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Science Advances Jun 2023Junctional folds are unique membrane specializations developed progressively during the postnatal maturation of vertebrate neuromuscular junctions (NMJs), but how they...
Junctional folds are unique membrane specializations developed progressively during the postnatal maturation of vertebrate neuromuscular junctions (NMJs), but how they are formed remains elusive. Previous studies suggested that topologically complex acetylcholine receptor (AChR) clusters in muscle cultures undergo a series of transformations, resembling the postnatal maturation of NMJs in vivo. We first demonstrated the presence of membrane infoldings at AChR clusters in cultured muscles. Live-cell super-resolution imaging further revealed that AChRs are gradually redistributed to the crest regions and spatially segregated from acetylcholinesterase along the elongating membrane infoldings over time. Mechanistically, lipid raft disruption or caveolin-3 knockdown not only inhibits membrane infolding formation at aneural AChR clusters and delays agrin-induced AChR clustering in vitro but also affects junctional fold development at NMJs in vivo. Collectively, this study demonstrated the progressive development of membrane infoldings via nerve-independent, caveolin-3-dependent mechanisms and identified their roles in AChR trafficking and redistribution during the structural maturation of NMJs.
Topics: Caveolin 3; Acetylcholinesterase; Neuromuscular Junction; Receptors, Cholinergic; Muscles
PubMed: 37327338
DOI: 10.1126/sciadv.adg0183 -
Pharmacological Research Aug 2023Lung cancer is the main reason for cancer-associated death globally, and lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer. Recently, AGRN is...
Lung cancer is the main reason for cancer-associated death globally, and lung adenocarcinoma (LUAD) is the most prevalent subtype of lung cancer. Recently, AGRN is considered playing an vital role in the development of some cancers. However, the regulatory effects and mechanisms of AGRN in LUAD remain elusive. In this study, we clarified the significant upregulation of AGRN expression in LUAD by single-cell RNA sequencing combined with immunohistochemistry. Besides, we confirmed that LUAD patients with high AGRN expression are more susceptible to lymph node metastases and have a worse prognosis by a retrospective study of 120 LUAD patients. Next, we demonstrated that AGRN directly interact with NOTCH1, which results in the release of the intracellular structural domain of NOTCH1 and the subsequent activation of the NOTCH pathway. Moreover, we also found that AGRN promotes proliferation, migration, invasion, EMT and tumorigenesis of LUAD cells in vitro and in vivo, and that these effects are reversed by blocking the NOTCH pathway. Furthermore, we prepared several antibodies targeting AGRN, and clarify that Anti-AGRN antibody treatment could significantly inhibit proliferation and promote apoptosis of tumor cells. Our study highlights the important role and regulatory mechanism of AGRN in LUAD development and progression, and suggests that antibodies targeting AGRN have therapeutic potential for LUAD. We also provide theoretical and experimental evidence for further development of monoclonal antibodies targeting AGRN.
Topics: Humans; Adenocarcinoma of Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Lung Neoplasms; Retrospective Studies; Signal Transduction; Agrin; Receptor, Notch1
PubMed: 37321467
DOI: 10.1016/j.phrs.2023.106819 -
Zhongguo Ying Yong Sheng Li Xue Za Zhi... Nov 2022To investigate the protective effects of erythropoietin derived peptide, also known as spiral B surface peptide (HBSP), on kidney and aggregated proteins (Agrin) levels...
