Neurofibromin 1


NF1 interaction with Ras

The neurofibromatosis type 1 (NF1) gene encodes, neurofibromin, a protein that contains a GTPase-activating protein-related domain (GRD) that stimulates intrinsic GTPase activity of Ras protein. By screening a randomly mutagenized NF1-GRD library in Saccharomyces cerevisiae, two NF1-GRD mutants (NF201 and NF204) with single amino acid substitutions were isolated that suppress the heat shock-sensitive phenotype of the RAS2(G19V) mutant. The NF1-GRD mutants also suppress the oncogenic Ras-induced transformation of NIH 3T3 mouse fibroblasts. The molecular mechanism of inhibition of the transforming Ras-specific function by the NF1-GRD mutants in mammalian cells was investigated. In human embryonic kidney (HEK) 293 cells, the mutant NF1-GRDs attenuate the stimulation of mitogen-activated protein kinase by Ras(G12V), but not by platelet-derived growth factor. In cell-free systems, purified recombinant NF1-GRD mutants show an inhibitory effect on the association of Ras.guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with Raf at several times lower concentrations than in wild type. It was revealed that the binding affinity of the mutant NF1-GRDs toward Ras.GTP gamma S is approximately 5-10 times higher than the wild type. These results suggest that the mutant NF1-GRDs tightly bind to an oncogenic Ras in its GTP-bound active conformation and block the interaction between Ras and its effector, Raf (Mori, 1995).

The kinetic properties for the enzymatic stimulation of the GTPase reaction of p21(ras) by the two GTPase-activating proteins (GAPs) p120(GAP) and neurofibromin are different. In order to understand these differences and since crystallization attempts have only been successful with truncated fragments, structure/function requirements of the catalytic core of these proteins were investigated. Differences in size of the minimal catalytic domains of these two proteins were found, determined by limited proteolysis. The minimal catalytic domain has a molecular mass of 30 kDa in the case of p120(GAP) and 26 kDa in the case of neurofibromin. Both catalytic domains contain the homology boxes as well as the residues perfectly conserved among all Ras GAPs. The C termini of these fragments are identical, whereas the N-terminal part of the minimal p120(GAP) domain is 47 amino acids longer that that of neurofibromin. These newly identified minimal catalytic fragments are as active in stimulating GTPase activity toward p21(ras) as the corresponding larger fragments GAP-334 and NF1-333 from which they have been generated via proteolytic digestion. Recently it was postulated that a fragment of 91 amino acids from neurofibromin located outside the conserved domain contains catalytic activity. This protein is shown to be unstable and has no catalytic activity. Thus, it is believed that the true minimal domains of p120(GAP) (GAP-273, residues Met714-His986) and neurofibromin (NF1-230, residues Asp1248-Phe1477) have been found (Ahmadian, 1996).

Neurofibromin plays a critical role in the downregulation of Ras proteins in neurons and Schwann cells. Thus, the ability of neurofibromin to interact with Ras is crucial for its function, since mutations in NF1 that abolish this interaction fail to maintain function. To investigate the neurofibromin-Ras interaction in a systematic manner, a yeast two-hybrid screen has been carried out using a mutant of H-ras (H-rasD92K) defective for interaction with the GTPase-activated protein-related domain (GRD) of NF1. Two screens of a randomly mutagenized NF1-GRD library led to the identification of seven novel NF1 mutants. Characterization of the NF1-GRD mutants reveals that one class of mutants are allele specific for H-raSD92K. These mutants exhibit increased affinity for H-raSD92K and significantly reduced affinity for wild-type H-ras protein. Furthermore, they do not interact with another H-ras mutant defective for interaction with GTPase-activating proteins. Another class of mutants are high-affinity mutants that exhibit dramatically increased affinity for both wild-type and mutant forms of Ras. They also exhibit a striking ability to suppress the heat shock sensitive traits of activated RAS2G19v in yeast cells. Five mutations cluster within a region encompassing residues 1391 to 1436 (region II). Three NF1 patient mutations have previously been identified in this region. Two mutations occur in a region encompassing residues 1262 to 1276 (region I). Combining high-affinity mutations from both regions results in even greater affinity for Ras. These results demonstrate that two distinct regions of NF1-GRD are involved in the Ras interaction and that single amino acid changes can affect NF1's affinity for Ras (Morcos, 1996).

