Neurofibromatosis type 2 tumor suppressor protein is expressed in oligodendrocytes and regulates cell proliferation and process formation.

The neurofibromatosis type 2 (NF2) tumor suppressor protein Merlin functions as a negative regulator of cell growth and actin dynamics in different cell types amongst which Schwann cells have been extensively studied. In contrast, the presence and the role of Merlin in oligodendrocytes, the myelin forming cells within the CNS, have not been elucidated. In this work, we demonstrate that Merlin immunoreactivity was broadly distributed in the white matter throughout the central nervous system. Following Merlin expression during development in the cerebellum, Merlin could be detected in the cerebellar white matter tract at early postnatal stages as shown by its co-localization with Olig2-positive cells as well as in adult brain sections where it was aligned with myelin basic protein containing fibers. This suggests that Merlin is expressed in immature and mature oligodendrocytes. Expression levels of Merlin were low in oligodendrocytes as compared to astrocytes and neurons throughout development. Expression of Merlin in oligodendroglia was further supported by its identification in either immortalized cell lines of oligodendroglial origin or in primary oligodendrocyte cultures. In these cultures, the two main splice variants of Nf2 could be detected. Merlin was localized in clusters within the nuclei and in the cytoplasm. Overexpressing Merlin in oligodendrocyte cell lines strengthened reduced impedance in XCELLigence measurements and Ki67 stainings in cultures over time. In addition, the initiation and elongation of cellular projections were reduced by Merlin overexpression. Consistently, cell migration was retarded in scratch assays done on Nf2-transfected oligodendrocyte cell lines. These data suggest that Merlin actively modulates process outgrowth and migration in oligodendrocytes.

Mtss1 promotes maturation and maintenance of cerebellar neurons via splice variant-specific effects.

Efficient coupling of the actin cytoskeleton to the cell membrane is crucial for histogenesis and maintenance of the nervous system. At this critical interface, BAR (Bin-Amphiphysin-Rvs) proteins regulate membrane bending, shown to be instrumental for mobility and morphogenesis of individual cells. Yet, the systemic significance of these proteins remains largely unexplored. Here, we probe the role of a prominent member of this protein family, the inverse-BAR protein Mtss1, for the development and function of a paradigmatic neuronal circuit, the cerebellar cortex. Mtss1-null mice show granule cell ectopias, dysmorphic Purkinje cells, malformed axons, and a protracted neurodegeneration entailing age-dependent motor deficits. In postmitotic granule cells, which transiently express Mtss1 while they migrate and form neurites, Mtss1 impinges on directional persistence and neuritogenesis. The latter effect can be specifically attributed to its exon 12a splice variant. Targeted re-expression of Mtss1 in Mtss1-null animals indicated that these pathologies were largely due to cell type-specific and intrinsic effects. Together, our results provide a mechanistic perspective on Mtss1 function for brain development and degeneration and relate it to structural features of this protein.

Neuron-Specific Deletion of the Nf2 Tumor Suppressor Impairs Functional Nerve Regeneration.

In contrast to axons of the central nervous system (CNS), axons of the peripheral nervous system (PNS) show better, but still incomplete and often slow regeneration following injury. The tumor suppressor protein merlin, mutated in the hereditary tumor syndrome Neurofibromatosis type 2 (NF2), has recently been shown to have RhoA regulatory functions in PNS neurons-in addition to its well-characterized, growth-inhibitory activity in Schwann cells. Here we report that the conditional knockout of merlin in PNS neurons leads to impaired functional recovery of mice following sciatic nerve crush injury, in a gene-dosage dependent manner. Gross anatomical or electrophysiological alterations of sciatic nerves could not be detected. However, correlating with attenuated RhoA activation due to merlin deletion, ultrastructural analysis of nerve samples indicated enhanced sprouting of axons with reduced caliber size and increased myelination compared to wildtype animals. We conclude that deletion of the tumor suppressor merlin in the neuronal compartment of peripheral nerves results in compromised functional regeneration after injury. This mechanism could explain the clinical observation that NF2 patients suffer from higher incidences of slowly recovering facial nerve paralysis after vestibular schwannoma surgery.

The importance of nerve microenvironment for schwannoma development.

