PRMT inhibitor promotes SMN2 exon 7 inclusion and synergizes with nusinersen to rescue SMA mice.

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality. The advent of approved treatments for this devastating condition has significantly changed SMA patients' life expectancy and quality of life. Nevertheless, these are not without limitations, and research efforts are underway to develop new approaches for improved and long-lasting benefits for patients. Protein arginine methyltransferases (PRMTs) are emerging as druggable epigenetic targets, with several small-molecule PRMT inhibitors already in clinical trials. From a screen of epigenetic molecules, we have identified MS023, a potent and selective type I PRMT inhibitor able to promote SMN2 exon 7 inclusion in preclinical SMA models. Treatment of SMA mice with MS023 results in amelioration of the disease phenotype, with strong synergistic amplification of the positive effect when delivered in combination with the antisense oligonucleotide nusinersen. Moreover, transcriptomic analysis revealed that MS023 treatment has minimal off-target effects, and the added benefit is mainly due to targeting neuroinflammation. Our study warrants further clinical investigation of PRMT inhibition both as a stand-alone and add-on therapy for SMA.

Antibody-oligonucleotide conjugate achieves CNS delivery in animal models for spinal muscular atrophy.

Antisense oligonucleotides (ASOs) have emerged as one of the most innovative new genetic drug modalities. However, their high molecular weight limits their bioavailability for otherwise-treatable neurological disorders. We investigated conjugation of ASOs to an antibody against the murine transferrin receptor, 8D3130, and evaluated it via systemic administration in mouse models of the neurodegenerative disease spinal muscular atrophy (SMA). SMA, like several other neurological and neuromuscular diseases, is treatable with single-stranded ASOs that modulate splicing of the survival motor neuron 2 (SMN2) gene. Administration of 8D3130-ASO conjugate resulted in elevated levels of bioavailability to the brain. Additionally, 8D3130-ASO yielded therapeutic levels of SMN2 splicing in the central nervous system of adult human SMN2-transgenic (hSMN2-transgenic) mice, which resulted in extended survival of a severely affected SMA mouse model. Systemic delivery of nucleic acid therapies with brain-targeting antibodies offers powerful translational potential for future treatments of neuromuscular and neurodegenerative diseases.

Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD.

Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. Here we have investigated the use of the Staphylococcus aureus CRISPR-Cas9 system and a double-cut strategy, delivered using a pair of adeno-associated virus serotype 9 (AAV9) vectors, for dystrophin restoration in the severely affected dystrophin/utrophin double-knockout (dKO) mouse. Single guide RNAs were designed to excise Dmd exon 23, with flanking intronic regions repaired by non-homologous end joining. Exon 23 deletion was confirmed at the DNA level by PCR and Sanger sequencing, and at the RNA level by RT-qPCR. Restoration of dystrophin protein expression was demonstrated by western blot and immunofluorescence staining in mice treated via either intraperitoneal or intravenous routes of delivery. Dystrophin restoration was most effective in the diaphragm, where a maximum of 5.7% of wild-type dystrophin expression was observed. CRISPR treatment was insufficient to extend lifespan in the dKO mouse, and dystrophin was expressed in a within-fiber patchy manner in skeletal muscle tissues. Further analysis revealed a plethora of non-productive DNA repair events, including AAV genome integration at the CRISPR cut sites. This study highlights potential challenges for the successful development of CRISPR therapies in the context of DMD.

Exon skipping induces uniform dystrophin rescue with dose-dependent restoration of serum miRNA biomarkers and muscle biophysical properties.

Therapies that restore dystrophin expression are presumed to correct Duchenne muscular dystrophy (DMD), with antisense-mediated exon skipping being the leading approach. Here we aimed to determine whether exon skipping using a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) conjugate results in dose-dependent restoration of uniform dystrophin localization, together with correction of putative DMD serum and muscle biomarkers. Dystrophin-deficient mdx mice were treated with a PPMO (Pip9b2-PMO) designed to induce Dmd exon 23 skipping at single, ascending intravenous doses (3, 6, or 12 mg/kg) and sacrificed 2 weeks later. Dose-dependent exon skipping and dystrophin protein restoration were observed, with dystrophin uniformly distributed at the sarcolemma of corrected myofibers at all doses. Serum microRNA biomarkers (i.e., miR-1a-3p, miR-133a-3p, miR-206-3p, miR-483-3p) and creatinine kinase levels were restored toward wild-type levels after treatment in a dose-dependent manner. All biomarkers were strongly anti-correlated with both exon skipping level and dystrophin expression. Dystrophin rescue was also strongly positively correlated with muscle stiffness (i.e., Young's modulus) as determined by atomic force microscopy (AFM) nanoindentation assay. These data demonstrate that PPMO-mediated exon skipping generates myofibers with uniform dystrophin expression and that both serum microRNA biomarkers and muscle AFM have potential utility as pharmacodynamic biomarkers of dystrophin restoration therapy in DMD.

