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International Journal of Molecular... Oct 2022Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4...
Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts.
Topics: Age Factors; Agrin; Bromodeoxyuridine; Cell Proliferation; Focal Adhesion Protein-Tyrosine Kinases; Heparan Sulfate Proteoglycans; Humans; LDL-Receptor Related Proteins; Motor Neurons; Myoblasts; Receptor Protein-Tyrosine Kinases
PubMed: 36233091
DOI: 10.3390/ijms231911784 -
Molecular Neurobiology Dec 2022Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its...
Neurotrypsin (NT) is a highly specific nervous system multi-domain serine protease best known for its selective processing of the potent synaptic organizer agrin. Its enzymatic activity is thought to influence processes of synaptic plasticity, with its deregulation causing accelerated neuromuscular junction (NMJ) degeneration or contributing to forms of mental retardation. These biological effects are likely to stem from NT-based regulation of agrin signaling. However, dissecting the exact biological implications of NT-agrin interplay is difficult, due to the scarce molecular detail regarding NT activity and NT-agrin interactions. We developed a strategy to reliably produce and purify a catalytically competent engineered variant of NT called "NT-mini" and a library of C-terminal agrin fragments, with which we performed a thorough biochemical and biophysical characterization of NT enzyme functionality. We studied the regulatory effects of calcium ions and heparin, identified NT's heparin-binding domain, and discovered how zinc ions induce modulation of enzymatic activity. Additionally, we investigated myotube differentiation and hippocampal neuron excitability, evidencing a dose-dependent increase in neuronal activity alongside a negative impact on myoblast fusion when using the active NT enzyme. Collectively, our results provide in vitro and cellular foundations to unravel the molecular underpinnings and biological significance of NT-agrin interactions.
Topics: Agrin; Muscle Fibers, Skeletal; Neurons; Heparin; Synapses
PubMed: 36197591
DOI: 10.1007/s12035-022-03056-2 -
Indian Journal of Endocrinology and... 2022Undetected onset of sarcopenia among individuals with chronic diseases especially Type 2 Diabetes Mellitus (T2D) makes it important to be evaluated. The feasibility of...
BACKGROUND
Undetected onset of sarcopenia among individuals with chronic diseases especially Type 2 Diabetes Mellitus (T2D) makes it important to be evaluated. The feasibility of diagnosing sarcopenia in a clinical setup might be a difficult task. Circulating markers including C-terminal agrin fragment (CAF) are emerging as an alternative. Hence, the objectives of the study were to compare circulating CAF levels between T2D, prediabetes (PD) and healthy controls and to study its association with sarcopenic index, muscle mass, strength and quality.
METHODS
Ninety-nine participants (n = 42, T2D; n = 33, PD; n = 24, healthy controls) aged 18 to 60 yrs were recruited. HOMA (homeostatic model assessment) indices were derived using plasma glucose and insulin. All participants underwent lipid profiling, muscle strength including quality (isokinetic dynamometer), body composition (Dual energy X-ray Absorptiometry (DXA)) and sarcopenic index (appendicular skeletal muscle mass/body weight) assessment. Serum samples were used to estimate CAF levelsusing enzyme-linked immunosorbent assay (ELISA).
RESULTS
Median CAF level was significantly higher among T2D group compared to PD and control groups (P < 0.0001). Circulating CAF levels correlated positively with age and glycated haemoglobin (HbA1c) (both, P < 0.001) and negatively with HOMA-B and muscle quality (both, P < 0.001), and sarcopenic index (P = 0.07). Multivariable analysis demonstrated that the odds of being in the highest tertile category was 7.67, 95% C.I. (2.10, 29.3) among T2D.
CONCLUSION
Circulating CAF levels were significantly higher among T2D compared to PD and control study groups along with reduced skeletal muscle quality. This suggests that the circulating CAF level has the potential to be considered as a clinical marker to evaluate sarcopenia among T2D.
PubMed: 36185967
DOI: 10.4103/ijem.ijem_507_21 -
Cureus Aug 2022Vesicle-associated membrane protein 2 () and Agrin () are crucial proteins in neurotransmission. is a vesicular protein that facilitates the exocytosis of...
