The repair of severe nerve injuries requires an autograft or conduit to bridge the gap and avoid axon dispersion. Several conduits are used routinely, but their effectiveness is comparable to that of an autograft only for short gaps.... more
The repair of severe nerve injuries requires an autograft or conduit to bridge the gap and avoid axon dispersion. Several conduits are used routinely, but their effectiveness is comparable to that of an autograft only for short gaps. Understanding nerve regeneration within short conduits could help improve their efficacy for longer gaps. Since Schwann cells are known to migrate on endothelial cells to colonize the “nerve bridge”, the new tissue spontaneously forming to connect the injured nerve stumps, here we aimed to investigate whether this migratory mechanism drives Schwann cells to also proceed within the nerve conduits used to repair large nerve gaps. Injured median nerves of adult female rats were repaired with 10 mm chitosan conduits and the regenerated nerves within conduits were analyzed at different time points using confocal imaging of sequential thick sections. Our data showed that the endothelial cells formed a dense capillary network used by Schwann cells to migrate f...
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Nerves are subjected to tensile forces in various paradigms such as injury and regeneration, joint movement, and rehabilitation treatments, as in the case of neurodynamic treatment (NDT). The NDT induces selective uniaxial repeated... more
Nerves are subjected to tensile forces in various paradigms such as injury and regeneration, joint movement, and rehabilitation treatments, as in the case of neurodynamic treatment (NDT). The NDT induces selective uniaxial repeated tension on the nerve and was described to be an effective treatment to reduce pain in patients. Nevertheless, the biological mechanisms activated by the NDT promoting the healing processes of the nerve are yet still unknown. Moreover, a dose–response analysis to define a standard protocol of treatment is unavailable. In this study, we aimed to define in vitro whether NDT protocols could induce selective biological effects on sensory and motor neurons, also investigating the possible involved molecular mechanisms taking a role behind this change. The obtained results demonstrate that NDT induced significant dose-dependent changes promoting cell differentiation, neurite outgrowth, and neuron survival, especially in nociceptive neurons. Notably, NDT signific...
After big loos of substances of peripheral nerves, in order to connect proximal with distal stump, it is possible to use, in alternative to autologous grafting, different kind of conduits. The chitosan conduit and the muscle in vein... more
After big loos of substances of peripheral nerves, in order to connect proximal with distal stump, it is possible to use, in alternative to autologous grafting, different kind of conduits. The chitosan conduit and the muscle in vein technique showed very good results in pre clinical and clinical settings. We compared in this study the efficacy of empty chitosan conduit versus chitosan conduit enriched with fresh muscle fibbers (MIT) to improve peripheral nerve regeneration. The median nerve of rat was repaired by means of empty chitosan conduit or MIT (nerve gam 6mm, conduit length 10 mm). As control group we used auto grafting technique. We performed analysis at short term (7,14,28 days) and at long term (12 weeks) in order to register bimolecular modification ( quantitative real time PCR and western blot), morphological modification (optic and electronic microscope) and functional changing (grasping test) . Bimolecular analysis showed that muscle fibbers produced and released Neur...
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The expression of the tyrosine kinase receptor ErbB4 confers to the neural progenitor cell line ST14A increased migratory activity. By gene expression profiling analysis, we examined the transcriptional changes associated with higher... more
The expression of the tyrosine kinase receptor ErbB4 confers to the neural progenitor cell line ST14A increased migratory activity. By gene expression profiling analysis, we examined the transcriptional changes associated with higher migratory activity mediated by specific ErbB4 isoforms, and found up-regulation of Eps8, a multimodular regulator of actin dynamics. Through in vitro experimental manipulation of the Eps8 expression level, we showed that Eps8 synergizes with ErbB4 to confer increased migratory capability to ST14A cells, whereas siRNA mediated Eps8 silencing impairs both basal and neuregulin1 (NRG1) stimulated migration. In vivo, ErbB4 is involved in regulating the migratory behaviour of a subpopulation of medial ganglionic eminence derived interneurons migrating towards the cortex, and of olfactory bulb neural precursors migrating from the subventricular zone (SVZ) towards the olfactory bulb (OB). We focused our attention on the SVZ-OB system, showing that Eps8 and diff...
