SMN protein is required throughout life to prevent spinal muscular atrophy disease progression.

Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient.

TSUNAMI: an antisense method to phenocopy splicing-associated diseases in animals.

Antisense oligonucleotides (ASOs) are versatile molecules that can be designed to specifically alter splicing patterns of target pre-mRNAs. Here we exploit this feature to phenocopy a genetic disease. Spinal muscular atrophy (SMA) is a motor neuron disease caused by loss-of-function mutations in the SMN1 gene. The related SMN2 gene expresses suboptimal levels of functional SMN protein due to alternative splicing that skips exon 7; correcting this defect-e.g., with ASOs-is a promising therapeutic approach. We describe the use of ASOs that exacerbate SMN2 missplicing and phenocopy SMA in a dose-dependent manner when administered to transgenic Smn(-/-) mice. Intracerebroventricular ASO injection in neonatal mice recapitulates SMA-like progressive motor dysfunction, growth impairment, and shortened life span, with α-motor neuron loss and abnormal neuromuscular junctions. These SMA-like phenotypes are prevented by a therapeutic ASO that restores correct SMN2 splicing. We uncovered starvation-induced splicing changes, particularly in SMN2, which likely accelerate disease progression. These results constitute proof of principle that ASOs designed to cause sustained splicing defects can be used to induce pathogenesis and rapidly and accurately model splicing-associated diseases in animals. This approach allows the dissection of pathogenesis mechanisms, including spatial and temporal features of disease onset and progression, as well as testing of candidate therapeutics.

Mutant Profilin1 transgenic mice recapitulate cardinal features of motor neuron disease.

The recent identification of profilin1 mutations in 25 familial ALS cases has linked altered function of this cytoskeleton-regulating protein to the pathogenesis of motor neuron disease. To investigate the pathological role of mutant profilin1 in motor neuron disease, we generated transgenic lines of mice expressing human profilin1 with a mutation at position 118 (hPFN1G118V). One of the mouse lines expressing high levels of mutant human PFN1 protein in the brain and spinal cord exhibited many key clinical and pathological features consistent with human ALS disease. These include loss of lower (ventral horn) and upper motor neurons (corticospinal motor neurons in layer V), mutant profilin1 aggregation, abnormally ubiquitinated proteins, reduced choline acetyltransferase (ChAT) enzyme expression, fragmented mitochondria, glial cell activation, muscle atrophy, weight loss, and reduced survival. Our investigations of actin dynamics and axonal integrity suggest that mutant PFN1 protein is associated with an abnormally low filamentous/globular (F/G)-actin ratio that may be the underlying cause of severe damage to ventral root axons resulting in a Wallerian-like degeneration. These observations indicate that our novel profilin1 mutant mouse line may provide a new ALS model with the opportunity to gain unique perspectives into mechanisms of neurodegeneration that contribute to ALS pathogenesis.

Pharmacologically induced mouse model of adult spinal muscular atrophy to evaluate effectiveness of therapeutics after disease onset.

Spinal muscular atrophy (SMA) is a genetic disease characterized by atrophy of muscle and loss of spinal motor neurons. SMA is caused by deletion or mutation of the survival motor neuron 1 (SMN1) gene, and the nearly identical SMN2 gene fails to generate adequate levels of functional SMN protein due to a splicing defect. Currently, several therapeutics targeted to increase SMN protein are in clinical trials. An outstanding issue in the field is whether initiating treatment in symptomatic older patients would confer a therapeutic benefit, an important consideration as the majority of patients with milder forms of SMA are diagnosed at an older age. An SMA mouse model that recapitulates the disease phenotype observed in adolescent and adult SMA patients is needed to address this important question. We demonstrate here that Δ7 mice, a model of severe SMA, treated with a suboptimal dose of an SMN2 splicing modifier show increased SMN protein, survive into adulthood and display SMA disease-relevant pathologies. Increasing the dose of the splicing modifier after the disease symptoms are apparent further mitigates SMA histopathological features in suboptimally dosed adult Δ7 mice. In addition, inhibiting myostatin using intramuscular injection of AAV1-follistatin ameliorates muscle atrophy in suboptimally dosed Δ7 mice. Taken together, we have developed a new murine model of symptomatic SMA in adolescents and adult mice that is induced pharmacologically from a more severe model and demonstrated efficacy of both SMN2 splicing modifiers and a myostatin inhibitor in mice at later disease stages.

