Mechanisms Promoting Phosphorylation Of The Nf2 Tumor Suppressor And Its Effects On Schwann Cell Development

Neurofibromatosis type 2 is an autosomal dominant disease characterized by the formation of schwannomas and other peripheral neuropathies. The nf2 gene encodes the protein Schwannomin, or merlin. Schwannomin (Sch) is a membrane-cytoskeletal linking protein that suppresses cell proliferation at high cell density and modulates cell shape. Sch\u27s tumor suppressive activity is regulated by its localization, conformation, and phosphorylation at serine 518 (S518). Sch\u27s localization is dependent on binding the scaffold protein, paxillin. Phosphorylation of Sch at S518 regulates its conformation and tumor suppressor function. In a negative feedback loop, unphosphorylated Sch restricts cell proliferation downstream of Rac and p21-activated kinase (Pak), whereas Pak-induced phosphorylation inactivates Sch\u27s ability to inhibit Pak and cell proliferation. Little is known about the function of the phosphorylated form of Sch, or the molecular mechanisms leading to its phosphorylation. Here we demonstrate that Sch-S518 phosphorylation is dependent on paxillin-binding and plasma membrane localization in SCs. Phosphorylation of Sch at the plasma membrane is mediated by Cdc42-Pak and results in altered SC morphology and polarity. Moreover, we have identified two extracellular stimuli that trigger Sch-S518 phosphorylation; these are neuregulin (NRG) and laminin, two potent activators of SC proliferation and myelination. NRG promotes Sch-S518 phosphorylation downstream of ErbB2/ErbB3 through PKA, whereas laminin-1 stimulation of β1 integrin promotes Pak- dependent phosphorylation of Sch-S518. Additionally, we find that Sch promotes process formation and elongation in primary and myelinating SCs, independent of Sch S518 phosphorylation. However, Sch phosphorylation was found to influence SC differentiation, as expression of an unphosphorylatable variant, Sch-S518A, facilitated SC myelination, whereas expression of a phospho-mimicking variant, Sch-S518D, reduced the SC\u27s ability to myelinate. Together, these findings have identified receptor-mediated and paxillin-dependent pathways that regulate phosphorylation and inactivation of Sch\u27s tumor suppressor function. Additionally, these results have elucidated novel normal functions for Sch during peripheral nerve development and myelination, and identify novel therapeutic targets for treatment of NF2 and other peripheral neuropathies

Organization and maintenance of molecular domains in myelinated axons

Over a century ago, Ramon y Cajal first proposed the idea of a directionality involved in nerve conduction and neuronal communication. Decades later, it was discovered that myelin, produced by glial cells, insulated axons with periodic breaks where nodes of Ranvier (nodes) form to allow for saltatory conduction. In the peripheral nervous system (PNS), Schwann cells are the glia that can either individually myelinate the axon from one neuron or ensheath axons of many neurons. In the central nervous system (CNS), oligodendrocytes are the glia that myelinate axons from different neurons. Review of more recent studies revealed that this myelination created polarized domains adjacent to the nodes. However, the molecular mechanisms responsible for the organization of axonal domains are only now beginning to be elucidated. The molecular domains in myelinated axons include the axon initial segment (AIS), where various ion channels are clustered and action potentials are initiated; the node, where sodium channels are clustered and action potentials are propagated; the paranode, where myelin loops contact with the axolemma; the juxtaparanode (JXP), where delayed-rectifier potassium channels are clustered; and the internode, where myelin is compactly wrapped. Each domain contains a unique subset of proteins critical for the domain’s function. However, the roles of these proteins in axonal domain organization are not fully understood. In this review, we highlight recent advances on the molecular nature and functions of some of the components of each axonal domain and their roles in axonal domain organization and maintenance for proper neuronal communication

Nodes of Ranvier Act as Barriers to Restrict Invasion of Flanking Paranodal Domains in Myelinated Axons

Accumulation of voltage gated sodium (Nav) channels at nodes of Ranvier is paramount for action potential propagation along myelinated fibers, yet the mechanisms governing nodal development, organization and stabilization remain unresolved. Here, we report that genetic ablation of the neuron-specific isoform of Neurofascin (NfascNF186) in vivo results in nodal disorganization, including loss of Nav channel and ankyrin-G (AnkG) enrichment at nodes in the peripheral (PNS) and central (CNS) nervous systems. Interestingly, the presence of paranodal domains failed to rescue nodal organization in the PNS and the CNS. Most importantly, using ultrastructural analysis, we demonstrate that the paranodal domains invade the nodal space in NfascNF186 mutant axons and occlude node formation. Our results suggest that NfascNF186-dependent assembly of the nodal complex acts as a molecular boundary to restrict the movement of flanking paranodal domains into the nodal area, thereby facilitating the stereotypic axonal domain organization and saltatory conduction along myelinated axons

