Neuromuscular Research Articles

STUDY ON ANATOMICAL CHARACTERIZATIONOF EXOTIC SPECIES AMHERSTIA NOBILIS WALL

Amherstia nobilis, known as the Pride of Burma and colloquially referred to as Simshipa-vriksham, is a remarkable and rare tropical tree celebrated in Indian mythology, especially within the context of the epic Ramayana. Often dubbed the Queen of Flowering Trees. The species was named in honour of Lady Sarah Amherst, an early collector of Asian plants, and it stands as the sole representative of its genus. This study involves microscopic examination of various parts of the plant, revealing intricate details about its cellular structure, vascular arrangements, and overall morphology. Mature leaves of A. nobilis, for instance, have been analyzed micro-morphologically, with an average leaf area calculated. The leaves are characterized by numerous rubiaceous stomata, as hypostomatic. In the leaf midrib, the vascular area appears oval and vascular elements are U-shaped. The leaf lamina flanked by highly cutinized upper and lower epidermis layers. The palisade tissue is bi-layered, while the lower leaf lamina features loosely packed spongy layers with numerous vein bundles and large air spaces and contains starch grains. Histological examination of the rachis reveals an oval shape with a slightly thick-walled epidermis and hypodermis containing air cavities. The vascular region is well-differentiated withxylem elements in linear multiples. The secondary phloem elementspossess numerous prismatic crystals and patches of phloem fibers. In the mature stem, periderm formation, with a highly reduced cortical region containing chlorenchymatous cells. The vascular region comprises phloem tissues with patches of phloem fibers. The xylem is with large vessels arranged in linear multiples. The pith is highly reduced with included phloem elements. Wood anatomy reveals the vessels displaying paratracheal parenchyma distribution, appearing as aliform or eyelet type. The vessel elements are arranged in linear multiples, and the radial longitudinal section shows large vessel elements with bordered pits separated by end plates. The bark showed uni- and multi-seriate ray cells, long and narrow phloem parenchyma, and prismatic crystal deposits in phloem ray-parenchyma cells. The parenchyma cells are traversed by numerous phloem fibers, with starch granules deposited in phloem parenchyma. In conclusion, Amherstia nobilis, the Pride of Burma, exemplifies a unique combination of aesthetic appeal and complex anatomical structure, making it a subject of significant interest in botanical and horticultural research. Detailed histological and anatomical studies provide crucial insights into the species unique adaptations and structural features, underscoring the need for continued conservation efforts to protect this majestic and rare tropical tree.

Insights from CD71 presentation and serum lipid peroxidation in myasthenia gravis – A small cohort study

Myasthenia gravis (MG) is a multifaceted autoimmune disorder affecting the postsynaptic neuromuscular junction. In this study, we examined CD4+ and CD8+ T lymphocyte levels and ratios within peripheral blood mononuclear cells (PBMCs) in MG patients. Additionally, we assessed lymphocytes for the expression of CD71, which functions as a transferrin receptor mediating the uptake of iron into the cells. Building on recent discussions regarding CD20 depletion treatments in MG, we also scrutinized lymphocytes for CD20 expression. Comparative analyses were conducted among healthy controls, newly diagnosed MG patients, those undergoing pyridostigmine treatment alone, and MG patients receiving combination therapies. In the patients, the ratio of CD3+CD4+ T lymphocytes to CD3+ T lymphocytes was found to be decreased compared to the healthy controls, while the ratio of CD3+CD8+ cells to CD3+CD4+ cells increased. An increase in the percentage of CD71-expressing lymphocytes was observed in MG patients compared to the healthy control group, while CD20+ lymphocytes exhibited no statistical changes. Moreover, heightened serum lipid peroxidation levels were found in MG patients. These results suggest a possible relationship between iron metabolism, levels of CD71-expressing cells, and lipid peroxidation in MG. Conversely, pyridostigmine treatment reduced the levels of CD71-expressing cells and lipid peroxidation, suggesting potential immunomodulatory and antioxidant impacts of pyridostigmine in MG, either directly or indirectly.

