Neurofibromin 1

DEVELOPMENTAL BIOLOGY

Embryonic

The Drosophila Nf1 gene is expressed in low amounts during all developmental stages (The, 1997)

Effects of Mutation or Deletion

Unlike Nf1-deficient mice, Drosophila Nf1 mutants are viable and fertile. Although heterozygotes have no obvious defects, homozygotes of either of two alleles are 20% to 25% smaller than flies of the parental strain during all postembryonic stages. This growth defect is not accompanied by delayed eclosion or bristle phenotypes that are observed with several Minute mutations. To determine whether reduced cell growth underlies the smaller size of Nf1 mutants, the wings of wild-type and mutant animals were compared. The linear dimensions of mutant wings are 20-25% smaller than those of wild-type flies. Because each wing epidermal cell secretes a single hair, cell densities can be determined by counting the number of hairs in a defined region. Both homozygous mutants have a 30% to 35% higher density than flies of the parental line. Since no difference in cell density is observed between multiple Nf1 induced clones and surrounding tissue, the reduced size of the wing cells reflects a nonautonomous requirement for Nf1, perhaps reflecting a hormonal deficiency or impaired nutrition or metabolism. the eyes of Nf1 mutants show a reduced number of ommatidia of normal size and structure. Nf1 deficient embryos are of normal size. Thus, loss of Nf1 affects the growth of various tissues in different ways (The, 1997).

Nf1 mutants differ from wild-tupe flies in an assay that determines the number of flies that fly away upon release form their containers, either spontaneously or after repeated prodding. About 15% of Nf1 mutant flies fail to respond, whereas only 3% of parental flies do not respond. the reduced escape rate does not reflect obvious anatomical defects of the peripheral nervous system or the musculature. Electrophysiological studies show that the mutants have a defect at the larval neuromuscular junction that is rescued by pharmacological manipulation of the cAMP-PKA pathway and that is insenstive to manipulation of Ras1-mediated signaling (The, 1997)

A circadian output in Drosophila mediated by neurofibromatosis-1 and Ras/MAPK

Output from the circadian clock controls rhythmic behavior through poorly understood mechanisms. In Drosophila, null mutations of the neurofibromatosis-1 (Nf1) gene produce abnormalities of circadian rhythms in locomotor activity. Mutant flies show normal oscillations of the clock genes period (per) and timeless (tim) and of their corresponding proteins, but altered oscillations and levels of a clock-controlled reporter. Mitogen-activated protein kinase (MAPK) activity is increased in Nf1 mutants, and the circadian phenotype is rescued by loss-of-function mutations in the Ras/MAPK pathway. Thus, Nf1 signals through Ras/MAPK in Drosophila. Immunohistochemical staining has revealed a circadian oscillation of phospho-MAPK in the vicinity of nerve terminals containing pigment-dispersing factor (PDF), a secreted output from clock cells, suggesting a coupling of PDF to Ras/MAPK signaling (Williams, 2001).

Nf1 controls perineural glial growth as part of interacting neurotransmitter-mediated signaling pathways

Drosophila peripheral nerves, similar structurally to the peripheral nerves of mammals, comprise a layer of axons and inner glia, surrounded by an outer perineurial glial layer. Although it is well established that intercellular communication occurs among cells within peripheral nerves, the signaling pathways used and the effects of this signaling on nerve structure and function remain incompletely understood. The Drosophila peripheral nerve is a favorable system for the study of intercellular signaling. Growth of the perineurial glia is controlled by interactions among five genes: inebriated (ine), which encodes a member of the Na⁺/Cl^--dependent neurotransmitter transporter family; ether a go-go (eag), which encodes a potassium channel; pushover (push), which encodes a large, Zn2+-finger-containing protein; amnesiac, which encodes a putative neuropeptide related to the pituitary adenylate cyclase activator peptide, and NF1, the Drosophila ortholog of the human gene responsible for type 1 neurofibromatosis. In other Drosophila systems, push and NF1 are required for signaling pathways mediated by Amn or the pituitary adenylate cyclase activator peptide. These results support a model in which the Amn neuropeptide, acting through Push and NF1, inhibits perineurial glial growth, whereas the substrate neurotransmitter of Ine promotes perineurial glial growth. Defective intercellular signaling within peripheral nerves might underlie the formation of neurofibromas, the hallmark of neurofibromatosis (Yager, 2001).

