George H. De Vries

Neurofibrosarcoma-derived Schwann cells overexpress platelet-derived growth factor (PDGF) receptors and are induced to proliferate by PDGF BB

Neurofibromatosis type 1 (NF1) is characterized by the formation of neurofibromas, benign tumors of the peripheral nerve consisting essentially of Schwann cells, which can sometimes turn malignant to form neurofibrosarcomas. The mechanism of progression toward a malignant phenotype remains largely unknown. In this report, we show that platelet‐derived growth factor (PDGF) BB, and to a lesser extent fibroblast growth factor 2, are mitogenic for two neurofibrosarcoma‐derived Schwann cell lines, but not for a Schwann cell line derived from a schwannoma (from a non‐NF1 patient) or for transformed rat Schwann cells. Levels of expression of both PDGF receptor α and β are significantly increased in the two neurofibrosarcoma‐derived cell lines compared to the non‐NF1 Schwann cell lines. The level of tyrosyl‐phosphorylated PDGF receptor β is strongly increased upon stimulation by PDGF BB. In comparison, only modest levels of tyrosyl‐phosphorylated PDGF receptor α are observed, upon stimulation by PDGF AA or PDGF BB. Accordingly, PDGF AA is only a weak mitogen for the neurofibrosarcoma‐derived cells by comparison to PDGF BB. These results indicate that the mitogenic effect of PDGF BB for the neurofibrosarcoma‐derived Schwann cell lines is primarily transduced by PDGF receptor β. Neu differentiation factor β, a potent mitogen for normal Schwann cells, was unable to stimulate proliferation of the transformed Schwann cell lines, due to a dramatic down‐regulation of the erbB3 receptor. Therefore, aberrant expression of growth factor receptors by Schwann cells, such as the PDGF receptors, could represent an important step in the process leading to Schwann cell hyperplasia in NF1. J. Cell. Physiol. 177:334–342, 1998.

Expression of Kit in neurofibromin-deficient human Schwann cells: role in Schwann cell hyperplasia associated with Type 1 Neurofibromatosis

Type 1 Neurofibromatosis (NF1) is characterized by the formation of neurofibromas, benign tumors composed mainly of Schwann cells, which can turn malignant to form neurofibrosarcomas. Neurofibromin, the protein product of the Nf1 gene, is believed to act as a tumor suppressor, accelerating the conversion of the oncogene Ras to its inactive form. The absence of neurofibromin could therefore lead to higher Ras activity in Schwann cells, resulting in uncontrolled growth through a cascade of events not yet elucidated. We describe the abnormal expression of high levels of the Kit tyrosine kinase receptor in both NF1-derived Schwann cell lines and tissue, as compared to primary Schwann cells or schwannoma-derived cells. High levels of Kit expression in the neurofibrosarcoma-derived Schwann cells correlate with a decrease in neurofibromin expression. Using inhibitors of tyrosine kinase receptors, we found that proliferation of the neurofibrosarcoma-derived cells is dependent upon activation of a subclass of tyrosine-kinase receptors. The proliferation of these cells is not dependent upon an autocrine loop involving typical Schwann cell mitogens. Finally, the proliferation of the neurofibrosarcoma-derived Schwann cells can be increased by stimulation with Kit ligand. These data implicate Kit as one of the components leading to the Schwann cell hyperplasia observed in NF1.

Angiogenic Expression Profile of Normal and Neurofibromin- Deficient Human Schwann Cells

Peripheral nerve sheath tumors from individuals with Neurofibromatosis Type 1 (NF1) are highly vascular and contain Schwann cells which are deficient in neurofibromin. This study examines the angiogenic expression profile of neurofibromin-deficient human Schwann cells relative to normal human Schwann cells, characterizing both pro-angiogenic and anti-angiogenic factors. Conditioned media from neurofibromin-deficient Schwann cell lines was pro-angiogenic as evidenced by its ability to stimulate endothelial cell proliferation and migration. Using gene array and protein array analysis, we found increased expression of pro-angiogenic factors and decreased expression of anti-angiogenic factors in neurofibromin-deficient Schwann cells relative to normal human Schwann cells. Neurofibromin-deficient Schwann cells also showed increased expression of several growth factor receptors and decreased expression of an integrin. We conclude that neurofibromin-deficient Schwann cells have dysregulated expression of pro-angiogenic factors, anti-angiogenic factors, growth factor receptors, and an integrin. These dysregulated molecules may contribute to the growth and progression of NF1 peripheral nerve sheath tumors.

Glial Cell Lines: An Overview

The importance and essential functions of glial cells in the nervous system are now beginning to be understood and appreciated. Glial cell lines have been instrumental in the elucidation of many of these properties. In this Overview, the origin and properties of most of the existing cell lines for the major glial types: oligodendroglia, astroglia, microglia and Schwann cells, are documented. Particular emphasis is given to the culture conditions for each cell line and the degree to which the line can differentiate in vitro and in vivo. The major molecular markers for each glial cell lines are indicated. Finally, methods by which the glial cell lines have been developed are noted and the future directions of glial cell line research are discussed.

