scholarly journals Knockdown of cathepsin D in zebrafish fertilized eggs determines congenital myopathy

2013 ◽  
Vol 33 (2) ◽  
Author(s):  
Carlo Follo ◽  
Matteo Ozzano ◽  
Claudia Montalenti ◽  
Massimo Mattia Santoro ◽  
Ciro Isidoro
Keyword(s):  
Cathepsin D ◽  
Muscle Development ◽  
Congenital Myopathy ◽  
Muscle Fibres ◽  
Lysosomal Hydrolase ◽  
Congenital Myopathies ◽  
Altered Expression ◽  
Vertebrate Model

CD (cathepsin D) is a ubiquitous lysosomal hydrolase involved in a variety of pathophysiological functions, including protein turnover, activation of pro-hormones, cell death and embryo development. CD-mediated proteolysis plays a pivotal role in tissue and organ homoeostasis. Altered expression and compartmentalization of CD have been observed in diseased muscle fibres. Whether CD is actively involved in muscle development, homoeostasis and dystrophy remains to be demonstrated. Zebrafish (Danio rerio) is emerging as a valuable ‘in vivo’ vertebrate model for muscular degeneration and congenital myopathies. In this work, we report on the perturbance of the somitic musculature development in zebrafish larvae caused by MPO (morpholino)-mediated silencing of CD in oocytes at the time of fertilization. Restoring CD expression, using an MPO-non-matching mutated mRNA, partially rescued the normal phenotype, confirming the indispensable role of CD in the correct development and integrity of the somitic musculature. This is the first report showing a congenital myopathy caused by CD deficiency in a vertebrate experimental animal model.

RyR1-mediated moderate endoplasmic reticulum stress is required for myogenic differentiation of myoblasts

2021 ◽  
Author(s):  
Kai Qiu ◽  
Yubo Wang ◽  
Doudou Xu ◽  
Linjuan He ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Fate ◽  
Muscle Development ◽  
Myogenic Differentiation ◽  
C2c12 Cells ◽  
Stress Signaling ◽  
Congenital Myopathies ◽  
Myotube Formation

Abstract BackgroundCytosolic Ca2+ plays vital roles in myogenesis and muscle development. Key mutations of ryanodine receptor 1 (RyR1), a major Ca2+ release channel of endoplasmic reticulum (ER), are main causes of severe congenital myopathies. The role of RyR1 in myogenic differentiation has attracted intense research interest, however, it remains unclear. MethodsThis study employed RyR1-knockdown myoblasts and CRISPR/Cas9-based RyR1-knockout myoblasts cells to explore the role of RyR1 in myogenic differentiation, myotube formation as well as the potential mechanism of RyR1-related myopathies.ResultsCytoplasmic Ca2+ concentration was significantly elevated during myogenic differentiation of both primary myogenic cells and myoblasts C2C12 cells, accompanied with a dramatic increase in RyR1 expression and resultant ER stress. Inhibition of RyR1 by siRNA-mediated silence or chemical inhibitor, dantrolene, significantly reduced cytosolic Ca2+, alleviated ER stress, and blocked multinucleated myotube formation. Moderate activation of ER stress effectively relieved myogenic differentiation stagnation induced by RyR1 suppression and demonstrated that RyR1 modulates myogenic differentiation via activation of Ca2+ -induced ER stress signaling. RyR1 knockout-induced Ca2+ leakage led to severe ER stress and excessive unfolded protein response, and drove cell fate from differentiation into apoptosis. ConclusionsTherefore, we concluded that dramatic increase in RyR1 expression is required for myogenic differentiation, and RyR1-mediated Ca2+ release leading to the activation of ER stress signaling serves a double-edged sword role during myogenic differentiation. This study contributes to a novel understanding of the role of RyR1 in muscle development and related congenital myopathies, and provides a potential target for regulation of muscle regeneration and tissue engineering.

scholarly journals Role of proteoglycans and glycosaminoglycans in Duchenne muscular dystrophy

