in-vivo-electrophysiological-measurements-on-mouse-sciatic-nerves

<em>In Vivo</em> Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Fritz Lipmann Institute

1. Effective intervention with survivors of suicide requires knowledge of the diverse sequelae of response including blame, anger, guilt, shame, search for why, and feelings of stigmatization. 2. Fearing blame and stigmatization, survivors of suicide may be reluctant to contact helping professionals. 3. Duration and intensity of bereavement is mediated by the survivors' nature of the relationship with the deceased and their perception of the preventability of the death. 4. Group counseling is an effective intervention because it addresses the issue of disenfranchised grief.

Survivors of suicide. Emerging counseling strategies.

Mutations in the neurofibromatosis 2 (NF2) gene with the resultant loss of expression of the NF2 tumor suppressor schwannomin are one of the most common causes of benign human brain tumors, including schwannomas and meningiomas. Previously we demonstrated that the hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) strongly interacts with schwannomin. HRS is a powerful regulator of receptor tyrosine kinase trafficking to the degradation pathway and HRS also binds STAM. Both of these actions for HRS potentially inhibit STAT activation. Therefore, we hypothesized that schwannomin inhibits STAT activation through interaction with HRS. We now show that both schwannomin and HRS inhibit Stat3 activation and that schwannomin suppresses Stat3 activation mediated by IGF-I treatment in the human schwannoma cell line STS26T. We also find that schwannomin inhibits Stat3 and Stat5 phosphorylation in the rat schwannoma cell line RT4. Schwannomin with the pathogenic missense mutation Q538P fails to bind HRS and does not inhibit Stat5 phosphorylation. These data are consistent with the hypothesis that schwannomin requires HRS interaction to be fully functionally active and to inhibit STAT activation.

Neurofibromatosis 2 (NF2) tumor suppressor schwannomin and its interacting protein HRS regulate STAT signaling.

Cell-to-cell spread is a fundamental step in the infection cycle of Listeria monocytogenes that strictly depends on the formation of bacteria-induced protrusions. Since Listeria actin tails in the protrusions are tightly associated with the plasma membrane, we hypothesised that membrane-cytoskeleton linkers would be required for initiating and sustaining their formation and the subsequent cell-to-cell spread. We have found that ezrin, a member of the ezrin, radixin and moesin (ERM) family that functions as a key membrane-cytoskeleton linker, accumulates at Listeria protrusions. The ability of Listeria to induce protrusions and effectively spread between adjacent cells depends on the interaction of ERM proteins with both a membrane component such as CD44 and actin filaments. Interfering with either of these interactions or with ERM proteins phosphorylation not only reduces the number of protrusions but also alters their morphology, resulting in the formation of short and collapsed protrusions. As a consequence, Listeria cell-to-cell spread is severely impaired. Thus, ERM proteins are exploited by Listeria to escape the host immune response and to succeed in the development of the infection.

Listeria monocytogenes exploits ERM protein functions to efficiently spread from cell to cell.

The tumour suppressor protein merlin (encoded by the neurofibromatosis type 2 gene NF2) is an important regulator of proliferation in many cell and tissue types. Merlin is activated by dephosphorylation at serine 518 (S518), which occurs on serum withdrawal or on cell-cell or cell-matrix contact. However, the relevant phosphatase that activates merlin's tumour suppressor function is unknown. Here we identify this enzyme as the myosin phosphatase (MYPT-1-PP1delta). The cellular MYPT-1-PP1delta-specific inhibitor CPI-17 causes a loss of merlin function characterized by merlin phosphorylation, Ras activation and transformation. Constitutively active merlin (S518A) reverses CPI-17-induced transformation, showing that merlin is the decisive substrate of MYPT-1-PP1delta in tumour suppression. In addition we show that CPI-17 levels are raised in several human tumour cell lines and that the downregulation of CPI-17 induces merlin dephosphorylation, inhibits Ras activation and abolishes the transformed phenotype. MYPT-1-PP1delta and its substrate merlin are part of a previously undescribed tumour suppressor cascade that can be hindered in two ways, by mutation of the NF2 gene and by upregulation of the oncoprotein CPI-17.

Tumorigenic transformation by CPI-17 through inhibition of a merlin phosphatase.

