Efficient systemic CNS delivery of a therapeutic antisense oligonucleotide with a blood-brain barrier-penetrating ApoE-derived peptide.

Antisense oligonucleotide (ASO) has emerged as a promising therapeutic approach for treating central nervous system (CNS) disorders by modulating gene expression with high selectivity and specificity. However, the poor permeability of ASO across the blood-brain barrier (BBB) diminishes its therapeutic success. Here, we designed and synthesized a series of BBB-penetrating peptides (BPP) derived from either the receptor-binding domain of apolipoprotein E (ApoE) or a transferrin receptor-binding peptide (THR). The BPPs were conjugated to phosphorodiamidate morpholino oligomers (PMO) that are chemically analogous to the 2'-O-(2-methoxyethyl) (MOE)-modified ASO approved by the FDA for treating spinal muscular atrophy (SMA). The BPP-PMO conjugates significantly increased the level of full-length SMN2 in the patient-derived SMA fibroblasts in a concentration-dependent manner with minimal to no toxicity. Furthermore, the systemic administration of the most potent BPP-PMO conjugates significantly increased the expression of full-length SMN2 in the brain and spinal cord of SMN2 transgenic adult mice. Notably, BPP8-PMO conjugate showed a 1.25-fold increase in the expression of full-length functional SMN2 in the brain. Fluorescence imaging studies confirmed that 78% of the fluorescently (Cy7)-labelled BPP8-PMO reached brain parenchyma, with 11% uptake in neuronal cells. Additionally, the BPP-PMO conjugates containing retro-inverso (RI) D-BPPs were found to possess extended half-lives compared to their L-counterparts, indicating increased stability against protease degradation while preserving the bioactivity. This delivery platform based on BPP enhances the CNS bioavailability of PMO targeting the SMN2 gene, paving the way for the development of systemically administered neurotherapeutics for CNS disorders.

Heat increases full-length SMN splicing: promise for splice-augmenting therapies for SMA.

Spinal muscular atrophy (SMA) is a debilitating neurodegenerative pediatric disease characterized by low levels of the survival motor protein (SMN). Humans have two SMN genes that produce identical SMN proteins, but they differ at a key nucleotide in exon 7 that induces differential mRNA splicing. SMN1 primarily produces full-length SMN protein, but due to the spliceosome's inability to efficiently recognize exon 7, SMN2 transcripts are often truncated. SMA occurs primarily through mutations or deletions in the SMN1 gene; therefore, current therapies use antisense oligonucleotides (ASOs) to target exon 7 inclusion in SMN2 mRNA and promote full-length SMN protein production. Here, we explore additional methods that can target SMN splicing and therapeutically increase full-length SMN protein. We demonstrate that in vitro heat treatment of cells increases exon 7 inclusion and relative abundance of full-length SMN2 mRNA and protein, a response that is modulated through the upregulation of the positive splicing factor TRA2 beta. We also observe that HSP90, but not HSP40 or HSP70, in the heat shock response is essential for SMN2 exon 7 splicing under hyperthermic conditions. Finally, we show that pulsatile heat treatments for one hour in vitro and in vivo are effective in increasing full-length SMN2 levels. These findings suggest that timed interval treatments could be a therapeutic alternative for SMA patients who do not respond to current ASO-based therapies or require a unique combination regimen.

U2AF65-Dependent SF3B1 Function in SMN Alternative Splicing.

Splicing factor 3b subunit 1 (SF3B1) is an essential protein in spliceosomes and mutated frequently in many cancers. While roles of SF3B1 in single intron splicing and roles of its cancer-linked mutant in aberrant splicing have been identified to some extent, regulatory functions of wild-type SF3B1 in alternative splicing (AS) are not well-understood yet. Here, we applied RNA sequencing (RNA-seq) to analyze genome-wide AS in SF3B1 knockdown (KD) cells and to identify a large number of skipped exons (SEs), with a considerable number of alternative 5' splice-site selection, alternative 3' splice-site selection, mutually exclusive exons (MXE), and retention of introns (RI). Among altered SEs by SF3B1 KD, survival motor neuron 2 (SMN2) pre-mRNA exon 7 splicing was a regulatory target of SF3B1. RT-PCR analysis of SMN exon 7 splicing in SF3B1 KD or overexpressed HCT116, SH-SY5Y, HEK293T, and spinal muscular atrophy (SMA) patient cells validated the results. A deletion mutation demonstrated that the U2 snRNP auxiliary factor 65 kDa (U2AF65) interaction domain of SF3B1 was required for its function in SMN exon 7 splicing. In addition, mutations to lower the score of the polypyrimidine tract (PPT) of exon 7, resulting in lower affinity for U2AF65, were not able to support SF3B1 function, suggesting the importance of U2AF65 in SF3B1 function. Furthermore, the PPT of exon 7 with higher affinity to U2AF65 than exon 8 showed significantly stronger interactions with SF3B1. Collectively, our results revealed SF3B1 function in SMN alternative splicing.