Vesicle-associated membrane protein 2 () and Agrin () are crucial proteins in neurotransmission. is a vesicular protein that facilitates the exocytosis of neurotransmitters. At the same time, plays a critical role in the maintenance and function of neuromuscular junctions. Mutations in the signaling pathway of and impair proper signaling between the presynaptic and postsynaptic neurons, and can result in neurodevelopmental conditions known as global developmental delay (GDD). This study highlights a presentation of GDD in a patient with concurrent mutations in and . A three-year-old female child presented with GDD characterized by hypotonia, intellectual disability, and dysphagia. Physical exam exhibited signs of developmental delay and severe muscle weakness. EEG findings were suggestive of a hypsarrhythmia pattern. The ophthalmological evaluation showed partial optic atrophy bilaterally. Therapeutic interventions included Keppra and Topamax, which proved ineffective. The patient's outcome was inconclusive as care was transferred to another facility. This case study reports the novel appearance of two concurrent mutations: p.Gln76Pro associated with and p.Gln970Glu associated with . Mutations in lead to a dysfunctional SNARE complex and inhibit exocytosis of neurotransmitters into the synaptic cleft. Mutations in impair the ability to form and activate postsynaptic nicotinic acetylcholine receptors. Improper signaling between presynaptic and postsynaptic neurons is an important determinant of GDD. We hope that accounting for this mutational pattern will contribute to understanding synapse assembly and help unravel the complex interplay of factors involved in the pathology of neuromuscular disorders and GDD.
PubMed: 36176870
DOI: 10.7759/cureus.28464 -
Frontiers in Medicine 2022Extracellular matrix (ECM) is a key component of the stem cell local microenvironment. Our study aims to explore the periglandular distribution of major components of...
PURPOSE
Extracellular matrix (ECM) is a key component of the stem cell local microenvironment. Our study aims to explore the periglandular distribution of major components of ECM in the Meibomian gland (MG).
METHODS
Human eyelids and mouse eyelids were collected and processed for immunofluorescence staining.
RESULTS
Human MG tissues stained positive for collagen IV α1, collagen IV α2, collagen IV α5, and collagen IV α6 around the acini and duct, but negative for collagen IV α3 and collagen IV α4. The mouse MG were stained positive for the same collagen IV subunits as early as postnatal day 15. Laminin α2, laminin β1 and perlecan stained the regions surrounding the acini and the acinar/ductal junction in the human MG, but not the region around the duct. Tenascin-C was found specifically located at the junctions between the acini and the central ducts. Neither agrin nor endostatin was found in the human MG tissues.
CONCLUSION
The ECM expresses specific components in different regions around the MG, which may play a role in MG stem cell regulation, renewal, and regeneration.
PubMed: 36148459
DOI: 10.3389/fmed.2022.981610 -
Cell Reports Sep 2022The neuromuscular junction (NMJ) is an essential synapse whose loss is a key hallmark of the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that...
The neuromuscular junction (NMJ) is an essential synapse whose loss is a key hallmark of the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that activity of the SMA-determining SMN protein in the assembly of U7 small nuclear ribonucleoprotein (snRNP)-which functions in the 3'-end processing of replication-dependent histone mRNAs-is required for NMJ integrity. Co-expression of U7-specific Lsm10 and Lsm11 proteins selectively enhances U7 snRNP assembly, corrects histone mRNA processing defects, and rescues key structural and functional abnormalities of neuromuscular pathology in SMA mice-including NMJ denervation, decreased synaptic transmission, and skeletal muscle atrophy. Furthermore, U7 snRNP dysfunction drives selective loss of the synaptic organizing protein Agrin at NMJs innervating vulnerable muscles of SMA mice. These findings reveal a direct contribution of U7 snRNP dysfunction to neuromuscular pathology in SMA and suggest a role for histone gene regulation in maintaining functional synaptic connections between motor neurons and muscles.
Topics: Agrin; Animals; Histones; Mice; Muscular Atrophy, Spinal; Neurodegenerative Diseases; Neuromuscular Junction; RNA, Messenger; Ribonucleoprotein, U7 Small Nuclear
PubMed: 36130491
DOI: 10.1016/j.celrep.2022.111393 -
Scientific Reports Sep 2022The interphotoreceptor matrix (IPM) is a specialized extracellular mesh of molecules surrounding the inner and outer segments of photoreceptor neurons....