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. A consensus Ras binding sequence shared among a subset of Ras effectors has now been defined. Peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, the demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents (Clark, 1996).

Neurofibromin acts as a GTPase activating protein (GAP) on Ras; based on homology to p120GAP, a segment spanning 250-400 aa and termed GAP-related domain (NF1GRD; 25-40 kDa) has been shown to be responsible for GAP activity and represents the only functionally defined segment of neurofibromin. Missense mutations found in NF1 patients map to NF1GRD, underscoring its importance for pathogenesis. X-ray crystallographic analysis of a proteolytically treated catalytic fragment of NF1GRD comprising residues 1198-1530 (NF1-333) of human neurofibromin reveals NF1GRD as a helical protein that resembles the corresponding fragment derived from p120GAP (GAP-334). A central domain (NF1c) containing all residues conserved among RasGAPs is coupled to an extra domain (NF1ex), which, despite very limited sequence homology, is surprisingly similar to the corresponding part of GAP-334. Numerous point mutations found in NF1 patients or derived from genetic screening protocols can be analysed on the basis of the three-dimensional structural model, which also allows identification of the site where structural changes in a differentially spliced isoform are to be expected. Based on the structure of the complex between Ras and GAP-334 described earlier, a model of the NF1GRD-Ras complex is proposed that is used to discuss the strikingly different properties of the Ras-p120GAP and Ras-neurofibromin interactions (Scheffzek, 1998).

Ras proteins are guanine-nucleotide binding proteins that have a low intrinsic GTPase activity that is enhanced 10(5)-fold by both the GTPase-activating proteins (GAPs) p120-GAP and neurofibromin. Comparison of the primary sequences of RasGAPs shows two invariant arginine residues (Arg1276 and Arg1391 of neurofibromin). In this study, site-directed mutagenesis was used to change each of these residues in the catalytic domain of neurofibromin (NF1-334) to alanine. The ability of the mutant proteins to bind to Ras.GTP and to stimulate their intrinsic GTPase rate was then determined by kinetic methods under single turnover conditions using a fluorescent analogue of GTP. The separate contributions of each of these residues to catalysis and binding affinity to Ras were then measured. Both the R1276A and the R1391A mutant NF1-334 proteins are 1000-fold less active than wild-type NF1-334 in activating the GTPase, when measured at saturating concentrations. In contrast, there is only a minor effect of either mutation on NF1-334 affinity for wild-type Ha-Ras. These data are consistent with both arginines being required for efficient catalysis. Neither arginine is absolutely essential, because the mutant NF1-334 proteins increase the intrinsic Ras.GTPase by at least 100-fold. The roles of Arg1276 and Arg1391 in neurofibromin are consistent with proposals based on the recently published x-ray structure of p120-GAP complexed with Ras (Sermon, 1998).

NF1 effects downstream of Ras

Primary leukaemic cells from children with NF1 show a selective decrease in NF1-like GTPase activating protein (GAP) activity for Ras but retain normal cellular GAP activity. Leukaemic cells also show an elevated percentage of Ras in the GTP-bound conformation. JCML cells are hypersensitive to granulocyte-macrophage colony stimulating factor (GM-CSF), and a similar pattern of aberrant growth in haematopoietic cells from Nf1-/- mouse embryos is observed. These data define a specific role for neurofibromin in negatively regulating GM-CSF signaling through Ras in haematopoietic cells and they suggest that hypersensitivity to GM-CSF may be a primary event in the development of JCML (Bollag, 1996).

Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by loss of the NF1 gene, is characterized clinically by neurofibromas and more rarely by neurofibrosarcomas. Neurofibromin, the protein encoded by NF1, possesses an intrinsic GTPase accelerating activity for the Ras proto-oncogene. Through this activity, neurofibromin is a negative regulator of Ras. The Pak protein kinase (see Drosophila PAK-kinase) is a candidate for a downstream signaling protein that may mediate Ras signals because it is activated by Rac and Cdc42, two small G proteins required for Ras signaling. Pak mutants have been used to explore the role of Pak in Ras signaling in Schwann cells, the cells affected in NF1. Whereas an activated Pak mutant does not transform cells, dominant negative Pak mutants are potent inhibitors of Ras transformation of rat Schwann cells and of a neurofibrosarcoma cell line from an NF1 patient. Although activated Pak stimulates jun-N-terminal kinase, inhibition of Ras transformation by dominant negative Pak does not require inhibition of jun-N-terminal kinase. Instead, the Pak mutants appear to inhibit transformation by preventing Ras activation of the ERK/mitogen-activated protein kinase cascade. These results have implications for understanding of NF1 because a neurofibrosarcoma cell line derived from a patient with NF1 was reverted by stable expression of the Pak dominant negative mutants (Tang, 1998).

Alternative splicing and RNA editing of NF1 mRNA

Neurofibromatosis 1 (NF1) is a common autosomal dominant disorder in which affected individuals develop benign and malignant tumors as well as non-tumor-related abnormalities, such as seizures and learning disabilities. An NF1 isoform arising from the alternative splicing of exon 9a with predominant central nervous system (CNS) expression is reported. Exon 9a expression is enriched in neurons of the forebrain, specifically septum, striatum, cortex, hippocampus and olfactory bulb with significantly less expression in brainstem, cerebellum and spinal cord. This pattern of NF1 exon 9a expression correlates with the postnatal maturation of these neurons and suggests a role for NF1 in neuronal differentiation (Geist, 1996).

A functional mooring sequence, known to be required for apolipoprotein B (apoB) mRNA editing, exists in the mRNA encoding the neurofibromatosis type I (NF1) tumor suppressor. Editing of NF1 mRNA modifies cytidine in an arginine codon (CGA) at nucleotide 2914 to a uridine (UGA), creating an in-frame translation stop codon. NF1 editing occurs in normal tissue but is several-fold higher in tumors. In vitro editing and transfection assays demonstrate that apoB and NF1 RNA editing take place in both neural tumor and hepatoma cells. Unlike apoB, NF1 editing does not demonstrate dependence on rate-limiting quantities of APOBEC-1 (the apoB editing catalytic subunit), suggesting that different trans-acting factors may be involved in the two editing processes (Skuse, 1996).

Post-transcriptional regulation of NF1

3'-untranslated regions of various mRNAs have been shown to contain sequence motifs that control mRNA stability, translatability, and efficiency of translation as well as intracellular localization. Protein binding regions have been sought for the long and highly conserved 3'UTR of the mRNA coding for neurofibromin, a well-known tumor suppressor protein, whose genetic deficiency causes the autosomal dominant disease neurofibromatosis type 1 (NF1). Five RNA fragments are able to undergo specific binding to proteins from cell lysates (NF1-PBRs, NF1-protein-binding regions). Additionally the Elav-like protein HuR binding to NF1-PBR1, has been identified. HuR interacts with AU-rich elements in the 3'UTR of many protooncogenes, cytokines, and transcription factors, thereby regulating the expression of these mRNAs on the posttranscriptional level. Transfection assays with a CAT reporter construct reveal reduced expression of the reporter, suggesting that HuR may be involved in the fine-tuning of the expression of the NF1 gene (Haeussler, 2000).

Effects of NF1 mutation

A potential model has been developed for Schwann cell tumor formation in neurofibromatosis type 1 (NF1). 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. 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 diminishes both clone formation and hyperproliferation of null mutant cells, but not invasion; farnesyl transferase inhibitors could be useful in treating benign manifestations of NF1 (Kim, 1997).