Schwannomas are predominantly benign nerve sheath neoplasms caused by Nf2 gene inactivation. Presently, treatment options are mainly limited to surgical tumor resection due to the lack of effective pharmacological drugs. Although the mechanistic understanding of Nf2 gene function has advanced, it has so far been primarily restricted to Schwann cell-intrinsic events. Extracellular cues determining Schwann cell behavior with regard to schwannoma development remain unknown. Here we show pro-tumourigenic microenvironmental effects on Schwann cells where an altered axonal microenvironment in cooperation with injury signals contribute to a persistent regenerative Schwann cell response promoting schwannoma development. Specifically in genetically engineered mice following crush injuries on sciatic nerves, we found macroscopic nerve swellings in mice with homozygous nf2 gene deletion in Schwann cells and in animals with heterozygous nf2 knockout in both Schwann cells and axons. However, patient-mimicking schwannomas could only be provoked in animals with combined heterozygous nf2 knockout in Schwann cells and axons. We identified a severe re-myelination defect and sustained macrophage presence in the tumor tissue as major abnormalities. Strikingly, treatment of tumor-developing mice after nerve crush injury with medium-dose aspirin significantly decreased schwannoma progression in this disease model. Our results suggest a multifactorial concept for schwannoma formation-emphasizing axonal factors and mechanical nerve irritation as predilection site for schwannoma development. Furthermore, we provide evidence supporting the potential efficacy of anti-inflammatory drugs in the treatment of schwannomas.

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

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.

Merlin isoform 2 in neurofibromatosis type 2-associated polyneuropathy.

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 inhibits neurite outgrowth in the CNS.

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.

Transport of a hyaluronan-binding protein in brain tissue.

Hyaluronan is an unsulfated linear glycosaminoglycan with the ability to nucleate extracellular matrices by the formation of aggregates with lecticans. These matrices are essential during development of the central nervous system. In the prospective white matter of the developing brain hyaluronan is organized into fiber-like structures according to confocal microscopy of fixed slices which may guide the migration of neural precursor cells [Baier, C., S.L. Baader, J. Jankowski, V. Gieselmann, K. Schilling, U. Rauch, and J. Kappler. 2007. Hyaluronan is organized into fiber-like structures along migratory pathways in the developing mouse cerebellum. Matrix Biol. 26: 348-58]. By using plasmon surface resonance, microinjection into brain slices and fluorescence correlation spectroscopy, we show that the brain-specific lecticans bind to, but also dissociate rather rapidly from hyaluronan. After microinjection into native cerebellar slices a GFP-tagged hyaluronan-binding neurocan fragment was enriched at binding sites in the prospective white matter, which had a directional orientation and formed local stationary concentration gradients in areas where binding sites are abundant. Fluorescence correlation spectroscopy measurements at fixed brain slices revealed that fiber-bound neurocan-GFP was mobile with D(fiber(neurocan-GFP))=4x10(-10)cm(2)/s. Therefore, we propose that hyaluronan-rich fibers in the prospective white matter of the developing mouse cerebellum can guide the diffusion of lecticans. Since lecticans bind a variety of growth and mobility factors, their guided diffusion may contribute to the transport of these polypeptides and to the formation of concentration gradients. This mechanism could serve to encode positional information during development.

Developmental expression and differentiation-related neuron-specific splicing of metastasis suppressor 1 (Mtss1) in normal and transformed cerebellar cells.

BACKGROUND: Mtss1 encodes an actin-binding protein, dysregulated in a variety of tumors, that interacts with sonic hedgehog/Gli signaling in epidermal cells. Given the prime importance of this pathway for cerebellar development and tumorigenesis, we assessed expression of Mtss1 in the developing murine cerebellum and human medulloblastoma specimens. RESULTS: During development, Mtss1 is transiently expressed in granule cells, from the time point they cease to proliferate to their synaptic integration. It is also expressed by granule cell precursor-derived medulloblastomas. In the adult CNS, Mtss1 is found exclusively in cerebellar Purkinje cells. Neuronal differentiation is accompanied by a switch in Mtss1 splicing. Whereas immature granule cells express a Mtss1 variant observed also in peripheral tissues and comprising exon 12, this exon is replaced by a CNS-specific exon, 12a, in more mature granule cells and in adult Purkinje cells. Bioinformatic analysis of Mtss1 suggests that differential exon usage may affect interaction with Fyn and Src, two tyrosine kinases previously recognized as critical for cerebellar cell migration and histogenesis. Further, this approach led to the identification of two evolutionary conserved nuclear localization sequences. These overlap with the actin filament binding site of Mtss1, and one also harbors a potential PKA and PKC phosphorylation site. CONCLUSION: Both the pattern of expression and splicing of Mtss1 is developmentally regulated in the murine cerebellum. These findings are discussed with a view on the potential role of Mtss1 for cytoskeletal dynamics in developing and mature cerebellar neurons.

Aldolase C/zebrin II is released to the extracellular space after stroke and inhibits the network activity of cortical neurons.