To investigate the protective effects of erythropoietin derived peptide, also known as spiral B surface peptide (HBSP), on kidney and aggregated proteins (Agrin) levels in acute skeletal muscle strain rats. Forty SPF grade SD male rats were randomly divided into control group, injury group, HBSP group and EPO group, with 10 rats in each group. Acute skeletal muscle strain animal models were established except the control group. After successful modeling, the rats in HBSP group and EPO group were intraperitoneally injected with 60 μg/kg HBSP and 5 000 U/kg recombinant human erythropoietin (rhEPO), and the rats in the control group and the injured group were intraperitoneally injected with 0.9% normal saline. Renal function was monitored with relevant kits; Hematoxylin-eosin staining was used to observe the pathological morphology of kidney tissue and skeletal muscle strain tissue. The apoptosis rate of renal tissue cells was detected by in situ terminal transferase labeling (TUNEL). Western blot and quantitative polymerase chain reaction (Q-PCR) were used to determine the expressions of Agrin and muscular-specific kinase (MuSK) in the injured skeletal muscle of rats in each group. Compared with the control group, the renal function indexes serum creatinine (Cr), urea nitrogen (BUN) and 24 h urinary protein (UP24) levels of rats in injured group were increased (< 0.05), but the levels of BUN, Cr and UP24 of rats in HBSP group were decreased (<0.05). Compared with HBSP group, there were no significant differences in the above indexes in EPO group (>0.05). In the control group, the muscle fiber structure was intact, the shape and structure of the fiber bundles were normal, and there was no infiltration of red blood cells and inflammatory cells in the interstitium, and no fibrohyperplasia. In the injured group, the muscle tissue showed sparse and irregular arrangement, and the interstitial widened with a large number of inflammatory cells and red blood cell infiltration. Erythrocytes and inflammatory cells were reduced in HBSP group and EPO group, and the transverse and longitudinal lines of muscle were clear. The glomerular structure of the rats in the fibrohyperplasia control group was intact and no lesions were observed. In the injured group, glomerular hypertrophy and significant matrix hyperplasia were observed, as well as the expansion of renal cysts with vacuolar and significant inflammatory infiltration were observed, and the inflammatory infiltration was reduced in the HBSP and EPO groups. Glomerular hypertrophy and hyperplasia were alleviated. The apoptosis rates of kidney cells in control group, injured group, HBSP group and EPO group were (4.05±0.51) %, (26.30±2.05) %, (14.28±1.62) % and (16.03±1.77) %, respectively, and there were significant differences among these groups (<0.05). Compared with control group, the levels of Agrin and MuSK in skeletal muscle pulled tissue were significantly decreased (<0.05), and those of HBSP group and EPO group were significantly increased compared with injured group (<0.05), but there was no significant difference between HBSP group and EPO group (>0.05). Erythropoietin derived peptide (HBSP) has obvious intervention effects on renal function injury in rats with acute skeletal muscle strain, and its mechanisms may be related to reducing the apoptosis rate of renal tissue cells and activating Agrin and MuSK expression.
Topics: Humans; Male; Animals; Rats; Hyperplasia; Agrin; Erythropoietin; Kidney; Muscle, Skeletal; Peptides
PubMed: 37308405
DOI: 10.12047/j.cjap.6348.2022.112 -
Proteomics Sep 2023Cardiovascular diseases (CVDs) are among the most morbid and deadly types of diseases worldwide, while the existing therapeutic approaches all have their limitations....
Cardiovascular diseases (CVDs) are among the most morbid and deadly types of diseases worldwide, while the existing therapeutic approaches all have their limitations. Mouse heart undergoes a very complex postnatal developmental process, including the 1-week window in which cardiomyocytes (CMs) maintain relatively high cell activity. The underlying mechanism provides an attractive direction for CVDs treatment. Herein, we collected ventricular tissues from mice of different ages from E18.5D to P8W and performed iTRAQ-based quantitative proteomics to characterize the atlas of cardiac development. A total of 3422 proteins were quantified at all selected time points, revealing critical proteomic changes related to cardiac developmental events such as the metabolic transition from glycolysis to beta-oxidation. A cluster of significantly dysregulated proteins containing proteins that have already been reported to be associated with cardiac regeneration (Erbb2, Agrin, and Hmgb) was identified. Meanwhile, the peroxisome proliferator-activated receptor (PPAR) signaling pathway (Cpt1α, Hmgcs2, Plin2, and Fabp4) was also found specifically enriched. We further revealed that bezafibrate, a pan-activator of PPAR signaling pathway markedly enhanced H9C2 cardiomyocyte activity via enhancing Cpt1α expression. This work provides new hint that activation of PPAR signaling pathway could potentially be a therapeutic strategy for the treatment of CVDs.
Topics: Mice; Animals; Myocytes, Cardiac; Peroxisome Proliferator-Activated Receptors; Animals, Newborn; Proteomics; Signal Transduction; Cardiovascular Diseases
PubMed: 37271885
DOI: 10.1002/pmic.202200330 -
Proceedings of the National Academy of... Jun 2023MuSK is a receptor tyrosine kinase (RTK) that plays essential roles in the formation and maintenance of the neuromuscular junction. Distinct from most members of RTK...