Glycobiology ◽  
2018 ◽  
Vol 29 (2) ◽  
pp. 110-123 ◽  
Author(s):  
Laurino Carmen ◽  
Vadala’ Maria ◽  
Julio Cesar Morales-Medina ◽  
Annamaria Vallelunga ◽  
Beniamino Palmieri ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mammalian Cells ◽  
Muscle Development ◽  
Glycoprotein Complex ◽  
Experimental Therapies ◽  
Dystrophin Protein

Abstract Duchenne muscular dystrophy (DMD) is an inherited fatal X-linked myogenic disorder with a prevalence of 1 in 3500 male live births. It affects voluntary muscles, and heart and breathing muscles. DMD is characterized by continuous degeneration and regeneration cycles resulting in extensive fibrosis and a progressive reduction in muscle mass. Since the identification of a reduction in dystrophin protein as the cause of this disorder, numerous innovative and experimental therapies, focusing on increasing the levels of dystrophin, have been proposed, but the clinical improvement has been unsatisfactory. Dystrophin forms the dystrophin-associated glycoprotein complex and its proteins have been studied as a promising novel therapeutic target to treat DMD. Among these proteins, cell surface glycosaminoglycans (GAGs) are found almost ubiquitously on the surface and in the extracellular matrix (ECM) of mammalian cells. These macromolecules interact with numerous ligands, including ECM constituents, adhesion molecules and growth factors that play a crucial role in muscle development and maintenance. In this article, we have reviewed in vitro, in vivo and clinical studies focused on the functional role of GAGs in the pathophysiology of DMD with the final aim of summarizing the state of the art of GAG dysregulation within the ECM in DMD and discussing future therapeutic perspectives.

scholarly journals In vivo NCL targeting affects breast cancer aggressiveness through miRNA regulation

2013 ◽  
Vol 210 (5) ◽  
pp. 951-968 ◽  
Author(s):  
Flavia Pichiorri ◽  
Dario Palmieri ◽  
Luciana De Luca ◽  
Jessica Consiglio ◽  
Jia You ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Target Genes ◽  
Human Cancer ◽  
Mirna Regulation ◽  
Altered Expression ◽  
Specific Subset ◽  
Cancer Aggressiveness

Numerous studies have described the altered expression and the causal role of microRNAs (miRNAs) in human cancer. However, to date, efforts to modulate miRNA levels for therapeutic purposes have been challenging to implement. Here we find that nucleolin (NCL), a major nucleolar protein, posttranscriptionally regulates the expression of a specific subset of miRNAs, including miR-21, miR-221, miR-222, and miR-103, that are causally involved in breast cancer initiation, progression, and drug resistance. We also show that NCL is commonly overexpressed in human breast tumors and that its expression correlates with that of NCL-dependent miRNAs. Finally, inhibition of NCL using guanosine-rich aptamers reduces the levels of NCL-dependent miRNAs and their target genes, thus reducing breast cancer cell aggressiveness both in vitro and in vivo. These findings illuminate a path to novel therapeutic approaches based on NCL-targeting aptamers for the modulation of miRNA expression in the treatment of breast cancer.

scholarly journals Targeting Mitochondrial Ion Channels to Fight Cancer

2018 ◽  
Vol 19 (7) ◽  
pp. 2060 ◽  
Author(s):  
Magdalena Bachmann ◽  
Roberto Costa ◽  
Roberta Peruzzo ◽  
Elena Prosdocimi ◽  
Vanessa Checchetto ◽  
...  
Keyword(s):  
Ion Channels ◽  
Tumor Development ◽  
Cancer Development ◽  
Cancer Growth ◽  
Altered Expression ◽  
New Frontier ◽  
Near Future

In recent years, several experimental evidences have underlined a new role of ion channels in cancer development and progression. In particular, mitochondrial ion channels are arising as new oncological targets, since it has been proved that most of them show an altered expression during tumor development and the pharmacological targeting of some of them have been demonstrated to be able to modulate cancer growth and progression, both in vitro as well as in vivo in pre-clinical mouse models. In this scenario, pharmacology of mitochondrial ion channels would be in the near future a new frontier for the treatment of tumors. In this review, we discuss the new advances in the field, by focusing our attention on the improvements in new drug developments to target mitochondrial ion channels.