Spatiotemporal ablation of myelinating glia-specific neurofascin (Nfasc NF155 ) in mice reveals gradual loss of paranodal axoglial junctions and concomitant disorganization of axonal domains

The evolutionary demand for rapid nerve impulse conduction led to the process of myelination-dependent organization of axons into distinct molecular domains. These domains include the node of Ranvier flanked by highly specialized paranodal domains where myelin loops and axolemma orchestrate the axoglial septate junctions. These junctions are formed by interactions between a glial isoform of neurofascin (NfascNF155) and axonal Caspr and Cont. Here we report the generation of myelinating glia-specific NfascNF155 null mouse mutants. These mice exhibit severe ataxia, motor paresis, and death before the third postnatal week. In the absence of glial NfascNF155, paranodal axoglial junctions fail to form, axonal domains fail to segregate, and myelinated axons undergo degeneration. Electrophysiological measurements of peripheral nerves from NfascNF155 mutants revealed dramatic reductions in nerve conduction velocities. By using inducible PLP-CreER recombinase to ablate NfascNF155 in adult myelinating glia, we demonstrate that paranodal axoglial junctions disorganize gradually as the levels of NfascNF155 protein at the paranodes begin to drop. This coincides with the loss of the paranodal region and concomitant disorganization of the axonal domains. Our results provide the first direct evidence that the maintenance of axonal domains requires the fence function of the paranodal axoglial junctions. Together, our studies establish a central role for paranodal axoglial junctions in both the organization and the maintenance of axonal domains in myelinated axons

Decreased Axon Caliber Underlies Loss of Fiber Tract Integrity, Disproportional Reductions in White Matter Volume, and Microcephaly in Angelman Syndrome Model Mice

Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of function of the maternally inherited UBE3A allele. It is currently unclear how the consequences of this genetic insult unfold to impair neurodevelopment. We reasoned that by elucidating the basis of microcephaly in AS, a highly penetrant syndromic feature with early postnatal onset, we would gain new insights into the mechanisms by which maternal UBE3A loss derails neurotypical brain growth and function. Detailed anatomical analysis of both male and female maternal Ube3a-null mice reveals that microcephaly in the AS mouse model is primarily driven by deficits in the growth of white matter tracts, which by adulthood are characterized by densely packed axons of disproportionately small caliber. Our results implicate impaired axon growth in the pathogenesis of AS and identify noninvasive structural neuroimaging as a potentially valuable tool for gauging therapeutic efficacy in the disorder

The ClinGen Epilepsy Gene Curation Expert Panel—Bridging the divide between clinical domain knowledge and formal gene curation criteria

The field of epilepsy genetics is advancing rapidly and epilepsy is emerging as a frequent indication for diagnostic genetic testing. Within the larger ClinGen framework, the ClinGen Epilepsy Gene Curation Expert Panel is tasked with connecting two increasingly separate fields: the domain of traditional clinical epileptology, with its own established language and classification criteria, and the rapidly evolving area of diagnostic genetic testing that adheres to formal criteria for gene and variant curation. We identify critical components unique to the epilepsy gene curation effort, including: (a) precise phenotype definitions within existing disease and phenotype ontologies; (b) consideration of when epilepsy should be curated as a distinct disease entity; (c) strategies for gene selection; and (d) emerging rules for evaluating functional models for seizure disorders. Given that de novo variants play a prominent role in many of the epilepsies, sufficient genetic evidence is often awarded early in the curation process. Therefore, the emphasis of gene curation is frequently shifted toward an iterative precuration process to better capture phenotypic associations. We demonstrate that within the spectrum of neurodevelopmental disorders, gene curation for epilepsy-associated genes is feasible and suggest epilepsy-specific conventions, laying the groundwork for a curation process of all major epilepsy-associated genes

Beyond gene-disease validity: capturing structured data on inheritance, allelic requirement, disease-relevant variant classes, and disease mechanism for inherited cardiac conditions

Background: As the availability of genomic testing grows, variant interpretation will increasingly be performed by genomic generalists, rather than domain-specific experts. Demand is rising for laboratories to accurately classify variants in inherited cardiac condition (ICC) genes, including secondary findings. // Methods: We analyse evidence for inheritance patterns, allelic requirement, disease mechanism and disease-relevant variant classes for 65 ClinGen-curated ICC gene-disease pairs. We present this information for the first time in a structured dataset, CardiacG2P, and assess application in genomic variant filtering. // Results: For 36/65 gene-disease pairs, loss of function is not an established disease mechanism, and protein truncating variants are not known to be pathogenic. Using the CardiacG2P dataset as an initial variant filter allows for efficient variant prioritisation whilst maintaining a high sensitivity for retaining pathogenic variants compared with two other variant filtering approaches. // Conclusions: Access to evidence-based structured data representing disease mechanism and allelic requirement aids variant filtering and analysis and is a pre-requisite for scalable genomic testing

Clinical validity assessment of genes frequently tested on intellectual disability/autism sequencing panels.

