Activation of Muscle-Specific Kinase (MuSK) Reduces Neuromuscular Defects in the Delta7 Mouse Model of Spinal Muscular Atrophy (SMA).

Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.

Minor snRNA gene delivery improves the loss of proprioceptive synapses on SMA motor neurons.

Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder caused by reduced expression of the survival motor neuron (SMN) protein. SMN has key functions in multiple RNA pathways, including the biogenesis of small nuclear ribonucleoproteins that are essential components of both major (U2-dependent) and minor (U12-dependent) spliceosomes. Here we investigated the specific contribution of U12 splicing dysfunction to SMA pathology through selective restoration of this RNA pathway in mouse models of varying phenotypic severity. We show that virus-mediated delivery of minor snRNA genes specifically improves select U12 splicing defects induced by SMN deficiency in cultured mammalian cells, as well as in the spinal cord and dorsal root ganglia of SMA mice without increasing SMN expression. This approach resulted in a moderate amelioration of several parameters of the disease phenotype in SMA mice, including survival, weight gain, and motor function. Importantly, minor snRNA gene delivery improved aberrant splicing of the U12 intron-containing gene Stasimon and rescued the severe loss of proprioceptive sensory synapses on SMA motor neurons, which are early signatures of motor circuit dysfunction in mouse models. Taken together, these findings establish the direct contribution of U12 splicing dysfunction to synaptic deafferentation and motor circuit pathology in SMA.

Nerve sprouting capacity in a pharmacologically induced mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by loss-of-function mutations in the survival of motoneuron gene 1 (SMN1). SMA is characterized by motoneuron death, skeletal muscle denervation and atrophy. Disease severity inversely correlates with copy number of a second gene (SMN2), which harbors a splicing defect that causes the production of inadequate levels of functional SMN protein. Small molecules that modify SMN2 splicing towards increased production of functional SMN significantly ameliorate SMA phenotypes in mouse models of severe SMA. At suboptimal doses, splicing modifiers, such as SMN-C1, have served to generate mice that model milder SMA, referred to as pharmacological SMA mice, which survive into early adulthood. Nerve sprouting at endplates, known as terminal sprouting, is key to normal muscle fiber reinnervation following nerve injury and its promotion might mitigate neuromuscular symptoms in mild SMA. Sprouting has been difficult to study in severe SMA mice due to their short lifespan. Here, we show that pharmacological SMA mice are capable of terminal sprouting following reinnervation that is largely SMN-C1 dose-independent, but that they display a reinnervation delay that is critically SMN-C1 dose-dependent. Data also suggest that SMN-C1 can induce by itself a limited terminal sprouting response in SMA and wild-type normally-innervated endplates.

Development and characterization of an SMN2-based intermediate mouse model of Spinal Muscular Atrophy.

Spinal Muscular Atrophy (SMA) is due to the loss of the survival motor neuron gene 1 (SMN1), resulting in motor neuron (MN) degeneration, muscle atrophy and loss of motor function. While SMN2 encodes a protein identical to SMN1, a single nucleotide difference in exon 7 causes most of the SMN2-derived transcripts to be alternatively spliced resulting in a truncated and unstable protein (SMNΔ7). SMA patients retain at least one SMN2 copy, making it an important target for therapeutics. Many of the existing SMA models are very severe, with animals typically living less than 2 weeks. Here, we present a novel intermediate mouse model of SMA based upon the human genomic SMN2 gene. Genetically, this model is similar to the well-characterized SMNΔ7 model; however, we have manipulated the SMNΔ7 transgene to encode a modestly more functional protein referred to as SMN read-through (SMN(RT)). By introducing the SMN(RT) transgene onto the background of a severe mouse model of SMA (SMN2(+/+);Smn(-/-)), disease severity was significantly decreased based upon a battery of phenotypic parameters, including MN pathology and a significant extension in survival. Importantly, there is not a full phenotypic correction, allowing for the examination of a broad range of therapeutics, including SMN2-dependent and SMN-independent pathways. This novel animal model serves as an important biological and therapeutic model for less severe forms of SMA and provides an in vivo validation of the SMN(RT) protein.

Replacement of huntingtin exon 1 by trans-splicing.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). HD offers unique opportunities for promising RNA-based therapeutic approaches aimed at reducing mutant HTT expression, since the HD mutation is considered to be a "gain-of-function" mutation. Allele-specific strategies that preserve expression from the wild-type allele and reduce the levels of mutant protein would be of particular interest. Here, we have conducted proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically repair the HTT allele. We employed a dual plasmid transfection system consisting of a pre-mRNA trans-splicing module (PTM) containing HTT exon 1 and a HTT minigene to demonstrate that HTT exon 1 can be replaced in trans. We detected the presence of the trans-spliced RNA in which PTM exon 1 was correctly joined to minigene exons 2 and 3. Furthermore, exon 1 from the PTM was trans-spliced to the endogenous HTT pre-mRNA in cultured cells as well as disease-relevant models, including HD patient fibroblasts and primary neurons from a previously described HD mouse model. These results suggest that the repeat expansion of HTT can be repaired successfully not only in the context of synthetic minigenes but also within the context of HD neurons. Therefore, pre-mRNA trans-splicing may be a promising approach for the treatment of HD and other dominant genetic disorders.

