Neuronal hydrogen peroxide promotes nerve terminals regeneration at neuromuscular junction

The neuromuscular junction (NMJ) is the site of transmission of the electrical impulses from the motor axon terminal to the muscle; the anatomical organization of this highly dynamic system also includes the perisynaptic Schwann cells (PSCs), and therefore the NMJ has to be considered structurally and functionally as a tripartite system. These non-myelinating SCs are intimately associated with the nerve muscle contact and act as dynamic partners at the synapse: they are involved in many physiological functions including the embryonic development and the maintenance of adult NMJs. Moreover, they are able to detect and reciprocally modulate synaptic activity, through the activation of muscarinin and purinergic receptors present on their surface. In addition, non-traditional roles for PSCs in the recovery after nerve injury are being recognized. Following denervation or reduced synaptic activity, PSCs de-differentiate to an earlier developmental stage, becoming “reactive” PSCs, and start proliferating. These reactive PSCs actively participate in the process of nerve degeneration and regeneration: they undergo changes in their gene expression and acquire macrophagic-like activities, thus contributing to the removal of nerve debris as well as to the recruitment of macrophages, by releasing cytokines and chemokines. Moreover, following nerve terminals degeneration, PSCs at denervated end-plates extend long processes that induce and guide nerve regrowth. Given the increasing incidence of non cell-autonomous and dying-back axonopathies - such as amyotrophic lateral sclerosis (ALS) and autoimmune neuropathies - which affect predominantly motor axons terminals, it becomes very important to characterize the crosstalk between degenerating nerve terminals and adjacent PSCs at the NMJ; in particular, the identification of molecular mediators involved in PSCs activation and in nerve terminals regeneration would be crucial for the improvement of therapeutic strategies. This is the general aim of the present thesis and with this purpose in mind, we have adopted an innovative experimental approach, alternative to the traditional cut/crush surgical model employed till now. To confine the nerve damage to the sole motor axon terminal, thus avoiding the involvement of many cell types and inflammatory mediators, we exploited our knowledge on the mechanism of action of two classes of animal presynaptic neurotoxins: α-Ltx, a pore forming toxin of the venom of black widow spiders, and some snake neurotoxins endowed with phospholipase A2 activity called SPANs. Both kinds of neurotoxins induce an acute and highly reproducible motor axon terminal degeneration, which is followed in few days by complete regeneration: thus, this model represents an appropriate and controlled system to dissect the molecular mechanisms underlying de- and re-generation of peripheral nerve terminals, and to define how PSCs contribute to such processes. We have previously shown that nerve terminals exposed to spider or snake neurotoxins degenerate owing to calcium overload and mitochondrial failure. Here, we found that toxin-treated cultured neurons increase their mitochondrial production of hydrogen peroxide (H2O2), which can easily diffuse across membranes, thus acting as a paracrine signal on neighbouring cellS. Indeed, exposure of cultured SCs to H2O2 leads to ERK phosphorylation and to the activation of downstream pathways. The ERK signalling pathway plays a central role in controlling SCs plasticity during nerve repair in-vivo, but so far the molecular mediators responsible for its activation were unknown: neurons-derived H2O2 represents an ideal candidate for this role. In support of this hypothesis, we observed that ERK phosphorylation is reduced in intoxicated neurons-SCs co-cultures pre-incubated with catalase - which converts H2O2 to oxygen and water -, indicating that H2O2 produced inside neurons diffuses to reach nearby SCs, contributing to ERK activation in their cytosol. ERK phosphorylation takes place also in PSCs at intoxicated NMJs in-vivo. To confirm the involvement of H2O2 in promoting nerve regeneration, we performed electrophysiological recordings and immunohistochemistry on intoxicated muscles, and we found that co-injection of catalase together with neurotoxins delays nerve regeneration, confirming the prominent role of H2O2 in promoting NMJ recovery. Injection of the MAP kinase inhibitor PD98059 also impairs nerve repair in a way similar to that observed with catalase, supporting the finding that H2O2 enhances nerve terminals regeneration through the activation of ERK pathway in PSC

Snake and spider toxins induce a rapid recovery of function of botulinum neurotoxin paralysed neuromuscular junction

