The neuromuscular junction (NMJ) is the synapse between a motor neuron and skeletal muscle. Defects in NMJ transmission cause muscle weakness, termed myasthenia. The muscle protein Dok-7 is essential for activation of the receptor kinase MuSK, which governs NMJ formation, and DOK7 mutations underlie familial limb-girdle myasthenia (DOK7 myasthenia), a neuromuscular disease characterized by small NMJs. Here, we show in a mouse model of DOK7 myasthenia that therapeutic administration of an adeno-associated virus (AAV) vector encoding the human DOK7 gene resulted in an enlargement of NMJs and substantial increases in muscle strength and life span. When applied to model mice of another neuromuscular disorder, autosomal dominant Emery-Dreifuss muscular dystrophy, DOK7 gene therapy likewise resulted in enlargement of NMJs as well as positive effects on motor activity and life span. These results suggest that therapies aimed at enlarging the NMJ may be useful for a range of neuromuscular disorders.
Smo contributes to intestinal tumorigenesis by increasing Wnt signaling. SMO might be a good therapeutic target for patients with colorectal polyps and carcinomas, even in the absence of Hh signal activation.
Histiocytic sarcoma (HS), a rare hematological malignancy, is an aggressive neoplasm that responds poorly to therapy. The molecular etiology and pathology of this disease remain unclear, hampering the development of an effective therapy, and there remains a need for more, and more realistic, animal models. HS cells typically show a histiocytic (ie, tissue macrophage-like) morphology and express histiocyte/macrophage markers in the absence of lymphocyte markers. In this study, we report that Dok-1 À/À Dok-2 À/À Dok-3 À/À mice develop HS, but do not exhibit elevated incidence of other types of tumors. These mutant mice showed earlier mortality than wild-type (WT) or the other mutant mice, and this mortality was associated with HS. In total, 17 of 21 tumor-bearing Dok-1À/À Dok-3 À/À mice necropsied at 25-66 weeks of age showed multiple organ spread, with osteolytic lesions and orthotopic invasion from the bone marrow to skeletal muscle. Tumors from the mice were transplantable. In addition, all Dok-1À/À Dok-3 À/À mice, but only a small proportion of Dok-3 À/À mice and no Dok-1 À/À Dok-2 À/À mice, exhibited abnormal accumulation of macrophages in the lung on necropsy at 8-12 weeks of age. Macrophages derived from Dok-1 À/À Dok-2 À/À Dok-3 À/À mice displayed an exaggerated proliferative response to macrophage colony-stimulating factor (M-CSF) or granulocytemacrophage colony-stimulating factor (GM-CSF) compared with WT and mutant controls. Together, these findings indicate that Dok-1, Dok-2, and Dok-3 cooperatively suppress aggressive HS, and commend Dok-1 À/À Dok-2 À/À Dok-3 À/À mice as a useful model for the study of this neoplasia.
Amyotrophic lateral sclerosis (ALS) is a progressive, multifactorial motor neurodegenerative disease with severe muscle atrophy. The glutamate release inhibitor riluzole is the only medication approved by the FDA, and prolongs patient life span by a few months, testifying to a strong need for new treatment strategies. In ALS, motor neuron degeneration first becomes evident at the motor nerve terminals in neuromuscular junctions (NMJs), the cholinergic synapse between motor neuron and skeletal muscle; degeneration then progresses proximally, implicating the NMJ as a therapeutic target. We previously demonstrated that activation of muscle‐specific kinase MuSK by the cytoplasmic protein Dok‐7 is essential for NMJ formation, and forced expression of Dok‐7 in muscle activates MuSK and enlarges NMJs. Here, we show that therapeutic administration of an adeno‐associated virus vector encoding the human DOK7 gene suppressed motor nerve terminal degeneration at NMJs together with muscle atrophy in the SOD1‐G93A ALS mouse model. Ultimately, we show that DOK7 gene therapy enhanced motor activity and life span in ALS model mice.
Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect, and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and E2F target genes enriched in collagen vs. Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Additionally, experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.
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