Effectiveness and Safety of Mycophenolate Mophetil in Myasthenia Gravis: A Real-Life Multicenter Experience.

Myasthenia gravis (MG) is an autoimmune disease characterized by fluctuating muscle weakness due to autoantibodies targeting neuromuscular junction proteins. Mycophenolate mofetil (MMF), an immunosuppressive therapy, has shown potential for managing MG with fewer side effects compared to other treatments. This study aims to evaluate the effectiveness and safety of MMF in MG patients in a real-life multicenter setting. A retrospective cohort study was conducted on generalized MG patients, refractory to azathioprine (AZA) and treated with MMF alone or with steroids, at three Italian centers from January 2011 to February 2024. Patients were assessed using the Myasthenia Gravis Foundation of America (MGFA) classification, MG composite score (MGCS), and MG activity of daily living (MGADL) scores at baseline, 6, 12, 18, and 24 months. Statistical analyses included the Spearman correlation, the Friedman test, and ANOVA. Thirty-two patients were enrolled (13 males, mean age 66.5 ± 11.5 years). Significant improvements in MGADL and MGCS scores were observed at 6 and 12 months (p < 0.001), with continued improvement over 24 months. Side effects were reported in 12% of patients. MMF showed a faster onset of symptom control compared to azathioprine, with a significant improvement noted within 6 months. A recent study found that MMF and AZA were equally effective in improving patients' quality of life, but because AZA had more serious adverse events than MMF, lower doses of AZA were therefore recommended to reduce the adverse events while maintaining efficacy. Conversely, results showed that MMF is effective and well-tolerated in the long-term management of MG, providing faster symptom control and a favorable safety profile. Future prospective studies with larger cohorts are needed to confirm these findings and explore sex differences in response to MMF treatment.

Structural switch in acetylcholine receptors in developing muscle.

During development, motor neurons originating in the brainstem and spinal cord form elaborate synapses with skeletal muscle fibres1. These neurons release acetylcholine (ACh), which binds to nicotinic ACh receptors (AChRs) on the muscle, initiating contraction. Two types of AChR are present in developing muscle cells, and their differential expression serves as a hallmark of neuromuscular synapse maturation2-4. The structural principles underlying the switch from fetal to adult muscle receptors are unknown. Here, we present high-resolution structures of both fetal and adult muscle nicotinic AChRs, isolated from bovine skeletal muscle in developmental transition. These structures, obtained in the absence and presence of ACh, provide a structural context for understanding how fetal versus adult receptor isoforms are tuned for synapse development versus the all-or-none signalling required for high-fidelity skeletal muscle contraction. We find that ACh affinity differences are driven by binding site access, channel conductance is tuned by widespread surface electrostatics and open duration changes result from intrasubunit interactions and structural flexibility. The structures further reveal pathogenic mechanisms underlying congenital myasthenic syndromes.

Subxiphoid thoracoscopic thymectomy in a young female with seropositive Myasthenia Gravis: A case report

Case: Myasthenia Gravis (MG) is an autoimmune disease that occurs at the neuromuscular junction and characterized by weakness of voluntary skeletal muscles which may be generalized or localized with an incidence of 4.1 to 30 cases per million person-years. MG is diagnosed clinically and by measuring the level of acetylcholine receptor antibodies, Muscle specific kinase antibodies and Low-density lipoprotein receptor related protein-4 antibodies. Patient is managed conservatively with cholinesterase inhibitor (pyridostigmine), prednisone, Mycophenolate mofetil and surgically by thymectomy. Here we present a case of a 27-year female who was known case of seropositive Myasthenia gravis for five years who underwent a novel technique of subxiphoid thymectomy.

Experimental, numerical, and analytical studies of asymmetric bolted square hollow section splices in bending

The flexural behaviour of two types of asymmetric bolted square hollow section (SHS) splices is investigated in this paper. The asymmetric bolted SHS splices are derived from the traditional bolted end plate SHS joints but with the end plate flushed to the SHS surface on one side or two adjacent sides, where a cover plate connects two SHSs. Firstly, four-point bending tests are conducted for two configurations under different loading conditions. Next, finite element (FE) analysis is carried out to simulate the experiments. To reduce the computational time, a simplified FE model is developed, which is further employed for a parametric study. Finally, the component method, which was extended for the asymmetric bolted SHS splices under tensile load, is verified against the FE models in the parametric study. The results show that the extended component method could effectively predict the stiffness and the resistance of bolted asymmetric SHS splices under different bending loads.

