Potential limitations of micro-dystrophin gene therapy for Duchenne muscular dystrophy.

Clinical trials delivering high doses of adeno-associated viruses (AAVs) expressing truncated dystrophin molecules (micro-dystrophins) are underway for individuals with Duchenne muscular dystrophy (DMD). We examined the efficiency and efficacy of this strategy with four micro-dystrophin constructs (three in clinical trials and a variant of the largest clinical construct), in a severe mouse model of DMD, using doses of AAV comparable to those used in the clinical trials. We achieved high levels of micro-dystrophin expression in striated muscle with cardiac expression ~10 fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of the skeletal muscle disease was observed. Surprisingly, a lethal acceleration of cardiac disease progression occurred with two of the micro-dystrophins. The detrimental impact on the heart appears to be caused by the high levels of micro-dystrophin resulting in variable competition (dependent on the design of the micro-dystrophin) between micro-dystrophin and utrophin at the cardiomyocyte membrane. There may also be a contribution from an overloading of protein degradation. The significance of these observations for patients currently being treated with AAV-micro-dystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown. However, it suggests that micro-dystrophin treatments need to avoid excessively high levels of expression in the heart and cardiac function should be carefully monitored in these patients.

Myospreader improves gene editing in skeletal muscle by myonuclear propagation.

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

Myo10 tail is crucial for promoting long filopodia.

Filopodia are slender cellular protrusions containing parallel actin bundles involved in environmental sensing and signaling, cell adhesion and migration, and growth cone guidance and extension. Myosin 10 (Myo10), an unconventional actin-based motor protein, was reported to induce filopodial initiation with its motor domain. However, the roles of the multifunctional tail domain of Myo10 in filopodial formation and elongation remain elusive. Herein, we generated several constructs of Myo10-full-length Myo10, Myo10 with a truncated tail (Myo10 HMM), and Myo10 containing four mutations to disrupt its coiled-coil domain (Myo10 CC mutant). We found that the truncation of the tail domain decreased filopodial formation and filopodial length, while four mutations in the coiled-coil domain disrupted the motion of Myo10 toward filopodial tips and the elongation of filopodia. Furthermore, we found that filopodia elongated through multiple elongation cycles, which was supported by the Myo10 tail. These findings suggest that Myo10 tail is crucial for promoting long filopodia.

Myosin VI powers self-organization of branched contractile actin network.

The actomyosin cytoskeletal network is responsible for a variety of fundamental cellular processes. Assembly and maintenance of actin networks involve an array of associated regulatory proteins for polymerization, branching, crosslinking and contractility-driven self-organization. In this study, we make the unexpected discovery in vitro that myosin VI and myosin X, motor proteins specialized in vesicle transport and filopodia formation, are capable of crosslinking and self-organizing actin into higher-order contractile structures in the absence of other actin-associated proteins. Moreover, myosin VI alone can initiate actin elongation and branching, and assemble branched force-generating networks from crosslinked actin polymers. Additional architectural control is provided by the actin crosslinking proteins α-actinin and fascin. Our data identify critical stages of tension-mediated connectivity in network development and provide a model system for further exploration of the nonequilibrium mechanics of actomyosin self-organization.

Myospreader improves gene editing in skeletal muscle by myonuclear propagation.

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader", a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

How myosin VI traps its off-state, is activated and dimerizes.

Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the proper functioning of Myo6 relies on precise spatiotemporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6. TOM1 and Dab2 cannot bind the off-state, while GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation.

Direct electrical stimulation impacts on neuromuscular junction morphology on both stimulated and unstimulated contralateral soleus.

BACKGROUND: There is increasing evidence of crosstalk between organs. The neuromuscular junction (NMJ) is a peripheral chemical synapse whose function and morphology are sensitive to acetylcholine (ACh) release and muscle depolarization. In an attempt to improve our understanding of NMJ plasticity and muscle crosstalk, the effects of unilateral direct electrical stimulation of a hindlimb muscle on the NMJ were investigated in rats exposed long-term post-synaptic neuromuscular blockade. METHODS: Sprague Dawley rats were subjected to post-synaptic blockade of neuromuscular transmission by systemic administration of α-cobrotoxin and mechanically ventilated for up to 8 days and compared with untreated sham operated controls and animals exposed to unilateral chronic electrical stimulation 12 h/day for 5 or 8 days. RESULTS: NMJs produced axonal and glial sprouts (growth of processes that extend beyond the confines of the synapse defined by high-density aggregates of acetylcholine receptors [AChRs]) in response to post-synaptic neuromuscular blockade, but less than reported after peripheral denervation or pre-synaptic blockade. Direct electrical soleus muscle stimulation reduced the terminal Schwann cell (tSC) and axonal sprouting in both stimulated and non-stimulated contralateral soleus. Eight days chronic stimulation reduced (P < 0.001) the number of tSC sprouts on stimulated and non-stimulated soleus from 6.7 ± 0.5 and 6.9 ± 0.5 sprouts per NMJ, respectively, compared with 10.3 ± 0.9 tSC per NMJ (P < 0.001) in non-stimulated soleus from rats immobilized for 8 days. A similar reduction of axonal sprouts (P < 0.001) was observed in stimulated and non-stimulated contralateral soleus in response to chronic electrical stimulation. RNAseq-based gene expression analyses confirmed a restoring effect on both stimulated and unstimulated contralateral muscle. The cross-over effect was paralleled by increased cytokine/chemokine levels in stimulated and contralateral unstimulated muscle as well as in plasma. CONCLUSIONS: Motor axon terminals and terminal Schwann cells at NMJs of rats subjected to post-synaptic neuromuscular blockade exhibited sprouting responses. These axonal and glial responses were likely dampened by a muscle-derived myokines released in an activity-dependent manner with both local and systemic effects.