Mutations in two genes that affect neuronal excitability also affect the structure of the peripheral nerve: double mutants defective in ine, and push exhibit an extremely thickened nerve, which is a phenotype that is clearly visible with the dissecting microscope. To understand the cellular basis for this phenotype, transmission electron microscopy was performed on cross-sections of peripheral nerves. This analysis demonstrated that the push¹ and ine¹;push¹ double mutants exhibit a normal axon and peripheral glial layer, but a thickened perineurial glial layer. This increased perineurial thickness is expressed only moderately in push¹ but very strongly in the ine¹;push¹ double mutant. This increase in thickness is accompanied by an increase in the number of mitochondria within perineurial glial thin sections, suggesting that an increase in cell material accompanies this increased thickness. The ine¹;push¹ phenotype is significantly rescued in transgenic larvae expressing the 943-aa Ine isoform, called Ine-P1, under the transcriptional control of the heat-shock promoter. In particular, perineurial glial thickness in ine¹ push¹; hs-ine-P1 larvae, even in the absence of heat shock, was reduced to 2.0 ± 0.2 µm from 3.1 ± 0.3 in ine¹;push¹. The observed synergistic interaction between ine and push mutations suggests that each gene controls perineurial glial growth through partially redundant pathways (Yager, 2001).

In certain respects, mutations in ine confer phenotypes similar to mutations in the K⁺ channel structural gene eag. In particular, both eag and ine mutations interact synergistically with mutations in the K⁺ channel encoded by Shaker to cause a characteristic 'indented thorax and down-turned wings' phenotype, which is not exhibited by any of the single mutants. Because of this phenotypic similarity, the possibility that eag mutations might also affect perineurial glial thickness was tested. eag¹ resembles ine¹ in the control of perineurial glial growth: eag¹;push¹ double mutants, but not the eag¹ single mutant, exhibit strongly potentiated perineurial glial growth. This increased growth is similar to, but less extreme than, what is observed in ine¹;push¹. Double mutants for eag¹; push² also exhibit a thickened perineurial glial layer. In contrast, eag and ine mutations fail to display a comparable synergistic interaction (Yager, 2001).

Mutations in push were identified independently on the basis of defective segregation of nonrecombinant chromosomes in the female meiosis. push was implicated in this process as an intermediate in a signaling pathway mediated by the PACAP-like neuropeptide encoded by amn (S. Hawley, personal communication to Yager, 2001). This observation raised the possibility that push likewise affects perineurial glial growth by acting as an intermediate from an Amn signal. Consistent with this hypothesis, the amn^X8 deletion mutation increases perineurial glial thickness, and this increase is significantly rescued in transgenic flies expressing amn⁺ (Yager, 2001).

A second signaling pathway mediated by a PACAP-like neuropeptide has been identified in Drosophila. In this pathway, the larval muscle responds to application of PACAP by activating a voltage-gated potassium channel. This activation requires NF1, the ortholog of the human gene responsible for type 1 neurofibromatosis. The possibility was tested that NF1 might affect perineurial glial growth. The NF1^P2-null mutant exhibits strong potentiation of perineurial glial thickness in combination with ine¹. This thickness is much greater than the thickness observed in ine¹ mutants carrying K33, the NF1⁺ parent chromosome of NF1^P2. The increased glial thickness of ine¹; NF1^P2 is fully rescued by heat-shock-induced expression of the NF1⁺ transgene. However, unlike push, the phenotype of NF1^P2 is potentiated only moderately by the eag¹ mutation. In contrast, perineurial glial thickness in the push¹; NF1^P2 double mutant was 2.1 ± 0.15 µm, which is significantly thicker than either push¹ or NF1^P2, but not significantly different from amn^X8. These results are consistent with the possibility that push and NF1 mediate the amn signal through parallel partially redundant pathways (Yager, 2001).