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Skeletal muscle atrophy represents one of the main causes of poor outcome of microsurgical nerve reconstruction. Recent studies have pointed to the importance of the neuregulin/ErbB signaling pathway in the development and regeneration of... more
Skeletal muscle atrophy represents one of the main causes of poor outcome of microsurgical nerve reconstruction. Recent studies have pointed to the importance of the neuregulin/ErbB signaling pathway in the development and regeneration of the neuromuscular system. Here, we show by immunohistochemistry, RT-PCR, and Western blotting analyses, in an in vivo model of adult skeletal muscle denervation/reinnervation, that expression of Neuregulin1 (NRG1) and ErbB receptors is regulated by the innervation condition. We found out that a significant upregulation of the alpha-, but not beta-, isoform of NRG1, as well as of ErbB2, ErbB3, and ErbB4-cyt1 isoform occurs as a consequence of denervation of flexor digitorum muscles of the rat forelimb by median nerve transection. Moreover, after tubulization median nerve repair, and consequent muscle reinnervation, all messengers of the NRG1/ErbB system are promptly downregulated. Therefore, our results suggest the existence of a alpha-NRG1-mediated autocrine and/or paracrine trophic loop in skeletal muscles that is activated after denervation and promptly deactivated after nerve reconstruction. This myotrophic loop is a promising therapeutic target for the prevention of muscle atrophy. Yet, the recent demonstration of a similar alpha-NRG1-mediated gliotrophic loop in denervated Schwann cells provides a possible explanation for the effectiveness of muscle conduits for tubulization nerve repair.
Research Interests: Microsurgery, Immunohistochemistry, Confocal Microscopy, Medicine, Signal Transduction, and 15 moreFemale, Animals, Glycoproteins, Nerve Regeneration, Clinical Sciences, mRna expression levels, Skeletal Muscle, Rats, Sensitivity and Specificity, Reference Values, Genetic Markers, Median Nerve, Random Allocation, Recovery of Function, and reverse transcriptase polymerase chain reaction
Research Interests: Biological Chemistry, Biological Sciences, Cell Differentiation, Animals, Biological, and 15 moreChemotaxis, Phosphorylation, Plasmids, Neurons, Immunoprecipitation, CHEMICAL SCIENCES, Rats, Protein isoforms, Cell Proliferation, Protein Binding, Dimerization, Molecular Sequence Data, Cell Membrane, reverse transcriptase polymerase chain reaction, and Medical and Health Sciences
Stable expression of the tyrosine kinase receptor ErbB4 confers increased migratory behavior to the neuronal progenitor cell line ST14A, in response to neuregulin 1 (NRG1) stimulation. We used gene expression profiling analysis to... more
Stable expression of the tyrosine kinase receptor ErbB4 confers increased migratory behavior to the neuronal progenitor cell line ST14A, in response to neuregulin 1 (NRG1) stimulation. We used gene expression profiling analysis to identify transcriptional changes associated with higher migratory activity caused by the activation of a specific ErbB4 isoform, and found constitutive up-regulation of the epidermal growth factor receptor pathway substrate 8 (Eps8), a multimodular regulator of actin dynamics. We confirmed the increase of Eps8, both at the mRNA and at the protein level, in stable clones expressing two different ErbB4 isoforms, both characterized by high migratory activity. Using Transwell assays and experimental manipulation of Eps8 expression level, we demonstrated that Eps8 synergizes with ErbB4 to increase both basal and ligand induced cell migration, whereas siRNA mediated Eps8 silencing strongly impairs cell motility and NRG1 induced actin cytoskeleton remodeling. By transient knockdown of Eps8 through in vivo siRNA electroporation, followed by explant primary cultures, we demonstrated that Eps8 down-regulation affects migration of normal neuronal precursors. In conclusion, our data demonstrate that Eps8 is a key regulator of motility of neuronal progenitor cells expressing ErbB4, both in basal conditions and in response to external motogenic cues.