[en] PURPOSE: Neurodevelopmental disorders (NDDs), such as intellectual disability (ID) and autism spectrum disorder (ASD), exhibit genetic and phenotypic heterogeneity, making them difficult to differentiate without a molecular diagnosis. The Clinical Genome Resource Intellectual Disability/Autism Gene Curation Expert Panel (GCEP) uses systematic curation to distinguish ID/ASD genes that are appropriate for clinical testing (ie, with substantial evidence supporting their relationship to disease) from those that are not. METHODS: Using the Clinical Genome Resource gene-disease validity curation framework, the ID/Autism GCEP classified genes frequently included on clinical ID/ASD testing panels as Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Known Disease Relationship. RESULTS: As of September 2021, 156 gene-disease pairs have been evaluated. Although most (75%) were determined to have definitive roles in NDDs, 22 (14%) genes evaluated had either Limited or Disputed evidence. Such genes are currently not recommended for use in clinical testing owing to the limited ability to assess the effect of identified variants. CONCLUSION: Our understanding of gene-disease relationships evolves over time; new relationships are discovered and previously-held conclusions may be questioned. Without periodic re-examination, inaccurate gene-disease claims may be perpetuated. The ID/Autism GCEP will continue to evaluate these claims to improve diagnosis and clinical care for NDDs

Phosphorylation Of The Nf2 Tumor Suppressor In Schwann Cells Is Mediated By Cdc42-Pak And Requires Paxillin Binding

Mutations in the Neurofibromatosis type 2 tumor suppressor gene that encodes Schwannomin causes formation of benign schwannomas. Schwannoma cells lose their characteristic bipolar shape and become rounded with excessive ruffling membranes. Schwannomin is phosphorylated at serine 518 (S518) by p21 activated kinase (Pak). Unphosphorylated schwannomin is associated with growth inhibition but little is known about the function of the phosphorylated form, or the molecular events leading to its phosphorylation. Here, we report in SCs that schwannomin S518 phosphorylation requires binding to paxillin and targeting to the plasma membrane. Phospho-S518-schwannomin is enriched in the peripheral-most aspects of membrane specializations where paxillin, activated Pak, Cdc42 but not Rac are highly expressed. Schwannomin and Pak phosphorylation levels are not reduced in response to lowering Rac-GTP levels with NSC23766. Expression of schwannomin S518A/D-GFP variants each distinctively altered Schwann cell shape and polarity. These results are consistent with tight spatial regulation of S518 phosphorylation at the plasma membrane in a paxillin and Cdc42-Pak dependent manner that leads to local reorganization of the SC cytoskeleton. © 2006 Elsevier Inc. All rights reserved

The Nursing Dimension of Providing Palliative Care to Children and Adolescents with Cancer

Palliative care for children and adolescents with cancer includes interventions that focus on the relief of suffering, optimization of function, and improvement of quality of life at any and all stages of disease. This care is most effectively provided by a multidisciplinary team. Nurses perform an integral role on that team by identifying symptoms, providing care coordination, and assuring clear communication. Several basic tenets appear essential to the provision of optimal palliative care. First, palliative care should be administered concurrently with curative therapy beginning at diagnosis and assuming a more significant role at end of life. This treatment approach, recommended by many medical societies, has been associated with numerous benefits including longer survival. Second, realistic, objective goals of care must be developed. A clear understanding of the prognosis by the patient, family, and all members of the medical team is essential to the development of these goals. The pediatric oncology nurse is pivotal in developing these goals and assuring that they are adhered to across all specialties. Third, effective therapies to prevent and relieve the symptoms of suffering must be provided. This can only be accomplished with accurate and repeated assessments. The pediatric oncology nurse is vital in providing these assessments and must possess a working knowledge of the most common symptoms associated with suffering. With a basic understanding of these palliative care principles and competency in the core skills required for this care, the pediatric oncology nurse will optimize quality of life for children and adolescents with cancer