scholarly journals Role for Chitin and Chitooligomers in the Capsular Architecture of Cryptococcus neoformans

2009 ◽  
Vol 8 (10) ◽  
pp. 1543-1553 ◽  
Author(s):  
Fernanda L. Fonseca ◽  
Leonardo Nimrichter ◽  
Radames J. B. Cordero ◽  
Susana Frases ◽  
Jessica Rodrigues ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Pathogenic Fungus ◽  
Monosaccharide Composition ◽  
Altered Expression ◽  
Functional Activities ◽  
Differential Reactivity ◽  
Human Pathogenic Fungus

ABSTRACT Molecules composed of β-1,4-linked N-acetylglucosamine (GlcNAc) and deacetylated glucosamine units play key roles as surface constituents of the human pathogenic fungus Cryptococcus neoformans. GlcNAc is the monomeric unit of chitin and chitooligomers, which participate in the connection of capsular polysaccharides to the cryptococcal cell wall. In the present study, we evaluated the role of GlcNAc-containing structures in the assembly of the cryptococcal capsule. The in vivo expression of chitooligomers in C. neoformans varied depending on the infected tissue, as inferred from the differential reactivity of yeast forms to the wheat germ agglutinin (WGA) in infected brain and lungs of rats. Chromatographic and dynamic light-scattering analyses demonstrated that glucuronoxylomannan (GXM), the major cryptococcal capsular component, interacts with chitin and chitooligomers. When added to C. neoformans cultures, chitooligomers formed soluble complexes with GXM and interfered in capsular assembly, as manifested by aberrant capsules with defective connections with the cell wall and no reactivity with a monoclonal antibody to GXM. Cultivation of C. neoformans in the presence of an inhibitor of glucosamine 6-phosphate synthase resulted in altered expression of cell wall chitin. These cells formed capsules that were loosely connected to the cryptococcal wall and contained fibers with decreased diameters and altered monosaccharide composition. These results contribute to our understanding of the role played by chitin and chitooligosaccharides on the cryptococcal capsular structure, broadening the functional activities attributed to GlcNAc-containing structures in this biological system.

249 Targeting IFNβ-regulated secretory profiles to overcome acquired anti-PD-L1 resistance

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A270-A270
Author(s):  
Yuhao Shi ◽  
Melissa Dolan ◽  
Michalis Mastri ◽  
Kevin Eng ◽  
John Ebos
Keyword(s):  
Treatment Failure ◽  
Acquired Resistance ◽  
Secreted Proteins ◽  
Tumor Growth Inhibition ◽  
Type I ◽  
Altered Expression ◽  
The Impact

BackgroundTherapeutic targeting of programmed cell death protein ligand 1 (PD-L1) has led to durable benefits for many cancer patients; however, the development of acquired resistance is common. Dysregulated type II interferon (IFN) signaling on tumor cells can contribute to resistance via altered expression of IFN stimulated genes (ISGs), which include cytokines and growth factors capable of immune-suppression and tumor promotion. However, the role of type I IFNs, including IFNα and IFNβ, in acquired resistance remain understudied. Here we examined the impact of chronic PD-L1 blockade to evaluate the role of IFN-related secretory changes in preclinical models of resistance.MethodsUsing a mouse breast EMT6 orthotopic tumor model, we selected PD-L1 drug resistant (PDR) cells from tumors initially responsive to PD-L1 blockade, but that later relapsed. Using transcriptomic and proteomic approaches, we evaluated secreted proteins associated with IFN signaling. To test for direct connections between PD-L1 and IFN signaling in secretory profile modulation, genetic and therapeutic disruption of PD-L1/IFNAR1 were conducted in vitro.ResultsWe identified a unique gene signature for secreted proteins following acquired resistance to PD-L1 blockade that associated with IFN signaling. This secretory signature was validated using publicly available datasets derived from preclinical tumors and clinical biopsies after anti-PD-L1 treatment failure. Interestingly, genetic and antibody inhibition of PD-L1 in vitro enhanced PDR secretory signatures following IFNβ stimulation suggesting PD-L1 tumor-intrinsic functions may regulate IFN responses following acquired resistance. To test whether secretory profiles impact tumor growth, inhibition of specific ISGs (IL-6) or ISG regulators (IFNAR1) were examined and found to inhibit PDR tumors in vivo, compared to parental controls.ConclusionsTogether, these findings identify a secretory profile associated with acquired resistance to PD-L1 blockade that may be modulated, at least in part, by IFNβ. Selective targeting of secreted ISGs may provide a benefit for patients after anti-PD-L1 treatment failure.

