neuron degeneration are not well understood. Although SMA is degenerative in nature, SMN function during embryonic and early postnatal development appears to be essential for motor neuron survival in animal models and humans. Notwithstanding, how developmental defects contribute to the subversion of postnatal and adult motor function remains elusive. Here, in a Drosophila SMA model, we show that neurodevelopmental defects precede gross locomotor dysfunction in larvae. Furthermore, to specifically address the relevance of SMN during neurogenesis and in neurogenic cell types, we show that SMN knockdown using neuroblast-specific and pan-neuronal drivers, but not differentiated neuron or glial cell drivers, impairs adult motor function. Using targeted knockdown, we further restricted SMN manipulation in neuroblasts to a defined time window. Our aim was to express specifically in the neuronal progenitor cell types that have not formed synapses, and thus a time that precedes neuromuscular junction formation and maturation. By restoring SMN levels in these distinct neuronal population, we partially rescue the larval locomotor defects of Smn mutants. Finally, combinatorial SMN knockdown in immature and mature neurons synergistically enhances the locomotor and survival phenotypes. Our in-vivo study is the first to directly rescue the motor defects of an SMA model by expressing Smn in an identifiable population of Drosophila neuroblasts and developing neurons, highlighting that neuronal sensitivity to SMN loss may arise before synapse establishment and nerve cell maturation.     

A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy

5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.     

Ribonucleoprotein assembly defects correlate with spinal muscular atrophy severity and preferentially affect a subset of spliceosomal snRNPs

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival motor neuron (SMN) protein. SMN together with Gemins2-8 and unrip proteins form a macromolecular complex that functions in the assembly of small nuclear ribonucleoproteins (snRNPs) of both the major and the minor splicing pathways. It is not known whether the levels of spliceosomal snRNPs are decreased in SMA. Here we analyzed the consequence of SMN deficiency on snRNP metabolism in the spinal cord of mouse models of SMA with differing phenotypic severities. We demonstrate that the expression of a subset of Gemin proteins and snRNP assembly activity are dramatically reduced in the spinal cord of severe SMA mice. Comparative analysis of different tissues highlights a similar decrease in SMN levels and a strong impairment of snRNP assembly in tissues of severe SMA mice, although the defect appears smaller in kidney than in neural tissue. We further show that the extent of reduction in both Gemin proteins expression and snRNP assembly activity in the spinal cord of SMA mice correlates with disease severity. Remarkably, defective SMN complex function in snRNP assembly causes a significant decrease in the levels of a subset of snRNPs and preferentially affects the accumulation of U11 snRNP--a component of the minor spliceosome--in tissues of severe SMA mice. Thus, impairment of a ubiquitous function of SMN changes the snRNP profile of SMA tissues by unevenly altering the normal proportion of endogenous snRNPs. These findings are consistent with the hypothesis that SMN deficiency affects the splicing machinery and in particular the minor splicing pathway of a rare class of introns in SMA.     

Quantitative Analyses of SMN1 and SMN2 Based on Real-Time LightCycler PCR: Fast and Highly Reliable Carrier Testing and Prediction of Severity of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans, caused by homozygous absence of the survival motor neuron gene 1 (SMN1). SMN2, a copy gene, influences the severity of SMA and may be used in somatic gene therapy of patients with SMA in the future. We present a new, fast, and highly reliable quantitative test, based on real-time LightCycler PCR that amplifies either SMN1 or SMN2. The SMN1 copies were determined and validated in 329 carriers and controls. The specificity of the test is 100%, whereas the sensitivity is 96.2%. The quantitative analysis of SMN2 copies in 375 patients with type I, type II, or type III SMA showed a significant correlation between SMN2 copy number and type of SMA as well as duration of survival. Thus, 80% of patients with type I SMA carry one or two SMN2 copies, and 82% of patients with type II SMA carry three SMN2 copies, whereas 96% of patients with type III SMA carry three or four SMN2 copies. Among 113 patients with type I SMA, 9 with one SMN2 copy lived <11 mo, 88/94 with two SMN2 copies lived <21 mo, and 8/10 with three SMN2 copies lived 33-66 mo. On the basis of SMN2 copy number, we calculated the posterior probability that a child with homozygous absence of SMN1 will develop type I, type II, or type III SMA.     