In several types of cells, the activation of the receptor tyrosine kinase c-Met by its ligand hepatocyte growth factor (HGF) requires the coreceptor CD44v6. The CD44 extracellular domain is necessary for c-Met autophosphorylation, whereas the intracellular domain is required for signal transduction. We have already shown that the CD44 cytoplasmic tail recruits ezrin, radixin and moesin (ERM) proteins to the complex of CD44v6, c-Met, and HGF. We have now defined the function of the ERM proteins and the step they promote in the signaling cascade. The association of ERM proteins to the coreceptor is absolutely required to mediate the HGF-dependent activation of Ras by the guanine nucleotide exchange factor Sos. The ERM proteins need, in addition, to be linked to the actin cytoskeleton to catalyze the activation of Ras. Thus, we describe here a new function of the cytoskeleton. It is part of a "signalosome" complex that organizes the activation of Ras by Sos. So far the cytoskeleton has mainly been identified as a "responder" to signal transduction. Here, we show now that F-actin acts as an "inducer" that actively organizes the signaling cascade.

Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin.

The small G-protein Ras is a tightly controlled regulator of cell fate. Prolonged or persistent arrest in the activated GTP-loaded state by mutation of Ras as in lung cancer or in a Ras-GTPase-activating protein as in neurofibromatosis type 1 promotes tumorigenesis. We now show that the tumor-suppressor protein merlin (mutated in neurofibromatosis type 2) also controls Ras activity. Systematic analysis of growth factor signaling located the step of merlin interference to the activation of Ras and Rac. Merlin independently uncouples both Ras and Rac from growth factor signals. In the case of Ras, merlin acts downstream of the receptor tyrosine kinase-growth factor receptor binding protein 2 (Grb2)-SOS complex. However, merlin does not bind either SOS or Ras, but it counteracts the ERM (ezrin, radixin, moesin)-dependent activation of Ras, which correlates with the formation of a complex comprising ERM proteins, Grb2, SOS, Ras, and filamentous actin. Because efficient signaling from Ras requires Rac-p21-activated kinase-dependent phosphorylations of Raf and mitogen-activated protein/extracellular signal-regulated kinase kinase, merlin can also inhibit signal transfer from dominantly active Ras mutants. We propose that the interference of merlin with Ras- and Rac-dependent signal transfer represents part of the tumor-suppressive action of merlin.

Merlin/neurofibromatosis type 2 suppresses growth by inhibiting the activation of Ras and Rac.

Ezrin, radixin and moesin are a family of proteins that provide a link between the plasma membrane and the cortical actin cytoskeleton. The regulated targeting of ezrin to the plasma membrane and its association with cortical F-actin are more than likely functions necessary for a number of cellular processes, such as cell adhesion, motility, morphogenesis and cell signalling. The interaction with F-actin was originally mapped to the last 34 residues of ezrin, which correspond to the last three helices (alphaB, alphaC and alphaD) of the C-terminal tail. We set out to identify and mutate the ezrin/F-actin binding site in order to pinpoint the role of F-actin interaction in morphological processes as well as signal transduction. We report here the generation of an ezrin mutant defective in F-actin binding. We identified four actin-binding residues, T576, K577, R579 and I580, that form a contiguous patch on the surface of the last helix, alphaD. Interestingly, mutagenesis of R579 also eliminated the interaction of band four-point one, ezrin, radixin, moesin homology domains (FERM) and the C-terminal tail domain, identifying a hotspot of the FERM/tail interaction. In vivo expression of the ezrin mutant defective in F-actin binding and FERM/tail interaction (R579A) altered the normal cell surface structure dramatically and inhibited cell migration. Further, we showed that ezrin/F-actin binding is required for the receptor tyrosine kinase signal transfer to the Ras/MAP kinase signalling pathway. Taken together, these observations highlight the importance of ezrin/F-actin function in the development of dynamic membrane/actin structures critical for cell shape and motility, as well as signal transduction.

Properties of an ezrin mutant defective in F-actin binding.