Analysis of Intervertebral Disc Degeneration Induced by Endplate Drilling or Needle Puncture in Complement C6-Sufficient and C6-Deficient Rabbits.

Previous studies indicate an implication of the terminal complement complex (TCC) in disc degeneration (DD). To investigate the functional role of TCC in trauma-induced DD in vivo, the model of endplate (EP) drilling was first applied in rabbits using a C6-deficient rabbit strain in which no TCC formation was possible. In parallel the model of needle puncture was investigated. Using a minimally invasive surgical intervention, lumbar rabbit intervertebral discs (IVDs) were treated with EP drilling or needle puncture. Degenerative effects of both surgical interventions were assessed by Pfirrmann grading and T2 quantification of the IVDs based on high-resolution MRI (11.7 T), as well as radiographic determination of disc height index. Pfirrmann grading indicated significant degenerative effects after EP drilling. Contrary to our assumption, no evidence was found that the absence of TCC formation in C6-deficient rabbits reduces the development of DD compared to C6-sufficient animals. EP drilling was proven to be suitable for application in rabbits. However, results of the present study do not provide clear evidence of a central functional role of TCC within DD and suggest that TCC deposition in DD patients may be primarily considered as a marker of complement activation during DD progression.

Electroacupuncture alleviates motor dysfunction by regulating neuromuscular junction disruption and neuronal degeneration in SOD1G93A mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the progressive destruction of the neuromuscular junction (NMJ) and the degeneration of motor neurons, eventually leading to atrophy and paralysis of voluntary muscles responsible for motion and breathing. NMJs, synaptic connections between motor neurons and skeletal muscle fibers, are extremely fragile in ALS. To determine the effects of early electroacupuncture (EA) intervention on nerve reinnervation and regeneration following injury, a model of sciatic nerve injury (SNI) was first established using SOD1G93A mice, and early electroacupuncture (EA) intervention was conducted at Baihui (DU20), and bilateral Zusanli (ST36). The results revealed that EA increased the Sciatic nerve Functional Index, the structural integrity of the gastrocnemius muscles, and the cross-sectional area of muscle fibers, as well as up-regulated the expression of acetylcholinesterase and facilitated the co-location of α7 nicotinic acetate choline receptors and α-actinin. Overall, these results suggested that EA can promote the repair and regeneration of injured nerves and delay NMJ degeneration in SOD1G93A-SNI mice. Moreover, analysis of the cerebral cortex demonstrated that EA alleviated cortical motor neuron damage in SOD1G93A mice, potentially attributed to the inhibition of the cyclic GMP-AMP synthase-stimulator of interferon genes pathway and the release of interferon-β suppressing the activation of natural killer cells and the secretion of interferon-γ, thereby further inhibiting microglial activation and the expression of inflammatory factors. In summary, EA delayed the degeneration of NMJ and mitigated the loss of cortical motor neurons, thus delaying disease onset, accompanied by alleviation of muscle atrophy and improvements in motor function in SOD1G93A mice.

Component tests and numerical simulations of 3D steel frame structures for progressive collapse

This paper presents a comprehensive study on three-dimensional steel frame structures subjected to progressive collapse, drawing insights from a full-scale steel frame substructure test. The investigation encompasses connection component tests and numerical simulations, focusing on extended end plate and double-angle cleat connections employed in the substructure test. The mechanical properties of the connection components were tested, forming a basis for defining component properties in subsequent connection models. Finite element (FE) models of the test substructure were developed, utilizing hybrid elements for the steel frame part, which include beam, connector, and spring elements based on the component method. To simulate reinforced concrete slabs, a combination of refined solid elements and simplified shell elements was employed. The former accurately captures detailed failure modes, while the latter efficiently simulates the collapse behavior of large-scale steel frame structures. Validation of the established models against test results encompassed load-displacement responses, internal forces in structural members, and failure modes. The validated FE models were then utilized to analyze and discuss the contributions of various structural components in resisting progressive collapse. Specific focus was placed on the development of load-resisting mechanisms in the floor slab and the influence of beam-column connection types on structural behavior. The paper explores the role of bracing systems in a building in resisting progressive collapse. Additionally, it evaluates the effectiveness of the restraint systems used in the test, shedding light on their ability to accurately reflect real restraint effects from the surrounding structure. The findings presented herein contribute valuable insights to the understanding of progressive collapse behavior in steel frame structures, with implications for robustness design of multi-story steel buildings.

