Increasing LRP4 diminishes neuromuscular deficits in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease characterized by progressive wasting of skeletal muscles. The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, critical for the control of muscle contraction. The NMJ decline is observed in DMD patients, but the mechanism is unclear. LRP4 serves as a receptor for agrin, a proteoglycan secreted by motor neurons to induce NMJ, and plays a critical role in NMJ formation and maintenance. Interestingly, we found that protein levels of LRP4 were reduced both in muscles of the DMD patients and DMD model mdx mice. We explored whether increasing LRP4 is beneficial for DMD and crossed muscle-specific LRP4 transgenic mice with mdx mice (mdx; HSA-LRP4). The LRP4 transgene increased muscle strength, together with improved neuromuscular transmission in mdx mice. Furthermore, we found the LRP4 expression mitigated NMJ fragments and denervation in mdx mice. Mechanically, we showed that overexpression of LRP4 increased the activity of MuSK and expression of dystrophin-associated glycoprotein complex proteins in the mdx mice. Overall, our findings suggest that increasing LRP4 improves both function and structure of NMJ in the mdx mice and Agrin signaling might serve as a new therapeutic strategy in DMD.

Post-Graphene 2D Materials: Structures, Properties, and Cancer Therapy Applications.

Cancer is one of the most serious diseases challenging human health and life span. Cancer has claimed millions of lives worldwide. Early diagnosis and effective treatment of cancer are very important for the survival of patients. In recent years, 2D nanomaterials have shown great potential in the development of anticancer treatment by combining their inherent physicochemical properties after surface modification. 2D nanomaterials have attracted great interest due to their unique nanosheet structure, large surface area, and extraordinary physicochemical properties. This article reviews the advantages and application status of emerging 2D nanomaterials for targeted tumor synergistic therapy compared with traditional therapeutic strategies. In order to investigate novel potential anticancer strategies, this paper focuses on the surface modification, cargo delivery capability, and unique optical properties of emerging 2D nanomaterials. Finally, the current problems and challenges in cancer treatment are summarized and prospected.

Schottky heterojunction CeO2@MXene nanosheets with synergistic type I and type II PDT for anti-osteosarcoma.

Photodynamic therapy (PDT) has shown great potential for tumor treatment as the method is noninvasive, highly selective, and causes minimal side effects. However, conventional type II PDT, which relies on 1O2, presents poor therapeutic efficacy for hypoxic tumors due to its reliance on oxygen. Here, CeO2/Ti3C2-MXene (CeO2@MXene) hybrids were successfully designed by growing CeO2in situ using Ti3C2-MXene (MXene) nanosheets. CeO2@MXene serves as a reduction-oxidation (REDOX) center due to the presence of Ce in the lattice of CeO2 nanoparticles. This REDOX center reacts with H2O2 to generate oxygen and weakens the hypoxic tumor cell environment, achieving type II PDT. At the same time, many other ROS (such as ⋅O2- and ⋅OH) can be produced via a type I photodynamic mechanism (electron transfer process). The CeO2@MXene heterojunction performs nanoenzymatic functions for synergistic type I and type II PDT, which improves cancer treatment.

Deficiency of skeletal muscle Agrin contributes to the pathogenesis of age-related sarcopenia in mice.

Sarcopenia, a progressive and prevalent neuromuscular disorder, is characterized by age-related muscle wasting and weakening. Despite its widespread occurrence, the molecular underpinnings of this disease remain poorly understood. Herein, we report that levels of Agrin, an extracellular matrix (ECM) protein critical for neuromuscular formation, were decreased with age in the skeletal muscles of mice. The conditional loss of Agrin in myogenic progenitors and satellite cells (SCs) (Pax7 Cre:: Agrin flox/flox) causes premature muscle aging, manifesting a distinct sarcopenic phenotype in mice. Conversely, the elevation of a miniaturized form of Agrin in skeletal muscle through adenovirus-mediated gene transfer induces enhanced muscle capacity in aged mice. Mechanistic investigations suggest that Agrin-mediated improvement in muscle function occurs through the stimulation of Yap signaling and the concurrent upregulation of dystroglycan expression. Collectively, our findings underscore the pivotal role of Agrin in the aging process of skeletal muscles and propose Agrin as a potential therapeutic target for addressing sarcopenia.