Research Interests: Migration, Cell Migration, Gene expression, Mice, Animals, and 15 moreProteins, Actin Cytoskeleton, Actin Dynamics, Clinical Sciences, Epidermal Growth Factor Receptor, Rats, Microarray Analysis, RMS, Neural Stem Cells, Protein isoforms, Primary Culture, qRT PCR, Biochemistry and cell biology, Cell Motility, and reverse transcriptase polymerase chain reaction
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Research Interests: Cognitive Science, Biology, Medicine, Neural Development, Brain, and 15 moreMice, Animals, Astrocyte, Alternative splicing, Neuronal Development, Neurons, Radial Glia, Age Factors, Olfactory Bulb, Alternative Splicing, Protein isoforms, Neurosciences, Granule cell layer, Differential expression, and reverse transcriptase polymerase chain reaction
Background A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium... more
Background A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium fluctuations play central roles in neuronal motility. Stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation. In this work we analyzed the potential interactions between the NRG1/ErbB4 system and NMDARs in the ST14A migratory process as well as its calcium dependence. Results RT-PCR studies have shown that both native ST14A cells (non-expressing ErbB4), as well as ErbB4-transfected cells express low levels of a restricted number of NMDAR subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form Ca2+ channels characterized by low Mg2+-sensitivity and low Ca2+-permeability, generating small, long-lasting currents. Ca2+-imaging experiments showed slow [Ca2+]i incr...
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REGULATION OF THE NRG1/ERBB SYSTEM IN CMT1A PERIPHERAL NERVES / Gambarotta Giovanna; Fornasari Benedetta Elena; Ronchi Giulia; Pascal Davide; Raimondo Stefania; El Soury Marwa; Budau Claudia Alexandra; Visigalli Davide; Nobbio Lucilla;... more
REGULATION OF THE NRG1/ERBB SYSTEM IN CMT1A PERIPHERAL NERVES / Gambarotta Giovanna; Fornasari Benedetta Elena; Ronchi Giulia; Pascal Davide; Raimondo Stefania; El Soury Marwa; Budau Claudia Alexandra; Visigalli Davide; Nobbio Lucilla; Schenone Angelo; Geuna Stefano. In: JOURNAL OF THE PERIPHERAL NERVOUS SYSTEM. ISSN 1529-8027. 21:3(2016), pp. 258-258. ((Intervento presentato al convegno 6th International Charcot-Marie-Tooth and Related Neuropathy Consortium (CMTR) Meeting tenutosi a Venezia nel 8-10 September 2016. Original Citation: REGULATION OF THE NRG1/ERBB SYSTEM IN CMT1A PERIPHERAL NERVES
Neurogranin (Ng) is a brain-specific postsynaptic protein, whose role in modulating Ca2+/calmodulin signaling in glutamatergic neurons has been linked to enhancement in synaptic plasticity and cognitive functions. Accordingly, Ng... more
Neurogranin (Ng) is a brain-specific postsynaptic protein, whose role in modulating Ca2+/calmodulin signaling in glutamatergic neurons has been linked to enhancement in synaptic plasticity and cognitive functions. Accordingly, Ng knock-out (Ng-ko) mice display hippocampal-dependent learning and memory impairments associated with a deficit in long-term potentiation induction. In the adult olfactory bulb (OB), Ng is expressed by a large population of GABAergic granule cells (GCs) that are continuously generated during adult life, undergo high synaptic remodeling in response to the sensory context, and play a key role in odor processing. However, the possible implication of Ng in OB plasticity and function is yet to be investigated. Here, we show that Ng expression in the OB is associated with the mature state of adult-born GCs, where its active-phosphorylated form is concentrated at post-synaptic sites. Constitutive loss of Ng in Ng-ko mice resulted in defective spine density in adult...