Sugamadex na reversão da anafilaxia induzida por rocurônio em paciente pediátrico: relato de caso e revisão de literatura

INTRODUCTION: Among neuromuscular blockers (NMB), rocuronium is the most notably cited as a cause of anaphylactic reaction. Rocuronium-induced anaphylaxis is a serious and potentially fatal complication, requiring immediate and effective management. The advent of the specific antagonist, sugammadex, has widely spread its use because it reduces the incidence of residual neuromuscular blockade and is indicated in the protocol for the treatment and reversal of refractory anaphylaxis. This study reports an episode of anaphylaxis induced by rocuronium and successfully reversed after administration of sugammadex. CASE REPORT: A 6-year-old child, weighing 30 kg, scheduled for video-assisted thoracoscopy under general anesthesia presented with tachycardia, severe hypotension, and diffuse skin rash after induction of general anesthesia and administration of rocuronium. All therapeutic measures were immediately taken for the treatment of a severe allergic reaction, however there was no clinical improvement, and the patient progressed to sinus bradycardia. At this point, cardiopulmonary resuscitation maneuvers were initiated, and adrenaline was administered, but without satisfactory clinical response. In this context, the possibility of the causative agent being rocuronium was raised. Sugammadex was administered 16mg/kg, and the patient immediately returned to sinus rhythm and later presented a favorable clinical evolution and no sequelae. DISCUSSION: Sugammadex is a molecule that encapsulates rocuronium and removes it from both plasma and neuromuscular junction, and then excretes it inertly in the urine. Some authors suggest that sugammadex removes rocuronium molecules from the circulation, whether free or IgE-bound. This mechanism is extremely important, especially in grade III and IV anaphylactic reactions, which can lead to death. In view of the scarcity of publications, this report showed the role of sugammadex in the reversal of rocuronium-induced anaphylaxis, refractory to conventional treatment.

Congenital myasthenic syndromes: increasingly complex.

Congenital myasthenia syndromes (CMS) are treatable, inherited disorders affecting neuromuscular transmission. We highlight that the involvement of an increasing number of proteins is making the understanding of the disease mechanisms and potential treatments progressively more complex. Although early studies identified mutations of proteins directly involved in synaptic transmission at the neuromuscular junction, recently, next-generation sequencing has facilitated the identification of many novel mutations in genes that encode proteins that have a far wider expression profile, some even ubiquitously expressed, but whose defective function leads to impaired neuromuscular transmission. Unsurprisingly, mutations in these genes often causes a wider phenotypic disease spectrum where defective neuromuscular transmission forms only one component. This has implications for the management of CMS patients. Given the widening nonneuromuscular junction phenotypes in the newly identified forms of CMS, new therapies need to include disease-modifying approaches that address not only neuromuscular weakness but also the multisystem involvement. Whilst the current treatments for CMS are highly effective for many subtypes there remains, in a proportion of CMS patients, an unmet need for more efficacious therapies.