Transcription Factor Hb9 Is Expressed in Glial Cell Lineages in the Developing Mouse Spinal Cord.

Hb9 (Mnx1) is a transcription factor described as a spinal cord motor neuron (MN)-specific marker and critical factor for the postmitotic specification of these cells. To date, expression of Hb9 in other cell types has not been reported. We performed a fate-mapping approach to examine distributions of Hb9-expressing cells and their progeny ("Hb9-lineage cells") within the embryonic and adult spinal cord of Hb9cre;Ai14 mice. We found that Hb9-lineage cells are distributed in a gradient of increasing abundance throughout the rostrocaudal spinal cord axis during embryonic and postnatal stages. Furthermore, although the majority of Hb9-lineage cells at cervical spinal cord levels are MNs, at more caudal levels, Hb9-lineage cells include small-diameter dorsal horn neurons, astrocytes, and oligodendrocytes. In the peripheral nervous system, we observed a similar phenomenon with more abundant Hb9-lineage Schwann cells in muscles of the lower body versus upper body muscles. We cultured spinal cord progenitors in vitro and found that gliogenesis was increased by treatment with the caudalizing factor FGF-8B, while glial tdTomato expression was increased by treatment with both FGF-8B and GDF-11. Together, these observations suggest that early and transient expression of Hb9 in spinal cord neural progenitors may be induced by caudalizing factors such as FGF and GDF signaling. Furthermore, our work raises the possibility that early Hb9 expression may influence the development of spinal cord macroglia and Schwann cells, especially at caudal regions. Together, these findings highlight the importance of using caution when designing experiments using Hb9cre mice to perform spinal cord MN-specific manipulations.

Demodex Blepharitis: An Analysis of Nine Patients.

Inflammatory eyelid symptoms are common in primary care and there have been several reports on Demodex blepharitis. In the present study, we evaluate the 9 patients with Demodex blepharitis, who showed inflammation of the eyelids, dry eye, and cylindrical dandruff at the base of the eyelashes. The causative species from all patients was Demodex folliculorum of either the adult or nymph stage. Two patients had recurrent chalazion and 3 patients had keratitis. Weekly lid scrubs with 50% tee tree oil were performed for 6 weeks. After treatment, the symptoms of blepharitis and keratitis had improved in all patients. This case report provides clinical reference source for the proper treatment of ocular demodicosis.

Evaluation of the DBA/2J mouse as a potential background strain for genetic models of cardiomyopathy.

The potential use of the D2.mdx mouse (the mdx mutation on the DBA/2J genetic background) as a preclinical model of the cardiac aspects of Duchenne muscular dystrophy (DMD) has been criticized based on speculation that the DBA/2J genetic background displays an inherent hypertrophic cardiomyopathy (HCM) phenotype. Accordingly, the goal of the current study was to further examine the cardiac status of this mouse strain over a 12-month period to determine if observable signs of HCM develop, including histopathology and pathological enlargement of the myocardium. Previous reports have documented heightened TGFß signaling in the DBA2/J striated muscles, as compared to the C57 background, which, as expected, is manifested as increased cardiomyocyte size, wall thickness, and heart mass as compared to the C57 background. While normalized heart mass is larger in the DBA/2J mice, compared to age-matched C57/BL10 mice, both strains similarly increase in size from 4 to 12 months of age. We also report that DBA/2J mice contain equivalent amounts of left ventricular collagen as healthy canine and human samples. In a longitudinal echocardiography study, neither sedentary nor exercised DBA/2J mice demonstrated left ventricular wall thickening or cardiac functional deficits. In summary, we find no evidence of HCM, nor any other cardiac pathology, and thus propose that it is an appropriate background strain for genetic modeling of cardiac diseases, including the cardiomyopathy associated with DMD.