Pharmacokinetics, pharmacodynamics, and efficacy of a small-molecule SMN2 splicing modifier in mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a ∼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment.

Severe neuromuscular denervation of clinically relevant muscles in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA), a motoneuron disease caused by a deficiency of the survival of motor neuron (SMN) protein, is characterized by motoneuron loss and muscle weakness. It remains unclear whether widespread loss of neuromuscular junctions (NMJs) is involved in SMA pathogenesis. We undertook a systematic examination of NMJ innervation patterns in >20 muscles in the SMNΔ7 SMA mouse model. We found that severe denervation (<50% fully innervated endplates) occurs selectively in many vulnerable axial muscles and several appendicular muscles at the disease end stage. Since these vulnerable muscles were located throughout the body and were comprised of varying muscle fiber types, it is unlikely that muscle location or fiber type determines susceptibility to denervation. Furthermore, we found a similar extent of neurofilament accumulation at NMJs in both vulnerable and resistant muscles before the onset of denervation, suggesting that neurofilament accumulation does not predict subsequent NMJ denervation. Since vulnerable muscles were initially innervated, but later denervated, loss of innervation in SMA may be attributed to defects in synapse maintenance. Finally, we found that denervation was amendable by trichostatin A (TSA) treatment, which increased innervation in clinically relevant muscles in TSA-treated SMNΔ7 mice. Our findings suggest that neuromuscular denervation in vulnerable muscles is a widespread pathology in SMA, and can serve as a preparation for elucidating the biological basis of synapse loss, and for evaluating therapeutic efficacy.

Transcriptional control of different acetylcholinesterase subunits in formation and maintenance of vertebrate neuromuscular junctions.

Acetylcholinesterase (AChE; EC 3.1.1.7) is a highly polymorphic enzyme (Massoulié, 2002). Asingle ACHE gene produces several types of catalytic subunits by alternative splicing, but a single splice variant, called type T (AChET), is expressed in adult mammalian muscle and brain. Catalytic subunits of AChET produce amphiphilic monomers and dimers, nonamphiphilic homotetramers, as well as heteromeric associations with anchoring proteins, ColQ (collagenous subunit) and PRiMA (proline-rich membrane anchor), which allow their functional localization in cholinergic synapses (Massoulié, 2002). ColQ characterizes the collagen-tailed forms (Aforms) of AChE and butyrylcholinesterase (BChE), which are localized in the basal lamina at neuromuscular junctions (NMJs) of vertebrates (Krejci et al., 1999); in these molecules (A4, A8, A12), one, two, or three tetramers of catalytic subunits are disulfide-linked to the strands of a triple helix of ColQ collagen. The cDNAs encoding ColQ, which have two transcripts, have been cloned: ColQ-1a predominantly in fast-twitch muscle, and ColQ-1 predominantly in slow-twitch muscle. The tetrameric globular (G4) form of AChE is characterized by linkage to PRiMA. PRiMAcDNA encodes a single-pass approximately 20-kDa type-I transmembrane protein and, similar to that of ColQ, contains a short PRAD (proline-rich attachment domain) that is able to organize AChE catalytic subunits into tetramers and anchor the enzyme at the surface of neuron and muscle (Massoulié, 2002).

ATP acts via P2Y1 receptors to stimulate acetylcholinesterase and acetylcholine receptor expression: transduction and transcription control.

At the vertebrate neuromuscular junction ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, the P2Y1 receptor, is localized at the junction, and we propose that this mediates a trophic role for synaptic ATP there. Evidence in support of this and on its mechanism is given here. With the use of chick or mouse myotubes expressing promoter-reporter constructs from genes of acetylcholinesterase (AChE) or of the acetylcholine receptor subunits, P2Y1 receptor agonists were shown to stimulate the transcription of each of those genes. The pathway to activation of the AChE gene was shown to involve protein kinase C and intracellular Ca 2+ release. Application of dominant-negative or constitutively active mutants, or inhibitors of specific kinases, showed that it further proceeds via some of the known intermediates of extracellular signal-regulated kinase phosphorylation. In both chick and mouse myotubes this culminates in activation of the transcription factor Elk-1, confirmed by gel mobility shift assays and by the nuclear accumulation of phosphorylated Elk-1. All of the aforementioned activations by agonist were amplified when the content of P2Y1 receptors was boosted by transfection, and the activations were blocked by a P2Y1-selective antagonist. Two Elk-1 binding site sequences present in the AChE gene promoter were jointly sufficient to drive ATP-induced reporter gene transcription. Thus ATP regulates postsynaptic gene expression via a pathway to a selective transcription factor activation.