Phosphorene hydrogel conduits as "neurotrophin reservoirs" for promoting regeneration of peripheral nerves.

Treatment of large gaps in peripheral nerves is a major clinical challenge. Artificial nerve guidance conduits (NGCs) have provided new opportunities for guiding nerve regeneration. In this study, multifunctional black phosphorus (BP) hydrogel NGCs loaded with neuregulin 1 (Nrg1) were fabricated to support peripheral-nerve regeneration: they exhibited good flexibility and nerve regeneration-related cell induction, promoted Schwann-cell proliferation and accelerated neuron-branch elongation. Nrg1 induced the proliferation and migration of Schwann cells, which had beneficial roles in promoting nerve regeneration. In vivo immunofluorescence studies revealed BP hydrogel NGCs loaded with Nrg1 promoted sciatic-nerve regeneration and axon remyelination. Our method has great potential for promoting treatment of peripheral-nerve injuries.

Subtractive Nanopore Engineered MXene Photonic Nanomedicine with Enhanced Capability of Photothermia and Drug Delivery for Synergistic Treatment of Osteosarcoma.

Two-dimensional (2D) nanomaterials as drug carriers and photosensitizers have emerged as a promising antitumor strategy. However, our understanding of 2D antitumor nanomaterials is limited to intrinsic properties or additive modification of different materials. Subtractive structural engineering of 2D nanomaterials for better antitumor efficacy is largely overlooked. Here, subtractively engineered 2D MXenes with uniformly distributed nanopores are synthesized. The nanoporous defects endowed MXene with enhanced surface plasmon resonance effect for better optical absorbance performance and strong exciton-phonon coupling for higher photothermal conversion efficiency. In addition, porous structure improves the binding ability between drug and unsaturated bonds, thus promoting drug-loading capacity and reducing uncontrolled drug release. Furthermore, the porous structure provides adhesion sites for filopodia, thereby promoting the cellular internalization of the drug. Clinically, osteosarcoma is the most common bone malignancy routinely treated with doxorubicin-based chemotherapy. There have been no significant treatment advances in the past decade. As a proof-of-concept, nanoporous MXene loaded with doxorubicin is developed for treating human osteosarcoma cells. The porous MXene platform results in a higher amount of doxorubicin-loading, faster near-infrared (NIR)-controlled doxorubicin release, higher photothermal efficacy under NIR irradiation, and increased cell adhesion and internalization. This facile method pioneers a new paradigm for enhancing 2D material functions and is attractive for tumor treatment.

HKUST-1 loaded few-layer Ti3C2Tx for synergistic chemo-photothermal effects to enhance antibacterial activity.

The drug resistance of bacteria seriously reduces the recovery rate of general disease and endangers human health. Consequently, it is urgent to investigate a non-antibiotic antibacterial material. Recently, two-dimensional MXene has shown good antibacterial properties and received extensive attention due to the large number of active sites, extremely high thermal conversion efficiency, excellent cytocompatibility and ability to penetrate pellicula. However, the antibacterial activity of Ti3C2Tx is greatly affected by the morphology and concentration. Herein, an organic-inorganic hybrid of HKUST-1@Ti3C2Tx with high specific surface area and photothermal effect was designed and fabricated. By adjusting the content of Cu(CO2CH3)2·H2O and 1,3,5-benzenetricarboxylic acid, the photothermal properties of the material can be adjusted, and the release of Cu2+ can be easily reduced. The morphological characterization and fluorescent staining of bacteria which were co-incubated with HKUST-1@Ti3C2Tx confirmed that Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) had adhered to the material. NIR irradiation has enabled induced hyperthermia and the release of Cu2+ ions, causing the disruption of the bacterial membrane, resulting in cytoplasmic leakage. Furthermore, HKUST-1@Ti3C2Tx with synergistic antibacterial effect not only exhibits an excellent bactericidal rate (over 99%) but also greatly improves cytocompatibility with the reduction of the Cu2+ ion release. Therefore, organic-inorganic composites have potential for synergistic effects and non-antibiotic antibacterial activity.