Comparisons were made of whole cell voltage clamp recordings from cultures of normal Schwann cells (SC) from three human subjects and from three neurofibrosarcoma cell lines. The whole cell K+ (K) currents of normal and tumor cells can be divided into three types based on voltage activation range, pharmacology, and macroscopic inactivation: A type current, tetraethylammonium- (TEA-) only-sensitive current, and inward rectifier current. The most conspicuous difference between normal and tumor cells is the nature of K currents present. Normal SC K currents are inactivating; A type currents are blocked by extracellular 4-aminopyridine (4-AP; 5 mM). The whole cell K currents of tumor cells are noninactivating due to the presence of non-inactivating A current, or non-inactivating, TEA-only sensitive current, or both, despite the presence of inactivating A current in some tumor cells. TEA-only-sensitive currents, which are 4-AP-insensitive and noninactivating, are common in all three tumor cell lines, but are not observed in normal SC. Inward rectifier K currents are a conspicuous feature of two of the tumor cells lines but are rarely observed in whole cell recordings of normal SC. The properties of Na+ currents recorded in both normal and tumor cells are not significantly different. Treatment of normal SC with a membrane-permeant analog of cyclic AMP (cAMP) results in functional expression of the TEA-only-sensitive K currents typical of tumor cells. These results establish the abnormal ion channel profile of neurofibromatosis type 1 (NF1)-tumor cells and suggest that regulation of ionic currents by second messengers may involve the NF1 gene (Fieber, 1998).

Endocardial cushions are the precursors of the cardiac valves and form by a process of epithelial-mesenchymal transformation. Secreted growth factors from myocardium induce endocardial cells to transform into mesenchyme and invade the overlying extracellular matrix. The product of the Nf1 neurofibromatosis gene is required to regulate this event. In the absence of neurofibromin, mouse embryo hearts develop overabundant endocardial cushions due to hyperproliferation and lack of normal apoptosis. Neurofibromin deficiency in explant cultures is reproduced by activation of ras signaling pathways, and the Nf1(-/-) mutant phenotype is prevented by inhibiting ras in vitro. These results indicate that neurofibromin normally acts to modulate epithelial-mesenchymal transformation and proliferation in the developing heart by down regulating ras activity (Lakkis, 1998).

Neurofibromatosis type I (NF1) is one of the most commonly inherited neurological disorders in humans, affecting approximately one in 4,000 individuals. NF1 results in a complex cluster of developmental and tumour syndromes that include benign neurofibromas, hyperpigmentation of melanocytes and hamartomas of the iris. Some NF1 patients may also show neurologic lesions, such as optic pathway gliomas, dural ectasia and aqueduct stenosis. Importantly, learning disabilities occur in 30% to 45% of patients with NF1, even in the absence of any apparent neural pathology. The learning disabilities may include a depression in mean IQ scores, visuoperceptual problems and impairments in spatial cognitive abilities. Spatial learning has been assessed with a variety of cognitive tasks and the most consistent spatial learning deficits have been observed with the Judgement of Line Orientation test. It is important to note that some of these deficits could be secondary to developmental abnormalities and other neurological problems, such as poor motor coordination and attentional deficits. Previous studies have suggested a role for neurofibromin in brain function. (1) The expression of the Nf1 gene is largely restricted to neuronal tissues in the adult. (2) This GTPase activating protein may act as a negative regulator of neurotrophin-mediated signaling. (3) Immunohistochemical studies suggest that activation of astrocytes may be common in the brain of NF1 patients. The Nf1+/- mutation also affects learning and memory in mice. As in humans, the learning and memory deficits of the Nf1+/- mice are restricted to specific types of learning; they are not fully penetrant; they can be compensated for with extended training, and they do not involve deficits in simple associative learning (Silva, 1997).