LCM-seq reveals unique transcriptional adaptation mechanisms of resistant neurons and identifies protective pathways in spinal muscular atrophy.

Somatic motor neurons are selectively vulnerable in spinal muscular atrophy (SMA), which is caused by a deficiency of the ubiquitously expressed survival of motor neuron protein. However, some motor neuron groups, including oculomotor and trochlear (ocular), which innervate eye muscles, are for unknown reasons spared. To reveal mechanisms of vulnerability and resistance in SMA, we investigate the transcriptional dynamics in discrete neuronal populations using laser capture microdissection coupled with RNA sequencing (LCM-seq). Using gene correlation network analysis, we reveal a TRP53-mediated stress response that is intrinsic to all somatic motor neurons independent of their vulnerability, but absent in relatively resistant red nucleus and visceral motor neurons. However, the temporal and spatial expression analysis across neuron types shows that the majority of SMA-induced modulations are cell type-specific. Using Gene Ontology and protein network analyses, we show that ocular motor neurons present unique disease-adaptation mechanisms that could explain their resilience. Specifically, ocular motor neurons up-regulate (1) Syt1, Syt5, and Cplx2, which modulate neurotransmitter release; (2) the neuronal survival factors Gdf15, Chl1, and Lif; (3) Aldh4, that protects cells from oxidative stress; and (4) the caspase inhibitor Pak4. Finally, we show that GDF15 can rescue vulnerable human spinal motor neurons from degeneration. This confirms that adaptation mechanisms identified in resilient neurons can be used to reduce susceptibility of vulnerable neurons. In conclusion, this in-depth longitudinal transcriptomics analysis in SMA reveals novel cell type-specific changes that, alone and combined, present compelling targets, including Gdf15, for future gene therapy studies aimed toward preserving vulnerable motor neurons.

CRISPR artificial splicing factors.

Alternative splicing allows expression of mRNA isoforms from a single gene, expanding the diversity of the proteome. Its prevalence in normal biological and disease processes warrant precise tools for modulation. Here we report the engineering of CRISPR Artificial Splicing Factors (CASFx) based on RNA-targeting CRISPR-Cas systems. We show that simultaneous exon inclusion and exclusion can be induced at distinct targets by differential positioning of CASFx. We also create inducible CASFx (iCASFx) using the FKBP-FRB chemical-inducible dimerization domain, allowing small molecule control of alternative splicing. Finally, we demonstrate the activation of SMN2 exon 7 splicing in spinal muscular atrophy (SMA) patient fibroblasts, suggesting a potential application of the CASFx system.

Activation of Cryptic 3' Splice-Sites by SRSF2 Contributes to Cassette Exon Skipping.

Here we show that the serine/arginine rich splicing factor 2 (SRSF2) promotes cryptic 3' splice-site (3'AG') usage during cassette exon exclusion in survival of motor neuron (SMN2) minigenes. Deletion of the 3'AG' (3'AG'1), its associated branch point (BP') and polypyrimidine tract (PPT') sequences directs SRSF2 to promote a second 3'AG' (3'AG'2) with less conserved associated region for intron splicing. Furthermore, deletion of both 3'AG'1 and 3'AG'2 and their associated sequences triggered usage of a third 3'AG'3 that has very weak associated sequences. Interestingly, when intron splicing was directed to the 3'AG' cryptic splice-sites, intron splicing from the canonical 3'AG splice-site was reduced along with a decrease in cassette exon inclusion. Moreover, multiple SRSF2 binding sites within the intron are responsible for 3'AG' activation. We conclude that SRSF2 facilitates exon exclusion by activating a cryptic 3'AG' and inhibiting downstream intron splicing.

