Nancy Ratner

Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

Membrane‐bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin‐based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule‐stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

The Nf2 Tumor Suppressor, Merlin, Functions in Rac-Dependent Signaling

Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis. The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton:membrane linkers. However, the molecular basis for the tumor- and metastasis- suppressing activity of merlin is unknown. We have now placed merlin in a signaling pathway downstream of the small GTPase Rac. Expression of activated Rac induces phosphorylation and decreased association of merlin with the cytoskeleton. Furthermore, merlin overexpression inhibits Rac-induced signaling in a phosphorylation-dependent manner. Finally, Nf2-/- cells exhibit characteristics of cells expressing activated alleles of Rac. These studies provide insight into the normal cellular function of merlin and how Nf2 mutation contributes to tumor initiation and progression.

The protein product of the neurofibromatosis type 1 gene is expressed at highest abundance in neurons, Schwann cells, and oligodendrocytes

von Recklinghausens neurofibromatosis (NF1) is a common inherited human disease. The events leading to patient symptoms from inheritance of a defective NF1 gene are unknown. Since knowledge of the distribution of the normal NF1 gene product should improve understanding of the pathogenesis of the disease, we raised antibodies against peptides coded by portions of the recently cloned human NF1 cDNA. These antibodies specifically recognize a 220 kd protein (neurofibromin) in both human and rat spinal cord. Neurofibromin is most abundant in the nervous system. Immunostaining of tissue sections indicates that neurons, oligodendrocytes, and nonmyelinating Schwann cells contain neurofibromin while astrocytes and myelinating Schwann cells do not. These results suggest a function for neurofibromin in the normal nervous system. Some NF1 disease manifestations, such as Schwann cell tumors and learning disabilities, may result from abnormalities in the cells that express neurofibromin.

Cognitive function and academic performance in neurofibromatosis. 1: consensus statement from the NF1 Cognitive Disorders Task Force.

Neurofibromatosis type 1 (NF1) is the most common single gene disorder to affect the human nervous system; it is inherited in an autosomal dominant manner with an estimated incidence of 1 in 3,500.l The physical features of NF1 are well characterized and include multiple cafe-au-lait spots, skinfold freckling, iris hamartomas (Lisch nodules), and benign and malignant neural tumors (e.g., neurofibromas, pheochromocytomas, and neurofibrosarcomas).’X2 CNS lesions include optic pathway gliomas, dural ectasia, and aqueduct stenosis. In addition to these specific pathologic lesions, cognitive impairment is common. Learning disabilities occur in at least 30 to 45% of children with NF1 and can be responsible for significant lifetime m~rb id i ty .~ ,~ The NF1 gene on human chromosome 17 has been ~ l o n e d ~ ~ and its protein product neurofibromin identified.*b9 The NF1 gene is usually classified as a tumor suppressor gene, as mutations in both NF1 alleles are detectable in malignant tumors associated with NF1lOJ1 and in benign tumors such as neurofibromas.12 The effects of the disorder on higher cortical function and the relationship between NF1 gene mutations, cognitive deficits, and intracranial pathology are less well understood. This consensus statement summarizes our current understanding of the frequency and nature of cognitive deficits and learning disability in children with NF1, provides recommendations for assessment and management, and examines the putative relationship between cognitive deficits and MRI signal abnormalities. We review possible pathogenetic mechanisms and future directions for research.

Large-Scale Molecular Comparison of Human Schwann Cells to Malignant Peripheral Nerve Sheath Tumor Cell Lines and Tissues

Malignant peripheral nerve sheath tumors (MPNST) are highly invasive soft tissue sarcomas that arise within the peripheral nerve and frequently metastasize. To identify molecular events contributing to malignant transformation in peripheral nerve, we compared eight cell lines derived from MPNSTs and seven normal human Schwann cell samples. We found that MPNST lines are heterogeneous in their in vitro growth rates and exhibit diverse alterations in expression of pRb, p53, p14(Arf), and p16(INK4a) proteins. All MPNST cell lines express the epidermal growth factor receptor and lack S100beta protein. Global gene expression profiling using Affymetrix oligonucleotide microarrays identified a 159-gene molecular signature distinguishing MPNST cell lines from normal Schwann cells, which was validated in Affymetrix microarray data generated from 45 primary MPNSTs. Expression of Schwann cell differentiation markers (SOX10, CNP, PMP22, and NGFR) was down-regulated in MPNSTs whereas neural crest stem cell markers, SOX9 and TWIST1, were overexpressed in MPNSTs. Previous studies have implicated TWIST1 in apoptosis inhibition, resistance to chemotherapy, and metastasis. Reducing TWIST1 expression in MPNST cells using small interfering RNA did not affect apoptosis or chemoresistance but inhibited cell chemotaxis. Our results highlight the use of gene expression profiling in identifying genes and molecular pathways that are potential biomarkers and/or therapeutic targets for treatment of MPNST and support the use of the MPNST cell lines as a primary analytic tool.

Epidermal growth factor receptor expression in neurofibromatosis type 1–related tumors and NF1 animal models

We have found that EGF-R expression is associated with the development of the Schwann cell-derived tumors characteristic of neurofibromatosis type 1 (NF1) and in animal models of this disease. This is surprising, because Schwann cells normally lack EGF-R and respond to ligands other than EGF. Nevertheless, immunoblotting, Northern analysis, and immunohistochemistry revealed that each of 3 malignant peripheral nerve sheath tumor (MPNST) cell lines from NF1 patients expressed the EGF-R, as did 7 of 7 other primary MPNSTs, a non-NF1 MPNST cell line, and the S100(+) cells from each of 9 benign neurofibromas. Furthermore, transformed derivatives of Schwann cells from NF1(-/-) mouse embryos also expressed the EGF-R. All of the cells or cell lines expressing EGF-R responded to EGF by activation of downstream signaling pathways. Thus, EGF-R expression may play an important role in NF1 tumorigenesis and Schwann cell transformation. Consistent with this hypothesis, growth of NF1 MPNST lines and the transformed NF1(-/-) mouse embryo Schwann cells was greatly stimulated by EGF in vitro and could be blocked by agents that antagonize EGF-R function.

MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors

Neurofibromatosis type 1 (NF1) patients develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). These incurable peripheral nerve tumors result from loss of NF1 tumor suppressor gene function, causing hyperactive Ras signaling. Activated Ras controls numerous downstream effectors, but specific pathways mediating the effects of hyperactive Ras in NF1 tumors are unknown. We performed cross-species transcriptome analyses of mouse and human neurofibromas and MPNSTs and identified global negative feedback of genes that regulate Ras/Raf/MEK/ERK signaling in both species. Nonetheless, ERK activation was sustained in mouse and human neurofibromas and MPNST. We used a highly selective pharmacological inhibitor of MEK, PD0325901, to test whether sustained Ras/Raf/MEK/ERK signaling contributes to neurofibroma growth in a neurofibromatosis mouse model (Nf1(fl/fl);Dhh-Cre) or in NF1 patient MPNST cell xenografts. PD0325901 treatment reduced aberrantly proliferating cells in neurofibroma and MPNST, prolonged survival of mice implanted with human MPNST cells, and shrank neurofibromas in more than 80% of mice tested. Our data demonstrate that deregulated Ras/ERK signaling is critical for the growth of NF1 peripheral nerve tumors and provide a strong rationale for testing MEK inhibitors in NF1 clinical trials.

Nf1-deficient mouse Schwann cells are angiogenic and invasive and can be induced to hyperproliferate: reversion of some phenotypes by an inhibitor of farnesyl protein transferase.

We have developed a potential model of Schwann cell tumor formation in neurofibromatosis type 1 (NF1). We show that mouse Schwann cells heterozygous or null at Nf1 display angiogenic and invasive properties, mimicking the behavior of Schwann cells from human neurofibromas. Mutations at Nf1 are insufficient to promote Schwann cell hyperplasia. Here we show that Schwann cell hyperplasia can be induced by protein kinase A activation in mutant cells. Removal of serum from the culture medium also stimulates hyperplasia, but only in some mutant cells. After serum removal, clones of hyperproliferating Schwann cells lose contact with axons in vitro, develop growth factor-independent proliferation, and exhibit decreased expression of the cell differentiation marker P0 protein; hyperproliferating cells develop after a 1-week lag in Schwann cells heterozygous at Nf1. The experiments suggest that events subsequent to Nf1 mutations are required for development of Schwann cell hyperplasia. Finally, an anti-Ras farnesyl protein transferase inhibitor greatly diminished both clone formation and hyperproliferation of null mutant cells, but not invasion; farnesyl transferase inhibitors could be useful in treating benign manifestations of NF1.

Plexiform and Dermal Neurofibromas and Pigmentation Are Caused by Nf1 Loss in Desert Hedgehog-Expressing Cells

Neurofibromatosis type 1 (Nf1) mutation predisposes to benign peripheral nerve (glial) tumors called neurofibromas. The point(s) in development when Nf1 loss promotes neurofibroma formation are unknown. We show that inactivation of Nf1 in the glial lineage in vitro at embryonic day 12.5 + 1, but not earlier (neural crest) or later (mature Schwann cell), results in colony-forming cells capable of multilineage differentiation. In vivo, inactivation of Nf1 using a DhhCre driver beginning at E12.5 elicits plexiform neurofibromas, dermal neurofibromas, and pigmentation. Tumor Schwann cells uniquely show biallelic Nf1 inactivation. Peripheral nerve and tumors contain transiently proliferating Schwann cells that lose axonal contact, providing insight into early neurofibroma formation. We suggest that timing of Nf1 mutation is critical for neurofibroma formation.

Single cell Ras-GTP analysis reveals altered Ras activity in a subpopulation of neurofibroma Schwann cells but not fibroblasts

Neurofibromatosis type 1 (NF1) is a common genetic disorder characterized by multiple neurofibromas, peripheral nerve tumors containing mainly Schwann cells and fibroblasts. TheNF1 gene encodes neurofibromin, a tumor suppressor postulated to function in part as a Ras GTPase-activating protein. The roles of different cell types and of elevated Ras-GTP in neurofibroma formation are unclear. To determine which neurofibroma cell type has altered Ras-GTP regulation, we developed an immunocytochemical assay for active, GTP-bound Ras. In NIH 3T3 cells, the assay detected overexpressed, constitutively activated K-, N-, and Ha-Ras and insulin-induced endogenous Ras-GTP. In dissociated neurofibroma cells from NF1 patients, Ras-GTP was elevated in Schwann cells but not fibroblasts. Twelve to 62% of tumor Schwann cells showed elevated Ras-GTP, unexpectedly revealing neurofibroma Schwann cell heterogeneity. Increased basal Ras-GTP did not correlate with increased cell proliferation. Normal human Schwann cells, however, did not demonstrate elevated basal Ras activity. Furthermore, compared with cells from wild type littermates, Ras-GTP was elevated in all mouseNf1 −/− Schwann cells but never inNf1 −/− mouse fibroblasts. Our results indicate that Ras activity is detectably increased in only some neurofibroma Schwann cells and suggest that neurofibromin is not an essential regulator of Ras activity in fibroblasts.