The NF Conference is the largest annual gathering of researchers and clinicians focused on neurofibromatosis and has been convened by the Children's Tumor Foundation for over 20 years. The 2009 NF Conference was held in Portland, Oregon from June 13 to June 16, 2009 and co-chaired by Kathryn North from the University of Sydney and The Children's Hospital at Westmead, Sydney, Australia; and Joseph Kissil from the Wistar Institute, Philadelphia. The Conference included 80 platform presentations in 9 sessions over 4 days; over 100 abstracts presented as posters; and three Keynote presentations. To date, there have been tremendous advances in basic research in the pathogenesis of neurofibromatosis, and more recently in progress toward identifying effective drug therapies and the commencement of neurofibromatosis clinical trials. The NF Conference attendees have significantly increased (doubling from 140 in 2005 to 280 attending in 2009) with a significant increase in attendance of physicians and clinical researchers. Correspondingly the NF Conference scope has expanded to include translational research, clinical trials and clinical management issues while retaining a core of basic research. These themes are reflected in the highlights from the 2009 NF Conference presented here.

What's new in neurofibromatosis? Proceedings from the 2009 NF Conference: new frontiers.

The neurofibromatosis type 2 gene product merlin is known to provoke gliogenic tumors as a result of its mutagenic loss. Merlin's physiological anti-mitogenic function makes it unique among its ezrin-radixin-moesin (ERM) family members. Although ERM proteins and merlin are known to be expressed in glial cells of the peripheral nervous system and CNS, the neuronal expression pattern and function of merlin have been less well investigated. We report here expression of merlin in developing and mature neurons of the murine CNS. Within cerebellar Purkinje cells (PCs), merlin was localized in the soma, sprouting dendrites and axons. Merlin expression in PCs was high during the period of initial dendrite regression and declined during later phases of dendrite elongation. Consistently, merlin expression in vivo was increased in Engrailed-2-overexpressing PCs, which are characterized by a reduced dendritic extension. Furthermore, overexpression of merlin in dissociated cerebellar cultures and in neurogenic P19 cells caused a significant decline in neurite outgrowth, while, conversely, inhibition of merlin expression increased process formation. This effect was dependent on phosphorylation of serine 518 and involved the inactivation of the growth-promoting GTPase Rac. We thus provide evidence that merlin plays a pivotal role in controlling the neuronal wiring in the developing CNS.

Merlin inhibits neurite outgrowth in the CNS.

Oncogenic Ras mutations are rare in gastric cancer, indicating that other mechanisms may be responsible for aberrant Ras activation in this type of cancer. Ezrin is critical to Ras activation by remodeling cortical actin cy-toskeleton. In this study, we aimed to illustrate the relevance and regulation of ezrin in gastric cancer. Ezrin was upregulated in gastric cancer cells. Ezrin siRNA inhibited Ras activation, cell growth and cell migration. Ezrin overex-pression was correlated with a poor outcome of gastric cancer patients (n=150, p<0.01). Cox regression analysis revealed a significant value of ezrin expression in prognosis prediction of gastric cancer (relative risk: 2.37, 95% confidence interval: 1.24-4.56, p<0.01). MiR-204, which was predicted to target ezrin, was downregulated in gastric cancer cells and gastric carcinomas (n=22, p<0.01). MiR-204 inhibited ezrin expression, Ras activation, cell growth and cell migration. Importantly, miR-204 suppressed the expression of luciferase controlled by wild-type but not mutated ezrin 3'-UTR. In conclusion, ezrin is important to Ras activation in gastric cancer. Its upregulation is an independent prognosis prediction factor for gastric cancer. By contributing to ezrin upregulation, miR-204 downregulation represents a novel mechanism for aberrant Ras activation in gastric carcinogenesis.

A microRNA contribution to aberrant Ras activation in gastric cancer.

The 2011 annual meeting of the Children's Tumor Foundation, the annual gathering of the neurofibromatosis (NF) research and clinical communities, was attended by 330 participants who discussed integration of new signaling pathways into NF research, the appreciation for NF mutations in sporadic cancers, and an expanding pre-clinical and clinical agenda. NF1, NF2, and schwannomatosis collectively affect approximately 100,000 persons in US, and result from mutations in different genes. Benign tumors of NF1 (neurofibroma and optic pathway glioma) and NF2 (schwannoma, ependymoma, and meningioma) and schwannomatosis (schwannoma) can cause significant morbidity, and there are no proven drug treatments for any form of NF. Each disorder is associated with additional manifestations causing morbidity. The research presentations described in this review covered basic science, preclinical testing, and results from clinical trials, and demonstrate the remarkable strides being taken toward understanding of and progress toward treatments for these disorders based on the close interaction among scientists and clinicians.