These results are consistent with a model in which two neurotransmitter-mediated signaling pathways exert opposing effects on perineurial glial growth. One pathway, mediated by the Amn neuropeptide, inhibits perineurial glial growth. This pathway requires NF1 and Push activity. The second pathway, mediated by the substrate neurotransmitter of Ine (which will be called NT here), promotes perineurial glial growth. In this pathway, mutations in ine or eag each increase signaling by NT: ine mutations increase NT signaling by eliminating the NT reuptake transporter thus increasing NT persistence, whereas eag mutations increase NT signaling by increasing NT release as a consequence of increased excitability. These pathways interact such that the most extreme effects on perineurial glial growth are observed when the NT pathway is overstimulated and the Amn pathway is disrupted simultaneously. The genetic interactions that form the basis for this interpretation require that the mutations under investigation be null. Although the eag¹ mutation tested has not been characterized molecularly, the mutations in each of the other four genes analyzed are known to be or are strongly suspected to be null. Direct neuron-perineurial glia signaling is unlikely because the peripheral glia, which form the blood-brain barrier, are expected to be an impervious barrier to intercellular traffic. Two alternative mechanisms could underlie this signaling. In the first mechanism (direct peripheral glia-perineurial glia signaling), the peripheral glia release each neurotransmitter, and the perineurial glia respond. In the second mechanism (indirect signaling), each neurotransmitter is released by neurons, and the peripheral glia respond by regulating the release of a trophic factor that acts on perineurial glia (Yager, 2001).

Although direct signaling seems to be the simplest possibility, indirect signaling is most consistent with previous studies. As described above, both invertebrate and mammalian motor neurons can release small molecule and peptide neurotransmitters that affect properties of Schwann cells. A similar motor nerve terminal-peripheral glia communication could occur in Drosophila, because first boutons at the larval neuromuscular junction are covered by peripheral glia. This observation raises the possibility that Drosophila peripheral glia might respond to Amn and NT released from motor nerve terminals, and propagate these signals along the length of the nerve via gap junctions. However, the alternative possibility of NT release from along the length of axons, as has been suggested in other systems, cannot be ruled out. In addition, mammalian Schwann cells release trophic factors such as Desert hedgehog (Dhh) to induce growth of the surrounding perineurium, and astrocytes can respond to glutamate application by releasing a substance that affects blood vessels. This model predicts that peripheral glia release a trophic factor that behaves similarly to Dhh. The prediction that Drosophila NF1 acts within peripheral glia is consistent with the likelihood that mammalian NF1 acts within Schwann cells as well (Yager, 2001).

The possible effects of the thickened perineurial glia on motor neuron function are unclear. Mutations in four of the genes that affect perineurial glial thickness (eag, NF1, ine, and push) were each shown in previous studies to increase either neuronal or muscle membrane excitability, which raises the possibility of a correlation between excitability and perineurial glial growth. However, no increases in neuronal excitability have been detected in the amn mutant or the ine; NF1 double mutant (greater than that conferred by the ine mutation alone), despite the presence of greatly thickened perineurial glia in these genotypes. It is possible that the effects on neuronal excitability of these genotypes might be subtler than the assays can detect, or that the participation of these genes in both perineurial glial growth and excitability is coincidental (Yager, 2001).

These results are consistent with the previous observations that push and NF1 act downstream of the Amn/PACAP receptor. However, the precise nature of the interactions among these proteins is unknown. Thus, it is possible that the interactions are direct, and that Push, the NF1-encoded protein Neurofibromin, and the Amn receptor bind to each other in a macromolecular complex. Alternatively, it is possible that Push and Neurofibromin mediate the effects of Amn only indirectly. In either case, the observation that the push¹; NF1^P2 double mutant exhibits a perineurial glial thickness much greater than push¹ or NF1^P2 alone is consistent with the possibility that Push and Neurofibromin mediate the Amn signal through parallel partially redundant pathways (Yager, 2001).

The indirect signaling model could explain the partial cell-nonautonomy of NF1 in neurofibroma formation. Neurofibromas most likely initiate in individuals heterozygous for NF1 mutations by loss of the NF1⁺ allele in Schwann cells. However, neurofibromas contain, in addition to Schwann cells, cells derived from fibroblasts, perineurial cells, and neurons, which are thought to remain phenotypically NF1⁺. It is suggested that NF1 mutant Schwann cells cause the overproliferation of their wild-type neighbors by oversecreting trophic factors, and that this oversecretion might ultimately occur as a consequence of defective receipt of a neurotransmitter signal from neurons (Yager, 2001).