Dysregulation of Tweak and Fn14 in skeletal muscle of spinal muscular atrophy mice.

BACKGROUND: Spinal muscular atrophy (SMA) is a childhood neuromuscular disorder caused by depletion of the survival motor neuron (SMN) protein. SMA is characterized by the selective death of spinal cord motor neurons, leading to progressive muscle wasting. Loss of skeletal muscle in SMA is a combination of denervation-induced muscle atrophy and intrinsic muscle pathologies. Elucidation of the pathways involved is essential to identify the key molecules that contribute to and sustain muscle pathology. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/TNF receptor superfamily member fibroblast growth factor-inducible 14 (Fn14) pathway has been shown to play a critical role in the regulation of denervation-induced muscle atrophy as well as muscle proliferation, differentiation, and metabolism in adults. However, it is not clear whether this pathway would be important in highly dynamic and developing muscle. METHODS: We thus investigated the potential role of the TWEAK/Fn14 pathway in SMA muscle pathology, using the severe Taiwanese Smn-/-; SMN2 and the less severe Smn2B/- SMA mice, which undergo a progressive neuromuscular decline in the first three post-natal weeks. We also used experimental models of denervation and muscle injury in pre-weaned wild-type (WT) animals and siRNA-mediated knockdown in C2C12 muscle cells to conduct additional mechanistic investigations. RESULTS: Here, we report significantly dysregulated expression of Tweak, Fn14, and previously proposed downstream effectors during disease progression in skeletal muscle of the two SMA mouse models. In addition, siRNA-mediated Smn knockdown in C2C12 myoblasts suggests a genetic interaction between Smn and the TWEAK/Fn14 pathway. Further analyses of SMA, Tweak-/-, and Fn14-/- mice revealed dysregulated myopathy, myogenesis, and glucose metabolism pathways as a common skeletal muscle feature, providing further evidence in support of a relationship between the TWEAK/Fn14 pathway and Smn. Finally, administration of the TWEAK/Fn14 agonist Fc-TWEAK improved disease phenotypes in the two SMA mouse models. CONCLUSIONS: Our study provides mechanistic insights into potential molecular players that contribute to muscle pathology in SMA and into likely differential responses of the TWEAK/Fn14 pathway in developing muscle.

Glycine-induced NMDA receptor internalization provides neuroprotection and preserves vasculature following ischemic stroke.

Ischemic stroke is the second leading cause of death worldwide. Following an ischemic event, neuronal death is triggered by uncontrolled glutamate release leading to overactivation of glutamate sensitive N-methyl-d-aspartate receptor (NMDAR). For gating, NMDARs require not only the binding of glutamate, but also of glycine or a glycine-like compound as a co-agonist. Low glycine doses enhance NMDAR function, whereas high doses trigger glycine-induced NMDAR internalization (GINI) in vitro. Here, we report that following an ischemic event, in vivo, GINI also occurs and provides neuroprotection in the presence of a GlyT1 antagonist (GlyT1-A). Mice pretreated with a GlyT1-A, which increases synaptic glycine levels, exhibited smaller stroke volume, reduced cell death, and minimized behavioral deficits following stroke induction by either photothrombosis or endothelin-1. Moreover, we show evidence that in ischemic conditions, GlyT1-As preserve the vasculature in the peri-infarct area. Therefore, GlyT1 could be a new target for the treatment of ischemic stroke.