MuSK is a receptor tyrosine kinase (RTK) that plays essential roles in the formation and maintenance of the neuromuscular junction. Distinct from most members of RTK family, MuSK activation requires not only its cognate ligand agrin but also its coreceptors LRP4. However, how agrin and LRP4 coactivate MuSK remains unclear. Here, we report the cryo-EM structure of the extracellular ternary complex of agrin/LRP4/MuSK in a stoichiometry of 1:1:1. This structure reveals that arc-shaped LRP4 simultaneously recruits both agrin and MuSK to its central cavity, thereby promoting a direct interaction between agrin and MuSK. Our cryo-EM analyses therefore uncover the assembly mechanism of agrin/LRP4/MuSK signaling complex and reveal how MuSK receptor is activated by concurrent binding of agrin and LRP4.
Topics: Receptors, Cholinergic; Agrin; LDL-Receptor Related Proteins; Signal Transduction; Neuromuscular Junction; Receptor Protein-Tyrosine Kinases
PubMed: 37252960
DOI: 10.1073/pnas.2300453120 -
Respiratory Medicine 2023The relevant data about the effects and the associated mechanisms of statins on muscle strength and physical capacity is inconsistent. We investigated the potential...
Degradation of neuromuscular junction contributes to muscle weakness but not physical compromise in chronic obstructive pulmonary disease patients taking lipids-lowering medications.
OBJECTIVES
The relevant data about the effects and the associated mechanisms of statins on muscle strength and physical capacity is inconsistent. We investigated the potential contribution of neuromuscular junction (NMJ) degradation to muscle weakness and physical compromise in patients with chronic obstructive pulmonary disease (COPD) on statins.
METHOD
We recruited male COPD patients (age range = 63-75 years, n = 150) as nonusers (n = 71) and users of statin medications (n = 79) along with age-matched controls (n = 76). The COPD patients were evaluated at baseline and one year later. The data about handgrip strength (HGS), body composition, short physical performance battery (SPPB), and plasma c-terminal agrin fragment-22 (CAF22) as a marker of NMJ disintegration was collected at two time points.
RESULTS
We observed lower HGS, SPPB scores, and higher CAF22 levels in all COPD patients than controls, irrespective of the treatment status (all p < 0.05). Statins further reduced HGS and elevated CAF22 in COPD patients (both p < 0.05). The decline in SPPB was relatively modest in statin users (≈3.7%, p = 0.032) than in nonusers (≈8.7%, p = 0.002). The elevated plasma CAF22 exhibited robust negative correlations with a reduction in HGS but not with SPPB in COPD patients taking statins. We also found a reduction in markers of inflammation and no increase in oxidative stress markers following statin use in COPD patients.
CONCLUSION
Altogether, statin-induced NMJ degradation exacerbates muscle decline but does not contribute to physical compromise in COPD patients.
Topics: Humans; Male; Middle Aged; Aged; Hand Strength; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Pulmonary Disease, Chronic Obstructive; Muscle Weakness; Neuromuscular Junction; Lipids
PubMed: 37245649
DOI: 10.1016/j.rmed.2023.107298 -
Scientific Reports May 2023The study aimed to evaluate the impact of selected exerkines concentration induced by folk-dance and balance training on physical performance, insulin resistance, and... (Randomized Controlled Trial)
Randomized Controlled Trial
The study aimed to evaluate the impact of selected exerkines concentration induced by folk-dance and balance training on physical performance, insulin resistance, and blood pressure in older adults. Participants (n = 41, age 71.3 ± 5.5 years) were randomly assigned to folk-dance (DG), balance training (BG), or control group (CG). The training was performed 3 times a week for 12 weeks. Physical performance tests-time up and go (TUG) and 6-min walk test (6MWT), blood pressure, insulin resistance, and selected proteins induced by exercise (exerkines) were assessed at baseline and post-exercise intervention. Significant improvement in TUG (p = 0.006 for BG and 0.039 for DG) and 6MWT tests (in BG and DG p = 0.001), reduction of systolic blood pressure (p = 0.001 for BG and 0.003 for DG), and diastolic blood pressure (for BG; p = 0.001) were registered post-intervention. These positive changes were accompanied by the drop in brain-derived neurotrophic factor (p = 0.002 for BG and 0.002 for DG), the increase of irisin concentration (p = 0.029 for BG and 0.022 for DG) in both groups, and DG the amelioration of insulin resistance indicators (HOMA-IR p = 0.023 and QUICKI p = 0.035). Folk-dance training significantly reduced the c-terminal agrin fragment (CAF; p = 0.024). Obtained data indicated that both training programs effectively improved physical performance and blood pressure, accompanied by changes in selected exerkines. Still, folk-dance had enhanced insulin sensitivity.