Bifunctional RNAs targeting the intronic splicing silencer N1 increase SMN levels and reduce disease severity in an animal model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients. Although the SMN2 coding sequence has the potential to produce full-length SMN, nearly 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein. SMN2, however, is an excellent therapeutic target. Previously, we developed antisense-based oligonucleotides (bifunctional RNAs) that specifically recruit SR/SR-like splicing factors and target a negative regulator of SMN2 exon-7 inclusion within intron-6. As a means to optimize the antisense sequence of the bifunctional RNAs, we chose to target a potent intronic repressor downstream of SMN2 exon 7, called intronic splicing silencer N1 (ISS-N1). We developed RNAs that specifically target ISS-N1 and concurrently recruit the modular SR proteins SF2/ASF or hTra2ß1. RNAs were directly injected in the brains of SMA mice. Bifunctional RNA injections were able to elicit robust induction of SMN protein in the brain and spinal column of neonatal SMA mice. Importantly, hTra2ß1-ISS-N1 and SF2/ASF-ISS-N1 bifunctional RNAs significantly extended lifespan and increased weight in the SMNΔ7 mice. This technology has direct implications for SMA therapy and provides similar therapeutic strategies for other diseases caused by aberrant splicing.

Down-regulated in adenoma Cl/HCO3 exchanger couples with Na/H exchanger 3 for NaCl absorption in murine small intestine.

BACKGROUND & AIMS: Electroneutral NaCl absorption across small intestine contributes importantly to systemic fluid balance. Disturbances in this process occur in both obstructive and diarrheal diseases, eg, cystic fibrosis, secretory diarrhea. NaCl absorption involves coupling of Cl(-)/HCO(3)(-) exchanger(s) primarily with Na(+)/H(+) exchanger 3 (Nhe3) at the apical membrane of intestinal epithelia. Identity of the coupling Cl(-)/HCO(3)(-) exchanger(s) was investigated using mice with gene-targeted knockout (KO) of Cl(-)/HCO(3)(-) exchangers: Slc26a3, down-regulated in adenoma (Dra) or Slc26a6, putative anion transporter-1 (Pat-1). METHODS: Intracellular pH (pH(i)) of intact jejunal villous epithelium was measured by ratiometric microfluoroscopy. Ussing chambers were used to measure transepithelial (22)Na(36)Cl flux across murine jejunum, a site of electroneutral NaCl absorption. Expression was estimated using immunofluorescence and quantitative polymerase chain reaction. RESULTS: Basal pH(i) of DraKO epithelium, but not Pat-1KO epithelium, was alkaline, whereas pH(i) in the Nhe3KO was acidic relative to wild-type. Altered pH(i) was associated with robust Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange activity in the DraKO and Nhe3KO villous epithelium, respectively. Contrary to genetic ablation, pharmacologic inhibition of Nhe3 in wild-type did not alter pH(i) but coordinately inhibited Dra. Flux studies revealed that Cl(-) absorption was essentially abolished (>80%) in the DraKO and little changed (<20%) in the Pat-1KO jejunum. Net Na(+) absorption was unaffected. Immunofluorescence demonstrated modest Dra expression in the jejunum relative to large intestine. Functional and expression studies did not indicate compensatory changes in relevant transporters. CONCLUSIONS: These studies provide functional evidence that Dra is the major Cl(-)/HCO(3)(-) exchanger coupled with Nhe3 for electroneutral NaCl absorption across mammalian small intestine.

The inhibitor ABIN-2 disrupts the interaction of receptor-interacting protein with the kinase subunit IKKgamma to block activation of the transcription factor NF-kappaB and potentiate apoptosis.

NF-kappaB (nuclear factor kappaB) proteins are key transcription factors that regulate gene expression in response to various extracellular stimuli. The pathway leading to the activation of NF-kappaB involves a complicated network that includes a number of signalling molecules. The recent identification of a wide range of negative regulators of NF-kappaB has given another layer of complexity in NF-kappaB activation. We and others have previously identified the protein ABIN-2 (A20 binding inhibitor of NF-kappaB 2) as an inhibitor of NF-kappaB activation. In the present paper, we demonstrate that ABIN-2 exerts its inhibitory function by blocking the interaction of RIP (receptor-interacting protein) with the downstream effector IKKgamma, a non-kinase component of the IkappaB (inhibitory kappaB) kinase complex. When overexpressed in cells, ABIN-2 bound to IKKgamma and prevented the association of IKKgamma with RIP. By a deletion mapping, a stretch of 50 amino acids on ABIN-2 is found to be essential for its interaction with IKKgamma. The ABIN-2 mutant that lacked these 50 amino acids did not interact with IKKgamma and, consequently, failed to inhibit NF-kappaB activation. Strikingly, a portion of RIP, which is similar to this 50-residue domain of ABIN-2, is also essential for RIP interaction with IKKgamma. The RIP mutant with deletion of this similar region did not associate with IKKgamma and had substantial reduction of its ability to mediate NF-kappaB activation. Taken together, these conserved 50 residues of ABIN-2 and RIP define a novel structural domain in mediating a key step in the NF-kappaB signalling pathway through the interaction with IKKgamma. Finally, the signalling pathway of NF-kappaB activation is known to promote survival in many cellular events. The mechanism for decision between cell death and survival is under fine regulation. In the present paper, we demonstrated further that the expression of ABIN-2 could promote the RIP-mediated apoptosis by presumably suppressing the anti-apoptotic effect of NF-kappaB.