Cell death after stroke involves apoptotic, autophagocytic and necrotic mechanisms which may cause the release of cytosolic proteins to the extracellular space. Aldolase C (AldC) is the brain specific isoform of the glycolytic enzyme fructose-1,6-bisphosphate aldolase. According to its characteristic striped expression pattern in the adult cerebellum AldC is also termed zebrin II. Here, we demonstrate release of AldC into the cerebrospinal fluid (CSF) after stroke in vivo. Studies with cell cultures confirmed that AldC is released to the extracellular space after hypoxia. Moreover, addition of purified recombinant AldC to networks of cortical neurons plated on multielectrode arrays reversibly inhibited the spontaneous generation of action potentials at AldC concentrations which can be expected to occur after lesions of the human cerebral cortex. This mechanism could be relevant in the pathogenesis of the electrophysiological changes in the penumbra region after stroke.

Collapsin response mediator protein-4 regulates F-actin bundling.

Collapsin response mediator proteins (CRMPs) form a family of cytosolic phosphoproteins which are involved in the signal transduction of semaphorin 3A leading to growth cone collapse. These proteins interact with a variety of cytosolic proteins including tubulin heterodimers. Here, we show that CRMP-4 co-localizes with F-actin in regular rib-like structures within lamellipodia of B35 neuroblastoma cells. Furthermore, depolymerization of actin fibers changed the distribution of GFP-CRMP-4 in vivo. In vitro, recombinant CRMP-4 formed homo-oligomers, bound to F-actin and organized F-actin into tight bundles. Both oligomerization and F-actin bundling depended on the C-terminal part of CRMP-4. The stoichiometry of actin and CRMP-4 in bundles was approximately 1:1 and the apparent equilibrium constant of the microfilament-CRMP-4 interaction was estimated from bundling assays as K(app) = 730 mM(-1). CRMP-4 was abundant in the cytosol of B35 neuroblastoma cells and its concentration was measured as approximately 1.7 microM. Overexpression of CRMP-4 inhibited the migration of B35 neuroblastoma cells, while knockdown of CRMP-4 enhanced cell migration and disturbed rib-like actin-structures in lamellipodia. Taken together, our data indicate that CRMP-4 promotes bundling of F-actin in vitro, that it is an important component of rib-like actin bundles in lamellipodia in vivo and that it functionally regulates the actin cytoskeleton in motile cells. These findings suggest a specific regulatory role of CRMP-4 towards the actin cytoskeleton which may by be relevant for growth cone collapse.

Neurocan-GFP fusion protein: a new approach to detect hyaluronan on tissue sections and living cells.

Hyaluronan is an unsulfated glycosaminoglycan (GAG) that is ubiquitously expressed in the extracellular matrix (ECM) of all vertebrates, where hyaluronan rich matrices constitute a particular permissive environment for the development of complex biological structures and also for tumor progression. Because of its conserved structure and ubiquitous expression, antibodies for its histochemical detection cannot be produced. We have engineered a fusion protein, neurocan-GFP, and expressed it as a secreted molecule in mammalian cells. Neurocan-GFP fusion protein specifically binds to hyaluronan and directly visualizes hyaluronan on tissue sections, revealing a very detailed picture of hyaluronan distribution. The fluorescent fusion protein can be used in combination with antibodies and nuclear markers for double or triple staining. In addition, it is suitable to visualize hyaluronan on living cells by time-lapse video microscopy. The successful production and application of the neurocan-GFP fusion protein opens up new perspectives for using GFP fusion proteins as detection tools in histological and cytological studies complementing conventional antibody and biotin/avidin techniques.

Expression patterns and different subcellular localization of the growth factors HDGF (hepatoma-derived growth factor) and HRP-3 (HDGF-related protein-3) suggest functions in addition to their mitogenic activity.

HDGF (hepatoma-derived growth factor) and the HRPs (HDGF-related proteins) comprise a family of six proteins which display high identity in their N-terminus, but differ at the C-terminus. Here we investigate the patterns of expression of HDGF and HRP-3, by generating antisera specifically recognizing each growth factor. Whereas HRP-3 protein is expressed only in brain, HDGF can be found in a broad range of tissues, with highest levels in brain, testis, lung and spleen. The expression of HDGF and HRP-3 was found to be regulated during brain development, with highest levels around birth, followed by a decline until postnatal day 9. Interestingly, expression of HRP-3 increases again in adult brain. In situ hybridization and immunohistochemistry of cerebellar, cerebral and hippocampal brain slices showed that expression of both growth factors is not limited to areas of high proliferative activity. Both mRNAs and proteins are expressed in neuronal as well as glial cells. Immunocytochemistry of cultured neocortical neurons revealed that HDGF and HRP-3 can be found in the nucleus as well as the cytoplasm. HDGF is restricted to the neuronal soma, whereas HRP-3 can also be found in neurites. Thus the expression of HDGF and HRP-3 in differentiated cells, post-mitotic neurons and primary cultures of rat neocortex points to functions in brain that might not be limited to proliferation. In addition, their simultaneous expression in the same cell and their different subcellular localization in cultured neurons suggest different functions of HDGF and HRP-3 within single cells.