The Neuregulin1/ErbB system is selectively regulated during peripheral nerve degeneration and regeneration / Ronchi, Giulia; Haastert-Talini, Kirsten; Fornasari, Benedetta Elena; Perroteau, Isabelle; Geuna, Stefano; Gambarotta, Giovanna.... more
The Neuregulin1/ErbB system is selectively regulated during peripheral nerve degeneration and regeneration / Ronchi, Giulia; Haastert-Talini, Kirsten; Fornasari, Benedetta Elena; Perroteau, Isabelle; Geuna, Stefano; Gambarotta, Giovanna. In: EUROPEAN JOURNAL OF NEUROSCIENCE. ISSN 0953-816X. 43:-(2016), pp. 351-364. Original Citation: The Neuregulin1/ErbB system is selectively regulated during peripheral nerve degeneration and regeneration
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Peripheral nerve injuries are a common condition in which a nerve is damaged, affecting more than one million people every year. There are still no efficient therapeutic treatments for these injuries. Artificial scaffolds can offer new... more
Peripheral nerve injuries are a common condition in which a nerve is damaged, affecting more than one million people every year. There are still no efficient therapeutic treatments for these injuries. Artificial scaffolds can offer new opportunities for nerve regeneration applications; in this framework, chitosan is emerging as a promising biomaterial. Here, we set up a simple and effective method for the production of micro-structured chitosan films by solvent casting, with high fidelity in the micro-pattern reproducibility. Three types of chitosan directional micro-grooved patterns, presenting different levels of symmetricity, were developed for application in nerve regenerative medicine: gratings (GR), isosceles triangles (ISO) and scalene triangles (SCA). The directional patterns were tested with a Schwann cell line. The most asymmetric topography (SCA), although it polarized the cell shaping less efficiently, promoted higher cell proliferation and a faster cell migration, both ...
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Peripheral nerve injury treatment is a relevant problem because of nerve lesion high incidence and because of unsatisfactory regeneration after severe injuries, thus resulting in a reduced patient’s life quality. To repair severe nerve... more
Peripheral nerve injury treatment is a relevant problem because of nerve lesion high incidence and because of unsatisfactory regeneration after severe injuries, thus resulting in a reduced patient’s life quality. To repair severe nerve injuries characterized by substance loss and to improve the regeneration outcome at both motor and sensory level, different strategies have been investigated. Although autograft remains the gold standard technique, a growing number of research articles concerning nerve conduit use has been reported in the last years. Nerve conduits aim to overcome autograft disadvantages, but they must satisfy some requirements to be suitable for nerve repair. A universal ideal conduit does not exist, since conduit properties have to be evaluated case by case; nevertheless, because of their high biocompatibility and biodegradability, natural-based biomaterials have great potentiality to be used to produce nerve guides. Although they share many characteristics with synthetic biomaterials, natural-based biomaterials should also be preferable because of their extraction sources; indeed, these biomaterials are obtained from different renewable sources or food waste, thus reducing environmental impact and enhancing sustainability in comparison to synthetic ones. This review reports the strengths and weaknesses of natural-based biomaterials used for manufacturing peripheral nerve conduits, analyzing the interactions between natural-based biomaterials and biological environment. Particular attention was paid to the description of the preclinical outcome of nerve regeneration in injury repaired with the different natural-based conduits.