Nerve growth factor (NGF) is a required differentiation and survival factor for sympathetic neurons and a majority of neural crest-derived sensory neurons in the developing vertebrate peripheral nervous system. Although much is known about the function of NGF, the intracellular signaling cascade that it uses continues to be a subject of intense study. p21 ras signaling is considered necessary for sensory neuron survival. How additional intermediates downstream or in parallel may function has not been fully understood as yet. Two intracellular signaling cascades, extra cellular regulated kinase (erk) and phosphatidylinositol-3 (PI 3) kinase, transduce NGF signaling in the pheochromocytoma cell line PC12. To elucidate the role these cascades play in survival and differentiation, a combination of recombinant adenoviruses and chemical inhibitors were used to perturb these pathways in sensory neurons from wild-type mice and mice deficient for neurofibromin, in which the survival and differentiation pathway is constitutively active. Ras activity is both necessary and sufficient for the survival of embryonic sensory neurons. Downstream of ras, however, the erk cascade is neither required nor sufficient for neuron survival or overall differentiation. Instead, the activity of PI 3 kinase is necessary for the survival of the wild-type and neurofibromin-deficient neurons. Therefore, it is concluded that in sensory neurons, NGF acts via a signaling pathway, which includes both ras and PI 3 kinase (Klesse, 1998).

Neural tube defects are common and serious human congenital anomalies. These malformations have a multifactorial etiology and can be reproduced in mouse models by mutations of numerous individual genes and by perturbation of multiple environmental factors. The identification of specific genetic interactions affecting neural tube closure will facilitate an understanding of molecular pathways regulating normal neural development and will enhance the ability to predict and modify the incidence of spina bifida and other neural tube defects. A genetic interaction is reported between Nf1, encoding the intracellular signal transduction protein neurofibromin, and Pax3, a transcription factor gene mutated in the Splotch mouse. Both Pax3 and Nf1 are important for the development of neural crest-derived structures and the central nervous system. Splotch is an established model of folate-sensitive neural tube defects, and homozygous mutant embryos develop spina bifida and sometimes exencephaly. Neural development is grossly normal in heterozygotes and neural tube defects are not seen. In contrast, a low incidence of neural tube defects is found in heterozygous Splotch mice that also harbor a mutation in one Nf1 allele. All compound homozygotes have severe neural tube defects and die earlier in embryogenesis than either Nf1(-/-) or Sp(-/-) embryos. Occasional exencephaly occurs in Nf1(-/-) mice and more subtle CNS abnormalities are identified in normal-appearing Nf1(-/-) embryos. Though other genetic loci and environmental factors affect the incidence of neural tube defects in Splotch mice, these results establish Nf1 as the first known gene to act as a modifier of neural tube defects in Splotch. It seems most likely that Pax3 and Nf1 function in parallel pathways where both contribute to normal neural tube closure (Lakkis, 1999).

Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder that affects growth properties of neural-crest-derived cell populations. In addition, approximately one-half of NF1 patients exhibit learning disabilities. To characterize NF1 function both in vitro and in vivo, the embryonic lethality of NF1 null mouse embryos was circumvented by generating a conditional mutation in the NF1 gene using Cre/loxP technology. Introduction of a Synapsin I promoter driven Cre transgenic mouse strain into the conditional NF1 background ablates NF1 function in most differentiated neuronal populations. These mice show abnormal development of the cerebral cortex, which suggests that NF1 plays an indispensable role in this aspect of CNS development. Furthermore, although they are tumor free, these mice display extensive astrogliosis in the absence of conspicuous neurodegeneration or microgliosis. These results indicate that NF1-deficient neurons are capable of inducing reactive astrogliosis via a non-cell autonomous mechanism (Zhu, 2001).

Neural crest-derived melanocyte precursors (MPs) in avian and murine embryos emerge from the dorsal neural tube into a migration staging area (MSA). MPs subsequently migrate from the MSA on a dorsolateral pathway between the dermamyotome and the overlying epithelium. In mouse embryos, MPs express the receptor tyrosine kinase, KIT, and require its cognate ligand, Mast cell growth factor (MGF), for survival and differentiation. Prior to the onset of MP migration, MGF is expressed on the dorsolateral pathway at some distance from cells in the MSA and appears to be required for normal MP development. To learn if MGF is required solely for MP survival on this pathway, or if it also provides directional cues for migration, survival was uncoupled from chemoattractive or motogenic functions of this ligand using mice that carry a targeted mutation at the Neurofibromin (Nf1) locus and consequently lack RAS-GAP function. Nf1-mutant MPs survive in the absence of MGF in vitro and in vivo and Nf1-mutant MPs disperse normally on the lateral migration pathway in the presence of MGF. In contrast, Nf1-mutant MPs persist in the location of the MSA but are not observed on the lateral migration pathway in double-mutant mice that also lack MGF. It is concluded that MGF/KIT function provides a signal required for directed migration of the MPs on the lateral pathway in vivo, independent of its function in survival. It is further suggested that the MGF mediates MP migration through a signaling pathway that does not involve RAS (Wehrle-Haller, 2001).

