Present State and Future Perspectives of Prostaglandins as a Differentiation Factor in Motor Neurons

Spinal motor neurons have the longest axons that innervate the skeletal muscles of the central nervous system. Motor neuron diseases caused by spinal motor neuron cell death are incurable due to the unique and irreplaceable nature of their neural circuits. Understanding the mechanisms of neurogenesis, neuritogenesis, and synaptogenesis in motor neurons will allow investigators to develop new in vitro models and regenerative therapies for motor neuron diseases. In particular, small molecules can directly reprogram and convert into neural stem cells and neurons, and promote neuron-like cell differentiation. Prostaglandins are known to have a role in the differentiation and tissue regeneration of several cell types and organs. However, the involvement of prostaglandins in the differentiation of motor neurons from neural stem cells is poorly understood. The general cell line used in research on motor neuron diseases is the mouse neuroblastoma and spinal motor neuron fusion cell line NSC-34. Recently, our laboratory reported that prostaglandin E₂ and prostaglandin D₂ enhanced the conversion of NSC-34 cells into motor neuron-like cells with neurite outgrowth. Moreover, we found that prostaglandin E₂-differentiated NSC-34 cells had physiological and electrophysiological properties of mature motor neurons. In this review article, we provide contemporary evidence on the effects of prostaglandins, particularly prostaglandin E₂ and prostaglandin D₂, on differentiation and neural conversion. We also discuss the potential of prostaglandins as candidates for the development of new therapeutic drugs for motor neuron diseases.

     

PPP4R2 regulates neuronal cell differentiation and survival, functionally cooperating with SMN

Spinal muscular atrophy (SMA) is a human disease caused by reduced levels of the Survival of Motor Neuron (SMN) protein, leading to progressive loss of motor neurons and muscular paralysis. However, it is still not very clear why these cells are specifically sensitive to SMN levels. Therefore, understanding which proteins may functionally interact with SMN in a neuronal context is a very important issue. PPP4R2, a regulatory subunit of the protein phosphatase 4 (PPP4C), was previously identified as a functional interactor of the SMN complex, but has never been studied in neuronal cells. In this report, we show that PPP4R2 displays a very dynamic intracellular localization in mouse and rat neuronal cell lines and in rat primary hippocampal neurons, strongly correlating with differentiation. More importantly, we found that PPP4R2 loss of function impairs the differentiation of the mouse motor-neuronal cell line NSC-34, an effect that can be counteracted by SMN overexpression. In addition, we show that PPP4R2 may specifically protect NSC-34 cells from DNA damage-induced apoptosis and that it is capable to functionally cooperate with SMN in this activity. Our data indicate that PPP4R2 is a SMN partner that may modulate the differentiation and survival of neuronal cells.     

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.     

In vitro and in cellulo evidences for association of the survival of motor neuron complex with the fragile X mental retardation protein

Spinal muscular atrophy (SMA) is caused by reduced levels of the survival of motor neuron (SMN) protein. Although the SMN complex is essential for assembly of spliceosomal U small nuclear RNPs, it is still not understood why reduced levels of the SMN protein specifically cause motor neuron degeneration. SMN was recently proposed to have specific functions in mRNA transport and translation regulation in neuronal processes. The defective protein in Fragile X mental retardation syndrome (FMRP) also plays a role in transport of mRNPs and in their translation. Therefore, we examined possible relationships of SMN with FMRP. We observed granules containing both transiently expressed red fluorescent protein(RFP)-tagged SMN and green fluorescent protein(GFP)-tagged FMRP in cell bodies and processes of rat primary neurons of hypothalamus in culture. By immunoprecipitation experiments, we detected an association of FMRP with the SMN complex in human neuroblastoma SH-SY5Y cells and in murine motor neuron MN-1 cells. Then, by in vitro experiments, we demonstrated that the SMN protein is essential for this association. We showed that the COOH-terminal region of FMRP, as well as the conserved YG box and the region encoded by exon 7 of SMN, are required for the interaction. Our findings suggest a link between the SMN complex and FMRP in neuronal cells.     

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.     