Glyphosate-based herbicides (GBH) are the most widely used pesticides worldwide. Despite considerable progress in describing the neurotoxic potential of GBH, the harmful effects on brain cytoarchitecture and behavior are still unclear.... more
Glyphosate-based herbicides (GBH) are the most widely used pesticides worldwide. Despite considerable progress in describing the neurotoxic potential of GBH, the harmful effects on brain cytoarchitecture and behavior are still unclear. Here, we addressed the developmental impact of GBH by exposing female mice to 250 or 500 mg/kg doses of GBH during both pregnancy and lactation and then examined the downstream effects at the behavioral, neurochemical and molecular levels. We show that pre- and neonatal exposure to GBH impairs fertility and reproduction parameters as well as maternal behavior of exposed mothers. In offspring, GBH was responsible for a global delay in innate reflexes and a deficit in motor development. At the adult age, exposed animals showed a decrease of locomotor activity, sociability, learning and short- and long-term memory associated with alterations of cholinergic and dopaminergic systems. Furthermore, GBH-activated microglia and astrocytes, sign of neuroinflammation event in the medial prefrontal cortex and hippocampus. At the molecular level, a down-regulation of brain-derived neurotrophic factor (BDNF) expression and an up-regulation of tyrosine-related kinase receptor (TrkB), NR1 subunit of NMDA receptor as well as tumor necrosis factor α (TNFα) were found in the brain of GBH-exposed mice. The present work demonstrates that GBH induces numerous behavioral and cognitive abnormalities closely associated with significant histological, neurochemical and molecular impairments. It also raises fundamental concerns about the ability of current safety testing to assess risks of pesticide exposure during developmental periods of central nervous system.
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Conduits for the repair of peripheral nerve gaps are a good alternative to autografts as they provide a protected environment and a physical guide for axonal re-growth. Conduits require colonization by cells involved in nerve regeneration... more
Conduits for the repair of peripheral nerve gaps are a good alternative to autografts as they provide a protected environment and a physical guide for axonal re-growth. Conduits require colonization by cells involved in nerve regeneration (Schwann cells, fibroblasts, endothelial cells, macrophages) while in the autograft many cells are resident and just need to be activated. Since it is known that soluble Neuregulin1 (sNRG1) is released after injury and plays an important role activating Schwann cell dedifferentiation, its expression level was investigated in early regeneration steps (7, 14, 28 days) inside a 10 mm chitosan conduit used to repair median nerve gaps in Wistar rats. In vivo data show that sNRG1, mainly the isoform α, is highly expressed in the conduit, together with a fibroblast marker, while Schwann cell markers, including NRG1 receptors, were not. Primary culture analysis shows that nerve fibroblasts, unlike Schwann cells, express high NRG1α levels, while both expres...
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Muscle-in-vein conduit is successfully employed for repairing nerve injuries: the vein prevents muscle fiber dispersion, while the muscle prevents the vein collapse and creates a favorable environment for Schwann cell migration and axon... more
Muscle-in-vein conduit is successfully employed for repairing nerve injuries: the vein prevents muscle fiber dispersion, while the muscle prevents the vein collapse and creates a favorable environment for Schwann cell migration and axon regrowth. However, it requires microsurgical skills. In this study we show a simple strategy to improve the performance of a chitosan hollow tube by the introduction of fresh skeletal muscle fibers. The hypothesis is to overcome the technical issue of the muscle-in-vein preparation and to take advantage of fiber muscle properties to create an easy and effective conduit for nerve regeneration. Rat median nerve gaps were repaired with chitosan tubes filled with skeletal muscle fibers (muscle-in-tube graft), hollow chitosan tubes, or autologous nerve grafts. Our results demonstrate that the fresh skeletal muscle inside the conduit is an endogenous source of soluble Neuregulin 1, a key factor for Schwann cell survival and dedifferentiation, absent in the...
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Neuregulin 1 (NRG1) is a growth factor produced by both peripheral nerves and skeletal muscle. In muscle, it regulates neuromuscular junction gene expression, acetylcholine receptor number, muscle homeostasis and satellite cell survival.... more
Neuregulin 1 (NRG1) is a growth factor produced by both peripheral nerves and skeletal muscle. In muscle, it regulates neuromuscular junction gene expression, acetylcholine receptor number, muscle homeostasis and satellite cell survival. NRG1 signalling is mediated by the tyrosine kinase receptors ErbB3 and ErbB4 and their co-receptors ErbB1 and ErbB2. The NRG1/ErbB system is well studied in nerve tissue after injury, but little is known about this system in skeletal muscle after denervation/reinnervation processes. Here, we performed a detailed time-course expression analysis of several NRG1 isoforms and ErbB receptors in the rat superficial digitorum flexor muscle after three types of median nerve injuries of different severities. We found that ErbB receptor expression was correlated with the innervated state of the muscle, with upregulation of ErbB2 clearly associated with the denervation state. Interestingly, the NRG1 isoforms were differently regulated depending on the nerve in...