Neurofibromatosis 2011: a report of the Children's Tumor Foundation annual meeting.

Receptor tyrosine kinases (RTKs) participate in a multitude of signaling pathways, some of them via the small G-protein Ras. An important component in the activation of Ras is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras.

Activation of Ras requires the ERM-dependent link of actin to the plasma membrane.

The autosomal dominant disorder neurofibromatosis type 2 (NF2) is a hereditary tumor syndrome caused by inactivation of the NF2 tumor suppressor gene, encoding merlin. Apart from tumors affecting the peripheral and central nervous systems, most NF2 patients develop peripheral neuropathies. This peripheral nerve disease can occur in the absence of nerve-damaging tumors, suggesting an etiology that is independent of gross tumor burden. We discovered that merlin isoform 2 (merlin-iso2) has a specific function in maintaining axonal integrity and propose that reduced axonal NF2 gene dosage leads to NF2-associated polyneuropathy. We identified a merlin-iso2-dependent complex that promotes activation of the GTPase RhoA, enabling downstream Rho-associated kinase to promote neurofilament heavy chain phosphorylation. Merlin-iso2-deficient mice exhibited impaired locomotor capacities, delayed sensory reactions and electrophysiological signs of axonal neuropathy. Sciatic nerves from these mice and sural nerve biopsies from NF2 patients revealed reduced phosphorylation of the neurofilament H subunit, decreased interfilament spacings and irregularly shaped axons.

Merlin isoform 2 in neurofibromatosis type 2-associated polyneuropathy.

Receptor tyrosine kinases participate in several signaling pathways through small G proteins such as Ras (rat sarcoma). An important component in the activation of these G proteins is Son of sevenless (SOS), which catalyzes the nucleotide exchange on Ras. For optimal activity, a second Ras molecule acts as an allosteric activator by binding to a second Ras-binding site within SOS. This allosteric Ras-binding site is blocked by autoinhibitory domains of SOS. We have reported recently that Ras activation also requires the actin-binding proteins ezrin, radixin, and moesin. Here we report the mechanism by which ezrin modulates SOS activity and thereby Ras activation. Active ezrin enhances Ras/MAPK signaling and interacts with both SOS and Ras in vivo and in vitro. Moreover, in vitro kinetic assays with recombinant proteins show that ezrin also is important for the activity of SOS itself. Ezrin interacts with GDP-Ras and with the Dbl homology (DH)/pleckstrin homology (PH) domains of SOS, bringing GDP-Ras to the proximity of the allosteric site of SOS. These actions of ezrin are antagonized by the neurofibromatosis type 2 tumor-suppressor protein merlin. We propose an additional essential step in SOS/Ras control that is relevant for human cancer as well as all physiological processes involving Ras.

Regulation of Son of sevenless by the membrane-actin linker protein ezrin.

Axonal surface proteins encompass a group of heterogeneous molecules, which exert a variety of different functions in the highly interdependent relationship between axons and Schwann cells. We recently revealed that the tumour suppressor protein merlin, mutated in the hereditary tumour syndrome neurofibromatosis type 2, impacts significantly on axon structure maintenance in the peripheral nervous system. We now report on a role of neuronal merlin in the regulation of the axonal surface protein neuregulin 1 important for modulating Schwann cell differentiation and myelination. Specifically, neuregulin 1 type III expression is reduced in sciatic nerve tissue of neuron-specific knockout animals as well as in biopsies from seven patients with neurofibromatosis type 2. In vitro experiments performed on both the P19 neuronal cell line and primary dorsal root ganglion cells demonstrate the influence of merlin on neuregulin 1 type III expression. Moreover, expression of ERBB2, a Schwann cell receptor for neuregulin 1 ligands is increased in nerve tissue of both neuron-specific merlin knockout animals and patients with neurofibromatosis type 2, demonstrating for the first time that axonal merlin indirectly regulates Schwann cell behaviour. Collectively, we have identified that neuronally expressed merlin can influence Schwann cell activity in a cell-extrinsic manner.