Effect of neurofibromatosis type I mutations on a novel pathway for adenylyl cyclase activation requiring neurofibromin and Ras

Neurofibromatosis type I (NFI) is a common genetic disorder that causes nervous system tumors, and learning and memory defects in human and in other animal models. A novel growth factor stimulated adenylyl cyclase (AC) pathway has been identified in the Drosophila brain, which is disrupted by mutations in the epidermal growth factor receptor (EGFR), neurofibromin (NF1) and Ras, but not Galpha_s. This is the first demonstration in a metazoan that a receptor tyrosine kinase (RTK) pathway, acting independently of the heterotrimeric G-protein subunit Galpha_s, can activate AC. This study also shows that Galpha_s is the major Galpha isoform in fly brains, and a second AC pathway is defined stimulated by serotonin and histamine requiring NF1 and Galpha_s. A third, classical Galpha_s-dependent AC pathway, is stimulated by Phe-Met-Arg-Phe-amide (FMRFamide) and dopamine. Using mutations and deletions of the human NF1 protein (hNF1) expressed in Nf1 mutant flies, it is shown that Ras activation by hNF1 is essential for growth factor stimulation of AC activity. Further, it is demonstrated that sequences in the C-terminal region of hNF1 are sufficient for NF1/Galpha_s-dependent neurotransmitter stimulated AC activity, and for rescue of body size defects in Nf1 mutant flies (Hannan, 2006).

This study defines three separate pathways for AC activation: (1) a novel pathway for AC activation, downstream of growth factor stimulation of EGFR that requires both Ras and NF1, but not Galpha_s; (2) an NF1/Galpha_s-dependent AC pathway operating through the Rutabaga-AC (Rut-AC) and stimulated by serotonin and histamine, as observed in the larval brain; (3) a classical G-protein coupled receptor-stimulated AC pathway operating through Galpha_s alone. The Rut-AC pathway may also be stimulated by PACAP38 at the larval neuromuscular junction and in adult heads as shown in previous studies. The AC activated by NF1/Ras (AC-X), or Galpha_s (AC-Y), has not yet been identified (Hannan, 2006).

This study shows for the first time that Ras can stimulate AC in an NF1-dependent manner in higher organisms, via an RTK-coupled pathway that is independent of the Galpha_s G-protein. The functionality of human NF1 in the fly system, and the high degree of identity between human and fly NF1 (60%), suggests that similar pathways for AC activation may also operate in mammals. Previous studies failed to detect stimulation of AC by Ras in cultured vertebrate cell lines, and in Xenopus oocytes, however, these cell types may not contain sufficient NF1 to support NF1/Ras-dependent AC activation. This is consistent with the observation that levels of both Ras and NF1 are critical for stimulation of AC activity in adult head membranes. The reported EGF activation of AC in cardiac myocytes and other tissues requires both Galpha_s, and the juxtamembrane domain of the EGFR, which is not present in the Drosophila EGFR (Hannan, 2006).

Experiments with human NF1 mutants show that the GRD domain and the RasGAP activity of NF1 are both necessary and sufficient for growth factor-stimulated NF1/Ras-dependent AC activity. It is also concluded that C-terminal residues downstream of the GRD are critical for both body size regulation and neurotransmitter-stimulated NF1/Galpha_s-dependent AC activity, thus defining for the first time a region outside the GRD that contributes to this pathway. Interestingly, expression of a human NF1 GRD fragment in Nf1-/- astrocytes results in only partial restoration of NF1-mediated increases in cAMP levels in response to PACAP. Thus, regions outside the GRD also seem to be necessary for activation of AC in these mammalian cells (Hannan, 2006).

Thus, NF1, while being a negative regulator of Ras, is also actively involved in stimulation of AC activity. Moreover, it regulates AC activity through at least two different mechanisms, one of which depends on the RasGAP activity of NF1. The multifunctional nature of the NF1 protein illuminates its importance in nervous system development, tumor formation and behavioral plasticity, and may also explain the wide range of clinical manifestations in neurofibromatosis type I (Hannan, 2006).