Filopodia powered by class x myosin promote fusion of mammalian myoblasts.

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.

Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss.

The muscular dystrophy X-linked mouse (mdx) is the most commonly used preclinical model for Duchenne muscular dystrophy. Although disease progression in the mouse does not perfectly model the human disease, it shares many pathological features. Early characterizations of the model reported severe pathology through early adulthood followed by disease stabilization. As a result, research in the mdx mouse has largely focused on early adulthood. The overarching goal of this study is to improve the understanding of the mdx mouse model by tracking pathological features of the disease throughout life. We performed a thorough characterization of myofiber pathology in mdx mice from 2 weeks to 2 years of age. We report that individual mdx muscle fibers undergo progressive hypertrophy that continues through the lifespan. Despite massive hypertrophy on the myofiber level, we report no hypertrophy on the muscle level. These seemingly contradictory findings are explained by previously underappreciated myofiber loss in mdx mice. We conclude that due to myofiber loss, in combination with the progressive nature of other pathological features, aged mdx muscle tissue provides reliable benchmarks for disease progression that may be valuable in testing the efficacy of therapeutics for Duchenne muscular dystrophy.

Morphological remodeling during recovery of the neuromuscular junction from terminal Schwann cell ablation in adult mice.

Schwann cells (SCs) are integral to the formation and function of the peripheral nervous system (PNS). Exemplifying their importance, the loss or dysfunction of SCs is a feature of a myriad of diseases and conditions that compromise the PNS. Thus, it remains essential to understand the rules that govern the proliferation, differentiation and reconnection of Schwann cells with peripheral axons. Here, we examined the consequences of locally and acutely ablating terminal Schwann cells (tSCs) at the adult mouse neuromuscular junction (NMJ) by using mice expressing diphtheria toxin receptor (DTR) preferentially in tSCs compared to myelinating SCs followed by local application of diphtheria toxin (DTX). After DTX application, tSCs died but, importantly and contrary to expectations, their associated motor axons did not fully degenerate. Within 3 weeks, tSCs returned and reestablished coverage of the synapse with increased numbers. Furthermore, the post-synaptic muscle fibers displayed increased distinct clusters of acetylcholine receptors and axon terminals exhibited numerous terminal varicosities. The lack of degeneration of bare motor axon terminals and the morphological remodeling that occurs upon the return of tSCs to the NMJ may have wider implications for the mechanisms governing tSC occupancy of the adult NMJ and for conditions that adversely affect tSCs.

Exclusive vital labeling of myonuclei for studying myonuclear arrangement in mouse skeletal muscle tissue.

BACKGROUND: The arrangement of myonuclei in skeletal muscle tissue has long been used as a biomarker for muscle health, but there is a dearth of in vivo exploration of potential effects of myonuclear organization on the function and regeneration of skeletal muscle because traditional nuclear stains are performed on postmortem tissue. Therefore, we sought a transgenic method to produce a selective and persistent myonuclear label in whole muscles of living mice. METHODS: We bred together a mouse line with skeletal muscle fiber-selective expression of Cre recombinase and a second mouse line with a Cre-inducible fluorescently tagged histone protein to generate a mouse line that produces a myonuclear label suitable for vital imaging and histology of fixed tissue. We tested the effectiveness of this vital label in three conditions known to generate abnormal myonuclear positioning. First, we injured myofibers of young mice with cardiotoxin. Second, this nuclear label was bred into a murine model of Duchenne muscular dystrophy. Finally, we examined old mice from this line that have undergone the natural aging process. Welch's t test was used to compare wild type and transgenic mice. RESULTS: The resulting mouse line transgenically produces a vital red fluorescent label of myonuclei, which facilitates their in vivo imaging in skeletal muscle tissue. Transgenic fluorescent labeling of myonuclei has no significant effect on skeletal muscle function, as determined by twitch and tetanic force recordings. In each muscle examined, including those under damaged, dystrophic, and aged conditions, the labeled myonuclei exhibit morphology consistent with established literature, and reveal a specialized arrangement of subsynaptic myonuclei at the neuromuscular junction. CONCLUSIONS: Taken together, our results demonstrate that this mouse line provides a versatile tool to selectively visualize myonuclei within both living and fixed preparations of healthy, injured, diseased, and aged muscles.

Intestinal Flukes Recovered from a Herring Gull, Larus argentatus, in the Republic of Korea.