Combining multiomics and drug perturbation profiles to identify muscle-specific treatments for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss of survival motor neuron (SMN) protein. While SMN restoration therapies are beneficial, they are not a cure. We aimed to identify potentially novel treatments to alleviate muscle pathology combining transcriptomics, proteomics, and perturbational data sets. This revealed potential drug candidates for repurposing in SMA. One of the candidates, harmine, was further investigated in cell and animal models, improving multiple disease phenotypes, including lifespan, weight, and key molecular networks in skeletal muscle. Our work highlights the potential of multiple and parallel data-driven approaches for the development of potentially novel treatments for use in combination with SMN restoration therapies.

Muscle overexpression of Klf15 via an AAV8-Spc5-12 construct does not provide benefits in spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the survival motor neuron (SMN) gene. While there are currently two approved gene-based therapies for SMA, availability, high cost, and differences in patient response indicate that alternative treatment options are needed. Optimal therapeutic strategies will likely be a combination of SMN-dependent and -independent treatments aimed at alleviating symptoms in the central nervous system and peripheral muscles. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates key metabolic and ergogenic pathways in muscle. We have recently reported significant downregulation of Klf15 in muscle of presymptomatic SMA mice. Importantly, perinatal upregulation of Klf15 via transgenic and pharmacological methods resulted in improved disease phenotypes in SMA mice, including weight and survival. In the current study, we designed an adeno-associated virus serotype 8 (AAV8) vector to overexpress a codon-optimized Klf15 cDNA under the muscle-specific Spc5-12 promoter (AAV8-Klf15). Administration of AAV8-Klf15 to severe Taiwanese Smn-/-;SMN2 or intermediate Smn2B/- SMA mice significantly increased Klf15 expression in muscle. We also observed significant activity of the AAV8-Klf15 vector in liver and heart. AAV8-mediated Klf15 overexpression moderately improved survival in the Smn2B/- model but not in the Taiwanese mice. An inability to specifically induce Klf15 expression at physiological levels in a time- and tissue-dependent manner may have contributed to this limited efficacy. Thus, our work demonstrates that an AAV8-Spc5-12 vector induces high gene expression as early as P2 in several tissues including muscle, heart, and liver, but highlights the challenges of achieving meaningful vector-mediated transgene expression of Klf15.

Light modulation ameliorates expression of circadian genes and disease progression in spinal muscular atrophy mice.

Physiology and behaviour are critically dependent on circadian regulation via a core set of clock genes, dysregulation of which leads to metabolic and sleep disturbances. Metabolic and sleep perturbations occur in spinal muscular atrophy (SMA), a neuromuscular disorder caused by loss of the survival motor neuron (SMN) protein and characterized by motor neuron loss and muscle atrophy. We therefore investigated the expression of circadian rhythm genes in various metabolic tissues and spinal cord of the Taiwanese Smn-/-;SMN2 SMA animal model. We demonstrate a dysregulated expression of the core clock genes (clock, ARNTL/Bmal1, Cry1/2, Per1/2) and clock output genes (Nr1d1 and Dbp) in SMA tissues during disease progression. We also uncover an age- and tissue-dependent diurnal expression of the Smn gene. Importantly, we observe molecular and phenotypic corrections in SMA mice following direct light modulation. Our study identifies a key relationship between an SMA pathology and peripheral core clock gene dysregulation, highlights the influence of SMN on peripheral circadian regulation and metabolism and has significant implications for the development of peripheral therapeutic approaches and clinical care management of SMA patients.

Interventions Targeting Glucocorticoid-Krüppel-like Factor 15-Branched-Chain Amino Acid Signaling Improve Disease Phenotypes in Spinal Muscular Atrophy Mice.

The circadian glucocorticoid-Krüppel-like factor 15-branched-chain amino acid (GC-KLF15-BCAA) signaling pathway is a key regulatory axis in muscle, whose imbalance has wide-reaching effects on metabolic homeostasis. Spinal muscular atrophy (SMA) is a neuromuscular disorder also characterized by intrinsic muscle pathologies, metabolic abnormalities and disrupted sleep patterns, which can influence or be influenced by circadian regulatory networks that control behavioral and metabolic rhythms. We therefore set out to investigate the contribution of the GC-KLF15-BCAA pathway in SMA pathophysiology of Taiwanese Smn-/-;SMN2 and Smn2B/- mouse models. We thus uncover substantial dysregulation of GC-KLF15-BCAA diurnal rhythmicity in serum, skeletal muscle and metabolic tissues of SMA mice. Importantly, modulating the components of the GC-KLF15-BCAA pathway via pharmacological (prednisolone), genetic (muscle-specific Klf15 overexpression) and dietary (BCAA supplementation) interventions significantly improves disease phenotypes in SMA mice. Our study highlights the GC-KLF15-BCAA pathway as a contributor to SMA pathogenesis and provides several treatment avenues to alleviate peripheral manifestations of the disease. The therapeutic potential of targeting metabolic perturbations by diet and commercially available drugs could have a broader implementation across other neuromuscular and metabolic disorders characterized by altered GC-KLF15-BCAA signaling.