Botulinum neurotoxins (BoNTs) and some animal neurotoxins (-Bungarotoxin, -Btx, from elapid snakes and -Latrotoxin, -Ltx, from black widow spiders) are pre-synaptic neurotoxins that paralyse motor axon terminals with similar clinical outcomes in patients. However, their mechanism of action is different, leading to a largely-different duration of neuromuscular junction (NMJ) blockade. BoNTs induce a long-lasting paralysis without nerve terminal degeneration acting via proteolytic cleavage of SNARE proteins, whereas animal neurotoxins cause an acute and complete degeneration of motor axon terminals, followed by a rapid recovery. In this study, the injection of animal neurotoxins in mice muscles previously paralyzed by BoNT/A or /B accelerates the recovery of neurotransmission, as assessed by electrophysiology and morphological analysis. This result provides a proof of principle that, by causing the complete degeneration, reabsorption, and regeneration of a paralysed nerve terminal, one could favour the recovery of function of a biochemically- or genetically-altered motor axon terminal. These observations might be relevant to dying-back neuropathies, where pathological changes first occur at the neuromuscular junction and then progress proximally toward the cell body

CXCL12/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axonterminals

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by -latrotoxin. CXCL12 acts via binding to the neuronal CXCR4 receptor. A CXCL12-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration invivo. Recombinant CXCL12 invivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons invitro. These findings indicate that the CXCL12-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage

CXCL12α/SDF‐1 from perisynaptic Schwann cells promotes regeneration of injured motor axon terminals

The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter‐cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12α, also abbreviated as stromal‐derived factor‐1 (SDF‐1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by α‐latrotoxin. CXCL12α acts via binding to the neuronal CXCR4 receptor. A CXCL12α‐neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration in vivo. Recombinant CXCL12α in vivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons in vitro. These findings indicate that the CXCL12α‐CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage

Metabolic recovery after weight loss surgery is reflected in serum microRNAs.

Funder: Ministerio de Educación, Cultura y Deporte - SpainFunder: Fondation Leducq; FundRef: http://dx.doi.org/10.13039/501100001674Funder: Marie Skłodowska-Curie Innovative Training Network TRAIN-HEARTFunder: National Institute of Health ResearchINTRODUCTION: Bariatric surgery offers the most effective treatment for obesity, ameliorating or even reverting associated metabolic disorders, such as type 2 diabetes. We sought to determine the effects of bariatric surgery on circulating microRNAs (miRNAs) that have been implicated in the metabolic cross talk between the liver and adipose tissue. RESEARCH DESIGN AND METHODS: We measured 30 miRNAs in 155 morbidly obese patients and 47 controls and defined associations between miRNAs and metabolic parameters. Patients were followed up for 12 months after bariatric surgery. Key findings were replicated in a separate cohort of bariatric surgery patients with up to 18 months of follow-up. RESULTS: Higher circulating levels of liver-related miRNAs, such as miR-122, miR-885-5 p or miR-192 were observed in morbidly obese patients. The levels of these miRNAs were positively correlated with body mass index, percentage fat mass, blood glucose levels and liver transaminases. Elevated levels of circulating liver-derived miRNAs were reversed to levels of non-obese controls within 3 months after bariatric surgery. In contrast, putative adipose tissue-derived miRNAs remained unchanged (miR-99b) or increased (miR-221, miR-222) after bariatric surgery, suggesting a minor contribution of white adipose tissue to circulating miRNA levels. Circulating levels of liver-derived miRNAs normalized along with the endocrine and metabolic recovery of bariatric surgery, independent of the fat percentage reduction. CONCLUSIONS: Since liver miRNAs play a crucial role in the regulation of hepatic biochemical processes, future studies are warranted to assess whether they may serve as determinants or mediators of metabolic risk in morbidly obese patients

Extracellular Matrix in Heart Failure: Role of ADAMTS5 in Proteoglycan Remodeling