Trematode specimens were collected from the intestine of a herring gull, Larus argentatus, which was found in a critical condition on the shore of a small island (Yubu-do, Seocheon-gun, Chungcheongnam-do) located at the western coast of the Korean peninsula. Total 11 specimens of intestinal flukes, including 3 Cryptocotyle lingua (Heterophyidae), 1 Himasthla alincia (Echinostomatidae), 5 Cardiocephaloides medioconiger (Strigeidae), and 2 Diplostomum spathaceum (Diplostomidae), were recovered. C. lingua was morphologically characterized by the presence of a large ventrogenital apparatus and 2 obliquely tandem testes. H. alincia had an elongated body and a head collar equipped with 31 collar spines. C. medioconiger had a bisegmented body and a voluminous copulatory bursa containing the seminal vesicle and ejaculatory duct. D. spathaceum also had a bisegmented body and its vitellaria extended up to the anterior border of the tribocytic organ. It is of note that C. lingua is potentially zoonotic that can occur in birds and humans. Three of them, i.e., C. lingua, C. medioconiger, and D. spathaceum, are new trematode fauna in Korea. Studies on trematode fauna of migratory birds should be continued in Korea.

Developmental neuromuscular synapse elimination: Activity-dependence and potential downstream effector mechanisms.

Synaptic connections initially formed during nervous system development undergo a significant transformation during nervous system maturation. Such maturation is essential for the proper architecture and function of the nervous system. Developmental synaptic transformation includes "synapse elimination," a process in which multiple immature presynaptic inputs converge at and compete for control of a common postsynaptic target. This developmental synaptic remodeling is best understood at mammalian neuromuscular junctions. It is well established that neuromuscular activity provides the impetus for the pruning of redundant motor axon inputs. Despite the dominant influence neuromuscular activity exerts on developmental synapse elimination, however, the downstream mechanisms of neuromuscular activity that affect synapse elimination remain poorly understood. Conversely, although several cellular and molecular effector mechanisms are known to impact synapse elimination, it is unclear whether they are modulated by neuromuscular activity. This review discusses how the motor neurons, synaptic glia and muscle fibers each contributes to the developmental phenomenon, and speculates how neuromuscular activity may modulate these contributions.

Differences in the constituent fiber types contribute to the intermuscular variation in the timing of the developmental synapse elimination.

The emergence of a mature nervous system requires a significant refinement of the synaptic connections initially formed during development. Redundant synaptic connections are removed in a process known as synapse elimination. Synapse elimination has been extensively studied at the rodent neuromuscular junction (NMJ). Although several axons initially converge onto each postsynaptic muscle fiber, all redundant inputs are removed during early postnatal development until a single motor neuron innervates each NMJ. Neuronal activity as well as synaptic glia influence the course of synapse elimination. It is, however, unclear whether target muscle fibers are more than naïve substrates in this process. I examined the influence of target myofiber contractile properties on synapse elimination. The timing of redundant input removal in muscles examined correlates strongly with their proportion of slow myofibers: muscles with more slow fibers undergo elimination more slowly. Moreover, this intermuscular difference in the timing of synapse elimination appears to result from local differences in the rate of elimination on fast versus slow myofibers. These results, therefore, imply that differences in the constituent fiber types help account for the variation in the timing of the developmental synapse elimination between muscles and show that the muscle plays a role in the process.

Dysregulation of the SNARE-binding protein Munc18-1 impairs BDNF secretion and synaptic neurotransmission: a novel interventional target to protect the aging brain.

Brain-derived neurotrophic factor (BDNF) has a central role in maintaining and strengthening neuronal connections and to stimulate neurogenesis in the adult brain. Decreased levels of BDNF in the aging brain are thought to usher cognitive impairment. BDNF is stored in dense core vesicles and released through exocytosis from the neurites. The exact mechanism for the regulation of BDNF secretion is not well understood. Munc18-1 (STXBP1) was found to be essential for the exocytosis of synaptic vesicles, but its involvement in BDNF secretion is not known. Interestingly, neurons lacking munc18-1 undergo severe degeneration in knock-out mice. Here, we report the effects of BDNF treatment on the presynaptic terminal using munc18-1-deficient neurons. Reduced expression of munc18-1 in heterozygous (+/-) neurons diminishes synaptic transmitter release, as tested here on individual synaptic connections with FM1-43 fluorescence imaging. Transduction of cultured neurons with BDNF markedly increased BDNF secretion in wild-type but was less effective in munc18-1 +/- cells. In turn, BDNF enhanced synaptic functions and restored the severe synaptic dysfunction induced by munc18-1 deficiency. The role of munc18-1 in the synaptic effect of BDNF is highlighted by the finding that BDNF upregulated the expression of munc18-1 in neurons, consistent with enhanced synaptic functions. Accordingly, this is the first evidence showing the functional effect of BDNF in munc18-1 deficient synapses and about the direct role of munc18-1 in the regulation of BDNF secretion. We propose a molecular model of BDNF secretion and discuss its potential as therapeutic target to prevent cognitive decline in the elderly.

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.