Topics: Humans; Aged; Dancing; Insulin Resistance; Physical Functional Performance; Homeostasis; Glucose
PubMed: 37237034
DOI: 10.1038/s41598-023-35583-w -
Molecular and Cellular Neurosciences Jun 2023One of the effects of hypercholesterolemia (Hch) exerted on the central nervous system (CNS) is damage to the blood-brain barrier (BBB). Increased permeability of BBB...
The extent of damage to the blood-brain barrier in the hypercholesterolemic LDLR/Apo E double knockout mice depends on the animal's age, duration of pathology and brain area.
One of the effects of hypercholesterolemia (Hch) exerted on the central nervous system (CNS) is damage to the blood-brain barrier (BBB). Increased permeability of BBB results from structural changes in the vascular wall, loss of the tight junctions and barrier function, as well as alterations in the concentration of proteins located in the layers of the vascular wall. These changes occur in the course of metabolic and neurodegenerative diseases. The important role in the course of these processes is attributed to agrin, matrix metalloproteinase-9, and aquaporin-4. In this study, we aimed to determine: 1) the extent of Hch-induced damage to the BBB during maturation, and 2) the distribution of the above-mentioned markers in the vascular wall. Immunohistochemical staining and confocal microscopy were used for vascular wall protein assessment. The size of BBB damage was studied based on perivascular leakage of fluorescently labeled dextran. Three- and twelve-month-old male LDLR/Apo E double knockout mice (EX) developing Hch were used in the study. Age-matched male wild-type (WT) C57BL/6 mice were used as a control group. Differences in the concentration of studied markers coexisted with BBB disintegration, especially in younger mice. A relationship between the maturation of the vascular system and reduction of the BBB damage was also observed. We conclude that the extent of BBB permeability depends on animal age, duration of Hch, and brain region. These may explain different susceptibility of various brain areas to Hch, and different presentation of this pathology depending on age and its duration.
Topics: Animals; Male; Mice; Apolipoproteins E; Blood-Brain Barrier; Brain; Mice, Inbred C57BL; Mice, Knockout; Receptors, LDL
PubMed: 37182573
DOI: 10.1016/j.mcn.2023.103860 -
Cells May 2023Recent studies demonstrate the adverse effects of cannabinoids on development, including via pathways shared with ethanol exposure. Our laboratory has shown that both...
Recent studies demonstrate the adverse effects of cannabinoids on development, including via pathways shared with ethanol exposure. Our laboratory has shown that both the nervous system and cardiac development are dependent on agrin modulation of sonic hedgehog (shh) and fibroblast growth factor (Fgf) signaling pathways. As both ethanol and cannabinoids impact these signaling molecules, we examined their role on zebrafish heart development. Zebrafish embryos were exposed to a range of ethanol and/or cannabinoid receptor 1 and 2 agonist concentrations in the absence or presence of morpholino oligonucleotides that disrupt or expression. In situ hybridization was employed to analyze cardiac marker gene expression. Exposure to cannabinoid receptor agonists disrupted midbrain-hindbrain boundary development, but had no effect on heart development, as assessed by the presence of cardiac edema or the altered expression of cardiac marker genes. In contrast, exposure to 1.5% ethanol induced cardiac edema and the altered expression of cardiac marker genes. Combined exposure to or morpholino and 0.5% ethanol disrupted the gene expression pattern, with the restoration of the normal expression following mRNA overexpression. These studies provide evidence that signaling pathways critical to heart development are sensitive to ethanol exposure but not cannabinoid during early zebrafish embryogenesis.
Topics: Animals; Zebrafish; Ethanol; Hedgehog Proteins; Agrin; Cannabinoids; Edema, Cardiac; Morpholinos; Heart
PubMed: 37174727
DOI: 10.3390/cells12091327 -
International Journal of Molecular... Apr 2023The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented....
The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman's layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell-matrix adhesion (β-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.
Topics: Swine; Humans; Animals; Limbus Corneae; Cornea; Conjunctiva; Extracellular Matrix; Sus scrofa; Epithelial Cells
PubMed: 37108705
DOI: 10.3390/ijms24087543