Reduced growth of Drosophila neurofibromatosis 1 mutants reflects a non-cell-autonomous requirement for GTPase-Activating Protein activity in larval neurons

Neurofibromatosis type 1 (NF1) is among the most common genetic disorders of humans and is caused by loss of neurofibromin, a large and highly conserved protein whose only known function is to serve as a GTPase-Activating Protein (GAP) for Ras. However, most Drosophila NF1 mutant phenotypes, including an overall growth deficiency, are not readily modified by manipulating Ras signaling strength, but are rescued by increasing signaling through the cAMP-dependent protein kinase A pathway. This has led to suggestions that NF1 has distinct Ras- and cAMP-related functions. This study reports that the Drosophila NF1 growth defect reflects a non-cell-autonomous requirement for NF1 in larval neurons that express the R-Ras ortholog Ras2, that NF1 is a GAP for Ras1 and Ras2, and that a functional NF1-GAP catalytic domain is both necessary and sufficient for rescue. Moreover, a Drosophila p120RasGAP ortholog, when expressed in the appropriate cells, can substitute for NF1 in growth regulation. These results show that loss of NF1 can give rise to non-cell-autonomous developmental defects, implicate aberrant Ras-mediated signaling in larval neurons as the primary cause of the NF1 growth deficiency, and argue against the notion that neurofibromin has separable Ras- and cAMP-related functions (Walker, 2006).

Enhancing the GTPase activity of Ras family members is the only known biochemical activity of neurofibromin, the protein defective in patients with NF1. This has focused much attention on manipulating Ras signaling as a way to correct the diverse symptoms of NF1. However, most Drosophila NF1 phenotypes lack dosage-sensitive genetic interactions with mutants that affect signaling by Ras1, the single fly ortholog of mammalian H-Ras, K-Ras, and N-Ras. Rather, an NF1 mutant growth deficiency, an electrophysiological defect, and a defect in olfactory learning are rescued by manipulations that increase signaling through the cAMP/PKA pathway. These findings have led to suggestions that neurofibromin may affect cAMP/PKA signaling in a Ras-independent manner, a hypothesis supported by a recent report that human NF1 suppresses Drosophila NF1 mutant size independent of GAP activity. In contrast, the current experiments using Drosophila NF1 transgenes suggest that loss of RasGAP activity is inseparable from the NF1 size defect. The reason for this discrepancy remains unclear, but may reflect inappropriate interactions between human neurofibromin and Drosophila GTPases or other proteins involved in growth regulation (Walker, 2006).

The current results show that the impaired growth of Drosophila mutants reflects a non-cell-autonomous role for NF1 in larval neurons. While runting is relatively common in mutant mice, it has been noted that mice engineered to specifically lack neuronal Nf1 expression are small. Growth in Drosophila proceeds during three larval instars that culminate in pupariation, pupation, and adult eclosion. As in other animals, growth is affected by feeding, which in Drosophila occurs during the first two and most of the third larval instar. Early in the third instar, larvae reach what is known as critical weight, a point at which holometabolous insects commit to metamorphosis and can develop without further feeding. Two neuroendocrine pathways have been implicated in coordinating feeding with Drosophila development and overall growth, but the results argue against obvious roles for NF1 in either one. Perhaps the best-understood growth-related pathway involves Drosophila insulin-like proteins (dILPs), three of which are produced -- two in a nutrient-dependent manner -- by bilateral symmetric groups of seven neurosecretory cells in the pars intercerebralis of the larval CNS. Ablating these cells causes a severe growth defect that is rescued by expression of a dILP2 transgene. In peripheral tissues, dILPs activate the insulin receptor, leading to the phosphorylation of Chico and the recruitment of a class I PI3 kinase, consisting of Dp110 catalytic and p60 regulatory subunits. Genetic manipulations that increase signaling through this pathway increase the size of peripheral tissues in a cell-autonomous manner, whereas loss-of-function mutations have the opposite effect. Recently, insulin was found to control developmental timing, but not body or organ size, during the period before Drosophila achieves critical weight, whereas after reaching this set point insulin no longer affected developmental timing, but only body and organ size. Analysis of mutant development and behavior found no differences in feeding or developmental timing between NF1 mutants and isogenic controls. Moreover, the lack of dosage-sensitive genetic interactions between NF1 and PI3 kinase p60 or Tor mutants, and the observation that dILP2-GAL4-driven UAS-NF1 expression in insulin-producing neuroendocrine cells does not modify NF1 size, all argue that insulin deficiency is not likely to be a major contributor to the NF1 size defect (Walker, 2006).