The interphotoreceptor matrix (IPM) is a specialized extracellular mesh of molecules surrounding the inner and outer segments of photoreceptor neurons. Interphotoreceptor matrix proteoglycan 1 and 2 (IMPG1 and IMPG2) are major components of the IPM. Both proteoglycans possess SEA (sperm protein, enterokinase and agrin) domains, which may support proteolysis. Interestingly, mutations in the SEA domains of IMPG1 and IMPG2 are associated with vision disease in humans. However, if SEA domains in IMPG molecules undergo proteolysis, and how this contributes to vision pathology is unknown. Therefore, we investigated SEA-mediated proteolysis of IMPG1 and IMPG2 and its significance to IPM physiology. Immunoblot analysis confirmed proteolysis of IMPG1 and IMPG2 in the retinas of wildtype mice. Point mutations mimicking human mutations in the SEA domain of IMPG1 that are associated with vision disease inhibited proteolysis. These findings demonstrate that proteolysis is part of the maturation of IMPG1 and IMPG2, in which deficits are associated with vision diseases. Further, immunohistochemical assays showed that proteolysis of IMPG2 generated two subunits, a membrane-attached peptide and an extracellular peptide. Notably, the extracellular portion of IMPG2 trafficked from the IPM around the inner segment toward the outer segment IPM by an IMPG1-dependent mechanism. This result provides the first evidence of a trafficking system that shuttles IMPG1 and IMPG2 from the inner to outer IPM in a co-dependent manner. In addition, these results suggest an interaction between IMPG1-IMPG2 and propose that mutations affecting one IMPG could affect the localization of the normal IMPG partner, contributing to the disease mechanism of vision diseases associated with defective IMPG molecules.
Topics: Agrin; Animals; Enteropeptidase; Extracellular Matrix Proteins; Eye Proteins; Humans; Male; Mice; Proteoglycans; Semen
PubMed: 36109576
DOI: 10.1038/s41598-022-19910-1 -
The Journal of Physiology Nov 2022Electrophysiological alterations of the neuromuscular junction (NMJ) and motor unit potential (MUP) with unloading are poorly studied. We aimed to investigate these...
Electrophysiological alterations of the neuromuscular junction (NMJ) and motor unit potential (MUP) with unloading are poorly studied. We aimed to investigate these aspects and the underlying molecular mechanisms with short-term unloading and active recovery (AR). Eleven healthy males underwent a 10-day unilateral lower limb suspension (ULLS) period, followed by 21-day AR based on resistance exercise. Quadriceps femoris (QF) cross-sectional area (CSA) and isometric maximum voluntary contraction (MVC) were evaluated. Intramuscular electromyographic recordings were obtained during 10% and 25% MVC isometric contractions from the vastus lateralis (VL). Biomarkers of NMJ molecular instability (serum c-terminal agrin fragment, CAF), axonal damage (neurofilament light chain) and denervation status were assessed from blood samples and VL biopsies. NMJ and ion channel transcriptomic profiles were investigated by RNA-sequencing. QF CSA and MVC decreased with ULLS. Increased CAF and altered NMJ transcriptome with unloading suggested the emergence of NMJ molecular instability, which was not associated with impaired NMJ transmission stability. Instead, increased MUP complexity and decreased motor unit firing rates were found after ULLS. Downregulation of ion channel gene expression was found together with increased neurofilament light chain concentration and partial denervation. The AR period restored most of these neuromuscular alterations. In conclusion, the human NMJ is destabilized at the molecular level but shows functional resilience to a 10-day unloading period at least at relatively low contraction intensities. However, MUP properties are altered by ULLS, possibly due to alterations in ion channel dynamics and initial axonal damage and denervation. These changes are fully reversed by 21 days of AR. KEY POINTS: We used integrative electrophysiological and molecular approaches to comprehensively investigate changes in neuromuscular integrity and function after a 10-day unilateral lower limb suspension (ULLS), followed by 21 days of active recovery in young healthy men, with a particular focus on neuromuscular junction (NMJ) and motor unit potential (MUP) properties alterations. After 10-day ULLS, we found significant NMJ molecular alterations in the absence of NMJ transmission stability impairment. These findings suggest that the human NMJ is functionally resilient against insults and stresses induced by short-term disuse at least at relatively low contraction intensities, at which low-threshold, slow-type motor units are recruited. Intramuscular electromyography analysis revealed that unloading caused increased MUP complexity and decreased motor unit firing rates, and these alterations could be related to the observed changes in skeletal muscle ion channel pool and initial and partial signs of fibre denervation and axonal damage. The active recovery period restored these neuromuscular changes.