[Abstract] Background: Remodeling of the extracellular matrix (ECM) is a hallmark of heart failure (HF). Our previous analysis of the secretome of murine cardiac fibroblasts returned ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5) as one of the most abundant proteases. ADAMTS5 cleaves chondroitin sulfate proteoglycans such as versican. The contribution of ADAMTS5 and its substrate versican to HF is unknown. Methods: Versican remodeling was assessed in mice lacking the catalytic domain of ADAMTS5 (Adamts5ΔCat). Proteomics was applied to study ECM remodeling in left ventricular samples from patients with HF, with a particular focus on the effects of common medications used for the treatment of HF. Results: Versican and versikine, an ADAMTS-specific versican cleavage product, accumulated in patients with ischemic HF. Versikine was also elevated in a porcine model of cardiac ischemia/reperfusion injury and in murine hearts after angiotensin II infusion. In Adamts5ΔCat mice, angiotensin II infusion resulted in an aggravated versican build-up and hyaluronic acid disarrangement, accompanied by reduced levels of integrin β1, filamin A, and connexin 43. Echocardiographic assessment of Adamts5ΔCat mice revealed a reduced ejection fraction and an impaired global longitudinal strain on angiotensin II infusion. Cardiac hypertrophy and collagen deposition were similar to littermate controls. In a proteomics analysis of a larger cohort of cardiac explants from patients with ischemic HF (n=65), the use of β-blockers was associated with a reduction in ECM deposition, with versican being among the most pronounced changes. Subsequent experiments in cardiac fibroblasts confirmed that β1-adrenergic receptor stimulation increased versican expression. Despite similar clinical characteristics, patients with HF treated with β-blockers had a distinct cardiac ECM profile. Conclusions: Our results in animal models and patients suggest that ADAMTS proteases are critical for versican degradation in the heart and that versican accumulation is associated with impaired cardiac function. A comprehensive characterization of the cardiac ECM in patients with ischemic HF revealed that β-blockers may have a previously unrecognized beneficial effect on cardiac chondroitin sulfate proteoglycan content.Dr Barallobre-Barreiro is a British Heart Foundation Intermediate Fellow (FS/19/33/34328). Drs Mayr and Shah are British Heart Foundation Chair Holders (CH/16/3/32406 and CH/1999001/11735, respectively) and received support from the British Heart Foundation Center for Vascular Regeneration With Edinburgh/Bristol (RM/17/3/33381). Dr Doménech’s work was supported by Project PI16/02049 integrated in the National Plan for Scientific Research, Development and Technological Innovation, 2013–2016, and funded by the ISCIII–General Subdirection of Assessment and Promotion of Research–European Regional Development Fund. Dr Merkely’s work was funded by the National Research, Development and Innovation Fund (NVKP_16-1–2016-0017) and the Thematic Excellence Program of the Ministry for Innovation and Technology (2020-4.1.1.-TKP2020), Hungary. Dr Radovits is supported by the National Research, Development and Innovation Office of Hungary (K134939)British Heart Foundation; FS/19/33/34328British Heart Foundation; CH/16/3/32406British Heart Foundation; CH/1999001/11735British Heart Foundation Center for Vascular Regeneration; RM/17/3/33381Hungría. Ministry for Innovation and Technology; NVKP_16-1–2016-0017Hungría. Ministry for Innovation and Technology; 2020-4.1.1.-TKP2020Hungría. National Research, Development and Innovation Office; K13493

Neuronal hydrogen peroxide promotes nerve terminals regeneration at neuromuscular junction