Rescue of spinal muscular atrophy mouse models with AAV9-Exon-specific U1 snRNA.

Spinal Muscular Atrophy results from loss-of-function mutations in SMN1 but correcting aberrant splicing of SMN2 offers hope of a cure. However, current splice therapy requires repeated infusions and is expensive. We previously rescued SMA mice by promoting the inclusion of a defective exon in SMN2 with germline expression of Exon-Specific U1 snRNAs (ExspeU1). Here we tested viral delivery of SMN2 ExspeU1s encoded by adeno-associated virus AAV9. Strikingly the virus increased SMN2 exon 7 inclusion and SMN protein levels and rescued the phenotype of mild and severe SMA mice. In the severe mouse, the treatment improved the neuromuscular function and increased the life span from 10 to 219 days. ExspeU1 expression persisted for 1 month and was effective at around one five-hundredth of the concentration of the endogenous U1snRNA. RNA-seq analysis revealed our potential drug rescues aberrant SMA expression and splicing profiles, which are mostly related to DNA damage, cell-cycle control and acute phase response. Vastly overexpressing ExspeU1 more than 100-fold above the therapeutic level in human cells did not significantly alter global gene expression or splicing. These results indicate that AAV-mediated delivery of a modified U1snRNP particle may be a novel therapeutic option against SMA.

Drug screening with human SMN2 reporter identifies SMN protein stabilizers to correct SMA pathology.

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, is caused by reduced levels of functional survival motor neuron (SMN) protein. To identify therapeutic agents for SMA, we established a versatile SMN2-GFP reporter line by targeting the human SMN2 gene. We then screened a compound library and identified Z-FA-FMK as a potent candidate. Z-FA-FMK, a cysteine protease inhibitor, increased functional SMN through inhibiting the protease-mediated degradation of both full-length and exon 7-deleted forms of SMN. Further studies reveal that CAPN1, CAPN7, CTSB, and CTSL mediate the degradation of SMN proteins, providing novel targets for SMA. Notably, Z-FA-FMK mitigated mitochondriopathy and neuropathy in SMA patient-derived motor neurons and showed protective effects in SMA animal model after intracerebroventricular injection. E64d, another cysteine protease inhibitor which can pass through the blood-brain barrier, showed even more potent therapeutic effects after subcutaneous delivery to SMA mice. Taken together, we have successfully established a human SMN2 reporter for future drug discovery and identified the potential therapeutic value of cysteine protease inhibitors in treating SMA via stabilizing SMN proteins.

Overexpression of SMN2 Gene in Motoneuron-Like Cells Differentiated from Adipose-Derived Mesenchymal Stem Cells by Ponasterone A.

Cell therapy and stem cell transplantation strategies have provided potential therapeutic approaches for the treatment of neurological disorders. Adipose-derived mesenchymal stem cells (ADMSCs) are abundant adult stem cells with low immunogenicity, which can be used for allogeneic cell replacement therapies. Differentiation of ADMSCs into acetylcholine-secreting motoneurons (MNs) is a promising treatment for MN diseases, such as spinal muscular atrophy (SMA), which is associated with the level of SMN1 gene expression. The SMN2 gene plays an important role in MN disorders, as it can somewhat compensate for the lack of SMN1 expression in SMA patients. Although the differentiation potential of ADMSCs into MNs has been previously established, overexpression of SMN2 gene in a shorter period with a longer survival has yet to be elucidated. Ponasterone A (PNA), an ecdysteroid hormone activating the PI3K/Akt pathway, was studied as a new steroid to promote SMN2 overexpression in MNs differentiated from ADMSCs. After induction with retinoic acid, sonic hedgehog, forskolin, and PNA, MN phenotypes were differentiated from ADMSCs, and immunochemical staining, specific for ß-tubulin, neuron-specific enolase, and choline acetyltransferase, was performed. Also, the results of real-time PCR assay indicated nestin, Pax6, Nkx2.2, Hb9, Olig2, and SMN2 expression in the differentiated cells. After 2 weeks of treatment, cultures supplemented with PNA showed a longer survival and a 1.2-fold increase in the expression of SMN2 (an overall 5.6-fold increase; *P ≤ 0.05), as confirmed by the Western blot analysis. The PNA treatment increased the levels of ChAT, Isl1, Hb9, and Nkx2 expression in MN-like cells. Our findings highlight the role of PNA in the upregulation of SMN2 genes from MSC-derived MN-like cells, which may serve as a potential candidate in cellular therapy for SMA patients.

