Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by the absence of dystrophin protein. One current DMD therapeutic strategy, exon skipping, produces a truncated dystrophin isoform using phosphorodiamidate morpholino oligomers (PMOs). However, the potential of exon skipping therapeutics has not been fully realized as increases in dystrophin protein have been minimal in clinical trials. Here, we investigate how miR-146a-5p, which is highly elevated in dystrophic muscle, impacts dystrophin protein levels. We find inflammation strongly induces miR-146a in dystrophic, but not wild-type myotubes. Bioinformatics analysis reveals that the dystrophin 3' UTR harbors a miR-146a binding site, and subsequent luciferase assays demonstrate miR-146a binding inhibits dystrophin translation. In dystrophin-null mdx52 mice, co-injection of miR-146a reduces dystrophin restoration by an exon 51 skipping PMO. To directly investigate how miR-146a impacts therapeutic dystrophin rescue, we generated mdx52 with body-wide miR-146a deletion (146aX). Administration of an exon skipping PMO via intramuscular or intravenous injection markedly increases dystrophin protein levels in 146aX vs. mdx52 muscles while skipped dystrophin transcript levels are unchanged supporting a post-transcriptional mechanism of action. Together, these data show that miR-146a expression opposes therapeutic dystrophin restoration, suggesting miR-146a inhibition warrants further research as a potential DMD exon skipping co-therapy.

Key features of the innate immune response is mediated by the immunoproteasome in microglia.

Microglia are the resident immune cells of the central nervous system (CNS). We and others have shown that the inflammatory response of microglia is partially regulated by the immunoproteasome, an inducible form of the proteasome responsible for the generation of major histocompatibility complex (MHC) class I epitopes. While the role of the proteasome in the adaptive immune system is well established, emerging evidence suggests the immunoproteasome may have discrete functions in the innate immune response. Here, we show that inhibiting the immunoproteasome reduces the IFNγ-dependent induction of complement activator C1q, suppresses phagocytosis, and alters the cytokine expression profile in a microglial cell line and microglia derived from human inducible pluripotent stem cells. Moreover, we show that the immunoproteasome regulates the degradation of IκBα, a modulator of NF-κB signaling. Finally, we demonstrate that NADH prevents induction of the immunoproteasome, representing a potential pathway to suppress immunoproteasome-dependent immune responses.

The glucocorticoid receptor acts locally to protect dystrophic muscle and heart during disease.

Absence of dystrophin results in muscular weakness, chronic inflammation and cardiomyopathy in Duchenne muscular dystrophy (DMD). Pharmacological corticosteroids are the DMD standard of care; however, they have harsh side effects and unclear molecular benefits. It is uncertain whether signaling by physiological corticosteroids and their receptors plays a modifying role in the natural etiology of DMD. Here, we knocked out the glucocorticoid receptor (GR, encoded by Nr3c1) specifically in myofibers and cardiomyocytes within wild-type and mdx52 mice to dissect its role in muscular dystrophy. Double-knockout mice showed significantly worse phenotypes than mdx52 littermate controls in measures of grip strength, hang time, inflammatory pathology and gene expression. In the heart, GR deletion acted additively with dystrophin loss to exacerbate cardiomyopathy, resulting in enlarged hearts, pathological gene expression and systolic dysfunction, consistent with imbalanced mineralocorticoid signaling. The results show that physiological GR functions provide a protective role during muscular dystrophy, directly contrasting its degenerative role in other disease states. These data provide new insights into corticosteroids in disease pathophysiology and establish a new model to investigate cell-autonomous roles of nuclear receptors and mechanisms of pharmacological corticosteroids.

Vamorolone improves Becker muscular dystrophy and increases dystrophin protein in bmx model mice.

There is no approved therapy for Becker muscular dystrophy (BMD), a genetic muscle disease caused by in-frame dystrophin deletions. We previously developed the dissociative corticosteroid vamorolone for treatment of the allelic, dystrophin-null disease Duchenne muscular dystrophy. We hypothesize vamorolone can treat BMD by safely reducing inflammatory signaling in muscle and through a novel mechanism of increasing dystrophin protein via suppression of dystrophin-targeting miRNAs. Here, we test this in the bmx mouse model of BMD. Daily oral treatment with vamorolone or prednisolone improves bmx grip strength and hang time phenotypes. Both drugs reduce myofiber size and decrease the percentage of centrally nucleated fibers. Vamorolone shows improved safety versus prednisolone by avoiding or reducing key side effects to behavior and growth. Intriguingly, vamorolone increases dystrophin protein in both heart and skeletal muscle. These data indicate that vamorolone, nearing approval for Duchenne, shows efficacy in bmx mice and therefore warrants clinical investigation in BMD.

Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by the absence of dystrophin protein. One current DMD therapeutic strategy, exon skipping, produces a truncated dystrophin isoform using phosphorodiamidate morpholino oligomers (PMOs). However, the potential of exon skipping therapeutics has not been fully realized as increases in dystrophin protein have been minimal in clinical trials. Here, we investigate how miR-146a-5p, which is highly elevated in dystrophic muscle, impacts dystrophin protein levels. We find inflammation strongly induces miR-146a in dystrophic, but not wild-type myotubes. Bioinformatics analysis reveals that the dystrophin 3'UTR harbors a miR-146a binding site, and subsequent luciferase assays demonstrate miR-146a binding inhibits dystrophin translation. In dystrophin-null mdx52 mice, co-injection of miR-146a reduces dystrophin restoration by an exon 51 skipping PMO. To directly investigate how miR-146a impacts therapeutic dystrophin rescue, we generated mdx52 with body-wide miR-146a deletion (146aX). Administration of an exon skipping PMO via intramuscular or intravenous injection markedly increases dystrophin protein levels in 146aX versus mdx52 muscles; skipped dystrophin transcript levels are unchanged, suggesting a post-transcriptional mechanism-of-action. Together, these data show that miR-146a expression opposes therapeutic dystrophin restoration, suggesting miR-146a inhibition warrants further research as a potential DMD exon skipping co-therapy.

The X-linked Becker muscular dystrophy (bmx) mouse models Becker muscular dystrophy via deletion of murine dystrophin exons 45-47.

BACKGROUND: Becker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype. METHODS: We use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000 bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography. RESULTS: Overall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P < 0.0001), wire hang (P = 0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P = 0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P < 0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P = 0.0017) and centrally located nuclei indicating degeneration/regeneration (P < 0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P < 0.0001), collagen deposition (+1.4-fold, P = 0.0217), and sarcolemmal damage measured by intracellular IgM (P = 0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes. CONCLUSIONS: The bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

Survival motor neuron deficiency slows myoblast fusion through reduced myomaker and myomixer expression.

BACKGROUND: Spinal muscular atrophy is an inherited neurodegenerative disease caused by insufficient levels of the survival motor neuron (SMN) protein. Recently approved treatments aimed at increasing SMN protein levels have dramatically improved patient survival and have altered the disease landscape. While restoring SMN levels slows motor neuron loss, many patients continue to have smaller muscles and do not achieve normal motor milestones. While timing of treatment is important, it remains unclear why SMN restoration is insufficient to fully restore muscle size and function. We and others have shown that SMN-deficient muscle precursor cells fail to efficiently fuse into myotubes. However, the role of SMN in myoblast fusion is not known. METHODS: In this study, we show that SMN-deficient myoblasts readily fuse with wild-type myoblasts, demonstrating fusion competency. Conditioned media from wild type differentiating myoblasts do not rescue the fusion deficit of SMN-deficient cells, suggesting that compromised fusion may primarily be a result of altered membrane dynamics at the cell surface. Transcriptome profiling of skeletal muscle from SMN-deficient mice revealed altered expression of cell surface fusion molecules. Finally, using cell and mouse models, we investigate if myoblast fusion can be rescued in SMN-deficient myoblast and improve the muscle pathology in SMA mice. RESULTS: We found reduced expression of the muscle fusion proteins myomaker (P = 0.0060) and myomixer (P = 0.0051) in the muscle of SMA mice. Suppressing SMN expression in C2C12 myoblast cells reduces expression of myomaker (35% reduction; P < 0.0001) and myomixer, also known as myomerger and minion, (30% reduction; P < 0.0001) and restoring SMN levels only partially restores myomaker and myomixer expression. Ectopic expression of myomixer improves myofibre number (55% increase; P = 0.0006) and motor function (35% decrease in righting time; P = 0.0089) in SMA model mice and enhances motor function (82% decrease in righting time; P < 0.0001) and extends survival (28% increase; P < 0.01) when administered in combination with an antisense oligonucleotide that increases SMN protein levels. CONCLUSIONS: Here, we identified reduced expression of muscle fusion proteins as a key factor in the fusion deficits of SMN-deficient myoblasts. This discovery provides a novel target to improve SMA muscle pathology and motor function, which in combination with SMN increasing therapy could enhance clinical outcomes for SMA patients.

A high-throughput genome-wide RNAi screen identifies modifiers of survival motor neuron protein.