Drosophila growth and development are also coordinated by a hormonal cascade involving juvenile hormone (JH), prothoracicotrophic hormone (PTTH), and ecdysone. JH and ecdysone are produced by the corpora allata and the thoracic gland, respectively, which together with the corpora cardiaca form the neuroendocrine ring gland. PTTH stimulates ecdysone release and is made by neurons that innervate the thoracic gland in response to a developmentally controlled reduction in JH titer. JH production, in turn, is controlled by insulin, explaining the developmental delay and increased longevity of some hypomorphic insulin pathway mutants. It has recently been reported that increasing the size of the prothoracic gland by manipulations that activate Ras1 or its Dp110 PI3 kinase effector impairs Drosophila growth, possibly through ecdysone-mediated attenuation of insulin signaling in peripheral tissues. Again, the inability to modify NF1 size by expressing UAS-NF1 in the prothoracic gland, in other parts of the ring gland, or in neurons that innervate the ring gland suggests that excess Ras activity resulting from a loss of NF1 in these cells or tissues does not provide an easy explanation for the impaired growth of NF1 mutants. Further arguing against such a role, no obvious NF1 expression was detected in the ring gland (Walker, 2006).

Ras2-GAL4 is among the most restricted drivers that rescue NF1 size when driving UAS-NF1. This fact, combined with the observation that neuronal but not glial drivers similarly rescue, suggests that Ras2-GAL4-expressing cells are neuronal. It remains unclear in what proportion of these cells NF1 is required to restore growth, but costaining experiments revealed substantial overlap between endogenous NF1 and Ras2-GAL4-driven UAS-GFP expression. Moreover, Ras2-GAL4-driven UAS-NF1 expression strongly suppressed the larval CNS p-ERK phenotype. Several other findings support the conclusion that a Ras signaling defect in Ras2-GAL4-expressing cells is the primary cause of the NF1 size defect. (1) Ras2-GAL4-driven expression of a functional NF1 GAP-related domain (GRD) is necessary and sufficient for rescue. (2) Ras2-GAL4-driven expression of activated Ras1 or Ras2 phenocopied the NF1 size defect. (3) Ras2-GAL4-driven expression of a Drosophila p120RasGAP ortholog also rescued, arguing that the ability to rescue reflects a property shared between NF1 and RasGAP. Interestingly, expression of a third Drosophila RasGAP, Gap1, did not rescue either size or p-ERK phenotypes. Whether the inability of Gap1 to substitute for NF1 reflects an inappropriate expression level or some other factor (such as different regulation, localization, or GTPase substrate specificity) remains to be determined (Walker, 2006).

Initial reports that increasing cAMP/PKA activity rescues Drosophila NF1 phenotypes has generated much interest, in part because cAMP plays a prominent role in learning, which is impaired in many children with NF1. However, subsequent studies showed that genetic or pharmacologic manipulations that attenuate Ras signaling restored learning in heterozygous Nf1 mutant mice. Altered Ras signaling in the CNS appears capable of regulating the growth of the larval epidermis and imaginal discs. This could occur by modulating the levels of diffusible growth factors or growth inhibitors. Conceivably, cAMP/PKA signaling could be of importance at a more downstream component of this pathway, such as the release of, or response to, such diffusible factors (Walker, 2006).

The current results also demonstrate that heterozygous loss of individual genes encoding canonical Ras pathway components is insufficient to restore p-ERK activity in homozygous null or hypomorphic NF1 mutants. Interestingly, combined loss of Raf and rl, Ras1 and Raf, and Ras2 and Raf fully rescued the larval p-ERK defect, while the former two double mutants partially restored pupal size. Thus, Ras1 and Ras2 may jointly contribute to ERK activation in NF1-deficient CNS. Whether Ras effectors other than Raf/ERK contribute to the NF1 size defect, and how enhanced PKA activity rescues NF1 phenotypes remain to be determined (Walker, 2006).

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date revised: 5 March 2006

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