Neuromuscular junction-specific genes screening by deep RNA-seq analysis.

BACKGROUND: Neuromuscular junctions (NMJs) are chemical synapses formed between motor neurons and skeletal muscle fibers and are essential for controlling muscle contraction. NMJ dysfunction causes motor disorders, muscle wasting, and even breathing difficulties. Increasing evidence suggests that many NMJ disorders are closely related to alterations in specific gene products that are highly concentrated in the synaptic region of the muscle. However, many of these proteins are still undiscovered. Thus, screening for NMJ-specific proteins is essential for studying NMJ and the pathogenesis of NMJ diseases. RESULTS: In this study, synaptic regions (SRs) and nonsynaptic regions (NSRs) of diaphragm samples from newborn (P0) and adult (3-month-old) mice were used for RNA-seq. A total of 92 and 182 genes were identified as differentially expressed between the SR and NSR in newborn and adult mice, respectively. Meanwhile, a total of 1563 genes were identified as differentially expressed between the newborn SR and adult SR. Gene Ontology (GO) enrichment analyses, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and gene set enrichment analysis (GSEA) of the DEGs were performed. Protein-protein interaction (PPI) networks were constructed using STRING and Cytoscape. Further analysis identified some novel proteins and pathways that may be important for NMJ development, maintenance and maturation. Specifically, Sv2b, Ptgir, Gabrb3, P2rx3, Dlgap1 and Rims1 may play roles in NMJ development. Hcn1 may localize to the muscle membrane to regulate NMJ maintenance. Trim63, Fbxo32 and several Asb family proteins may regulate muscle developmental-related processes. CONCLUSION: Here, we present a complete dataset describing the spatiotemporal transcriptome changes in synaptic genes and important synaptic pathways. The neuronal projection-related pathway, ion channel activity and neuroactive ligand-receptor interaction pathway are important for NMJ development. The myelination and voltage-gated ion channel activity pathway may be important for NMJ maintenance. These data will facilitate the understanding of the molecular mechanisms underlying the development and maintenance of NMJ and the pathogenesis of NMJ disorders.

Synapse-specific Lrp4 mRNA enrichment requires Lrp4/MuSK signaling, muscle activity and Wnt non-canonical pathway.

BACKGROUND: The neuromuscular junction (NMJ) is a peripheral synapse critical to muscle contraction. Like acetylcholine receptors (AChRs), many essential proteins of NMJ are extremely concentrated at the postjunctional membrane. However, the mechanisms of synapse-specific concentration are not well understood; furthermore, it is unclear whether signaling molecules critical to NMJ formation and maintenance are also locally transcribed. RESULTS: We studied the ß-gal activity encoded by a lacZ cassette driven by the promoter of the Lrp4 gene. As reported for Lrp4 mRNA, ß-gal was in the central region in embryonic muscles and at the NMJ after its formation. However, ß-gal was no longer in the central areas of muscle fibers in Lrp4 or MuSK mutant mice, indicating a requirement of Lrp4/MuSK signaling. This phenotype could be rescued by transgenic expression of LRP4 with a transmembrane domain but not soluble ECD in Lrp4 mutant mice. ß-gal and AChR clusters were distributed in a broader region in lacZ/ECD than that of heterozygous lacZ/+ mice, indicating an important role of the transmembrane domain in Lrp4 signaling. Synaptic ß-gal activity became diffused after denervation or treatment with µ-conotoxin, despite its mRNA was increased, indicating synaptic Lrp4 mRNA enrichment requires muscle activity. ß-gal was also diffused in aged mice but became re-concentrated after muscle stimulation. Finally, Lrp4 mRNA was increased in C2C12 myotubes by Wnt ligands in a manner that could be inhibited by RKI-1447, an inhibitor of ROCK in Wnt non-canonical signaling. Injecting RKI-1447 into muscles of adult mice diminished Lrp4 synaptic expression. CONCLUSIONS: This study demonstrates that synapse-specific enrichment of Lrp4 mRNA requires a coordinated interaction between Lrp4/MuSK signaling, muscle activity, and Wnt non-canonical signaling. Thus, the study provides a new mechanism for Lrp4 mRNA enrichment. It also provides a potential target for the treatment of NMJ aging and other NMJ-related diseases.