Impaired myogenic development, differentiation and function in hESC-derived SMA myoblasts and myotubes.

Spinal muscular atrophy (SMA) is a severe genetic disorder that manifests in progressive neuromuscular degeneration. SMA originates from loss-of-function mutations of the SMN1 (Survival of Motor Neuron 1) gene. Recent evidence has implicated peripheral deficits, especially in skeletal muscle, as key contributors to disease progression in SMA. In this study we generated myogenic cells from two SMA-affected human embryonic stem cell (hESC) lines with deletion of SMN1 bearing two copies of the SMN2 gene and recapitulating the molecular phenotype of Type 1 SMA. We characterized myoblasts and myotubes by comparing them to two unaffected, control hESC lines and demonstrate that SMA myoblasts and myotubes showed altered expression of various myogenic markers, which translated into an impaired in vitro myogenic maturation and development process. Additionally, we provide evidence that these SMN1 deficient cells display functional deficits in cholinergic calcium signaling response, glycolysis and oxidative phosphorylation. Our data describe a novel human myogenic SMA model that might be used for interrogating the effect of SMN depletion during skeletal muscle development, and as model to investigate biological mechanisms targeting myogenic differentiation, mitochondrial respiration and calcium signaling processes in SMA muscle cells.

High-affinity RNA targets of the Survival Motor Neuron protein reveal diverse preferences for sequence and structural motifs.

The Survival Motor Neuron (SMN) protein is essential for survival of all animal cells. SMN harbors a nucleic acid-binding domain and plays an important role in RNA metabolism. However, the RNA-binding property of SMN is poorly understood. Here we employ iterative in vitro selection and chemical structure probing to identify sequence and structural motif(s) critical for RNA-SMN interactions. Our results reveal that motifs that drive RNA-SMN interactions are diverse and suggest that tight RNA-SMN interaction requires presence of multiple contact sites on the RNA molecule. We performed UV crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to identify cellular RNA targets of SMN in neuronal SH-SY5Y cells. Results of HITS-CLIP identified a wide variety of targets, including mRNAs coding for ribosome biogenesis and cytoskeleton dynamics. We show critical determinants of ANXA2 mRNA for a direct SMN interaction in vitro. Our data confirms the ability of SMN to discriminate among close RNA sequences, and represent the first validation of a direct interaction of SMN with a cellular RNA target. Our findings suggest direct RNA-SMN interaction as a novel mechanism to initiate the cascade of events leading to the execution of SMN-specific functions.

Two breakthrough gene-targeted treatments for spinal muscular atrophy: challenges remain.

The motor neuron disease spinal muscular atrophy (SMA) is caused by recessive, loss-of-function mutations of the survival motor neuron 1 gene (SMN1). Alone, such mutations are embryonically lethal, but SMA patients retain a paralog gene, SMN2, that undergoes alternative pre-mRNA splicing, producing low levels of SMN protein. By mechanisms that are not well understood, reduced expression of the ubiquitously expressed SMN protein causes an early-onset motor neuron disease that often results in infantile or childhood mortality. Recently, striking clinical improvements have resulted from two novel treatment strategies to increase SMN protein by (a) modulating the splicing of existing SMN2 pre-mRNAs using antisense oligonucleotides, and (b) transducing motor neurons with self-complementary adeno-associated virus 9 (scAAV9) expressing exogenous SMN1 cDNA. We review the recently published clinical trial results and discuss the differing administration, tissue targeting, and potential toxicities of these two therapies. We also focus on the challenges that remain, emphasizing the many clinical and biologic questions that remain open. Answers to these questions will enable further optimization of these remarkable SMA treatments as well as provide insights that may well be useful in application of these therapeutic platforms to other diseases.

Intron-retained transcripts of the spinal muscular atrophy genes, SMN1 and SMN2.