Neuronal merlin influences ERBB2 receptor expression on Schwann cells through neuregulin 1 type III signalling.

The neurofibromatoses (NF) are autosomal dominant genetic disorders that encompass the rare diseases NF1, NF2, and schwannomatosis. The NFs affect more people worldwide than Duchenne muscular dystrophy and Huntington's disease combined. NF1 and NF2 are caused by mutations of known tumor suppressor genes (NF1 and NF2, respectively). For schwannomatosis, although mutations in SMARCB1 were identified in a subpopulation of schwannomatosis patients, additional causative gene mutations are still to be discovered. Individuals with NF1 may demonstrate manifestations in multiple organ systems, including tumors of the nervous system, learning disabilities, and physical disfigurement. NF2 ultimately can cause deafness, cranial nerve deficits, and additional severe morbidities caused by tumors of the nervous system. Unmanageable pain is a key finding in patients with schwannomatosis. Although today there is no marketed treatment for NF-related tumors, a significant number of clinical trials have become available. In addition, significant preclinical efforts have led to a more rational selection of potential drug candidates for NF trials. An important element in fueling this progress is the sharing of knowledge. For over 20 years the Children's Tumor Foundation has convened an annual NF Conference, bringing together NF professionals to share novel findings, ideas, and build collaborations. The 2012 NF Conference held in New Orleans hosted over 350 NF researchers and clinicians. This article provides a synthesis of the highlights presented at the conference and as such, is a "state-of-the-field" for NF research in 2012. © 2014 Wiley Periodicals, Inc.

CTF meeting 2012: Translation of the basic understanding of the biology and genetics of NF1, NF2, and schwannomatosis toward the development of effective therapies.

Article	Year
Survivors of suicide. Emerging counseling strategies. Journal of psychosocial nursing and mental health services\| PubMed ID: 11813351	2002
Neurofibromatosis 2 (NF2) tumor suppressor schwannomin and its interacting protein HRS regulate STAT signaling. Human molecular genetics\| PubMed ID: 12444102	2002
Listeria monocytogenes exploits ERM protein functions to efficiently spread from cell to cell. The EMBO journal\| PubMed ID: 15729356	2005
Tumorigenic transformation by CPI-17 through inhibition of a merlin phosphatase. Nature\| PubMed ID: 16885985	2006
Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin. Molecular biology of the cell\| PubMed ID: 17065554	2007
Merlin/neurofibromatosis type 2 suppresses growth by inhibiting the activation of Ras and Rac. Cancer research\| PubMed ID: 17234759	2007
Properties of an ezrin mutant defective in F-actin binding. Journal of molecular biology\| PubMed ID: 19084535	2009
What's new in neurofibromatosis? Proceedings from the 2009 NF Conference: new frontiers. American journal of medical genetics. Part A\| PubMed ID: 20082461	2010
Merlin inhibits neurite outgrowth in the CNS. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 20668201	2010
A microRNA contribution to aberrant Ras activation in gastric cancer. American journal of translational research\| PubMed ID: 21416062	2011
Neurofibromatosis 2011: a report of the Children's Tumor Foundation annual meeting. Acta neuropathologica\| PubMed ID: 22083253	2012
Activation of Ras requires the ERM-dependent link of actin to the plasma membrane. PloS one\| PubMed ID: 22132106	2011
Merlin isoform 2 in neurofibromatosis type 2-associated polyneuropathy. Nature neuroscience\| PubMed ID: 23455610	2013
Regulation of Son of sevenless by the membrane-actin linker protein ezrin. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24297905	2013
Neuronal merlin influences ERBB2 receptor expression on Schwann cells through neuregulin 1 type III signalling. Brain : a journal of neurology\| PubMed ID: 24309211	2014
CTF meeting 2012: Translation of the basic understanding of the biology and genetics of NF1, NF2, and schwannomatosis toward the development of effective therapies. American journal of medical genetics. Part A\| PubMed ID: 24443315	2014

Helen Morrison

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