The neuromuscular junction (NMJ) is the site of transmission of the electrical impulses from the motor axon terminal to the muscle; the anatomical organization of this highly dynamic system also includes the perisynaptic Schwann cells (PSCs), and therefore the NMJ has to be considered structurally and functionally as a tripartite system. These non-myelinating SCs are intimately associated with the nerve muscle contact and act as dynamic partners at the synapse: they are involved in many physiological functions including the embryonic development and the maintenance of adult NMJs. Moreover, they are able to detect and reciprocally modulate synaptic activity, through the activation of muscarinin and purinergic receptors present on their surface. In addition, non-traditional roles for PSCs in the recovery after nerve injury are being recognized. Following denervation or reduced synaptic activity, PSCs de-differentiate to an earlier developmental stage, becoming “reactive” PSCs, and start proliferating. These reactive PSCs actively participate in the process of nerve degeneration and regeneration: they undergo changes in their gene expression and acquire macrophagic-like activities, thus contributing to the removal of nerve debris as well as to the recruitment of macrophages, by releasing cytokines and chemokines. Moreover, following nerve terminals degeneration, PSCs at denervated end-plates extend long processes that induce and guide nerve regrowth. Given the increasing incidence of non cell-autonomous and dying-back axonopathies - such as amyotrophic lateral sclerosis (ALS) and autoimmune neuropathies - which affect predominantly motor axons terminals, it becomes very important to characterize the crosstalk between degenerating nerve terminals and adjacent PSCs at the NMJ; in particular, the identification of molecular mediators involved in PSCs activation and in nerve terminals regeneration would be crucial for the improvement of therapeutic strategies. This is the general aim of the present thesis and with this purpose in mind, we have adopted an innovative experimental approach, alternative to the traditional cut/crush surgical model employed till now. To confine the nerve damage to the sole motor axon terminal, thus avoiding the involvement of many cell types and inflammatory mediators, we exploited our knowledge on the mechanism of action of two classes of animal presynaptic neurotoxins: α-Ltx, a pore forming toxin of the venom of black widow spiders, and some snake neurotoxins endowed with phospholipase A2 activity called SPANs. Both kinds of neurotoxins induce an acute and highly reproducible motor axon terminal degeneration, which is followed in few days by complete regeneration: thus, this model represents an appropriate and controlled system to dissect the molecular mechanisms underlying de- and re-generation of peripheral nerve terminals, and to define how PSCs contribute to such processes. We have previously shown that nerve terminals exposed to spider or snake neurotoxins degenerate owing to calcium overload and mitochondrial failure. Here, we found that toxin-treated cultured neurons increase their mitochondrial production of hydrogen peroxide (H2O2), which can easily diffuse across membranes, thus acting as a paracrine signal on neighbouring cellS. Indeed, exposure of cultured SCs to H2O2 leads to ERK phosphorylation and to the activation of downstream pathways. The ERK signalling pathway plays a central role in controlling SCs plasticity during nerve repair in-vivo, but so far the molecular mediators responsible for its activation were unknown: neurons-derived H2O2 represents an ideal candidate for this role. In support of this hypothesis, we observed that ERK phosphorylation is reduced in intoxicated neurons-SCs co-cultures pre-incubated with catalase - which converts H2O2 to oxygen and water -, indicating that H2O2 produced inside neurons diffuses to reach nearby SCs, contributing to ERK activation in their cytosol. ERK phosphorylation takes place also in PSCs at intoxicated NMJs in-vivo. To confirm the involvement of H2O2 in promoting nerve regeneration, we performed electrophysiological recordings and immunohistochemistry on intoxicated muscles, and we found that co-injection of catalase together with neurotoxins delays nerve regeneration, confirming the prominent role of H2O2 in promoting NMJ recovery. Injection of the MAP kinase inhibitor PD98059 also impairs nerve repair in a way similar to that observed with catalase, supporting the finding that H2O2 enhances nerve terminals regeneration through the activation of ERK pathway in PSCsLa giunzione neuromuscolare (GNM) costituisce il sito di trasmissione di un impulso elettrico dal terminale del motoneurone alla fibra muscolare; l’organizzazione strutturale di questo sistema altamente dinamico è stata ulteriormente complicata dall’aggiunta delle cellule di Schwann perisinaptiche (CSPs), dando origine al concetto di sistema tripartito. Le CSPs sono cellule di Schwann non mielinizzanti strettamente adese alla zona di contatto tra nervo e muscolo; esse partecipano attivamente a molte funzioni fisiologiche della GNM, come il suo sviluppo embrionale ma anche il corretto mantenimento di GNMs adulte. Esse sono inoltre in grado di percepire e modulare l’attività sinaptica, mediante l’attivazione di recettori muscarinici e purinergici presenti sulla loro superficie. Studi più recenti hanno dimostrato che le CSPs sono coinvolte nei processi di recupero che hanno luogo in risposta ad un danno nervoso; in seguito a denervazione