BACKGROUND: The SMN genes, SMN1 and SMN2, are highly homologous genes which are related to the development or clinical severity of spinal muscular atrophy. Some alternative splicing patterns of the SMN genes have been well documented. In 2007, an SMN1 transcript with a full sequence of intron 3 was reported as the first intron-retained SMN transcript. METHODS: Intron-retained SMN transcripts in various cells and tissues were studied using reverse transcription (RT)-PCR. HeLa cells were used for subcellular localization of the transcripts and protein expression analysis with Western blotting. RESULTS: Two intron-retained SMN transcripts were detected, which contain full sequences of intron 2b or intron 3. These transcripts were produced from SMN1 and SMN2, and ubiquitously expressed in human cells and tissues. Western blotting analysis showed no proteins derived from the intron-retained transcripts. Fractionation analysis showed that these intron-retained transcripts were localized mainly in the nucleus. Contrary to our expectation, the intron-retained transcript levels decreased during the treatment of cycloheximide, an inhibitor of nonsense-mediated decay (NMD), suggesting that they were not targets of NMD. CONCLUSION: Intron 2b-retained SMN transcript and intron3-retained SMN transcript were ubiquitously expressed in human cells and tissues. The intron-retained transcripts were mainly localized in the nucleus and decreased through non-NMD pathway.

The water extract of Liuwei dihuang possesses multi-protective properties on neurons and muscle tissue against deficiency of survival motor neuron protein.

BACKGROUND: Deficiency of survival motor neuron (SMN) protein, which is encoded by the SMN1 and SMN2 genes, induces widespread splicing defects mainly in spinal motor neurons, and leads to spinal muscular atrophy (SMA). Currently, there is no effective treatment for SMA. Liuwei dihuang (LWDH), a traditional Chinese herbal formula, possesses multiple therapeutic benefits against various diseases via modulation of the nervous, immune and endocrine systems. Previously, we demonstrated water extract of LWDH (LWDH-WE) protects dopaminergic neurons and improves motor activity in models of Parkinson's disease. PURPOSE: This study aimed to investigate the potential protection of LWDH-WE on SMN deficiency-induced neurodegeneration and muscle weakness. STUDY DESIGN: The effects of LWDH-WE on SMN deficiency-induced neurotoxicity and muscle atrophy were examined by using SMN-deficient NSC34 motor neuron-like cells and SMA-like mice, respectively. METHODS: Inducible SMN-knockdown NSC34 motor neuron-like cells were used to mimic SMN-deficient condition. Doxycycline (1 µg/ml) was used to induce SMN deficiency in stable NSC34 cell line carrying SMN-specific shRNA. SMAΔ7 mice were used as a severe type of SMA mouse model. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Apoptotic cells and neurite length were observed by inverted microscope. Protein expressions were examined by western blots. Muscle strength of animals was evaluated by hind-limb suspension test. RESULTS: LWDH-WE significantly increased SMN protein level, mitochondrial membrane potential and cell viability of SMN-deficient NSC34 cells. LWDH-WE attenuated SMN deficiency-induced down-regulation of B-cell lymphoma-2 (Bcl-2) and up-regulation of cytosolic cytochrome c and cleaved caspase-3. Moreover, LWDH-WE prevented SMN deficiency-induced inhibition of neurite outgrowth and activation of Ras homolog gene family, member A (RhoA)/ Rho-associated protein kinase (ROCK2)/ phospho-LIM kinase (p-LIMK)/ phospho-cofilin (p-cofilin) pathway. Furthermore, in SMA-like mice, LWDH-WE improved muscle strength and body weight accompanied with up-regulation of SMN protein in spinal cord, brain, and gastrocnemius muscle tissues. CONCLUSION: The present study demonstrated that LWDH-WE protects motor neurons against SMN deficiency-induced neurodegeneration, and it also improves the muscle strength of SMA-like mice, suggesting the potential benefits of LWDH-WE as a complementary prescription for SMN deficiency-related diseases.

A-44G transition in SMN2 intron 6 protects patients with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by reduced expression of survival of motor neuron (SMN), a protein expressed in humans by two paralogous genes, SMN1 and SMN2. These genes are nearly identical, except for 10 single-nucleotide differences and a 5-nucleotide insertion in SMN2. SMA is subdivided into four main types, with type I being the most severe. SMN2 copy number is a key positive modifier of the disease, but it is not always inversely correlated with clinical severity. We previously reported the c.859G > C variant in SMN2 exon 7 as a positive modifier in several patients. We have now identified A-44G as an additional positive disease modifier, present in a group of patients carrying 3 SMN2 copies but displaying milder clinical phenotypes than other patients with the same SMN2 copy number. One of the three SMN2 copies appears to have been converted from SMN1, but except for the C6T transition, no other changes were detected. Analyzed with minigenes, SMN1C6T displayed a ∼20% increase in exon 7 inclusion, compared to SMN2. Through systematic mutagenesis, we found that the improvement in exon 7 splicing is mainly attributable to the A-44G transition in intron 6. Using RNA-affinity chromatography and mass spectrometry, we further uncovered binding of the RNA-binding protein HuR to the -44 region, where it acts as a splicing repressor. The A-44G change markedly decreases the binding affinity of HuR, resulting in a moderate increase in exon 7 inclusion.