Individuals with the neurofibromatosis 1 (NF1)-inherited tumor predisposition syndrome develop low-grade astrocytomas. The NF1 tumor suppressor gene product neurofibromin exhibits GTPase-activating activity (GAP) toward RAS, such that loss of neurofibromin expression leads to high levels of activated RAS and increased cell proliferation. Nf1 inactivation in astrocytes leads to increased cell proliferation in vitro and in vivo, accompanied by increased RAS pathway activation. Studies on Nf1 mutant Drosophila have suggested that neurofibromin might also regulate cAMP signaling. Because intracellular cAMP levels have profound effects on astrocyte growth control, the contribution of neurofibromin to astrocyte cAMP regulation was examined. Nf1 inactivation in astrocytes results in reduced cAMP generation in response to PACAP and attenuated calcium influx and Rap1 activation. Based on the differential effects of forskolin and dibutyryl-cAMP on Nf1-/- astrocytes, neurofibromin likely functions at the level of adenylyl cyclase activation. Last, the reintroduction of a fragment of neurofibromin containing residues sufficient for restoring RAS-GAP function in Nf1-/- cells resulted in only partial restoration of neurofibromin-mediated cAMP regulation. These results demonstrate that neurofibromin positively influences cAMP generation and activation of cAMP growth regulatory targets in astrocytes and expands the role of the NF1 gene in astrocyte growth regulation (Dasgupta, 2003).

The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. This study compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. MEK inhibitors were shown to be ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. Clone-specific retroviral integrations were cloned to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38alpha. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance (Lauchle, 2009).

Recent studies have shown that neuroglial progenitor/stem cells (NSCs) from different brain regions exhibit varying capacities for self-renewal and differentiation. This study used neurofibromatosis-1 (NF1) as a model system to elucidate a novel molecular mechanism underlying brain region-specific NSC functional heterogeneity. Nf1 loss leads to increased NSC proliferation and gliogenesis in the brainstem, but not in the cortex. Using Nf1 genetically engineered mice and derivative NSC neurosphere cultures, it was shown that this brain region-specific increase in NSC proliferation and gliogenesis results from selective Akt hyperactivation. The molecular basis for the increased brainstem-specific Akt activation in brainstem NSCs is the consequence of differential rictor expression, leading to region-specific mammalian target of rapamycin (mTOR)/rictor-mediated Akt phosphorylation and Akt-regulated p27 phosphorylation. Collectively, these findings establish mTOR/rictor-mediated Akt activation as a key driver of NSC proliferation and gliogenesis, and identify a unique mechanism for conferring brain region-specific responses to cancer-causing genetic changes (Lee, 2010).

Methylation NF1 gene

Recent reports of cytosine methylation occurring at CpA and CpT dinucleotides in murine ES cells as well as in Drosophila have renewed interest in methylation at sites other than CpGs. Examination of the murine neurofibromatosis type 1 gene by sodium bisulfite genomic sequencing has revealed non-CpG methylation primarily in the oocyte and the maternally derived allele of the 2-cell embryo, with markedly lower levels found in sperm. Non-CpG methylation is not found in later stages of embryo development or in adult tissue. These results suggest that maternal-specific de novo non-CpG methylation occurs sometime between ovulation and formation of the 2-cell embryo, while during the same period the paternally derived allele undergoes site-specific active demethylation. These data demonstrate both stage and parent-of-origin specific changes in methylation patterns within the neurofibromatosis type 1 coding region-involving cytosines located at both CpG and non-CpG dinucleotides (Haines, 2001).

Neurofibromin 1: Biological Overview | Developmental Biology | Effects of Mutation | References

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