Metformin improves skeletal muscle and physical capacity by stabilizing neuromuscular junction in older adults

ObjectivesMetformin is an anti-diabetic drug with protective effects on skeletal muscle and physical capacity. However, the relevant mechanisms of action on skeletal muscle remain poorly understood. We investigated the potential contribution of neuromuscular junction (NMJ) degradation to skeletal muscle and physical capacity in geriatric men taking metformin. MethodWe recruited geriatric men for placebo (Age=73.1 ± 4.2 years, n = 70) and metformin (Age=70.1 ± 4.5 years, n = 62) groups. The patients in the metformin group received 1700 mg of metformin twice a day for 16 weeks. We measured plasma c-terminal agrin-fragment-22 (CAF22) and neurofilament light chain (NfL) as markers of neuromuscular junction (NMJ) degradation and neurodegeneration, respectively, with relevance to handgrip strength (HGS) and short physical performance battery (SPPB; a marker of physical capacity) in older adults taking metformin. These findings were associated with reduced oxidative stress in the metformin group. ResultsAt baseline, both groups had similar HGS, gait speed, SPPB scores, and plasma biochemistry. Metformin improved HGS, gait speed, and cumulative SPPB scores in geriatric men (all p < 0.05). Metformin also reduced plasma CAF22 and NfL levels when compared to baseline. Similar observations were not found in the placebo group. Correlation analysis revealed significant correlations of plasma CAF22 with HGS, gait speed, and cumulative SPPB scores in the metformin group. These observations were associated with reduced oxidative stress in the metformin group. ConclusionAltogether, the restorative effects of metformin on skeletal muscle and physical capacity involve NMJ stabilization. Our data is clinically relevant for geriatric men with functional disabilities.

Therapy with botulinum neurotoxin for Parkinson's disease.

Botulinum neurotoxin (BoNT) has been in use since the 1970's. Its effect is reached mainly by inhibiting the release of acetylcholine in the synaptic gap of motor neurons or at the motor end plate and the parasympathetic ganglia. In the case of Parkinson's disease, it is used to treat several motor and non-motor symptoms. Within recent years increasingly numerous possible fields of application of BoNT have been found for the treatment of Parkinson's disease, and for some specific symptoms it has in fact become the therapy of choice, while for others it is but one of the therapeutic options that come into consideration when others are not sufficiently effective. In the following, we intend to outline the indications, the possible side effects and also the approvals for therapies with botulinum toxin in the primary and secondary symptoms of Parkinson's disease.

Muscle-specific lack of Gfpt1 triggers ER stress to alleviate misfolded protein accumulation.

Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in (KI) mouse model carrying a frameshift variant in Gfpt1 exon 9, simulating that found in a patient with CMS. As Gfpt1 exon 9 is exclusively expressed in striated muscles, Gfpt1-KI mice were deficient for Gfpt1 only in skeletal muscles. In Gfpt1-KI mice, (1) UDP-HexNAc, CMP-NeuAc and protein O-GlcNAcylation were reduced in skeletal muscles; (2) aged Gfpt1-KI mice showed poor exercise performance and abnormal neuromuscular junction structures; and (3) markers of the unfolded protein response (UPR) were elevated in skeletal muscles. Denervation-mediated enhancement of endoplasmic reticulum (ER) stress in Gfpt1-KI mice facilitated protein folding, ubiquitin-proteasome degradation and apoptosis, whereas autophagy was not induced and protein aggregates were markedly increased. Lack of autophagy was accounted for by enhanced degradation of FoxO1 by increased Xbp1-s/u proteins. Similarly, in Gfpt1-silenced C2C12 myotubes, ER stress exacerbated protein aggregates and activated apoptosis, but autophagy was attenuated. In both skeletal muscles in Gfpt1-KI mice and Gfpt1-silenced C2C12 myotubes, maladaptive UPR failed to eliminate protein aggregates and provoked apoptosis.

Engineered Regenerative Isolated Peripheral Nerve Interface for Targeted Reinnervation.

A regenerative peripheral nerve interface (RPNI) offers a therapeutic solution for nerve injury through reconstruction of the target muscle. However, implanting a transected peripheral nerve into an autologous skeletal muscle graft in RPNI causes donor-site morbidity, highlighting the need for tissue-engineered skeletal muscle constructs. Here, an engineered regenerative isolated peripheral nerve interface (eRIPEN) is developed using 3D skeletal cell printing combined with direct electrospinning to create a nanofiber membrane envelop for host nerve implantation. In this in vivo study, after over 8 months of RPNI surgery, the eRIPEN exhibits a minimum Feret diameter of 15-20µm with a cross-sectional area of 100-500 µm2, representing the largest distribution of myofibers. Furthermore, neuromuscular junction formation and muscle contraction with a force of ≈28 N are observed. Notably, the decreased hypersensitivity to mechanical/thermal stimuli and an improved tibial functional index from -77 to -56 are found in the eRIPEN group. The present novel concept of eRIPEN paves the way for the utilization and application of tissue-engineered constructs in RPNI, ultimately realizing neuroprosthesis control through synaptic connections.