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis.

Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca2+-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.

Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways.

The selection of 3΄ splice sites (3΄ss) is an essential early step in mammalian RNA splicing reactions, but the processes involved are unknown. We have used single molecule methods to test whether the major components implicated in selection, the proteins U2AF35 and U2AF65 and the U2 snRNP, are able to recognize alternative candidate sites or are restricted to one pre-specified site. In the presence of adenosine triphosphate (ATP), all three components bind in a 1:1 stoichiometry with a 3΄ss. Pre-mRNA molecules with two alternative 3΄ss can be bound concurrently by two molecules of U2AF or two U2 snRNPs, so none of the components are restricted. However, concurrent occupancy inhibits splicing. Stoichiometric binding requires conditions consistent with coalescence of the 5΄ and 3΄ sites in a complex (I, initial), but if this cannot form the components show unrestricted and stochastic association. In the absence of ATP, when complex E forms, U2 snRNP association is unrestricted. However, if protein dephosphorylation is prevented, an I-like complex forms with stoichiometric association of U2 snRNPs and the U2 snRNA is base-paired to the pre-mRNA. Complex I differs from complex A in that the formation of complex A is associated with the loss of U2AF65 and 35.

A novel human-specific splice isoform alters the critical C-terminus of Survival Motor Neuron protein.

Spinal muscular atrophy (SMA), a leading genetic disease of children and infants, is caused by mutations or deletions of Survival Motor Neuron 1 (SMN1) gene. SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of SMN1 due to skipping of exon 7. SMN2 predominantly produces SMNΔ7, an unstable protein. Here we report exon 6B, a novel exon, generated by exonization of an intronic Alu-like sequence of SMN. We validate the expression of exon 6B-containing transcripts SMN6B and SMN6BΔ7 in human tissues and cell lines. We confirm generation of SMN6B transcripts from both SMN1 and SMN2. We detect expression of SMN6B protein using antibodies raised against a unique polypeptide encoded by exon 6B. We analyze RNA-Seq data to show that hnRNP C is a potential regulator of SMN6B expression and demonstrate that SMN6B is a substrate of nonsense-mediated decay. We show interaction of SMN6B with Gemin2, a critical SMN-interacting protein. We demonstrate that SMN6B is more stable than SMNΔ7 and localizes to both the nucleus and the cytoplasm. Our finding expands the diversity of transcripts generated from human SMN genes and reveals a novel protein isoform predicted to be stably expressed during conditions of stress.

Differential induction of muscle atrophy pathways in two mouse models of spinal muscular atrophy.

Motor neuron loss and neurogenic atrophy are hallmarks of spinal muscular atrophy (SMA), a leading genetic cause of infant deaths. Previous studies have focused on deciphering disease pathogenesis in motor neurons. However, a systematic evaluation of atrophy pathways in muscles is lacking. Here, we show that these pathways are differentially activated depending on severity of disease in two different SMA model mice. Although proteasomal degradation is induced in skeletal muscle of both models, autophagosomal degradation is present only in Smn(2B/-) mice but not in the more severe Smn(-/-); SMN2 mice. Expression of FoxO transcription factors, which regulate both proteasomal and autophagosomal degradation, is elevated in Smn(2B/-) muscle. Remarkably, administration of trichostatin A reversed all molecular changes associated with atrophy. Cardiac muscle also exhibits differential induction of atrophy between Smn(2B/-) and Smn(-/-); SMN2 mice, albeit in the opposite direction to that of skeletal muscle. Altogether, our work highlights the importance of cautious analysis of different mouse models of SMA as distinct patterns of atrophy induction are at play depending on disease severity. We also revealed that one of the beneficial impacts of trichostatin A on SMA model mice is via attenuation of muscle atrophy through reduction of FoxO expression to normal levels.

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.