scholarly journals The role of TORC1 in muscle development in Drosophila.

2014 ◽  
Author(s):  
Isabelle Hatfield ◽  
Innocence Harvey ◽  
Erika R. Yates ◽  
JeAnna R. Redd ◽  
Lawrence T. Reiter ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Muscle Development ◽  
Myosin Heavy Chain Isoforms ◽  
C2c12 Cells ◽  
Important Process ◽  
Mtorc1 Pathway ◽  
And Function ◽  
Muscle Precursor Cells

Myogenesis is an important process during both development and muscle repair. Previous studies suggest that mTORC1 plays a role in the formation of mature muscle from immature muscle precursor cells. Here we show that gene expression for several myogenic transcription factors including Myf5, Myog and Mef2c but not MyoD and myosin heavy chain isoforms decrease when C2C12 cells are treated with rapamycin, supporting a role for mTORC1 pathway during muscle development. To investigate the possibility that mTORC1 can regulate muscle in vivo we ablated the essential dTORC1 subunit Raptor in Drosophila melanogaster and found that muscle-specific knockdown of Raptor causes flies to be too weak to emerge from their pupal cases during eclosion. Using a series of GAL4 drivers we also show that muscle-specific Raptor knockdown also causes shortened lifespan, even when eclosure is unaffected. Together these results highlight an important role for TORC1 in muscle development, integrity and function in both Drosophila and mammalian cells.

Insights into wild-type dynamin 2 and the consequences of DNM2 mutations from transgenic zebrafish

2019 ◽  
Vol 28 (24) ◽  
pp. 4186-4196 ◽  
Author(s):  
Mo Zhao ◽  
Lindsay Smith ◽  
Jonathan Volpatti ◽  
Lacramioara Fabian ◽  
James J Dowling
Keyword(s):  
Muscle Development ◽  
Neuromuscular Disorders ◽  
Transgenic Zebrafish ◽  
Wild Type ◽  
Charcot Marie Tooth Disease ◽  
Membrane Fission ◽  
First Time ◽  
Dynamin 2

Abstract Dynamin 2 (DNM2) encodes a ubiquitously expressed large GTPase with membrane fission capabilities that participates in the endocytosis of clathrin-coated vesicles. Heterozygous mutations in DNM2 are associated with two distinct neuromuscular disorders, Charcot–Marie–Tooth disease (CMT) and autosomal dominant centronuclear myopathy (CNM). Despite extensive investigations in cell culture, the role of dynamin 2 in normal muscle development is poorly understood and the consequences of DNM2 mutations at the molecular level in vivo are not known. To address these gaps in knowledge, we developed transgenic zebrafish expressing either wild-type dynamin 2 or dynamin 2 with either a CNM or CMT mutation. Taking advantage of the live imaging capabilities of the zebrafish embryo, we establish the localization of wild-type and mutant dynamin 2 in vivo, showing for the first time distinctive dynamin 2 subcellular compartments. Additionally, we demonstrate that CNM-related DNM2 mutations are associated with protein mislocalization and aggregation. Lastly, we define core phenotypes associated with our transgenic mutant fish, including impaired motor function and altered muscle ultrastructure, making them the ideal platform for drug screening. Overall, using the power of the zebrafish, we establish novel insights into dynamin 2 localization and dynamics and provide the necessary groundwork for future studies examining dynamin 2 pathomechanisms and therapy development.