AAV-NRIP gene therapy ameliorates motor neuron degeneration and muscle atrophy in ALS model mice

BackgroundAmyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS.MethodsWe used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice.ResultsSOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice.ConclusionsAAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS.

Does computed tomography-derived volumometry and densitometry of psoas muscle really correlate with complications in rectal cancer patients after elective surgery?

Although sarcopenia is recognized as one of the risk factors for increased morbidity after resection for colorectal cancer, the question of the most appropriate way to identify and quantify it is still unresolved. This is a retrospective unicentric study following patients undergoing elective resection of the rectum for carcinoma with available staging computed tomography (CT) of the trunk. Psoas muscle density (PMD) and its area relative to patient height psoas muscle index (PMI) at the level of inferior vertebral end plate of third lumbar vertebra (L3) were assessed using an initial staging CT scan of the trunk. Post-operative complications, evaluated according to the Clavien-Dindo classification, and blood samples on post-operative days (POD) 3 and 5 were also recorded in the study population. Patients were divided into groups with complicated and uncomplicated post-operative course, and observed parameters were then statistically compared. The correlation of PMI values with the development of post-operative complications was not confirmed in a data set of 206 patients. PMD values were found to be borderline statistically significant in patients with complicated post-operative course, while in the group of patients with severe complications (Clavien-Dindo III-IV), there was no statistically significant difference in PMI or PMD values. The same results were obtained when comparing patients with anastomotic leak (AL). It was confirmed that operations on the lower rectum are riskier for the development of post-operative complications. The secondary objective of our study regarding serum C-reactive protein (CRP) levels of 3rd and 5th POD gave us the answer in the form of cutoff values of 115.7 mg/L (3rd POD) and 76 mg/L (5th POD). PMD appears to be a promising tool for predicting post-operative morbidity in patients after rectal resection, but a clear consensus on the method of measurement, interpretation of results and cutoff values is needed. Lower rectal resections are burdened with a higher risk of post-operative complications, especially AL. Monitoring of CRP levels remains an important marker in the prediction of AL due to its negative predictive value.

BDNF/TrkB signalling, in cooperation with muscarinic signalling, retrogradely regulates PKA pathway to phosphorylate SNAP-25 and Synapsin-1 at the neuromuscular junction

BackgroundProtein kinase A (PKA) enhances neurotransmission at the neuromuscular junction (NMJ), which is retrogradely regulated by nerve-induced muscle contraction to promote Acetylcholine (ACh) release through the phosphorylation of molecules involved in synaptic vesicle exocytosis (SNAP-25 and Synapsin-1). However, the molecular mechanism of the retrograde regulation of PKA subunits and its targets by BDNF/TrkB pathway and muscarinic signalling has not been demonstrated until now. At the NMJ, retrograde control is mainly associated with BDNF/TrkB signalling as muscle contraction enhances BDNF levels and controls specific kinases involved in the neurotransmission. Neurotransmission at the NMJ is also highly modulated by muscarinic receptors M1 and M2 (mAChRs), which are related to PKA and TrkB signallings. Here, we investigated the hypothesis that TrkB, in cooperation with mAChRs, regulates the activity-dependent dynamics of PKA subunits to phosphorylate SNAP-25 and Synapsin-1.MethodsTo explore this, we stimulated the rat phrenic nerve at 1Hz (30 minutes), with or without subsequent contraction (abolished by µ-conotoxin GIIIB). Pharmacological treatments were conducted with the anti-TrkB antibody clone 47/TrkB for TrkB inhibition and exogenous h-BDNF; muscarinic inhibition with Pirenzepine-dihydrochloride and Methoctramine-tetrahydrochloride for M1 and M2 mAChRs, respectively. Diaphragm protein levels and phosphorylation’ changes were detected by Western blotting. Location of the target proteins was demonstrated using immunohistochemistry.ResultsWhile TrkB does not directly impact the levels of PKA catalytic subunits Cα and Cβ, it regulates PKA regulatory subunits RIα and RIIβ, facilitating the phosphorylation of critical exocytotic targets such as SNAP-25 and Synapsin-1. Furthermore, the muscarinic receptors pathway maintains a delicate balance in this regulatory process. These findings explain the dynamic interplay of PKA subunits influenced by BDNF/TrkB signalling, M1 and M2 mAChRs pathways, that are differently regulated by pre- and postsynaptic activity, demonstrating the specific roles of the BDNF/TrkB and muscarinic receptors pathway in retrograde regulation.ConclusionThis complex molecular interplay has the relevance of interrelating two fundamental pathways in PKA-synaptic modulation: one retrograde (neurotrophic) and the other autocrine (muscarinic). This deepens the fundamental understanding of neuromuscular physiology of neurotransmission that gives plasticity to synapses and holds the potential for identifying therapeutic strategies in conditions characterized by impaired neuromuscular communication.