Aberrant Subcellular Dynamics of Sigma-1 Receptor Mutants Underlying Neuromuscular Diseases.

The sigma-1 receptor (σ-1R) is an endoplasmic reticulum resident chaperone protein involved in a plethora of cellular functions, and whose disruption has been implicated in a wide range of diseases. Genetic analysis has revealed two σ-1R mutants involved in neuromuscular disorders. A point mutation (E102Q) in the ligand-binding domain results in the juvenile form of amyotrophic lateral sclerosis (ALS16), and a 20 amino-acid deletion (Δ31-50) in the putative cytosolic domain leads to a form of distal hereditary motor neuropathy. We investigated the localization and functional properties of these mutants in cell lines using confocal imaging and electrophysiology. The σ-1R mutants exhibited a significant increase in mobility, aberrant localization, and enhanced block of the inwardly rectifying K(+) channel Kir2.1, compared with the wild-type σ-1R. Thus, these σ-1R mutants have different functional properties that could contribute to their disease phenotypes.

Plasminogen is a critical regulator of cutaneous wound healing.

Wound healing is a complicated biological process that consist of partially overlapping inflammatory, proliferation and tissue remodelling phases. A successful wound healing depends on a proper activation and subsequent termination of the inflammatory phase. The failure to terminate the inflammation halts the completion of wound healing and is a known reason for formation of chronic wounds. Previous studies have shown that wound closure is delayed in plasminogen-deficient mice, and a role for plasminogen in dissection of extracellular matrix was suggested. However, our finding that plasminogen is transported to the wound by inflammatory cells early during the healing process, where it potentiates inflammation, indicates that plasminogen may also have other roles in the wound healing process. Here we report that plasminogen-deficient mice have extensive fibrin and neutrophil depositions in the wounded area long after re-epithelialisation, indicating inefficient debridement and chronic inflammation. Delayed formation of granulation tissue suggests that fibroblast function is impaired in the absence of plasminogen. Therefore, in addition to its role in the activation of inflammation, plasminogen is also crucial for subsequent steps, including resolution of inflammation and activation of the proliferation phase. Importantly, supplementation of plasminogen-deficient mice with human plasminogen leads to a restored healing process that is comparable to that in wild-type mice. Besides of being an activator of the inflammatory phase during wound healing, plasminogen is also required for the subsequent termination of inflammation. Based on these results, we propose that plasminogen may be an important future therapeutic agent for wound treatment.

Differential effects of N-acetyl-aspartyl-glutamate on synaptic and extrasynaptic NMDA receptors are subunit- and pH-dependent in the CA1 region of the mouse hippocampus.

Ischemic strokes cause excessive release of glutamate, leading to overactivation of N-methyl-d-aspartate receptors (NMDARs) and excitotoxicity-induced neuronal death. For this reason, inhibition of NMDARs has been a central focus in identifying mechanisms to avert this extensive neuronal damage. N-acetyl-aspartyl-glutamate (NAAG), the most abundant neuropeptide in the brain, is neuroprotective in ischemic conditions in vivo. Despite this evidence, the exact mechanism underlying its neuroprotection, and more specifically its effect on NMDARs, is currently unknown due to conflicting results in the literature. Here, we uncover a pH-dependent subunit-specific action of NAAG on NMDARs. Using whole-cell electrophysiological recordings on acute hippocampal slices from adult mice and on HEK293 cells, we found that NAAG increases synaptic GluN2A-containing NMDAR EPSCs, while effectively decreasing extrasynaptic GluN2B-containing NMDAR EPSCs in physiological pH. Intriguingly, the results of our study further show that in low pH, which is a physiological occurrence during ischemia, NAAG depresses GluN2A-containing NMDAR EPSCs and amplifies its inhibitory effect on GluN2B-containing NMDAR EPSCs, as well as upregulates the surface expression of the GluN2A subunit. Altogether, our data demonstrate that NAAG has differential effects on NMDAR function based on subunit composition and pH. These findings suggest that the role of NAAG as a neuroprotective agent during an ischemic stroke is likely mediated by its ability to reduce NMDAR excitation. The inhibitory effect of NAAG on NMDARs and its enhanced function in acidic conditions make NAAG a prime therapeutic agent for the treatment of ischemic events.