ATP potentiates the formation of AChR aggregate in the co-culture of NG108-15 cells with C2C12 myotubes.

The role of adenosine 5'-triphosphate (ATP) and P2Y(1) nucleotide receptor in potentiating agrin-induced acetylcholine receptor (AChR) aggregation is being demonstrated in a co-culture system of NG108-15 cell, a mouse neuroblastoma X rat glioma hybrid cell line that resembles spinal motor neuron, with C2C12 myotube. In the co-cultures, antagonized P2Y(1) receptors showed a reduction in NG108-15 cell-induced AChR aggregation. Parallel to this observation, cultured NG108-15 cell secreted ATP into the conditioned medium in a time-dependent manner. Enhancement of ATP release from the cultured NG108-15 cells by overexpression of active mutants of small GTPases increased the aggregation of AChRs in co-culturing with C2C12 myotubes. In addition, ecto-nucleotidase was revealed in the co-culture, which rapidly degraded the applied ATP. These results support the notion that ATP has a role in directing the formation of post-synaptic apparatus in vertebrate neuromuscular junctions.

Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.

Pathological impact of SMN2 mis-splicing in adult SMA mice.

Loss-of-function mutations in SMN1 cause spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. The related SMN2 gene expresses suboptimal levels of functional SMN protein, due to a splicing defect. Many SMA patients reach adulthood, and there is also adult-onset (type IV) SMA. There is currently no animal model for adult-onset SMA, and the tissue-specific pathogenesis of post-developmental SMN deficiency remains elusive. Here, we use an antisense oligonucleotide (ASO) to exacerbate SMN2 mis-splicing. Intracerebroventricular ASO injection in adult SMN2-transgenic mice phenocopies key aspects of adult-onset SMA, including delayed-onset motor dysfunction and relevant histopathological features. SMN2 mis-splicing increases during late-stage disease, likely accelerating disease progression. Systemic ASO injection in adult mice causes peripheral SMN2 mis-splicing and affects prognosis, eliciting marked liver and heart pathologies, with decreased IGF1 levels. ASO dose-response and time-course studies suggest that only moderate SMN levels are required in the adult central nervous system, and treatment with a splicing-correcting ASO shows a broad therapeutic time window. We describe distinctive pathological features of adult-onset and early-onset SMA.

Synaptic defects in the spinal and neuromuscular circuitry in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a major genetic cause of death in childhood characterized by marked muscle weakness. To investigate mechanisms underlying motor impairment in SMA, we examined the spinal and neuromuscular circuitry governing hindlimb ambulatory behavior in SMA model mice (SMNΔ7). In the neuromuscular circuitry, we found that nearly all neuromuscular junctions (NMJs) in hindlimb muscles of SMNΔ7 mice remained fully innervated at the disease end stage and were capable of eliciting muscle contraction, despite a modest reduction in quantal content. In the spinal circuitry, we observed a ∼28% loss of synapses onto spinal motoneurons in the lateral column of lumbar segments 3-5, and a significant reduction in proprioceptive sensory neurons, which may contribute to the 50% reduction in vesicular glutamate transporter 1(VGLUT1)-positive synapses onto SMNΔ7 motoneurons. In addition, there was an increase in the association of activated microglia with SMNΔ7 motoneurons. Together, our results present a novel concept that synaptic defects occur at multiple levels of the spinal and neuromuscular circuitry in SMNΔ7 mice, and that proprioceptive spinal synapses could be a potential target for SMA therapy.

ATP potentiates agrin-induced AChR aggregation in cultured myotubes: activation of RhoA in P2Y1 nucleotide receptor signaling at vertebrate neuromuscular junctions.