scholarly journals Dysregulation of NRAP degradation by KLHL41 contributes to pathophysiology in nemaline myopathy

2019 ◽  
Vol 28 (15) ◽  
pp. 2549-2560 ◽  
Author(s):  
Caroline Jirka ◽  
Jasmine H Pak ◽  
Claire A Grosgogeat ◽  
Michael Mario Marchetii ◽  
Vandana A Gupta
Keyword(s):  
Skeletal Muscle ◽  
Thin Filament ◽  
Muscle Development ◽  
Nemaline Myopathy ◽  
Therapeutic Interventions ◽  
Transgenic Zebrafish ◽  
Anchoring Protein ◽  
And Function

Abstract Nemaline myopathy (NM) is the most common form of congenital myopathy that results in hypotonia and muscle weakness. This disease is clinically and genetically heterogeneous, but three recently discovered genes in NM encode for members of the Kelch family of proteins. Kelch proteins act as substrate-specific adaptors for Cullin 3 (CUL3) E3 ubiquitin ligase to regulate protein turnover through the ubiquitin-proteasome machinery. Defects in thin filament formation and/or stability are key molecular processes that underlie the disease pathology in NM; however, the role of Kelch proteins in these processes in normal and diseases conditions remains elusive. Here, we describe a role of NM causing Kelch protein, KLHL41, in premyofibil-myofibil transition during skeletal muscle development through a regulation of the thin filament chaperone, nebulin-related anchoring protein (NRAP). KLHL41 binds to the thin filament chaperone NRAP and promotes ubiquitination and subsequent degradation of NRAP, a process that is critical for the formation of mature myofibrils. KLHL41 deficiency results in abnormal accumulation of NRAP in muscle cells. NRAP overexpression in transgenic zebrafish resulted in a severe myopathic phenotype and absence of mature myofibrils demonstrating a role in disease pathology. Reducing Nrap levels in KLHL41 deficient zebrafish rescues the structural and function defects associated with disease pathology. We conclude that defects in KLHL41-mediated ubiquitination of sarcomeric proteins contribute to structural and functional deficits in skeletal muscle. These findings further our understanding of how the sarcomere assembly is regulated by disease-causing factors in vivo, which will be imperative for developing mechanism-based specific therapeutic interventions.

Molecular expression of myostatin and MyoD is greater in double-muscled than normal-muscled cattle fetuses

2001 ◽  
Vol 280 (5) ◽  
pp. R1488-R1493 ◽  
Author(s):  
J. M. Oldham ◽  
J. A. K. Martyn ◽  
M. Sharma ◽  
F. Jeanplong ◽  
R. Kambadur ◽  
...  
Keyword(s):  
Feedback Loop ◽  
Muscle Development ◽  
Fiber Formation ◽  
Normal Muscle ◽  
Myostatin Gene ◽  
Relationship Of ◽  
Molecular Expression ◽  
Myod Expression

Excessive muscling in double-muscled cattle arises from mutations in the myostatin gene, but the role of myostatin in normal muscle development is unclear. The aim of this study was to measure the temporal relationship of myostatin and myogenic regulatory factors during muscle development in normal (NM)- and double-muscled (DM) cattle to determine the timing and possible targets of myostatin action in vivo. Myostatin mRNA peaked at the onset of secondary fiber formation ( P < 0.001) and was greater in DM ( P < 0.001) than in NM. MyoD expression was also elevated throughout primary and secondary fiber formation ( P < 0.001) and greater in DM ( P < 0.05). Expression of myogenin peaked later than MyoD ( P< 0.05); however, it did not differ between NM and DM. These data show that myostatin and MyoD increase coincidentally during formation of muscle fibers, indicating a coordinated role in the terminal differentiation and/or fusion of myoblasts. Myostatin mRNA is also consistently higher in DM than NM, suggesting that a feedback loop of regulation is also disrupted in the myostatin-deficient condition.

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