Nerve guidance channels facilitate nerve regeneration and represent an attractive alternative to nerve graft. Actually, nano- and microstructured biomaterials for nerve reconstruction have gained much attention, thanks to recent... more
Nerve guidance channels facilitate nerve regeneration and represent an attractive alternative to nerve graft. Actually, nano- and microstructured biomaterials for nerve reconstruction have gained much attention, thanks to recent discoveries about topography effects on cell behavior and morphology. Electrospun fibres have been proposed as filler or structural component for nerve guidance channels, principally due to their similarity with extracellular matrices which facilitate nerve regeneration. Among several tested biomaterials, gelatin has been used to prepare fibres able to support Schwann cell migration and neurite outgrowth. In this work, the effects of gelatin fibre size on axon elongation and Schwann cell migration have been tested using dorsal root ganglia cultures. Moreover, we analyzed how fibres might affect the expression of specific neuronal subtype markers in sensory neuron cultures and how the combined effect of substrate and biological cues affects neurite growth and...
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OBJECTIVE Multiple factors may affect functional recovery after peripheral nerve injury, among them the lesion site and the interval between the injury and the surgical repair. When the nerve segment distal to the lesion site undergoes... more
OBJECTIVE Multiple factors may affect functional recovery after peripheral nerve injury, among them the lesion site and the interval between the injury and the surgical repair. When the nerve segment distal to the lesion site undergoes chronic degeneration, the ensuing regeneration (when allowed) is often poor. The aims of the current study were as follows: 1) to examine the expression changes of the neuregulin 1/ErbB system during long-term nerve degeneration; and 2) to investigate whether a chronically denervated distal nerve stump can sustain nerve regeneration of freshly axotomized axons. METHODS This study used a rat surgical model of delayed nerve repair consisting of a cross suture between the chronically degenerated median nerve distal stump and the freshly axotomized ulnar proximal stump. Before the suture, a segment of long-term degenerated median nerve stump was harvested for analysis. Functional, morphological, morphometric, and biomolecular analyses were performed. RESU...
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Neuregulin1 (Nrg1) and its receptors ErbB are crucial for heart development and for adult heart structural maintenance and function and Nrg1 has been proposed for heart failure treatment. Infarct size is the major determinant of heart... more
Neuregulin1 (Nrg1) and its receptors ErbB are crucial for heart development and for adult heart structural maintenance and function and Nrg1 has been proposed for heart failure treatment. Infarct size is the major determinant of heart failure and the mechanism of action and the role of each ErbB receptor remain obscure, especially in the post-ischemic myocardium. We hypothesized that Nrg1 and ErbB are affected at transcriptional level early after ischemia/reperfusion (I/R) injury, and that the protective postconditioning procedure (PostC, brief cycles of ischemia/reperfusion carried out after a sustained ischemia) can influence this pathway. The Langendorff's heart was used as an ex-vivo model to mimic an I/R injury in the whole rat heart; after 30min of ischemia and 2h of reperfusion, with or without PostC, Nrg1 and ErbB expression were analysed by quantitative real-time PCR and Western blot. While no changes occur for ErbB2, ErbB4 and Nrg1, an increase of ErbB3 expression occu...
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Maggiora, P., Gambarotta, G., Olivero, M., Giordano, S., Di Renzo, MF and Comoglio, PM (1997), Control of invasive growth by the HGF receptor family. Journal of Cellular Physiology, 173: 183186. doi: 10.1002/(SICI) 1097-4652 (199711)... more
Maggiora, P., Gambarotta, G., Olivero, M., Giordano, S., Di Renzo, MF and Comoglio, PM (1997), Control of invasive growth by the HGF receptor family. Journal of Cellular Physiology, 173: 183186. doi: 10.1002/(SICI) 1097-4652 (199711) 173: 2< 183:: AID-...