At vertebrate neuromuscular junctions, ATP is known to stabilize acetylcholine in the synaptic vesicles and to be co-released with it. We have shown previously that a nucleotide receptor, P2Y(1) receptor, is localized at the nmjs, and we propose that this mediates a trophic role for synaptic ATP there. In cultured myotubes, the activation of P2Y(1) receptors modulated agrin-induced acetylcholine receptor (AChR) aggregation in a potentiation manner. This potentiation effect in agrin-induced AChR aggregation was reduced by antagonizing the P2Y(1) receptors. The guanosine triphosphatase RhoA was shown to be responsible for this P2Y(1)-potentiated effect. The localization of RhoA in rat and chicken skeletal muscles was restricted at the neuromuscular junctions. Application of P2Y(1) agonists in cultured myotubes induced RhoA activation, which showed an additive effect with agrin-induced RhoA activation. Over-expression of dominant-negative mutant of RhoA in cultured myotubes diminished the agrin-induced AChR aggregation, as well as the potentiation effect of P2Y(1)-specific agonist. Application of UTP in the cultures also triggered similar responses as did 2-methylthioadenosine 5'-diphosphate, suggesting the involvement of other subtypes of P2Y receptors. These results demonstrate that RhoA could serve as a downstream mediator of signaling mediated by P2Y(1) receptor and agrin, which therefore synergizes the effects of the two neuron-derived trophic factors in modulating the formation and/or maintenance of post-synaptic apparatus at the neuromuscular junctions.

P2Y2 receptor activation regulates the expression of acetylcholinesterase and acetylcholine receptor genes at vertebrate neuromuscular junctions.

At the vertebrate neuromuscular junction (nmj), ATP is known to be coreleased with acetylcholine from the synaptic vesicles. We have previously shown that the P2Y1 receptor is localized at the nmj. Here, we extend the findings to show that another nucleotide receptor, P2Y2, is also localized there and with P2Y1 jointly mediates trophic responses to ATP. The P2Y2 receptor mRNA in rat muscle increased during development and peaked in adulthood. The P2Y2 receptor protein was shown to become restricted to the nmjs during embryonic development, in chick and in rat. In both rat and chick myotubes, P2Y1 and P2Y2 are expressed, increasing with differentiation, but P2Y4 is absent. The P2Y2 agonist UTP stimulated there inositol trisphosphate production and phosphorylation of extracellular signal-regulated kinases, in a dose-dependent manner. These UTP-induced responses were insensitive to the P2Y1-specific antagonist MRS 2179 (2'-deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt). In differentiated myotubes, P2Y2 activation induced expression of acetylcholinesterase (AChE) protein (but not control alpha-tubulin). This was shown to arise from AChE promoter activation, mediated by activation of the transcription factor Elk-1. Two Elk-1-responsive elements, located in intron-1 of the AChE promoter, were found by mutation to act in this gene activation initiated at the P2Y2 receptor and also in that initiated at the P2Y1 receptor. Furthermore, the promoters of different acetylcholine receptor subunits were also stimulated by application of UTP to myotubes. These results indicate that ATP regulates postsynaptic gene expressions via a common pathway triggered by the activation of P2Y1 and P2Y2 receptors at the nmjs.

ATP induces post-synaptic gene expressions in vertebrate skeletal neuromuscular junctions.

In vertebrate neuromuscular junctions (nmjs), adenosine 5'-triphosphate (ATP) is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Several lines of evidence suggest that the synaptic ATP can act as a synapse-organizing factor at the nmjs, mediated by metabotropic P2Y(1) receptors. P2Y(1) receptor mRNAs in chicken and rat muscles are low in embryo but increases markedly in the adult, and decreased after denervation. The P2Y(1) receptor protein is restricted to the nmjs and co-localized with AChRs in adult muscles. The activation of P2Y(1) receptor by adenine nucleotides in cultured chick myotubes stimulated the accumulation of inositol phosphates, intracellular Ca(2+) mobilization, protein kinase C activity and phosphorylation of extracellular signal-regulated kinases. The receptor activation led to an increase in the expression of transcripts encoding AChE catalytic subunit and AChR subunits. The ATP-induced post-synaptic gene expression is possibly mediated by the activation of signaling cascades of mitogen-activated protein kinase. Therefore, a model is being proposed here that the synaptic ATP has a role of synergy with other regulatory signals, such as neuregulin, which act via their post-synaptic receptors to activate second signaling molecules locally to enhance the transcription of AChR/AChE genes specifically in the adjacent sub-synaptic nuclei during the formation and, especially, the maintenance of post-synaptic specializations at the nmjs.