Molecular Adaptations of BDNF/NT-4 Neurotrophic and Muscarinic Pathways in Ageing Neuromuscular Synapses.

Age-related conditions, such as sarcopenia, cause physical disabilities for an increasing section of society. At the neuromuscular junction, the postsynaptic-derived neurotrophic factors brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NT-4) have neuroprotective functions and contribute to the correct regulation of the exocytotic machinery. Similarly, presynaptic muscarinic signalling plays a fundamental modulatory function in this synapse. However, whether or not these signalling pathways are compromised in ageing neuromuscular system has not yet been analysed. The present study analyses, through Western blotting, the differences in expression and activation of the main key proteins of the BDNF/NT-4 and muscarinic pathways related to neurotransmission in young versus ageing Extensor digitorum longus (EDL) rat muscles. The main results show an imbalance in several sections of these pathways: (i) a change in the stoichiometry of BDNF/NT-4, (ii) an imbalance of Tropomyosin-related kinase B receptor (TrkB)-FL/TrkB-T1 and neurotrophic receptor p 75 (p75NTR), (iii) no changes in the cytosol/membrane distribution of phosphorylated downstream protein kinase C (PKC)βI and PKCε, (iv) a reduction in the M2-subtype muscarinic receptor and P/Q-subtype voltage-gated calcium channel, (v) an imbalance of phosphorylated mammalian uncoordinated-18-1 (Munc18-1) (S313) and synaptosomal-associated protein 25 (SNAP-25) (S187), and (vi) normal levels of molecules related to the management of acetylcholine (Ach). Based on this descriptive analysis, we hypothesise that these pathways can be adjusted to ensure neurotransmission rather than undergoing negative alterations caused by ageing. However, further studies are needed to assess this hypothetical suggestion. Our results contribute to the understanding of some previously described neuromuscular functional age-related impairments. Strategies to promote these signalling pathways could improve the neuromuscular physiology and quality of life of older people.

Therapeutics Targeting Skeletal Muscle in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a complex neuromuscular disease characterized by progressive motor neuron degeneration, neuromuscular junction dismantling, and muscle wasting. The pathological and therapeutic studies of ALS have long been neurocentric. However, recent insights have highlighted the significance of peripheral tissue, particularly skeletal muscle, in disease pathology and treatment. This is evidenced by restricted ALS-like muscle atrophy, which can retrogradely induce neuromuscular junction and motor neuron degeneration. Moreover, therapeutics targeting skeletal muscles can effectively decelerate disease progression by modulating muscle satellite cells for muscle repair, suppressing inflammation, and promoting the recovery or regeneration of the neuromuscular junction. This review summarizes and discusses therapeutic strategies targeting skeletal muscles for ALS treatment. It aims to provide a comprehensive reference for the development of novel therapeutics targeting skeletal muscles, potentially ameliorating the progression of ALS.