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The peripheral nervous system has an intrinsic capability to regenerate, crucially related to the ability of Schwann cells (SC) to create a permissive environment, for example, through production of regeneration-promoting neurotrophic... more
The peripheral nervous system has an intrinsic capability to regenerate, crucially related to the ability of Schwann cells (SC) to create a permissive environment, for example, through production of regeneration-promoting neurotrophic factors. Survival, proliferation, migration and differentiation of SC into a myelinating phenotype during development and after injury is regulated by different Neuregulin1 (NRG1) isoforms. This study investigates the expression of different NRG1 isoforms and of their ErbB receptors in distal rat median nerve samples under regenerating conditions after a mild (crush) and more severe (end-to-end repair) injury and under degenerating condition. The expression of the NRG1/ErbB system was evaluated at mRNA and protein level, and demonstrated to be specific for distinct and consecutive phases following nerve injury and regeneration or the progress in degeneration. For the first time a detailed analysis of expression profiles not only of soluble and transmembrane NRG1 isoforms, but also of alpha and beta as well as type a, b and c isoforms is presented. The results of mRNA and protein expression pattern analyses were related to nerve ultrastructure changes evaluated by electron microscopy. In particular, transmembrane NRG1 isoforms are differentially regulated and proteolytically processed under regeneration and degeneration conditions. Soluble NRG1 isoforms alpha and beta, as well as type a and b, are strongly upregulated during axonal regrowth, while type c NRG1 isoform is downregulated. This is accompanied by an upregulation of ErbB receptors. This accurate regulation suggests that each molecule plays a specific role that could be clinically exploited to improve nerve regeneration.
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MET, a potentially harmful oncogene controlling invasive growth, is overexpressed in a significant percentage of human cancers. Since amplification of the MET gene occurs only in a fraction of these cases, we investigated the... more
MET, a potentially harmful oncogene controlling invasive growth, is overexpressed in a significant percentage of human cancers. Since amplification of the MET gene occurs only in a fraction of these cases, we investigated the transcriptional mechanisms responsible for up-regulation of the promoter activity. The transcription driven by the 3.1 kbp DNA fragment containing the minimal promoter was studied by 5' progressive deletion analysis. The patterns of MET promoter activity suggest the presence of weak negative and positive elements in the region between 300 and 840 bp upstream to the transcription start site. The region encompassing the first 300 bp strongly up-regulates the promoter. This region contains four putative binding sites for members of the Ets transcription factor family, known to be involved in invasive growth. Transient co-expression of Ets1 resulted in a strong enhancement of the MET promoter activity. Increased expression of the Met protein was observed in cel...
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In epithelial cells, hepatocyte growth factor (HGF) activates a genetic program involving cell-cell dissociation ("scattering"), growth and invasiveness. The full program is not elicited by other growth factors like epidermal... more
In epithelial cells, hepatocyte growth factor (HGF) activates a genetic program involving cell-cell dissociation ("scattering"), growth and invasiveness. The full program is not elicited by other growth factors like epidermal growth factor, and is aberrantly activated during cancer progression to the invasive-metastatic phenotype. To identify genes involved in the onset of invasive growth, we explored by cDNA microarrays the in vitro transcriptional response to HGF of mouse embryo liver cells. We identified osteopontin (OPN), a secreted matrix protein, as a major HGF transcriptional target. The wave of OPN induction is maximal at 6 h, in concomitance with the initiation of scattering, and is specific, because no other matrix protein among those explored by the microarray is affected. Interestingly, HGF, but not epidermal growth factor, promotes cell adhesion to OPN via the CD44 receptor. Scattering is significantly impaired by antibodies against OPN and CD44; conversely, c...