Myelin basic protein (MBP) and MBP peptides are mitogens for cultured astrocytes.

After CNS demyelination, astrogliosis interferes with axonal regeneration and remyelination. We now provide evidence that myelin basic protein (MBP) can contribute to this observed astrocyte proliferation. We found that astrocytes grown in either serum‐containing or serum‐free medium proliferate in response to MBP. The mitogenic regions of MBP in both media were MBP1–44, MBP88–151 and MBP152–167. The mitogenic effect of these individual peptides was potentiated by simultaneous treatment with microglia conditioned media (CM). MBP‐induced proliferation was inhibited by suramin at concentrations known to block the fibroblast growth factor receptor (FGFR), whereas neither MBP1–44, MBP88–151 nor MBP152–167 were affected. Cholera toxin B, that binds to ganglioside GM1, inhibited the mitogenicity of MBP1–44 and had no significant effect on the mitogenicity of MBP, MBP88–151 or MBP152–167. Treatment of astrocytes with MBP and MBP152–167 caused a modest and transitory elevation of intracellular calcium, whereas treatment with MBP1–44 resulted in a substantial and sustained increase in intracellular calcium. These results suggest that for cultured astrocytes 1) FGFR and extracellular calcium play a major role in MBP mitogenicity; 2) MBP1–44, MBP88–151 and MBP152–167 are the mitogenic regions of MBP; 3) MBP1–44 and MBP152–167 interact with ganglioside GM1 and FGFR, respectively; 4) Component(s) present in microglial CM potentiate the mitogenicity of MBP1–44, MBP88–151 and MBP152–167. These data support the hypothesis that MBP related peptides in conjunction with microglial secreted factors may stimulate astrogliosis after demyelination in vivo. GLIA 29:81–90, 2000.

Neu differentiation factor regulates tau protein and mRNA in cultured neonatal oligodendrocytes

Axonal signals activate myelinogenesis via regulation of the extent to which oligodendrocyte (OLG) processes wrap around the axon. The cytoskeleton in OLG processes is actively involved in myelination and is a putative target for axonal regulation of myelination. The axon‐associated neuregulins may regulate the cytoskeleton extensions in OLG processes. Here, we report that the neuregulin neu differentiation factor (NDF) increases the expression of tau mRNA and tau protein in OLGs. Treatment of neonatal OLGs with α‐NDF or β‐NDF resulted in dramatic increases in the length of OLG processes, which appeared either as singular unbranched extensions or as a network of extensively branched processes. By immunoblot analysis with tau‐1 mAb, which recognizes the dephosphorylated form of the tau proteins, neonatal OLGs treated with α‐NDF or β‐NDF, had an increase in tau protein levels. The increase of tau levels in β‐NDF–treated cells is much greater than the twofold increase present in α‐NDF–treated cells. By immunoblot analysis with the phosphorylation‐insensitive tau‐5 mAb, β‐NDF–treated cells had a twofold increase in tau. Immunoblot analysis suggest that α‐NDF and β‐NDF promote a twofold increase in the tau protein levels in OLG, with the β‐factor also promoting a tau dephosphorylation. Using promoters spanning the amino‐terminal region of tau, we found that OLGs treated with α‐NDF or β‐NDF contained approximately twofold more tau mRNA than untreated cells. However, there was no qualitative difference between control and NDF‐treated cells in the pattern of tau mRNA isoforms expressed. A model is proposed in which the axonal NDF‐induced regulation of tau expression in OLGs may be part of the mechanism by which the axon regulates myelination. GLIA 35:147–155, 2001.

Schwann cells stimulated by axolemma-enriched fractions express cyclic AMP responsive element binding protein.

Both axolemma‐enriched fractions (AEF) and cyclic AMP have been shown to regulate the proliferation and differentiation of cultured primary Schwann cells (SC). We have evaluated the role of CREB, a transcription factor that binds to the cAMP‐responsive element, in mediating the AEF‐stimulated SC proliferation and differentiation. We detected CREB in nuclear extracts derived from SC stimulated with 40 μg/ml of AEF for 16, 24, 48, 72, and 96 hr, using a DNA‐electrophoretic mobility shift assay. Unstimulated quiescent SC contained low levels of CREB which increased to a maximal level after 48 hr of AEF treatment. Using anti‐CREB antibodies and Western blot analysis, after 24 hr of AEF treatment we first detected CREB as a 45 kDa protein which reached a maximal level of expression after 72 hr. Double labeled immunocytochemistry using anti‐CREB and anti‐5‐bromo‐2′‐deoxy‐uridine antibodies demonstrated maximal CREB expression after 72 hr of AEF treatment, closely coinciding with the temporal expression of SC proliferation. At all times examined, all AEF‐treated SC labeled by anti‐CREB antibodies were also labeled with anti‐BrdU antibodies. These observations are consistent with the view that CREB could play an important role in the induction of SC proliferation by AEF.

