1,720,982 research outputs found
Signaling and transcriptomics at the degenerating-regenerating neuromuscular junction
Full text linkThe neuromuscular junction (NMJ) is a specialized tripartite synapse that allows the transmission of an electrical impulse travelling along the axon to the muscle. It is composed of the motor axon terminal (MAT), covered by perisynaptic Schwann cells (PSCs), and the muscle fibre (MF), which are separated by a basal lamina. The NMJ is not protected by anatomical barriers: it can be therefore exposed to traumas, to the attack of many pathogens including neurotoxins, and affected by many neuromuscular diseases such as amyotrophic lateral sclerosis and immune-mediated disorders, such as the Guillain-Barré and Miller Fisher syndromes. For these reasons and for its essential role in life and survival the NMJ has retained throughout vertebrate evolution an intrinsic ability for repair and regeneration, differently from central synapses. After nerve injury the glial cells of the NMJ, the PSCs, acquire a regenerative phenotype and release a series of factors that act on the stump of the MAT, providing several cues to promote neuronal regeneration. Following peripheral nerve injury, many changes taking place at the NMJ have been reported so far, but the inter- and intra-cellular signaling that occur during MAT degeneration and, more importantly, those governing the ensuing regeneration are not completely understood.
We have recently established a model to study NMJ degeneration and regeneration in mice based on the specific action of -latrotoxin, a presynaptic neurotoxin isolated from the venom of the black widow spider, which targets specifically the presynaptic terminal causing its complete degeneration. Following intoxication and the subsequent clearing of MAT debris by PSCs, the axon stump regrows in few days in mice allowing complete NMJ recovery. This toxin represents therefore a simple and controlled method to induce an acute, localized and reversible nerve terminal degeneration not blurred by inflammation, and can help to identify molecules involved in the intra- and inter-cellular signalling governing NMJ regeneration.
In the search of candidate molecules involved in triggering and sustaining nerve recovery we choose to perform a transcriptomic analysis of the mouse NMJs at different time points after injection of -latrotoxin. This approach has been very challenging: to our knowledge a transcriptomic analysis of the sole NMJ was never reported before. We succeeded in collecting a number of NMJs suitable for RNA isolation and sequencing of both coding and non–coding RNAs. Among the coding transcripts we selected a series of messenger RNAs (mRNAs) that are expressed at low level in controls, at higher levels during regeneration, and then return to basal when substantial regeneration is attained and we selected the mRNA encoding for the chemokine CXCL12. We found that CXCL12 is produced by PSCs during nerve degeneration, and that intraperitoneal injection of a neutralizing antibody for CXCL12 slows down the regeneration process. Moreover, the exposure of primary motor neurons to the recombinant chemokine stimulates neurite growth.
These data suggest that CXCL12 is an important factor released by PSCs with a crucial role in the nerve terminal regeneration process.
Parallely, we looked for molecules released by injured neurons that could activate SCs and stimulate nerve regeneration. We found that ATP released by intoxicated neurons activates a series of intracellullar signaling pathways in SCs including Ca2+, adenylate cyclase, ERK 1/2 and CREB, that are of fundamental importance for the recovery of nerve function. We propose ATP as an important alarm signal partecipating in the cross-talk between degenerating nerve terminals and adjacent PSCs not only in a model of degeneration by a spider toxin, but also in different forms of neurodegeneration of the presynaptic nerve terminal
Extra-pineal melatonin in perisynaptic Schwann cell-muscle fiber cross talk at the regenerating neuromuscular junction
Full text linkImpact of ageing and disuse on neuromuscular junction and mitochondrial function and morphology: Current evidence and controversies
Full text linkInactivity and ageing can have a detrimental impact on skeletal muscle and the neuromuscular junction (NMJ). Decreased physical activity results in muscle atrophy, impaired mitochondrial function, and NMJ instability. Ageing is associated with a progressive decrease in muscle mass, deterioration of mitochondrial function in the motor axon terminals and in myofibres, NMJ instability and loss of motor units. Focusing on the impact of inactivity and ageing, this review examines the consequences on NMJ stability and the role of mitochondrial dysfunction, delving into their complex relationship with ageing and disuse. Evidence suggests that mitochondrial dysfunction can be a pathogenic driver for NMJ alterations, with studies revealing the role of mitochondrial defects in motor neuron degeneration and NMJ instability. Two perspectives behind NMJ instability are discussed: one is that mitochondrial dysfunction in skeletal muscle triggers NMJ deterioration, the other envisages dysfunction of motor terminal mitochondria as a primary contributor to NMJ instability. While evidence from these studies supports both perspectives on the relationship between NMJ dysfunction and mitochondrial impairment, gaps persist in the understanding of how mitochondrial dysfunction can cause NMJ deterioration. Further research, both in humans and in animal models, is essential for unravelling the mechanisms and potential interventions for age- and inactivity-related neuromuscular and mitochondrial alterations