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The MET oncogene, encoding the tyrosine kinase receptor for the hepatocyte growth factor/scatter factor, is expressed in epithelial cells and overexpressed in a significant proportion of human epithelial cancers, suggesting the occurrence... more
The MET oncogene, encoding the tyrosine kinase receptor for the hepatocyte growth factor/scatter factor, is expressed in epithelial cells and overexpressed in a significant proportion of human epithelial cancers, suggesting the occurrence of transcriptional alteration(s). To identify the MET promoter, we isolated recombinant cDNA clones encompassing the entire 5'-noncoding sequence of MET messenger RNAs. Using probes derived from this region, we cloned the entire genomic region spanning the first MET exon and the flanking regulatory sequences. The first exon, containing the entire untranslated sequence, is present in the MET mRNAs of 7.1, 5.9, and 4.6 kilobases, showing that the expression of the multiple transcripts is regulated by a single promoter. The start site of transcription was determined by primer extension and by rapid amplification of cDNA ends. We show that a 300-base pair fragment, containing sequences upstream from the start site, efficiently drives the expression...
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Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout... more
Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well-known anti-tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated β-galactosidase activity and enlarged γH2AX foci co-l...
Research Interests: Confocal Microscopy, DNA damage, Gene expression, DNA, Cell Differentiation, and 11 moreHumans, Mesenchymal Stem Cell, Clinical Sciences, Histones, Cellular and Molecular Medicine, Cell Survival, Mesenchymal Stromal Cells, Biochemistry and cell biology, Interleukin, Immunoblotting, and reverse transcriptase polymerase chain reaction
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Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization... more
Over the last five years, we have used the rat forelimb model for investigating neuromuscular recovery after microsurgical nerve reconstruction of median and ulnar nerves by end-to-side neurorrhaphy and muscle-vein-combined tubulization (using both straight and Y-shaped guides). The outcome of nerve repair at different postoperative times was assessed by functional, morphological and biomolecular analysis. Results showed that both end-to-side and tubulization repair of rat median and ulnar nerves led to successful axonal regeneration along the severed nerve trunk as well as to a partial recovery of the lost function as assessed by grasping test. Biomolecular analysis by means of reverse transcription polymerase chain reaction (RT-PCR) demonstrated early overexpression during nerve regeneration of the gliotrophic factor NRG1 and two of its receptors: erbB2 and erbB3. Finally, our experience also suggests that the rat forelimb experimental model is particularly appropriate for the study of microsurgical reconstruction of major mixed nerve trunks. Furthermore, since the forelimb model is less compromising for the animal, it should be preferred to the hindlimb model for many research purposes.
Research Interests: Microsurgery, Female, Animals, Glycoproteins, Nerve Regeneration, and 15 moreClinical Sciences, Research Purpose, Rats, Functional Recovery, Guided Tissue Regeneration, Peripheral Nerve, Neuromuscular Junction, Axons, Schwann Cell, Neurosciences, Median Nerve, Experimental Model, Recovery of Function, Reverse transcription polymerase chain reaction, and axon regeneration
The neuregulins (NRGs) are a family of signaling proteins that are ligands for receptor tyrosine kinase of the ErbB family (namely ErbB3 and ErbB4). To date, four different neuregulin genes have been identified (neuregulin1-4). While NRG1... more
The neuregulins (NRGs) are a family of signaling proteins that are ligands for receptor tyrosine kinase of the ErbB family (namely ErbB3 and ErbB4). To date, four different neuregulin genes have been identified (neuregulin1-4). While NRG1 isoforms have been extensively studied, little is yet known about the other genes of the family. We report the expression of recombinant NRG1beta1, NRG2alpha, NRG2beta, and NRG3 as recombinant fusion proteins in Escherichia coli. The cDNA encoding for the EGF-like domain of each protein was cloned from the mouse olfactory bulb and inserted into the pET-19b vector allowing for bacterial expression of the protein fused to an N-terminal His tag. The recombinant NRGs expressed in the inclusion bodies were solubilized under denaturing conditions, purified by affinity chromatography, and refolded via dialysis in the presence of reducing agents. Purified recombinant NRGs were active as they bound to their receptors and induced their phosphorylation. In particular, and in agreement with data on the native proteins, all the molecules were able to bind and activate ErbB4 while only the rNRG1 and the two rNRG2 (but not rNRG3) bound ErbB3.