Spinal muscular atrophy (SMA) is a debilitating neurological disorder marked by degeneration of spinal motor neurons and muscle atrophy. SMA results from mutations in survival motor neuron 1 (SMN1), leading to deficiency of survival motor neuron (SMN) protein. Current therapies increase SMN protein and improve patient survival but have variable improvements in motor function, making it necessary to identify complementary strategies to further improve disease outcomes. Here, we perform a genome-wide RNAi screen using a luciferase-based activity reporter and identify genes involved in regulating SMN gene expression, RNA processing, and protein stability. We show that reduced expression of Transcription Export complex components increases SMN levels through the regulation of nuclear/cytoplasmic RNA transport. We also show that the E3 ligase, Neurl2, works cooperatively with Mib1 to ubiquitinate and promote SMN degradation. Together, our screen uncovers pathways through which SMN expression is regulated, potentially revealing additional strategies to treat SMA.

SMN-deficiency disrupts SERCA2 expression and intracellular Ca2+ signaling in cardiomyocytes from SMA mice and patient-derived iPSCs.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of alpha motor neurons and skeletal muscle atrophy. The disease is caused by mutations of the SMN1 gene that result in reduced functional expression of survival motor neuron (SMN) protein. SMN is ubiquitously expressed, and there have been reports of cardiovascular dysfunction in the most severe SMA patients and animal models of the disease. In this study, we directly assessed the function of cardiomyocytes isolated from a severe SMA model mouse and cardiomyocytes generated from patient-derived IPSCs. Consistent with impaired cardiovascular function at the very early disease stages in mice, heart failure markers such as brain natriuretic peptide were significantly elevated. Functionally, cardiomyocyte relaxation kinetics were markedly slowed and the T50 for Ca2+ sequestration increased to 146 ± 4 ms in SMN-deficient cardiomyocytes from 126 ± 4 ms in wild type cells. Reducing SMN levels in cardiomyocytes from control patient IPSCs slowed calcium reuptake similar to SMA patent-derived cardiac cells. Importantly, restoring SMN increased calcium reuptake rate. Taken together, these results indicate that SMN deficiency impairs cardiomyocyte function at least partially through intracellular Ca2+ cycling dysregulation.

Neonatal mouse cortical but not isogenic human astrocyte feeder layers enhance the functional maturation of induced pluripotent stem cell-derived neurons in culture.

Human induced pluripotent stem (iPS) cell-derived neurons and astrocytes are attractive cellular tools for nervous system disease modeling and drug screening. Optimal utilization of these tools requires differentiation protocols that efficiently generate functional cell phenotypes in vitro. As nervous system function is dependent on networked neuronal activity involving both neuronal and astrocytic synaptic functions, we examined astrocyte effects on the functional maturation of neurons from human iPS cell-derived neural stem cells (NSCs). We first demonstrate human iPS cell-derived NSCs can be rapidly differentiated in culture to either neurons or astrocytes with characteristic cellular, molecular and physiological features. Although differentiated neurons were capable of firing multiple action potentials (APs), few cells developed spontaneous electrical activity in culture. We show spontaneous electrical activity was significantly increased by neuronal differentiation of human NSCs on feeder layers of neonatal mouse cortical astrocytes. In contrast, co-culture on feeder layers of isogenic human iPS cell-derived astrocytes had no positive effect on spontaneous neuronal activity. Spontaneous electrical activity was dependent on glutamate receptor-channel function and occurred without changes in INa , IK , Vm , and AP properties of iPS cell-derived neurons. These data demonstrate co-culture with neonatal mouse cortical astrocytes but not human isogenic iPS cell-derived astrocytes stimulates glutamatergic synaptic transmission between iPS cell-derived neurons in culture. We present RNA-sequencing data for an immature, fetal-like status of our human iPS cell-derived astrocytes as one possible explanation for their failure to enhance synaptic activity in our co-culture system.

The role of the immunoproteasome in interferon-γ-mediated microglial activation.

Microglia regulate the brain microenvironment by sensing damage and neutralizing potentially harmful insults. Disruption of central nervous system (CNS) homeostasis results in transition of microglia to a reactive state characterized by morphological changes and production of cytokines to prevent further damage to CNS tissue. Immunoproteasome levels are elevated in activated microglia in models of stroke, infection and traumatic brain injury, though the exact role of the immunoproteasome in neuropathology remains poorly defined. Using gene expression analysis and native gel electrophoresis we characterize the expression and assembly of the immunoproteasome in microglia following interferon-gamma exposure. Transcriptome analysis suggests that the immunoproteasome regulates multiple features of microglial activation including nitric oxide production and phagocytosis. We show that inhibiting the immunoproteasome attenuates expression of pro-inflammatory cytokines and suppresses interferon-gamma-dependent priming of microglia. These results imply that targeting immunoproteasome function following CNS injury may attenuate select microglial activity to improve the pathophysiology of neurodegenerative conditions or the progress of inflammation-mediated secondary injury following neurotrauma.