Topics: Male; Humans; Transcriptome; Muscle Contraction; Neuromuscular Junction; Muscle, Skeletal; Quadriceps Muscle; Electromyography
PubMed: 36071599
DOI: 10.1113/JP283381 -
BMC Neurology Sep 2022Few studies focused on the risk factors for hand rehabilitation of intracerebral hemorrhage (ICH) using of soft robotic hand therapy (SRHT). The aim of this study was to...
BACKGROUND
Few studies focused on the risk factors for hand rehabilitation of intracerebral hemorrhage (ICH) using of soft robotic hand therapy (SRHT). The aim of this study was to establish a predictive nomogram for soft robotic hand rehabilitation in patients with ICH.
METHODS
According to the Brunnstrom motor recovery (BMR) stage, the patients were grouped into poor and good motor function groups. The data of patient demographic information and serum level of C-terminal Agrin Fragment (CAF), S100B and neurofilament light (NfL) were collected. The logistic regression was used to analyze the risk factors for poor hand function.
RESULTS
Finally, we enrolled 102 and 103 patients in the control and SRHT groups. For the SRHT group, there were 17 and 86 cases with poor and good motor function at 6-months follow-up respectively. In the good motor function group, the Fugl-Meyer Assessment-Wrist and Hand (FMA-WH score) and BMR score at admission were all better than that in the poor motor function group respectively (p < 0.001). The mean serum level of CAF, S100B and NfL in the good motor function group were 2.5 ± 0.82 ng/mL, 286.6 ± 236.4 ng/L and 12.1 ± 10.4 pg/mL respectively, which were lower than that in the poor motor function group (p < 0.001, Table 3). The multivariate logistic regression showed that hematoma volume (OR = 1.47, p = 0.007), FMA-WH score admission (OR = 0.78, p = 0.02), S100B (OR = 1.32, p = 0.04), and NfL (OR = 1.24, p = 0.003) were all significant predictors of poor motor function.
CONCLUSIONS
We found that Soft robotic hands therapy benefited in hand function in patients with ICH and hematoma volume, FMA-WH score admission, S100B, and NfL were all significant predictors for poor motor function of patients with ICH.
Topics: Cerebral Hemorrhage; Hematoma; Humans; Nomograms; Recovery of Function; Robotics
PubMed: 36068493
DOI: 10.1186/s12883-022-02864-2 -
Cell & Bioscience Aug 2022Neuromuscular junctions (NMJs) are peripheral synapses connecting motoneurons and skeletal myofibers. At the postsynaptic side in myofibers, acetylcholine receptor...
BACKGROUND
Neuromuscular junctions (NMJs) are peripheral synapses connecting motoneurons and skeletal myofibers. At the postsynaptic side in myofibers, acetylcholine receptor (AChR) proteins are clustered by the neuronal agrin signal. Meanwhile, several nuclei in each myofiber are specially enriched around the NMJ for postsynaptic gene transcription. It remains mysterious that how gene expressions in these synaptic nuclei are systematically regulated, especially by motoneurons.
RESULTS
We found that synaptic nuclei have a distinctive chromatin structure and gene expression profiling. Synaptic nuclei are formed during NMJ development and maintained by motoneuron innervation. Transcriptome analysis revealed that motoneuron innervation determines the distinct expression patterns in the synaptic region and non-synaptic region in each multinucleated myofiber, probably through epigenetic regulation. Myonuclei in synaptic and non-synaptic regions have different responses to denervation. Weighted gene co-expression network analysis revealed that the histone lysine demethylases Kdm1a is a negative regulator of synaptic gene expression. Inhibition of Kdm1a promotes AChR expression but impairs motor functions.
CONCLUSION
These results demonstrate that motoneurons innervation determines the distinct gene expressions in multinucleated myofibers. Thus, dysregulation of nerve-controlled chromatin structure and muscle gene expression might cause muscle weakness and atrophy in motoneuron degenerative disorders.
PubMed: 36042463
DOI: 10.1186/s13578-022-00876-6