Electrophysiological Recordings of Evoked End-Plate Potential on Murine Neuro-muscular Synapse Preparations
Full text linkNeuromuscular junction (NMJ) is the specialized chemical synapse that mediates the transmission of the electrical impulse running along motor neuron axons to skeletal muscle fibers. NMJ is the best characterized chemical synapse and its study along many years of research has provided most of the general knowledge of synapse development, structure and functionality. Electrophysiology is the most accurate experimental procedure to study NMJ physiology and it largely contributed to the elucidation of synaptic transmission basic principles. Many electrophysiological techniques have been developed to study NMJ physiology and physiopathology. In this paper, we describe an ex vivo tissue preparation for electrophysiology that can be applied to investigate nerve-muscle transmission functionality in mice. It is routinely used in our laboratory to study presynaptic neurotoxins, antitoxins, and to monitor NMJ degeneration and regeneration. This is a broadly applicable technique which can also be adopted to investigate alterations of NMJ activity in mouse models of neuromuscular diseases, including peripheral neuropathies, motor neuron disorders and myasthenic syndromes
Mouse Phrenic Nerve Hemidiaphragm Assay (MPN)
Full text linkThe neuromuscular junction (NMJ) is the specialized synapse by which peripheral motor neurons innervate muscle fibers and control skeletal muscle contraction. The NMJ is the target of several xenobiotics, including chemicals, plant, animal and bacterial toxins, as well as of autoantibodies raised against NMJ antigens. Depending on their biochemical nature, the site they target (either the nerve or the muscle) and their mechanism of action, substances affecting NMJ produce very specific alterations of neuromuscular functionality. Here we provide a detailed protocol to isolate the diaphragmatic muscle from mice and to set up two autonomously innervated hemidiaphragms. This preparation can be used to study bioactive substances like toxins, venoms and neuroactive molecules of various origin, or to measure the force of skeletal muscle contraction. The 'mouse phrenic nerve hemidiaphragm assay' (MPN) is an established model of ex vivo NMJ and recapitulates the complexity of neuromuscular transmission in a system easy to control and to manipulate, thus representing a valuable tool to study both NMJ physiology and the mechanism of action of toxins and other molecules acting at this synapse
The node of Ranvier influences the in vivo axonal transport of mitochondria and signaling endosomes
Full text linkEfficient long-range axonal transport is essential for maintaining neuronal function, and perturbations in this process underlie severe neurological diseases. Nodes of Ranvier (NoR) are short, specialized unmyelinated axonal domains with a unique molecular and structural composition. Currently, it remains unresolved how the distinct molecular structures of the NoR impact axonal transport dynamics. Using intravital time-lapse microscopy of sciatic nerves in live, anesthetized mice, we reveal (1) similar morphologies of the NoR in fast and slow motor axons, (2) signaling endosomes and mitochondria accumulate specifically at the distal node, and (3) unique axonal transport profiles of signaling endosomes and mitochondria transiting through the NoR. Collectively, these findings provide important insights into the fundamental physiology of peripheral nerve axons, motor neuron subtypes, and diverse organelle dynamics at the NoR. Furthermore, this work has relevance for several pathologies affecting peripheral nerves and the NoR
Hydrogen Peroxide Modulates the Timely Activation of Jun and Erk in Schwann Cells at the Injury Site and Is Required for Motor Axon Regeneration
No full textPeripheral nervous system (PNS) neurons, including motor neurons (MNs), possess a remarkable ability to regenerate and reinnervate target muscles following nerve injury. This process is orchestrated by a combination of intrinsic neuronal properties and extrinsic factors, with Schwann cells (SCs) playing a central role. Upon injury, SCs transition into a repair phenotype that allows axonal regeneration through molecular signaling and structural guidance. However, the identity of the SCs’ reprogramming factors is only partially known. We previously identified hydrogen peroxide (H2O2) as an early and key driver of nerve repair, inducing gene expression rewiring in SCs to support nerve re-growth. In this study, we quantitatively assessed the role of H2O2 in the activation of key pro-regenerative signaling pathways in SCs following sciatic nerve compression, specifically the extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun, which are essential for functional nerve recovery. Notably, we found that H2O2 neutralization does not impact degeneration, but it significantly affects the regenerative response. Collectively, our findings establish H2O2 as a promising regulator of the Schwann cell injury response at the injury site, linking oxidative signaling to the molecular mechanisms governing nerve regeneration
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
- …