Antibodies to L-periaxin in sera of patients with peripheral neuropathy produce experimental sensory nerve conduction deficits

L‐Periaxin is a PDZ‐domain protein localized to the plasma membrane of myelinating Schwann cells and plays a key role in the stabilization of mature myelin in peripheral nerves. Mutations in L‐periaxin have recently been described in some patients with demyelinating peripheral neuropathy, suggesting that disruption of L‐periaxin function may result in nerve injury. In this study, we report the presence of autoantibodies to L‐periaxin in sera from two of 12 patients with diabetes mellitus (type 2)‐associated neuropathy and three of 17 patients with IgG monoclonal gammopathy of undetermined significance (MGUS) neuropathy, an autoimmune peripheral nerve disorder. By comparison, anti‐L‐periaxin antibodies were not present in sera from nine patients with IgM MGUS neuropathy or in sera from 10 healthy control subjects. The effect of anti‐L‐periaxin serum antibody on peripheral nerve function was tested in vivo by intraneural injection. Sera containing anti‐L‐periaxin antibody, but not sera from age‐matched control subjects, injected into the endoneurium of rat sciatic nerve significantly (pu2003<u20030.005, nu2003=u20033) attenuated sensory‐evoked compound muscle action potential (CMAP) amplitudes in the absence of temporal dispersion. In contrast, motor‐evoked CMAP amplitudes and latencies were not affected by intraneural injection of sera containing anti‐L‐periaxin antibody. Light and electron microscopy of anti‐L‐periaxin serum‐injected nerves showed morphologic evidence of demyelination and axon enlargement. Depleting sera of anti‐L‐periaxin antibodies neutralized the serum‐mediated effects on nerve function and nerve morphology. Together, these data support anti‐L‐periaxin antibody as the pathologic agent in these serum samples. We suggest that anti‐L‐periaxin antibodies, when present in sera of patients with IgG MGUS‐ or diabetes‐associated peripheral neuropathy, may elicit sensory nerve conduction deficits.

In vitro studies of pigment epithelium-derived factor in human Schwann cells after treatment with axolemma-enriched fraction

Pigment epithelium‐derived factor (PEDF) is a multifunctional protein with known anti‐angiogenic and trophic properties, capable of promoting the survival and growth of Schwann cells (SC). Normal rat SCs and ganglioneuroma‐derived human SCs secrete PEDF. The ability of normal SC to secrete a number of trophic factors is controlled by axonal contact. Normal human Schwann cells (HSC) and malignant peripheral nerve sheath tumors (MPNST) cell lines synthesize and secrete PEDF as determined by reverse transcription PCR analysis for PEDF mRNA, immunocytochemistry, and Western blot analysis for PEDF protein. Two MPNST cell lines secreted higher levels of PEDF than did HSC. A 90.3% decrease in PEDF mRNA and a 29.3% decrease in secreted PEDF were observed after treatment of HSC with axolemma‐enriched fraction (AEF, 100 μg/ml), a neuronal membrane fraction of the axonal plasma membrane used with cultured SC to mimic axonal contact in vitro. PEDF levels remained unchanged, however, in MPNST‐derived SC conditioned media under the same treatment paradigm. These results suggest that MPNST SC lose the ability to downregulate PEDF upon axonal contact, which is characteristic of HSC. The elevated PEDF levels expressed by MPNST cell lines may serve to promote their proliferation and survival.

Prostaglandin E(2) metabolism is activated in Schwann cell lines derived from human NF1 malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are characteristic of Neurofibromatosis type 1 (NF1), a human genetic disorder affecting approximately 1 in 3000 individuals. The absence of neurofibromin in Schwann cells results in hyperactivation of Ras, which contributes to Schwann cell hyperplasia. However, additional intracellular abnormalities in Schwann cells might contribute to the malignancy. We now report that cell lines derived from MPNSTs secrete elevated levels of prostaglandin E(2) (PGE(2)), express higher levels of phosphorylated mitogen-activated protein kinase (MAPK), phosphorylated cytosolic phospholipaseA(2) (cPLA(2)) and cyclooxygenase 2 (COX-2) when compared to normal adult human Schwann cells (nhSCs). PCR analysis reveals that NF1 MPNST cell lines express mRNA for both EP2 and EP4 prostaglandin E2 receptors, whereas nhSCs express only the EP4 receptor. COX-2 inhibitors and PGE(2) receptor antagonists decrease the proliferation of MPNST cell lines. These results indicate that prostaglandin metabolism is activated in MPNSTs and might contribute to tumor growth in NF1.