o a ridotta attività sinaptica, le CSPs de-differenziano, diventando CSPs “reattive”, ed iniziano a proliferare. Queste CSPs reattive partecipano attivamente ai processi di degenerazione e rigenerazione nervosa: esse subiscono variazioni nella loro espressione genica e acquisiscono attività simil-macrofagiche, contribuendo alla rimozione dei detriti neuronali e reclutando fagociti in seguito al rilascio di citochine e chemochine. Inoltre, in seguito alla degenerazione dei terminali nervosi, le CSPs presenti alle placche motrici denervate estendono lunghi processi citosolici in grado di indurre e guidare la ricrescita neuronale. Considerando la crescente incidenza di malattie neurodegenerative che inizialmente interessano in maniera selettiva i terminali dei motoneuroni – quali la SLA e le neuropatie autoimmuni -, sarebbe senz’altro utile caratterizzare in maniera più approfondita il crosstalk tra terminali nervosi in degenerazione e le adiacenti CSPs. In particolare, l’identificazione di mediatori molecolari coinvolti nell’attivazione delle CSPs e nel processo di rigenerazione nervosa potrebbe rivelarsi cruciale per lo sviluppo di nuovi approcci terapeutici. A tale scopo, abbiamo adottato un approccio sperimentale innovativo, alternativo al cut/crush del nervo sciatico tradizionalmente utilizzato fino ad oggi. Al fine di effettuare un danno localizzato ai soli terminali nervosi, evitando il coinvolgimento di molti tipi cellulari e mediatori dell’infiammazione come accade nel corso della degenerazione Walleriana, abbiamo deciso di sfruttare il meccanismo d’azione di due classi di neurotossine presinaptiche animali: α-Latrotoxin, una tossina formante poro presente nel veleno dei ragni del genere Latrodectus, ed alcune neurotossine di serpente dotate di attività fosfolipasica, denominate SPANs. Entrambi i tipi di neurotossine inducono un’acuta e altamente riproducibile degenerazione dei terminali nervosi dei motoneuroni, seguita entro pochi giorni da una rigenerazione completa: l’azione di tali neurotossine rappresenta quindi un sistema appropriato e controllato per esaminare i meccanismi molecolari alla base della degenerazione e rigenerazione nervosa, come anche il contributo delle CSPs a tali processi. Abbiamo precedentemente dimostrato che i terminali nervosi esposti ad α-Ltx e SPANs deegenerano a causa di un eccessivo influsso di calcio nel citosol, che a sua volta induce un danno mitocondriale. In questo lavoro, abbiamo dimostrato che neuroni primari intossicati aumentano la produzione di H2O2 a livello mitocondriale: il perossido di idrogeno è una molecola stabile e diffusibile attraverso membrane lipidiche, e potrebbe perciò agire come segnale paracrino su cellule adiacenti. Infatti, l’esposizione di cellule di Schwann (CSs) primarie in coltura a basse concentrazioni di H2O2 induce la fosforilazione di ERK, con la conseguente attivazione di pathways a valle. È stato recentemente dimostrato che la via di ERK gioca un ruolo fondamentale nel controllo della plasticità delle CSs durante la rigenerazione nervosa in vivo, ma fino ad oggi i mediatori molecolari responsabili per l’attivazione di tale pathway non sono ancora stati identificati: il perossido di idrogeno prodotto dai neuroni in degenerazione costituisce un buon candidato per tale ruolo. In supporto a tale ipotesi, abbiamo osservato che il livello di fosforilazione di ERK è ridotto in co-colture di neuroni e CSs intossicate e pre-incubate con catalasi, che converte rapidamente il perossido di idrogeno in ossigeno ed acqua: ciò conferma che il perossido di idrogeno prodotto dai neuroni diffonde effettivamente nel mezzo extracellulare fino a raggiungere le vicine CSs, nelle quali induce l’attivazione della via di ERK. Tale attivazione è riscontrata anche nelle CSPs alle GNMs intossicate in vivo. Per confermare il coinvolgimento del perossido di idrogeno nell’induzione della rigenerazione nervosa, abbiamo effettuato registrazioni elettrofisiologiche ed esperimenti di immunoistochimica, ed entrambi gli approcci sperimentali hanno dimostrato che in la somministrazione di catalasi in vivo ritarda il processo di rigenerazione nervosa in muscoli intossicati. Inoltre, il pre-trattamento con un inibitore della via di ERK - PD98059 – rallenta la il recupero dall’intossicazione con una cinetica molto simile a quella osservata in presenza di catalasi, supportando l’idea che in effetti il perossido di idrogeno promuova la rigenerazione nervosa attraverso l’attivazione della via di ERK nelle CSP

Calpains participate in nerve terminal degeneration induced by spider and snake presynaptic neurotoxins

alpha-latrotoxin and snake presynaptic phospholipases A2 neurotoxins target the presynaptic membrane of axon terminals of the neuromuscular junction causing paralysis. These neurotoxins display different biochemical activities, but similarly alter the presynaptic membrane permeability causing Ca2+ overload within the nerve terminals, which in turn induces nerve degeneration. Using different methods, here we show that the calcium-activated proteases calpains are involved in the cytoskeletal rearrangements that we have previously documented in neurons exposed to ch-latrotoxin or to snake presynaptic phospholipases A2 neurotoxins. These results indicate that calpains, activated by the massive calcium influx from the extracellular medium, target fundamental components of neuronal cytoskeleton such as spectrin and neurofilaments, whose cleavage is functional to the ensuing nerve terminal fragmentation. (C) 2013 Elsevier Ltd. All rights reserved