Metformin Increases Sarcolemma Integrity and Ameliorates Neuromuscular Deficits in a Murine Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disease characterized by progressive muscle weakness and wasting. Stimulation of AMP-activated protein kinase (AMPK) has been demonstrated to increase muscle function and protect muscle against damage in dystrophic mice. Metformin is a widely used anti-hyperglycemic drug and has been shown to be an indirect activator of AMPK. Based on these findings, we sought to determine the effects of metformin on neuromuscular deficits in mdx murine model of DMD. In this study, we found metformin treatment increased muscle strength accompanied by elevated twitch and tetanic force of tibialis anterior (TA) muscle in mdx mice. Immunofluorescence and electron microscopy analysis of metformin-treated mdx muscles revealed an improvement in muscle fiber membrane integrity. Electrophysiological studies showed the amplitude of miniature endplate potentials (mEPP) was increased in treated mice, indicating metformin also improved neuromuscular transmission of the mdx mice. Analysis of mRNA and protein levels from muscles of treated mice showed an upregulation of AMPK phosphorylation and dystrophin-glycoprotein complex protein expression. In conclusion, metformin can indeed improve muscle function and diminish neuromuscular deficits in mdx mice, suggesting its potential use as a therapeutic drug in DMD patients.

Ablation of Lrp4 in Schwann Cells Promotes Peripheral Nerve Regeneration in Mice.

Low-density lipoprotein receptor-related protein 4 (Lrp4) is a critical protein involved in the Agrin-Lrp4-MuSK signaling pathway that drives the clustering of acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). Many studies have shown that Lrp4 also functions in kidney development, bone formation, nervous system development, etc. However, whether Lrp4 participates in nerve regeneration in mammals remains unknown. Herein, we show that Lrp4 is expressed in SCs and that conditional knockout (cKO) of Lrp4 in SCs promotes peripheral nerve regeneration. In Lrp4 cKO mice, the demyelination of SCs was accelerated, and the proliferation of SCs was increased in the injured nerve. Furthermore, we identified that two myelination-related genes, Krox-20 and Mpz, were downregulated more dramatically in the cKO group than in the control group. Our results elucidate a novel role of Lrp4 in peripheral nerve regeneration and thereby provide a potential therapeutic target for peripheral nerve recovery.

Motoneuron Wnts regulate neuromuscular junction development.

The neuromuscular junction (NMJ) is a synapse between motoneurons and skeletal muscles to control motor behavior. Unlike extensively investigated postsynaptic differentiation, less is known about mechanisms of presynaptic assembly. Genetic evidence of Wnt in mammalian NMJ development was missing due to the existence of multiple Wnts and their receptors. We show when Wnt secretion is abolished from motoneurons by mutating the Wnt ligand secretion mediator (Wls) gene, mutant mice showed muscle weakness and neurotransmission impairment. NMJs were unstable with reduced synaptic junctional folds and fragmented AChR clusters. Nerve terminals were swollen; synaptic vesicles were fewer and mislocated. The presynaptic deficits occurred earlier than postsynaptic deficits. Intriguingly, these phenotypes were not observed when deleting Wls in muscles or Schwann cells. We identified Wnt7A and Wnt7B as major Wnts for nerve terminal development in rescue experiments. These observations demonstrate a necessary role of motoneuron Wnts in NMJ development, in particular presynaptic differentiation.