SMN Protein Can Be Reliably Measured in Whole Blood with an Electrochemiluminescence (ECL) Immunoassay: Implications for Clinical Trials

Spinal muscular atrophy (SMA) is caused by defects in the survival motor neuron 1 (SMN1) gene that encodes survival motor neuron (SMN) protein. The majority of therapeutic approaches currently in clinical development for SMA aim to increase SMN protein expression and there is a need for sensitive methods able to quantify increases in SMN protein levels in accessible tissues. We have developed a sensitive electrochemiluminescence (ECL)-based immunoassay for measuring SMN protein in whole blood with a minimum volume requirement of 5μL. The SMN-ECL immunoassay enables accurate measurement of SMN in whole blood and other tissues. Using the assay, we measured SMN protein in whole blood from SMA patients and healthy controls and found that SMN protein levels were associated with SMN2 copy number and were greater in SMA patients with 4 copies, relative to those with 2 and 3 copies. SMN protein levels did not vary significantly in healthy individuals over a four-week period and were not affected by circadian rhythms. Almost half of the SMN protein was found in platelets. We show that SMN protein levels in C/C-allele mice, which model a mild form of SMA, were high in neonatal stage, decreased in the first few weeks after birth, and then remained stable throughout the adult stage. Importantly, SMN protein levels in the CNS correlated with SMN levels measured in whole blood of the C/C-allele mice. These findings have implications for the measurement of SMN protein induction in whole blood in response to SMN-upregulating therapy.     

Stabilization of the survival motor neuron protein by ASK1

The survival motor neuron (SMN) is a spliceosomal snRNP-interacting protein that was initially identified as a defective molecule in spinal muscular atrophy (SMA). The disease severity of SMA is determined by SMN protein level. Here, we show that apoptosis signal-regulating kinase 1 (ASK1) stabilizes SMN protein by inhibiting SMN poly-ubiquitination, and that the kinase activity of ASK1 is less important than its ability to bind to SMN. Furthermore, depletion of ASK1 by RNA interference revealed that ASK1 modulates neurite outgrowth by regulating SMN protein level in NSC34 motor neuron-like cells. Collectively, our results suggest that ASK1 acts as a novel binding partner of SMN and controls the steady-state level of SMN through complex formation with SMN in neurite outgrowth.     

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.     

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.     

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.     

Infantile spinale Muskelatrophie: mehr als eine Motoneuronerkrankung?

Infantile spinal muscular atrophy (SMA) is characterized by loss of motor neurons in the ventral horn of the spinal cord leading to weakness and muscle atrophy and occurs as a result of homozygous deletions or mutations in the survival motor neuron (SMN 1) gene. Loss of SMN 1 leads to a dramatic reduction in survival motor neuron (SMN) protein in the motor neurons of the spinal cord and of the brain stem. The SMA disease severity ranges from extremely severe to a relatively mild adult onset form of proximal muscle atrophy. More recently, clinical case reports in patients and studies in animal models provided evidence that severe SMN protein deficiency not only results in loss of motor neurons but also to additional organ manifestations. These include the peripheral, central and autonomic nervous system, development and function of the heart and the digestive tract and metabolic deficiencies. Therefore, to develop the most efficient therapeutic approach and also prevent further complications in patients that may arise with extended survival following therapeutic interventions, it is necessary to investigate in detail the specific damage to every system independently. The comparison of the defects in SMA mouse models will provide valuable insights; however, phenotypic differences between mice and men still remain.     

A Motor Function for the DEAD-Box RNA Helicase,Gemin3, in Drosophila

The survival motor neuron (SMN) protein, the determining factor for spinal muscular atrophy (SMA), is complexed with a group of proteins in human cells. Gemin3 is the only RNA helicase in the SMN complex. Here, we report the identification of Drosophila melanogaster Gemin3 and investigate its function in vivo. Like in vertebrates, Gemin3 physically interacts with SMN in Drosophila. Loss of function of gemin3 results in lethality at larval and/or prepupal stages. Before they die, gemin3 mutant larvae exhibit declined mobility and expanded neuromuscular junctions. Expression of a dominant-negative transgene and knockdown of Gemin3 in mesoderm cause lethality. A less severe Gemin3 disruption in developing muscles leads to flightless adults and flight muscle degeneration. Our findings suggest that Drosophila Gemin3 is required for larval development and motor function.     

Restoration of SMN expression in mesenchymal stem cells derived from gene-targeted patient-specific iPSCs

Spinal muscular atrophy (SMA) is primarily a neurodegenerative disease caused by the homozygous deletion of the survival motor neuron 1 (SMN1) gene, thereby reducing SMN protein expression. Mesenchymal stem cells (MSCs) have been implicated in the treatment of SMA. In the present study, we overexpressed exogenous SMN1 at the ribosomal DNA (rDNA) locus of induced pluripotent stem cells (iPSCs) generated from a SMA patient using an rDNA-targeting vector. The gene-targeted patient iPSCs differentiated into MSCs (SMN1-MSCs). A 2.1-fold higher expression level of SMN protein was detected in SMN1-MSCs than that detected in MSCs derived from patient iPSCs, and the results of the immunofluorescence analysis showed no difference in the quantity of SMN nuclear structures (gems) between SMN1-MSCs and MSCs derived from normal human iPSCs (h-MSCs). These findings provide a novel strategy for obtaining gene-targeted MSCs for potential clinical applications in autologous cell-based therapy.