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.     

Targeting SR Proteins Improves SMN Expression in Spinal Muscular Atrophy Cells

Spinal muscular atrophy (SMA) is one of the most common inherited causes of pediatric mortality. SMA is caused by deletions or mutations in the survival of motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Humans have a centromeric copy of the survival of motor neuron gene, SMN2, which is nearly identical to SMN1. However, SMN2 cannot compensate for the loss of SMN1 because SMN2 has a single-nucleotide difference in exon 7, which negatively affects splicing of the exon. As a result, most mRNA produced from SMN2 lacks exon 7. SMN2 mRNA lacking exon 7 encodes a truncated protein with reduced functionality. Improving SMN2 exon 7 inclusion is a goal of many SMA therapeutic strategies. The identification of regulators of exon 7 inclusion may provide additional therapeutic targets or improve the design of existing strategies. Although a number of regulators of exon 7 inclusion have been identified, the function of most splicing proteins in exon 7 inclusion is unknown. Here, we test the role of SR proteins and hnRNP proteins in SMN2 exon 7 inclusion. Knockdown and overexpression studies reveal that SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF11, hnRNPA1/B1 and hnRNP U can inhibit exon 7 inclusion. Depletion of two of the most potent inhibitors of exon 7 inclusion, SRSF2 or SRSF3, in cell lines derived from SMA patients, increased SMN2 exon 7 inclusion and SMN protein. Our results identify novel regulators of SMN2 exon 7 inclusion, revealing potential targets for SMA therapeutics.     

Reorganization of Cajal bodies and nucleolar targeting of coilin in motor neurons of type I spinal muscular atrophy

Type I spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by loss or mutations of the survival motor neuron 1 (SMN1) gene. The reduction in SMN protein levels in SMA leads to degeneration and death of motor neurons. In this study, we have analyzed the nuclear reorganization of Cajal bodies, PML bodies and nucleoli in type I SMA motor neurons with homozygous deletion of exons 7 and 8 of the SMN1 gene. Western blot analysis revealed a marked reduction of SMN levels compared to the control sample. Using a neuronal dissociation procedure to perform a careful immunocytochemical and quantitative analysis of nuclear bodies, we demonstrated a severe decrease in the mean number of Cajal bodies per neuron and in the proportion of motor neurons containing these structures in type I SMA. Moreover, most Cajal bodies fail to recruit SMN and spliceosomal snRNPs, but contain the proteasome activator PA28γ, a molecular marker associated with the cellular stress response. Neuronal stress in SMA motor neurons also increases PML body number. The existence of chromatolysis and eccentric nuclei in SMA motor neurons correlates with Cajal body disruption and nucleolar relocalization of coilin, a Cajal body marker. Our results indicate that the Cajal body is a pathophysiological target in type I SMA motor neurons. They also suggest the Cajal body-dependent dysfunction of snRNP biogenesis and, therefore, pre-mRNA splicing in these neurons seems to be an essential component for SMA pathogenesis.     

Restoration of SMN to Emx-1 expressing cortical neurons is not sufficient to provide benefit to a severe mouse model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA), an autosomal recessive neuromuscular disorder, is a leading genetic cause of infant mortality. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). However, low, but essential, levels of SMN protein are produced by a nearly identical copy gene called SMN2. Detailed analysis of neuromuscular junctions in SMA mice has revealed a selective vulnerability in a subset of muscle targets, suggesting that while SMN is reduced uniformly, the functional deficits manifest sporadically. Additionally, in severe SMA models, it is becoming increasing apparent that SMA is not restricted solely to motor neurons. Rather, additional tissues including the heart, vasculature, and the pancreas contribute to the complete SMA-associated pathology. Recently, transgenic models have been utilized to examine the tissue-specific requirements of SMN, including selective depletion and restoration of SMN in motor neurons. To determine whether the cortical neuronal populations expressing the Emx-1 promoter are involved in SMA pathology, we generated a novel SMA mouse model in which SMN expression was specifically induced in Emx-1 expressing cortical neurons utilizing an Emx-1-Cre transgene. While SMN expression was robust in the central nervous system as expected, SMA mice did not live longer. Weight and time-to-right motor function were not significantly improved.