NMDA receptors are upregulated and trafficked to the plasma membrane after sigma-1 receptor activation in the rat hippocampus.

Sigma-1 receptors (σ-1Rs) are endoplasmic reticulum resident chaperone proteins implicated in many physiological and pathological processes in the CNS. A striking feature of σ-1Rs is their ability to interact and modulate a large number of voltage- and ligand-gated ion channels at the plasma membrane. We have reported previously that agonists for σ-1Rs potentiate NMDA receptor (NMDAR) currents, although the mechanism by which this occurs is still unclear. In this study, we show that in vivo administration of the selective σ-1R agonists (+)-SKF 10,047 [2S-(2α,6α,11R*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol hydrochloride (N-allylnormetazocine) hydrochloride], PRE-084 (2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate hydrochloride), and (+)-pentazocine increases the expression of GluN2A and GluN2B subunits, as well as postsynaptic density protein 95 in the rat hippocampus. We also demonstrate that σ-1R activation leads to an increased interaction between GluN2 subunits and σ-1Rs and mediates trafficking of NMDARs to the cell surface. These results suggest that σ-1R may play an important role in NMDAR-mediated functions, such as learning and memory. It also opens new avenues for additional studies into a multitude of pathological conditions in which NMDARs are involved, including schizophrenia, dementia, and stroke.

Expression and localization of the serine proteases high-temperature requirement factor A1, serine protease 23, and serine protease 35 in the mouse ovary.

Proteolytic degradation of extracellular matrix components has been suggested to play an essential role in the occurrence of ovulation. Recent studies in our laboratory have indicated that the plasminogen activator and matrix metalloproteinase systems, which were previously believed to be crucial for ovulation, are not required in this process. In this study we have used a microarray approach to identify new proteases that are involved in ovulation. We found three serine proteases that were relatively highly expressed during ovulation: high-temperature requirement factor A1 (HtrA1), which was not regulated much during ovulation; serine protease 23 (PRSS23), which was down-regulated by gonadotropins; and serine protease 35 (PRSS35), which was up-regulated by gonadotropins. We have further investigated the expression patterns of these proteases during gonadotropin-induced ovulation in immature mice and in the corpus luteum (CL) of pseudopregnant mice. We found that HtrA1 was highly expressed in granulosa cells throughout follicular development and ovulation, as well as in the forming and regressing CL. PRSS23 was highly expressed in atretic follicles, and it was expressed in the ovarian stroma and theca tissues just before ovulation. PRSS35 was expressed in the theca layers of developing follicles. It was also highly induced in granulosa cells of preovulatory follicles. PRSS35 was also expressed in the forming and regressing CL. These data suggest that HtrA1 and PRSS35 may be involved in ovulation and CL formation and regression, and that PRSS23 may play a role in follicular atresia.

Multivalent sialic acid conjugates inhibit adenovirus type 37 from binding to and infecting human corneal epithelial cells.

Adenovirus type 37 is one of the main causative agents of epidemic keratoconjunctivitis. In a series of publications, we have reported that this virus uses sialic acid as a cellular receptor. Here we demonstrate in vitro that on a molar basis, multivalent sialic acid conjugated to human serum albumin prevents adenovirus type 37 from binding to and infecting human corneal epithelial cells 1000-fold more efficiently than monosaccharidic sialic acid. We also demonstrate that the extraordinary inhibitory effect of multivalent sialic acid is due to the ability of this compound to aggregate virions. We conclude that multivalent sialic acid may be a potential new antiviral drug, for use in the treatment of epidemic keratoconjunctivitis caused by the adenoviruses that use sialic acid as cellular receptor.